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Farahani RM. Neural differentiation in perspective: mitochondria as early programmers. Front Neurosci 2025; 18:1529855. [PMID: 39844856 PMCID: PMC11751005 DOI: 10.3389/fnins.2024.1529855] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2024] [Accepted: 12/23/2024] [Indexed: 01/24/2025] Open
Abstract
Neural differentiation during development of the nervous system has been extensively studied for decades. These efforts have culminated in the generation of a detailed map of developmental events that appear to be associated with emergence of committed cells in the nervous system. In this review the landscape of neural differentiation is revisited by focusing on abiotic signals that play a role in induction of neural differentiation. Evidence is presented regarding a chimeric landscape whereby abiotic signals generated by mitochondria orchestrate early events during neural differentiation. This early stage, characterised by mitochondrial hyperactivity, in turn triggers a late stage of differentiation by reprogramming the activity of biotic signals.
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Affiliation(s)
- Ramin M. Farahani
- IDR/Research and Education Network, Westmead, NSW, Australia
- Faculty of Medicine and Health, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia
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2
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Romero-Moreno R, Czowski BJ, Harris L, Kuehn JF, White KA. Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells. J Biol Chem 2024; 300:107658. [PMID: 39128712 PMCID: PMC11489351 DOI: 10.1016/j.jbc.2024.107658] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Accepted: 07/31/2024] [Indexed: 08/13/2024] Open
Abstract
Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch homolog 1 protein expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch homolog 1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.
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Affiliation(s)
- Ricardo Romero-Moreno
- Harper Cancer Research Institute, South Bend, Indiana, USA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Brandon J Czowski
- Harper Cancer Research Institute, South Bend, Indiana, USA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Lindsey Harris
- Harper Cancer Research Institute, South Bend, Indiana, USA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Jessamine F Kuehn
- Harper Cancer Research Institute, South Bend, Indiana, USA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA
| | - Katharine A White
- Harper Cancer Research Institute, South Bend, Indiana, USA; Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
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Van Dyck PK, Piszkin L, Gorski EA, Nascimento ET, Abebe JA, Hoffmann LM, Peng JW, White KA. Ionizable networks mediate pH-dependent allostery in SH2 signaling proteins. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.08.21.608875. [PMID: 39229188 PMCID: PMC11370553 DOI: 10.1101/2024.08.21.608875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 09/05/2024]
Abstract
IntroductionTransient intracellular pH dynamics1regulate mammalian proliferation2,3, migration4, and differentiation5. However, for many pH-dependent cell processes, the molecular mediators are unknown6. Prior work identified histidine residues as molecular switches in pH-sensitive proteins, but how other ionizable residues contribute to pH-dependent protein allostery is understudied. Here, we develop anin silicocomputational pipeline to identify putative pH-sensitive proteins and their molecular mechanisms. We first apply this pipeline to SHP2, a known pH-sensitive signaling protein with an uncharacterized molecular mechanism. We show wild-type SHP2 phosphatase activity is pH-sensitivein vitroand in cells, and mutation of identified H116 and E252 to non-titratable alanine residues abolishes pH-sensitive function. We also show that c-Src is a previously unrecognized pH-dependent kinase, and mutation of the identified ionizable network again abolishes pH-sensitive activity. Constant pH molecular dynamics simulations support a conserved allosteric mechanism of pH-dependent binding of inhibitory SH2 domains to the functional catalytic domains of SHP2 and c-Src. We apply our computational pipeline across SH2 domain-containing signaling proteins and identify evolutionarily conserved putative pH-sensing networks. Our results reveal that pH is an allosteric regulator of SH2 domain-containing signaling proteins providing insight into normal pH-dependent cell biology and diseases where pHi is dysregulated, such as cancer.
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Affiliation(s)
- Papa Kobina Van Dyck
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
| | - Luke Piszkin
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
| | - Elijah A. Gorski
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
| | - Eduarda Tartarella Nascimento
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
- Saint Mary’s College, Notre Dame, IN 46556 USA
| | - Joshua A. Abebe
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
| | - Logan M. Hoffmann
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
| | - Jeffrey W. Peng
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
| | - Katharine A. White
- Department of Chemistry and Biochemistry, University of Notre Dame 251 Nieuwland Science Hall Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame 1234 N. Notre Dame Avenue South Bend, IN 46617 USA
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4
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Didan Y, Ghomlaghi M, Nguyen LK, Ng DCH. Stress pathway outputs are encoded by pH-dependent clustering of kinase components. Nat Commun 2024; 15:6614. [PMID: 39103333 DOI: 10.1038/s41467-024-50638-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2023] [Accepted: 07/10/2024] [Indexed: 08/07/2024] Open
Abstract
Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses.
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Affiliation(s)
- Yuliia Didan
- School of Biomedical Science, Faculty of Medicine, University of Queensland; St Lucia, Brisbane, Australia
| | - Milad Ghomlaghi
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia
- Biomedicine Discovery Institute, Monash University, Clayton, Australia
| | - Lan K Nguyen
- Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia
- Biomedicine Discovery Institute, Monash University, Clayton, Australia
| | - Dominic C H Ng
- School of Biomedical Science, Faculty of Medicine, University of Queensland; St Lucia, Brisbane, Australia.
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5
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Peralta J, DuPriest B, Orozco D, Pacheco JR, Martins L, Soriano RA, Wong A, Wong R, Grillo-Hill B. Drosophila Nhe2 overexpression induces autophagic cell death. Mol Biol Cell 2024; 35:br13. [PMID: 38696256 PMCID: PMC11244158 DOI: 10.1091/mbc.e24-02-0058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Revised: 04/19/2024] [Accepted: 04/24/2024] [Indexed: 05/04/2024] Open
Abstract
Autophagy is a conserved catabolic process where double membrane-bound structures form around macromolecules or organelles targeted for degradation. Autophagosomes fuse with lysosomes to facilitate degradation and macromolecule recycling for homeostasis or growth in a cell autonomous manner. In cancer cells, autophagy is often up-regulated and helps cancer cells survive nutrient deprivation and stressful growth conditions. Here, we propose that the increased intracellular pH (pHi) common to cancer cells is sufficient to induce autophagic cell death. We previously developed tools to increase pHi in the Drosophila eye via overexpression of DNhe2, resulting in aberrant patterning and reduced tissue size. We examined fly eyes at earlier stages of development and found fewer interommatidial cells. We next tested whether this decrease in cell number was due to increased cell death. We found that the DNhe2-induced cell death was caspase independent, which is inconsistent with apoptosis. However, this cell death required autophagy genes, which supports autophagy as the mode of cell death. We also found that expression of molecular markers supports increased autophagy. Together, our findings suggest new roles for ion transport proteins in regulating conserved, critical developmental processes and provide evidence for new paradigms in growth control.
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Affiliation(s)
- Jobelle Peralta
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Blake DuPriest
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Daniel Orozco
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Juan Reyna Pacheco
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Laura Martins
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Rachel Ann Soriano
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Alan Wong
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Ramy Wong
- Department of Biological Sciences, San José State University, San José, CA 95112
| | - Bree Grillo-Hill
- Department of Biological Sciences, San José State University, San José, CA 95112
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6
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Lund LM, Marchi AN, Alderfer L, Hall E, Hammer J, Trull KJ, Hanjaya-Putra D, White KA. Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.04.597454. [PMID: 38895391 PMCID: PMC11185592 DOI: 10.1101/2024.06.04.597454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/21/2024]
Abstract
Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize β-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in β-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.
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Affiliation(s)
- Leah M Lund
- Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
| | - Angelina N Marchi
- Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
| | - Laura Alderfer
- Bioengineering Graduate Program, Aerospace and Mechanical Engineering, University of Notre Dame, 153 Multidisciplinary Engineering Research Building, Notre Dame, IN 46556 USA
- Current: Vivodyne, Suite 775 601 Walnut Street, Philadelphia PA 19106 USA
| | - Eva Hall
- Bioengineering Graduate Program, Aerospace and Mechanical Engineering, University of Notre Dame, 153 Multidisciplinary Engineering Research Building, Notre Dame, IN 46556 USA
| | - Jacob Hammer
- Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
| | - Keelan J Trull
- Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
| | - Donny Hanjaya-Putra
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
- Bioengineering Graduate Program, Aerospace and Mechanical Engineering, University of Notre Dame, 153 Multidisciplinary Engineering Research Building, Notre Dame, IN 46556 USA
- Chemical and Biomolecular Engineering, University of Notre Dame, 250 Nieuwland Hall, Notre Dame, IN 46556 USA
| | - Katharine A White
- Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, IN 46556 USA
- Harper Cancer Research Institute, University of Notre Dame, 1234 N. Notre Dame Avenue, South Bend, IN 46617 USA
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Shi R, Lu W, Yang J, Ma S, Wang A, Sun L, Xia Q, Zhao P. Ectopic expression of BmeryCA in Bombyx mori increases silk yield and mechanical properties by altering the pH of posterior silk gland. Int J Biol Macromol 2024; 271:132695. [PMID: 38810858 DOI: 10.1016/j.ijbiomac.2024.132695] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2024] [Revised: 05/14/2024] [Accepted: 05/25/2024] [Indexed: 05/31/2024]
Abstract
The silk glands are the specialized tissue where silk protein synthesis, secretion, and conformational transitions take place, with pH playing a critical role in both silk protein synthesis and fiber formation. In the present study, we have identified erythrocyte carbonic anhydrase (BmeryCA) belonging to the α-CA class in the silk gland, which is a Zn2+ dependent metalloenzyme capable of efficiently and reversibly catalyzing the hydrated reaction of CO2 to HCO3-, thus participating in the regulation of acid-base balance. Multiple sequence alignments revealed that the active site of BmeryCA was highly conserved. Tissue expression profiling showed that BmeryCA had relatively high expression levels in hemolymph and epidermis but is barely expressed in the posterior silk gland (PSG). By specifically overexpressing BmeryCA in the PSG, we generated transgenic silkworms. Ion-selective microelectrode (ISM) measurements demonstrated that specifically overexpression of BmeryCA in the PSG led to a shift in pH from weakly alkaline to slightly neutral conditions. Moreover, the resultant PSG-specific BmeryCA overexpression mutant strain displayed a significant increase in both silk yield and silk fiber mechanical properties. Our research provided new insights into enhancing silk yield and improving the mechanical properties of silk fibers.
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Affiliation(s)
- Run Shi
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Wei Lu
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Jie Yang
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Sanyuan Ma
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Aoming Wang
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Le Sun
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Qingyou Xia
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China
| | - Ping Zhao
- Integrative Science Center of Germplasm Creation in Western China (CHONGQING) Science City, Biological Science Research Center, Southwest University, Chongqing, China.
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8
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Kisor KP, Ruiz DG, Jacobson MP, Barber DL. A role for pH dynamics regulating transcription factor DNA binding selectivity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.05.21.595212. [PMID: 38826444 PMCID: PMC11142074 DOI: 10.1101/2024.05.21.595212] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/04/2024]
Abstract
Intracellular pH (pHi) dynamics regulates diverse cell processes such as proliferation, dysplasia, and differentiation, often mediated by the protonation state of a functionally critical histidine residue in endogenous pH sensing proteins. How pHi dynamics can directly regulate gene expression and whether transcription factors can function as pH sensors has received limited attention. We tested the prediction that transcription factors with a histidine in their DNA binding domain (DBD) that forms hydrogen bonds with nucleotides can have pH-regulated activity, which is relevant to more than 85 transcription factors in distinct families, including FOX, KLF, SOX and MITF/Myc. Focusing on FOX family transcription factors, we used unbiased SELEX-seq to identify pH-dependent DNA binding motif preferences, then confirm pH-regulated binding affinities for FOXC2, FOXM1, and FOXN1 to a canonical FkhP DNA motif that are 2.5 to 7.5 greater at pH 7.0 compared with pH 7.5. For FOXC2, we also find greater activity for an FkhP motif at lower pHi in cells and that pH-regulated binding and activity are dependent on a conserved histidine (His122) in the DBD. RNA-seq with FOXC2 also reveals pH-dependent differences in enriched promoter motifs. Our findings identify pH-regulated transcription factor-DNA binding selectivity with relevance to how pHi dynamics can regulate gene expression for myriad cell behaviours.
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9
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Zimmerman SG, Berg CA. CO2 exposure drives a rapid pH response in live adult Drosophila. PLoS One 2024; 19:e0302240. [PMID: 38625910 PMCID: PMC11020609 DOI: 10.1371/journal.pone.0302240] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2023] [Accepted: 03/29/2024] [Indexed: 04/18/2024] Open
Abstract
CO2 anesthesia is the most common method for immobilizing Drosophila for research purposes. But CO2 exposure has consequences-it can impact fertility, behavior, morphogenesis, and cytoskeletal dynamics. In this respect, Drosophila is an outstanding model for studying the impact of CO2 exposure on tissues. In this study we explored the response of intracellular pH (pHi) to a one-minute CO2 pulse using a genetically encoded, ubiquitously expressed pH sensor, tpHusion, to monitor pHi within a live, intact, whole fly. We compared wild-type flies to flies lacking Imaginal disc growth factors (Idgfs), which are chitinase-like proteins that facilitate developmental processes and the innate immune response. Morphogenetic and cytoskeletal defects in Idgf-null flies are enhanced after CO2 exposure. We found that pHi drops sharply within seconds of the beginning of a CO2 pulse and recovers over several minutes. The initial profile was nearly identical in control and Idgf-null flies but diverged as the pHi returned to normal. This study demonstrates the feasibility of monitoring pH in live adult Drosophila. Studies exploring pH homeostasis are important for understanding human pathologies associated with pH dysregulation.
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Affiliation(s)
- Sandra G. Zimmerman
- Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America
| | - Celeste A. Berg
- Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America
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10
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Pedersen SHF. Acid-base transporters in the context of tumor heterogeneity. Pflugers Arch 2024; 476:689-701. [PMID: 38332178 DOI: 10.1007/s00424-024-02918-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 01/20/2024] [Accepted: 01/29/2024] [Indexed: 02/10/2024]
Abstract
The copious metabolic acid production and -extrusion by cancer cells render poorly vascularized regions of solid tumors highly acidic. A growing list of proton - and bicarbonate transporters has been suggested to contribute to net acid extrusion from cancer cells, and/or been shown to be dysregulated and favor malignant development in various cancers. The great majority of these roles have been studied at the level of the cancer cells. However, recent advances in understanding of the cellular and physicochemical heterogeneity of solid tumors both enable and necessitate a reexamination of the regulation and roles of acid-base transporters in such malignancies. This review will briefly summarize the state-of-the-art, with a focus on the SLC9A and SLC4A families, for which most evidence is available. This is followed by a discussion of key concepts and open questions arising from recent insights and of the challenges that need to be tackled to address them. Finally, opportunities and challenges in therapeutic targeting of the acid-base transportome in cancers will be addressed.
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Affiliation(s)
- Stine Helene Falsig Pedersen
- Section for Cell Biology and Physiology, Department of Biology, Faculty of Science, University of Copenhagen, Universitetsparken 13, 2100, Copenhagen, Denmark.
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11
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Czowski BJ, White KA. Intracellular pH regulates β-catenin with low pHi increasing adhesion and signaling functions. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.03.22.586349. [PMID: 38585883 PMCID: PMC10996556 DOI: 10.1101/2024.03.22.586349] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/09/2024]
Abstract
Intracellular pH (pHi) dynamics are linked to cell processes including proliferation, migration, and differentiation. The adherens junction (AJ) and signaling protein β-catenin has decreased abundance at high pHi due to increased proteasomal-mediated degradation. However, the effects of low pHi on β-catenin abundance and functions have not been characterized. Here, we show that low pHi stabilizes β-catenin in epithelial cells using population-level and single-cell assays. β-catenin abundance is increased at low pHi and decreased at high pHi. We also assay single-cell protein degradation rates to show that β-catenin half-life is longer at low compared to high pHi. Importantly, we show that AJs are not disrupted by β-catenin loss at high pHi due to rescue by plakoglobin. Finally, we show that low pHi increases β-catenin transcriptional activity in single cells and is indistinguishable from a Wnt-on state. This work characterizes pHi as a rheostat regulating β-catenin abundance, stability, and function and implicates β-catenin as a molecular mediator of pHi-dependent cell processes.
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Affiliation(s)
- Brandon J Czowski
- Department of Chemistry and Biochemistry, University of Notre Dame
- Harper Cancer Research Institute, University of Notre Dame
| | - Katharine A White
- Department of Chemistry and Biochemistry, University of Notre Dame
- Harper Cancer Research Institute, University of Notre Dame
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12
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Uttekar B, Verma RK, Tomer D, Rikhy R. Mitochondrial morphology dynamics and ROS regulate apical polarity and differentiation in Drosophila follicle cells. Development 2024; 151:dev201732. [PMID: 38345270 PMCID: PMC7616099 DOI: 10.1242/dev.201732] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 01/23/2024] [Indexed: 03/01/2024]
Abstract
Mitochondrial morphology dynamics regulate signaling pathways during epithelial cell formation and differentiation. The mitochondrial fission protein Drp1 affects the appropriate activation of EGFR and Notch signaling-driven differentiation of posterior follicle cells in Drosophila oogenesis. The mechanisms by which Drp1 regulates epithelial polarity during differentiation are not known. In this study, we show that Drp1-depleted follicle cells are constricted in early stages and present in multiple layers at later stages with decreased levels of apical polarity protein aPKC. These defects are suppressed by additional depletion of mitochondrial fusion protein Opa1. Opa1 depletion leads to mitochondrial fragmentation and increased reactive oxygen species (ROS) in follicle cells. We find that increasing ROS by depleting the ROS scavengers, mitochondrial SOD2 and catalase also leads to mitochondrial fragmentation. Further, the loss of Opa1, SOD2 and catalase partially restores the defects in epithelial polarity and aPKC, along with EGFR and Notch signaling in Drp1-depleted follicle cells. Our results show a crucial interaction between mitochondrial morphology, ROS generation and epithelial cell polarity formation during the differentiation of follicle epithelial cells in Drosophila oogenesis.
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Affiliation(s)
- Bhavin Uttekar
- Biology, Indian Institute of Science Education and Research, Homi Bhabha Road, Pashan, Pune 411008, India
| | - Rahul Kumar Verma
- Biology, Indian Institute of Science Education and Research, Homi Bhabha Road, Pashan, Pune 411008, India
| | - Darshika Tomer
- Biology, Indian Institute of Science Education and Research, Homi Bhabha Road, Pashan, Pune 411008, India
| | - Richa Rikhy
- Biology, Indian Institute of Science Education and Research, Homi Bhabha Road, Pashan, Pune 411008, India
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Abstract
Cancers undergo sequential changes to proton (H+) concentration and sensing that are consequences of the disease and facilitate its further progression. The impact of protonation state on protein activity can arise from alterations to amino acids or their titration. Indeed, many cancer-initiating mutations influence pH balance, regulation or sensing in a manner that enables growth and invasion outside normal constraints as part of oncogenic transformation. These cancer-supporting effects become more prominent when tumours develop an acidic microenvironment owing to metabolic reprogramming and disordered perfusion. The ensuing intracellular and extracellular pH disturbances affect multiple aspects of tumour biology, ranging from proliferation to immune surveillance, and can even facilitate further mutagenesis. As a selection pressure, extracellular acidosis accelerates disease progression by favouring acid-resistant cancer cells, which are typically associated with aggressive phenotypes. Although acid-base disturbances in tumours often occur alongside hypoxia and lactate accumulation, there is now ample evidence for a distinct role of H+-operated responses in key events underpinning cancer. The breadth of these actions presents therapeutic opportunities to change the trajectory of disease.
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Affiliation(s)
- Pawel Swietach
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK.
| | - Ebbe Boedtkjer
- Department of Biomedicine, Aarhus University, Aarhus, Denmark.
| | - Stine Falsig Pedersen
- Department of Biology, University of Copenhagen, University of Copenhagen, Faculty of Science, København, Denmark.
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Mieville V, Griffioen AW, Benamran D, Nowak-Sliwinska P. Advanced in vitro models for renal cell carcinoma therapy design. Biochim Biophys Acta Rev Cancer 2023; 1878:188942. [PMID: 37343729 DOI: 10.1016/j.bbcan.2023.188942] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2023] [Revised: 06/14/2023] [Accepted: 06/15/2023] [Indexed: 06/23/2023]
Abstract
Renal cell carcinoma (RCC) and its principal subtype, clear cell RCC, are the most diagnosed kidney cancer. Despite substantial improvement over the last decades, current pharmacological intervention still fails to achieve long-term therapeutic success. RCC is characterized by a high intra- and inter-tumoral heterogeneity and is heavily influenced by the crosstalk of the cells composing the tumor microenvironment, such as cancer-associated fibroblasts, endothelial cells and immune cells. Moreover, multiple physicochemical properties such as pH, interstitial pressure or oxygenation may also play an important role. These elements are often poorly recapitulated in in vitro models used for drug development. This inadequate recapitulation of the tumor is partially responsible for the current lack of an effective and curative treatment. Therefore, there are needs for more complex in vitro or ex vivo drug screening models. In this review, we discuss the current state-of-the-art of RCC models and suggest strategies for their further development.
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Affiliation(s)
- Valentin Mieville
- School of Pharmaceutical Sciences, Faculty of Sciences, University of Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland; Translational Research Center in Oncohaematology, Geneva, Switzerland
| | - Arjan W Griffioen
- Angiogenesis Laboratory, Department of Medical Oncology, Amsterdam UMC, Cancer Center Amsterdam, Amsterdam, The Netherlands
| | - Daniel Benamran
- Division of Urology, Geneva University Hospitals, Geneva, Switzerland
| | - Patrycja Nowak-Sliwinska
- School of Pharmaceutical Sciences, Faculty of Sciences, University of Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland; Translational Research Center in Oncohaematology, Geneva, Switzerland.
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15
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Di Molfetta D, Cannone S, Greco MR, Caroppo R, Piccapane F, Carvalho TMA, Altamura C, Saltarella I, Tavares Valente D, Desaphy JF, Reshkin SJ, Cardone RA. ECM Composition Differentially Regulates Intracellular and Extracellular pH in Normal and Cancer Pancreatic Duct Epithelial Cells. Int J Mol Sci 2023; 24:10632. [PMID: 37445810 DOI: 10.3390/ijms241310632] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2023] [Revised: 06/07/2023] [Accepted: 06/17/2023] [Indexed: 07/15/2023] Open
Abstract
Intracellular pH (pHi) regulation is a challenge for the exocrine pancreas, where the luminal secretion of bicarbonate-rich fluid is accompanied by interstitial flows of acid. This acid-base transport requires a plethora of ion transporters, including bicarbonate transporters and the Na+/H+ exchanger isoform 1 (NHE1), which are dysregulated in Pancreatic Ductal Adenocarcinoma (PDAC). PDAC progression is favored by a Collagen-I rich extracellular matrix (ECM) which exacerbates the physiological interstitial acidosis. In organotypic cultures of normal human pancreatic cells (HPDE), parenchymal cancer cells (CPCs) and cancer stem cells (CSCs) growing on matrices reproducing ECM changes during progression, we studied resting pHi, the pHi response to fluxes of NaHCO3 and acidosis and the role of NHE1 in pHi regulation. Our findings show that: (i) on the physiological ECM, HPDE cells have the most alkaline pHi, followed by CSCs and CPCs, while a Collagen I-rich ECM reverses the acid-base balance in cancer cells compared to normal cells; (ii) both resting pHi and pHi recovery from an acid load are reduced by extracellular NaHCO3, especially in HPDE cells on a normal ECM; (iii) cancer cell NHE1 activity is less affected by NaHCO3. We conclude that ECM composition and the fluctuations of pHe cooperate to predispose pHi homeostasis towards the presence of NaHCO3 gradients similar to that expected in the tumor.
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Affiliation(s)
- Daria Di Molfetta
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Stefania Cannone
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Maria Raffaella Greco
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Rosa Caroppo
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Francesca Piccapane
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | | | - Concetta Altamura
- Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy
| | - Ilaria Saltarella
- Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy
| | - Diana Tavares Valente
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
| | - Jean Francois Desaphy
- Department of Biomedical Sciences and Human Oncology, School of Medicine, University of Bari Aldo Moro, 70124 Bari, Italy
| | - Stephan J Reshkin
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
| | - Rosa Angela Cardone
- Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy
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16
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Liu Y, Reyes E, Castillo-Azofeifa D, Klein OD, Nystul T, Barber DL. Intracellular pH dynamics regulates intestinal stem cell lineage specification. Nat Commun 2023; 14:3745. [PMID: 37353491 PMCID: PMC10290085 DOI: 10.1038/s41467-023-39312-9] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Accepted: 06/06/2023] [Indexed: 06/25/2023] Open
Abstract
Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H+ efflux by Na+/H+ exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage.
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Affiliation(s)
- Yi Liu
- Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Efren Reyes
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA, 94143, USA
| | - David Castillo-Azofeifa
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA, 94143, USA
- Immunology Discovery, Genentech, Inc., South San Francisco, CA, 94080, USA
| | - Ophir D Klein
- Program in Craniofacial Biology and Department of Orofacial Sciences, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Todd Nystul
- Departments of Anatomy, University of California San Francisco, San Francisco, CA, 94143, USA.
| | - Diane L Barber
- Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA, 94143, USA.
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17
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Kumar S, Vassallo JD, Nattamai KJ, Hassan A, Karns R, Vollmer A, Soller K, Sakk V, Sacma M, Nemkov T, D'Alessandro A, Geiger H. pH regulates hematopoietic stem cell potential via polyamines. EMBO Rep 2023; 24:e55373. [PMID: 36943011 PMCID: PMC10157373 DOI: 10.15252/embr.202255373] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Revised: 02/21/2023] [Accepted: 03/03/2023] [Indexed: 03/23/2023] Open
Abstract
Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.
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Affiliation(s)
- Sachin Kumar
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA
- Pharmacology Division, CSIR-Central Drug Research Institute, Lucknow, India
| | - Jeffrey D Vassallo
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA
| | - Kalpana J Nattamai
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA
| | - Aishlin Hassan
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA
| | - Rebekah Karns
- Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH, USA
| | | | - Karin Soller
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Vadim Sakk
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Mehmet Sacma
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
| | - Travis Nemkov
- University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, USA
| | | | - Hartmut Geiger
- Institute of Molecular Medicine, Ulm University, Ulm, Germany
- Aging Research Center, Ulm University, Ulm, Germany
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18
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Sustar AE, Strand LG, Zimmerman SG, Berg CA. Imaginal disk growth factors are Drosophila chitinase-like proteins with roles in morphogenesis and CO2 response. Genetics 2023; 223:iyac185. [PMID: 36576887 PMCID: PMC9910413 DOI: 10.1093/genetics/iyac185] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 07/18/2022] [Accepted: 11/16/2022] [Indexed: 12/29/2022] Open
Abstract
Chitinase-like proteins (CLPs) are members of the family 18 glycosyl hydrolases, which include chitinases and the enzymatically inactive CLPs. A mutation in the enzyme's catalytic site, conserved in vertebrates and invertebrates, allowed CLPs to evolve independently with functions that do not require chitinase activity. CLPs normally function during inflammatory responses, wound healing, and host defense, but when they persist at excessive levels at sites of chronic inflammation and in tissue-remodeling disorders, they correlate positively with disease progression and poor prognosis. Little is known, however, about their physiological function. Drosophila melanogaster has 6 CLPs, termed Imaginal disk growth factors (Idgfs), encoded by Idgf1, Idgf2, Idgf3, Idgf4, Idgf5, and Idgf6. In this study, we developed tools to facilitate characterization of the physiological roles of the Idgfs by deleting each of the Idgf genes using the CRISPR/Cas9 system and assessing loss-of-function phenotypes. Using null lines, we showed that loss of function for all 6 Idgf proteins significantly lowers viability and fertility. We also showed that Idgfs play roles in epithelial morphogenesis, maintaining proper epithelial architecture and cell shape, regulating E-cadherin and cortical actin, and remarkably, protecting these tissues against CO2 exposure. Defining the normal molecular mechanisms of CLPs is a key to understanding how deviations tip the balance from a physiological to a pathological state.
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Affiliation(s)
- Anne E Sustar
- Department of Genome Sciences, University of Washington, Foege Bldg. S-250, 3720 15th Ave NE, Seattle, WA 98195-5065, USA
| | - Liesl G Strand
- Department of Genome Sciences, University of Washington, Foege Bldg. S-250, 3720 15th Ave NE, Seattle, WA 98195-5065, USA
| | - Sandra G Zimmerman
- Department of Genome Sciences, University of Washington, Foege Bldg. S-250, 3720 15th Ave NE, Seattle, WA 98195-5065, USA
| | - Celeste A Berg
- Department of Genome Sciences, University of Washington, Foege Bldg. S-250, 3720 15th Ave NE, Seattle, WA 98195-5065, USA
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19
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Song Y, Zhang Y, Qu Q, Zhang X, Lu T, Xu J, Ma W, Zhu M, Huang C, Xiong R. Biomaterials based on hyaluronic acid, collagen and peptides for three-dimensional cell culture and their application in stem cell differentiation. Int J Biol Macromol 2023; 226:14-36. [PMID: 36436602 DOI: 10.1016/j.ijbiomac.2022.11.213] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2022] [Revised: 11/17/2022] [Accepted: 11/21/2022] [Indexed: 11/27/2022]
Abstract
In recent decades, three-dimensional (3D) cell culture technologies have been developed rapidly in the field of tissue engineering and regeneration, and have shown unique advantages and great prospects in the differentiation of stem cells. Herein, the article reviews the progress and advantages of 3D cell culture technologies in the field of stem cell differentiation. Firstly, 3D cell culture technologies are divided into two main categories: scaffoldless and scaffolds. Secondly, the effects of hydrogels scaffolds and porous scaffolds on stem cell differentiation in the scaffold category were mainly reviewed. Among them, hydrogels scaffolds are divided into natural hydrogels and synthetic hydrogels. Natural materials include polysaccharides, proteins, and their derivatives, focusing on hyaluronic acid, collagen and polypeptides. Synthetic materials mainly include polyethylene glycol (PEG), polyacrylic acid (PAA), polyvinyl alcohol (PVA), etc. In addition, since the preparation techniques have a large impact on the properties of porous scaffolds, several techniques for preparing porous scaffolds based on different macromolecular materials are reviewed. Finally, the future prospects and challenges of 3D cell culture in the field of stem cell differentiation are reviewed. This review will provide a useful guideline for the selection of materials and techniques for 3D cell culture in stem cell differentiation.
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Affiliation(s)
- Yuanyuan Song
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Yingying Zhang
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Qingli Qu
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Xiaoli Zhang
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Tao Lu
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Jianhua Xu
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Wenjing Ma
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Miaomiao Zhu
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China
| | - Chaobo Huang
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China.
| | - Ranhua Xiong
- Joint Laboratory of Advanced Biomedical Materials (NFU-UGent), Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, College of Chemical Engineering, Nanjing Forestry University (NFU), Nanjing 210037, China.
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20
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Wang N, Zhou L, Shao CY, Wang XT, Zhang N, Ma J, Hu HL, Wang Y, Qiu M, Shen Y. Potassium channel K ir 4.1 regulates oligodendrocyte differentiation via intracellular pH regulation. Glia 2022; 70:2093-2107. [PMID: 35775976 DOI: 10.1002/glia.24240] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 06/06/2022] [Accepted: 06/24/2022] [Indexed: 11/10/2022]
Abstract
In humans, loss-of-function mutations of Kcnj10 in SeSAME/EAST syndrome, which encodes the inwardly rectifying K+ channel 4.1 (Kir 4.1), causes progressive neurological decline. Despite its rich expression in oligodendrocyte (OL) lineage cells and an emerging link with demyelinating disease, the function of Kir 4.1 in OLs is unclear. Here we show a novel role of Kir 4.1 in OL development. Kir 4.1 expression is markedly greater in OLs than in OL precursor cells (OPCs), and the down-regulation of Kir 4.1 impairs OL maturation by affecting OPC differentiation. Interestingly, Kir 4.1 regulates the intracellular pH of OPCs and OLs via the Na+ /H+ exchanger, which underlies impeded OPC differentiation by Kir 4.1 inhibition. Furthermore, Kir 4.1 regulates GSK3β and SOX10, two molecules critical to OPC development. Collectively, our work opens a new avenue to understanding the functions of Kir 4.1 and intracellular pH in OLs.
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Affiliation(s)
- Na Wang
- Department of Physiology and Department of Neurology of the First Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China
| | - Liang Zhou
- Department of Physiology and Department of Neurology of the First Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China.,Key Laboratory of Brain Science, Guizhou Institution of Higher Education, Zunyi Medical University, Zunyi, China
| | - Chong-Yu Shao
- Department of Physiology and Department of Neurology of the First Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China
| | - Xin-Tai Wang
- Department of Physiology and Department of Neurology of the First Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China
| | - Nan Zhang
- Key Laboratory of Cranial Cerebral Diseases, Department of Neurobiology of Basic Medical College, Ningxia Medical University, Yinchuan, China
| | - Jiao Ma
- Key Laboratory of Cranial Cerebral Diseases, Department of Neurobiology of Basic Medical College, Ningxia Medical University, Yinchuan, China
| | - Hai-Lan Hu
- Interdisciplinary Institute of Neuroscience and Technology, Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, China
| | - Yin Wang
- Key Laboratory of Cranial Cerebral Diseases, Department of Neurobiology of Basic Medical College, Ningxia Medical University, Yinchuan, China
| | - Mengsheng Qiu
- Institute of Life Sciences, Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China
| | - Ying Shen
- Department of Physiology and Department of Neurology of the First Affiliated Hospital, NHC and CAMS Key Laboratory of Medical Neurobiology, Zhejiang University School of Medicine, Hangzhou, China
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21
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Nikolovska K, Seidler UE, Stock C. The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity. Front Physiol 2022; 13:899286. [PMID: 35665228 PMCID: PMC9159811 DOI: 10.3389/fphys.2022.899286] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Accepted: 04/19/2022] [Indexed: 12/11/2022] Open
Abstract
The five plasma membrane Na+/H+ exchanger (NHE) isoforms in the gastrointestinal tract are characterized by distinct cellular localization, tissue distribution, inhibitor sensitivities, and physiological regulation. NHE1 (Slc9a1) is ubiquitously expressed along the gastrointestinal tract in the basolateral membrane of enterocytes, but so far, an exclusive role for NHE1 in enterocyte physiology has remained elusive. NHE2 (Slc9a2) and NHE8 (Slc9a8) are apically expressed isoforms with ubiquitous distribution along the colonic crypt axis. They are involved in pHi regulation of intestinal epithelial cells. Combined use of a knockout mouse model, intestinal organoid technology, and specific inhibitors revealed previously unrecognized actions of NHE2 and NHE8 in enterocyte proliferation and differentiation. NHE3 (Slc9a3), expressed in the apical membrane of differentiated intestinal epithelial cells, functions as the predominant nutrient-independent Na+ absorptive mechanism in the gut. The new selective NHE3 inhibitor (Tenapanor) allowed discovery of novel pathophysiological and drug-targetable NHE3 functions in cystic-fibrosis associated intestinal obstructions. NHE4, expressed in the basolateral membrane of parietal cells, is essential for parietal cell integrity and acid secretory function, through its role in cell volume regulation. This review focuses on the expression, regulation and activity of the five plasma membrane Na+/H+ exchangers in the gastrointestinal tract, emphasizing their role in maintaining intestinal homeostasis, or their impact on disease pathogenesis. We point to major open questions in identifying NHE interacting partners in central cellular pathways and processes and the necessity of determining their physiological role in a system where their endogenous expression/activity is maintained, such as organoids derived from different parts of the gastrointestinal tract.
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22
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Deguchi H, Yamashita T, Hiramoto N, Otsuki Y, Mukai A, Ueno M, Sotozono C, Kinoshita S, Hamuro J. Intracellular pH affects mitochondrial homeostasis in cultured human corneal endothelial cells prepared for cell injection therapy. Sci Rep 2022; 12:6263. [PMID: 35428816 PMCID: PMC9012833 DOI: 10.1038/s41598-022-10176-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 03/30/2022] [Indexed: 12/11/2022] Open
Abstract
This study aimed to uncover the mechanism responsible for the clinical efficacy of cell injection therapy with fully differentiated cultured cells. Analysis of polarized expression of ion transporters on cultured human corneal endothelial cells (CECs) subpopulations (SPs) was performed. The intracellular pH (pHi) between two CEC SPs, distinct in the proportion of differentiated cells, was measured, and the association with mitochondrial respiration homeostasis was investigated. The effects of the ion transporter inhibition by their selective inhibitors or siRNA transfection were also explored. Na+/K+-ATPase, Aquaporin 1, SLC4A11, NBCe1, NHE1 as transporters, and ZO-1, were all selectively expressed in differentiated SPs, but were almost null in the cell-state-transitioned SPs. We also confirmed that the pHi of CEC SPs affected their mitochondrial respiration by modulating the expression of these ion transporters via inhibitors or siRNA transfection. Ion and water transporters might participate in the maintenance of pHi and mitochondria homeostasis in differentiated SPs, which may contribute, combined with integral barrier functions, to efficient water efflux. The differences in intracellular pH between the two SPs is attributed to variations in the expression profile of specific ion transporters and mitochondrial functions, which may associate with the efficacy of the SPs in cell injection therapy.
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Affiliation(s)
- Hideto Deguchi
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Tomoko Yamashita
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Nao Hiramoto
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Yohei Otsuki
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Atsushi Mukai
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Morio Ueno
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Chie Sotozono
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan
| | - Shigeru Kinoshita
- Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Junji Hamuro
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Japan.
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23
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Hamuro J, Asada K, Ueno M, Yamashita T, Mukai A, Fujita T, Ito E, Hiramoto N, Toda M, Sotozono C, Kinoshita S. Repressed miR-34a Expression Dictates the Cell Fate to Corneal Endothelium Failure. Invest Ophthalmol Vis Sci 2022; 63:22. [PMID: 35475886 PMCID: PMC9055560 DOI: 10.1167/iovs.63.4.22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Purpose To reveal the mechanism triggering the functional disparity between degenerated and non-degenerated corneal endothelium cells in the water efflux from corneal stroma to the anterior chamber. Methods The varied levels of the microRNA (miR)-34, miR-378, and miR-146 family in human corneal endothelium and cultured cells thereof were investigated using 3D-Gene Human miRNA Oligo Chips. Concomitantly, CD44, p53, c-Myc, matrix metalloprotease (MMP)-2 expression, and Ras homolog gene family member A (Rho A) activity was correlated to the expression intensities of these microRNAs, partly complemented with their altered expression levels with the transfection of the corresponding mimics and inhibitors. The levels of miRs were further associated with intracellular pH (pHi) and mitochondrial energy homeostasis. Results P53-inducible miR-34a/b repressed CD44 expression, and CD44 was repressed with the elevated c-Myc. The repressed miR-34a activated the CD44 downstream factors Rho A and MMP-2. MiR-34a mimics downregulated pHi, inducing the skewing of mitochondrial respiration to oxidative phosphorylation. The oxidative stress (H2O2) induced on human corneal endothelial cells, which repressed miR-34a/b expression, may account for the impaired signaling cascade to mitochondrial metabolic homeostasis necessary for an efficient water efflux from the corneal stroma. Conclusions The upregulated expression of CD44, through repressed miR-34a/b by reactive oxygen species and elevated c-Myc by oxidative stress, may impair mitochondrial metabolic homeostasis, leading to human corneal endothelial failure.
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Affiliation(s)
- Junji Hamuro
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Kazuko Asada
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Morio Ueno
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Tomoko Yamashita
- Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Atsushi Mukai
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Tomoko Fujita
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Eiko Ito
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Nao Hiramoto
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Munetoyo Toda
- Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Chie Sotozono
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Shigeru Kinoshita
- Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
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Nikolovska K, Cao L, Hensel I, Di Stefano G, Seidler A, Zhou K, Qian J, Singh AK, Riederer B, Seidler U. Sodium/hydrogen-exchanger-2 modulates colonocyte lineage differentiation. Acta Physiol (Oxf) 2022; 234:e13774. [PMID: 34985202 DOI: 10.1111/apha.13774] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 10/12/2021] [Accepted: 01/01/2022] [Indexed: 12/11/2022]
Abstract
AIM The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport. METHODS Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence. RESULTS pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon. CONCLUSIONS The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.
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Affiliation(s)
- Katerina Nikolovska
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Li Cao
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
- Department of Gastroenterology Tongji Hospital Huazhong University Wuhan China
| | - Inga Hensel
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Gabriella Di Stefano
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Anna Elisabeth Seidler
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Kunyan Zhou
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Jiajie Qian
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
- Department of Transplantation and Hepatobiliary Surgery First Affiliated Hospital of Zheijang University Hangzhou China
| | - Anurag Kumar Singh
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
- Department of Physiological Chemistry University of Halle Halle (Saale) Germany
| | - Brigitte Riederer
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
| | - Ursula Seidler
- Department of Gastroenterology, Hepatology and Endocrinology Hannover Medical School Hannover Germany
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25
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Majane AC, Cridland JM, Begun DJ. Single-nucleus transcriptomes reveal evolutionary and functional properties of cell types in the Drosophila accessory gland. Genetics 2022; 220:iyab213. [PMID: 34849871 PMCID: PMC9097260 DOI: 10.1093/genetics/iyab213] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2021] [Accepted: 11/10/2021] [Indexed: 11/14/2022] Open
Abstract
Many traits responsible for male reproduction evolve quickly, including gene expression phenotypes in germline and somatic male reproductive tissues. Rapid male evolution in polyandrous species is thought to be driven by competition among males for fertilizations and conflicts between male and female fitness interests that manifest in postcopulatory phenotypes. In Drosophila, seminal fluid proteins secreted by three major cell types of the male accessory gland and ejaculatory duct are required for female sperm storage and use, and influence female postcopulatory traits. Recent work has shown that these cell types have overlapping but distinct effects on female postcopulatory biology, yet relatively little is known about their evolutionary properties. Here, we use single-nucleus RNA-Seq of the accessory gland and ejaculatory duct from Drosophila melanogaster and two closely related species to comprehensively describe the cell diversity of these tissues and their transcriptome evolution for the first time. We find that seminal fluid transcripts are strongly partitioned across the major cell types, and expression of many other genes additionally defines each cell type. We also report previously undocumented diversity in main cells. Transcriptome divergence was found to be heterogeneous across cell types and lineages, revealing a complex evolutionary process. Furthermore, protein adaptation varied across cell types, with potential consequences for our understanding of selection on male postcopulatory traits.
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Affiliation(s)
- Alex C Majane
- Department of Evolution and Ecology, University of California – Davis, Davis, CA 95616, USA
| | - Julie M Cridland
- Department of Evolution and Ecology, University of California – Davis, Davis, CA 95616, USA
| | - David J Begun
- Department of Evolution and Ecology, University of California – Davis, Davis, CA 95616, USA
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26
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Probing altered enzyme activity in the biochemical characterization of cancer. Biosci Rep 2022; 42:230680. [PMID: 35048115 PMCID: PMC8819661 DOI: 10.1042/bsr20212002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 01/10/2022] [Accepted: 01/19/2022] [Indexed: 11/30/2022] Open
Abstract
Enzymes have evolved to catalyze their precise reactions at the necessary rates, locations, and time to facilitate our development, to respond to a variety of insults and challenges, and to maintain a healthy, balanced state. Enzymes achieve this extraordinary feat through their unique kinetic parameters, myriad regulatory strategies, and their sensitivity to their surroundings, including substrate concentration and pH. The Cancer Genome Atlas (TCGA) highlights the extraordinary number of ways in which the finely tuned activities of enzymes can be disrupted, contributing to cancer development and progression often due to somatic and/or inherited genetic alterations. Rather than being limited to the domain of enzymologists, kinetic constants such as kcat, Km, and kcat/Km are highly informative parameters that can impact a cancer patient in tangible ways—these parameters can be used to sort tumor driver mutations from passenger mutations, to establish the pathways that cancer cells rely on to drive patients’ tumors, to evaluate the selectivity and efficacy of anti-cancer drugs, to identify mechanisms of resistance to treatment, and more. In this review, we will discuss how changes in enzyme activity, primarily through somatic mutation, can lead to altered kinetic parameters, new activities, or changes in conformation and oligomerization. We will also address how changes in the tumor microenvironment can affect enzymatic activity, and briefly describe how enzymology, when combined with additional powerful tools, and can provide us with tremendous insight into the chemical and molecular mechanisms of cancer.
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Tatapudy S, Peralta J, Nystul T. Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation. Development 2021; 148:dev199630. [PMID: 35020878 PMCID: PMC8645206 DOI: 10.1242/dev.199630] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Accepted: 10/13/2021] [Indexed: 04/05/2024]
Abstract
A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. Here, we found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and we found that it, as well as other JNK pathway genes, are essential for differentiation of FSC daughter cells. Our data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Also, we found that loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, we demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, our findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage.
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Affiliation(s)
| | | | - Todd Nystul
- Department of Anatomy and Department of OB/Gyn-RS, University of California, San Francisco, Center for Reproductive Sciences, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, 513 Parnassus Avenue, San Francisco, CA, 94143, USA
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28
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Ting MS, Travas-Sejdic J, Malmström J. Modulation of hydrogel stiffness by external stimuli: soft materials for mechanotransduction studies. J Mater Chem B 2021; 9:7578-7596. [PMID: 34596202 DOI: 10.1039/d1tb01415c] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Abstract
Mechanotransduction is an important process in determining cell survival, proliferation, migration and differentiation. The extracellular matrix (ECM) is the component of natural tissue that provides structural support and biochemical signals to adhering cells. The ECM is dynamic and undergoes physical and biochemical changes in response to various stimuli and there is an interest in understanding the effect of dynamic changes in stiffness on cell behaviour and fate. Therefore, stimuli-responsive hydrogels have been developed to mimic the cells' microenvironment in a controlled fashion. Herein, we review strategies for dynamic modulation of stiffness using various stimuli, such as light, temperature and pH. Special emphasis is placed on conducting polymer (CP) hydrogels and their fabrication procedures. We believe that the redox properties of CPs and hydrogels' biological properties make CPs hydrogels a promising substrate to investigate the effect of dynamic stiffness changes and mechanical actuation on cell fate in future studies.
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Affiliation(s)
- Matthew S Ting
- Department of Chemical and Materials Engineering, The University of Auckland, Auckland, New Zealand. .,MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand.,Polymer Biointerface Centre, School of Chemical Sciences, The University of Auckland, Auckland, New Zealand
| | - Jadranka Travas-Sejdic
- MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand.,Polymer Biointerface Centre, School of Chemical Sciences, The University of Auckland, Auckland, New Zealand
| | - Jenny Malmström
- Department of Chemical and Materials Engineering, The University of Auckland, Auckland, New Zealand. .,MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand.,Polymer Biointerface Centre, School of Chemical Sciences, The University of Auckland, Auckland, New Zealand
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An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity. Biochem J 2021; 477:2999-3018. [PMID: 32729927 DOI: 10.1042/bcj20200311] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Revised: 07/28/2020] [Accepted: 07/29/2020] [Indexed: 12/19/2022]
Abstract
Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to α-ketoglutarate (αKG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for αKG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue ∼12 Å from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.
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Response of Pluripotent Stem Cells to Environmental Stress and Its Application for Directed Differentiation. BIOLOGY 2021; 10:biology10020084. [PMID: 33498611 PMCID: PMC7912122 DOI: 10.3390/biology10020084] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 01/14/2021] [Accepted: 01/20/2021] [Indexed: 12/15/2022]
Abstract
Simple Summary Environmental changes in oxygen concentration, temperature, and mechanical stimulation lead to the activation of specific transcriptional factors and induce the expression of each downstream gene. In general, these responses are protective machinery against such environmental stresses, while these transcriptional factors also regulate cell proliferation, differentiation, and organ development in mammals. In the case of pluripotent stem cells, similar response mechanisms normally work and sometimes stimulate the differentiation cues. Up to now, differentiation protocols utilizing such environmental stresses have been reported to obtain various types of somatic cells from pluripotent stem cells. Basically, environmental stresses as hypoxia (low oxygen), hyperoxia, (high oxygen) and mechanical stress from cell culture plates are relatively safer than chemicals and gene transfers, which affect the genome irreversibly. Therefore, protocols designed with such environments in mind could be useful for the technology development of cell therapy and regenerative medicine. In this manuscript, we summarize recent findings of environmental stress-induced differentiation protocols and discuss their mechanisms. Abstract Pluripotent stem cells have unique characteristics compared to somatic cells. In this review, we summarize the response to environmental stresses (hypoxic, oxidative, thermal, and mechanical stresses) in embryonic stem cells (ESCs) and their applications in the differentiation methods directed to specific lineages. Those stresses lead to activation of each specific transcription factor followed by the induction of downstream genes, and one of them regulates lineage specification. In short, hypoxic stress promotes the differentiation of ESCs to mesodermal lineages via HIF-1α activation. Concerning mechanical stress, high stiffness tends to promote mesodermal differentiation, while low stiffness promotes ectodermal differentiation via the modulation of YAP1. Furthermore, each step in the same lineage differentiation favors each appropriate stiffness of culture plate; for example, definitive endoderm favors high stiffness, while pancreatic progenitor favors low stiffness during pancreatic differentiation of human ESCs. Overall, treatments utilizing those stresses have no genotoxic or carcinogenic effects except oxidative stress; therefore, the differentiated cells are safe and could be useful for cell replacement therapy. In particular, the effect of mechanical stress on differentiation is becoming attractive for the field of regenerative medicine. Therefore, the development of a stress-mediated differentiation protocol is an important matter for the future.
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31
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Amiri M, Seidler UE, Nikolovska K. The Role of pH i in Intestinal Epithelial Proliferation-Transport Mechanisms, Regulatory Pathways, and Consequences. Front Cell Dev Biol 2021; 9:618135. [PMID: 33553180 PMCID: PMC7862550 DOI: 10.3389/fcell.2021.618135] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Accepted: 01/04/2021] [Indexed: 01/07/2023] Open
Abstract
During the maturation of intestinal epithelial cells along the crypt/surface axis, a multitude of acid/base transporters are differentially expressed in their apical and basolateral membranes, enabling processes of electrolyte, macromolecule, nutrient, acid/base and fluid secretion, and absorption. An intracellular pH (pHi)-gradient is generated along the epithelial crypt/surface axis, either as a consequence of the sum of the ion transport activities or as a distinctly regulated entity. While the role of pHi on proliferation, migration, and tumorigenesis has been explored in cancer cells for some time, emerging evidence suggests an important role of the pHi in the intestinal stem cells (ISCs) proliferative rate under physiological conditions. The present review highlights the current state of knowledge about the potential regulatory role of pHi on intestinal proliferation and differentiation.
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32
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Kim N. pH variation impacts molecular pathways associated with somatic cell reprogramming and differentiation of pluripotent stem cells. Reprod Med Biol 2021; 20:20-26. [PMID: 33488280 PMCID: PMC7812493 DOI: 10.1002/rmb2.12346] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 07/27/2020] [Accepted: 08/10/2020] [Indexed: 12/19/2022] Open
Abstract
RATIONALE The study of somatic cell reprogramming and cell differentiation is essential for the application of recent techniques in regenerative medicine. It is, specifically, necessary to determine the appropriate conditions required for the induction of reprogramming and cell differentiation. METHODS Based on a comprehensive literature review, the effects of pH fluctuation on alternative splicing, mitochondria, plasma membrane, and phase separation, in several cell types are discussed. Additionally, the associated molecular pathways important for the induction of differentiation and reprogramming are reviewed. RESULTS While cells change their state, several factors such as cytokines and physical parameters affect cellular reprogramming and differentiation. As the extracellular and intracellular pH affects biophysical phenomena in a cell, the effects of pH fluctuation can ultimately decide the cell fate through molecular pathways. Though few studies have reported on the direct effects of culture pH on cell state, there is substantial information on the pathways related to stem cell differentiation and somatic cell reprogramming that can be stimulated by environmental pH. CONCLUSION Environmental pH fluctuations may decide cell fate through the molecular pathways associated with somatic cell reprogramming and cell differentiation.
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Affiliation(s)
- Narae Kim
- Nucleic Acid Chemistry and EngineeringOkinawa Institute of Science and Technology Graduate UniversityOkinawaJapan
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33
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Liu Y, White KA, Barber DL. Intracellular pH Regulates Cancer and Stem Cell Behaviors: A Protein Dynamics Perspective. Front Oncol 2020; 10:1401. [PMID: 32983969 PMCID: PMC7479815 DOI: 10.3389/fonc.2020.01401] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Accepted: 07/02/2020] [Indexed: 12/12/2022] Open
Abstract
The International Society of Cancer Metabolism (ISCaM) meeting on Cancer Metabolic Rewiring, held in Braga Portugal in October 2019, provided an outstanding forum for investigators to present current findings and views, and discuss ideas and future directions on fundamental biology as well as clinical translations. The first session on Cancer pH Dynamics was preceded by the opening keynote presentation from our group entitled Intracellular pH Regulation of Protein Dynamics: From Cancer to Stem Cell Behaviors. In this review we introduce a brief background on intracellular pH (pHi) dynamics, including how it is regulated as well as functional consequences, summarize key findings included in our presentation, and conclude with perspectives on how understanding the role of pHi dynamics in stem cells can be relevant for understanding how pHi dynamics enables cancer progression.
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Affiliation(s)
- Yi Liu
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, United States
| | - Katharine A White
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, United States
| | - Diane L Barber
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, United States
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Lee SP, Chao SC, Chou MF, Huang SF, Dai NT, Wu GJ, Tsai CS, Loh SH, Tsai YT. Characterization of intracellular buffering power in human induced pluripotent stem cells and the loss of pluripotency is delayed by acidic stimulation and increase of NHE1 activity. J Cell Physiol 2020; 236:1515-1528. [PMID: 32841374 DOI: 10.1002/jcp.29959] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 06/18/2020] [Accepted: 07/07/2020] [Indexed: 11/07/2022]
Abstract
The homeostasis of intracellular pH (pHi ) affects many cellular functions. Our previous study has established a functional and molecular model of the active pHi regulators in human induced pluripotent stem cells (hiPSCs). The aims of the present study were to further quantify passive pHi buffering power (β) and to investigate the effects of extracellular pH and Na+ -H+ exchanger 1 (NHE1) activity on pluripotency in hiPSCs. pHi was detected by microspectrofluorimetry with pH-sensitive dye-BCECF. Western blot, immunofluorescence staining, and flow cytometry were used to detect protein expression and pluripotency. Our study in hiPSCs showed that (a) the value of total (βtot ), intrinsic (βi ), and CO2 -dependent ( β C O 2 ) buffering power all increased while pHi increased; (b) during the spontaneous differentiation for 4 days, the β values of βtot and β C O 2 changed in a tendency of decrease, despite the absence of statistical significance; (c) an acidic cultured environment retained pluripotency and further upregulated expression and activity of NHE1 during spontaneous differentiation; (d) inhibition on NHE1 activity promoted the loss of pluripotency. In conclusion, we, for the first time, established a quantitative model of passive β during differentiation and demonstrated that maintenance of NHE1 at a higher level was of critical importance for pluripotency retention in hiPSCs.
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Affiliation(s)
- Shiao-Pieng Lee
- Department of Dentistry, School of Dentistry, Division of Oral and Maxillofacial Surgery, Tri-Service General Hospital and National Defense Medical Center, Taipei, Taiwan
| | - Shih-Chi Chao
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
| | - Mei-Fang Chou
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
| | - Shu-Fu Huang
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
| | - Niann-Tzyy Dai
- Department of Surgery, Division of Plastic and Reconstructive Surgery, Tri-Service General Hospital, Taipei, Taiwan
| | - Gwo-Jang Wu
- Department of Obstetrics and Gynecology, Tri-Service General Hospital, Taipei, Taiwan
| | - Chien-Sung Tsai
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
- Department of Pharmacology, National Defense Medical Center, Taipei, Taiwan
- Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University, Moscow, Russia
| | - Shih-Hurng Loh
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
- Department of Pharmacy Practice, Tri-Service General Hospital, Taipei, Taiwan
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Yi-Ting Tsai
- Department of Surgery, Division of Cardiovascular Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
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Wilson LT, Tipping WJ, Jamieson LE, Wetherill C, Henley Z, Faulds K, Graham D, Mackay SP, Tomkinson NCO. A new class of ratiometric small molecule intracellular pH sensors for Raman microscopy. Analyst 2020; 145:5289-5298. [PMID: 32672252 DOI: 10.1039/d0an00865f] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Intracellular pH (pHi) homeostasis is intertwined with a myriad of normal cellular behaviors as well as pathological processes. As such, small molecule probes for the measurement of pHi are invaluable tools for chemical biology, facilitating the study of the role of pH in cellular function and disease. The field of small molecule pHi sensors has traditionally been dominated with probes based on fluorescent scaffolds. In this study, a series of low molecular weight (<260) oligoyne compounds have been developed which exhibit pH sensitive alkyne stretching frequencies (νalkyne) in Raman spectroscopy. The modular design of the compounds enabled tuneability of their pKa(H) through simple structural modification, such that continuous pH sensitivity is achieved over the range 2-10. Alkyne stretching bands reside in the 'cell-silent' region of the Raman spectrum (1800-2600 cm-1) and are readily detectable in a cellular environment with subcellular spatial resolution. This enabled the application of a pH sensitive oligoyne compound to the ratiometric sensing of pHi in prostate cancer (PC3) cells in response to drug treatment. We propose that probes based on Alkyne Tag Raman Imaging offer an entirely new platform for the sensing of pHi, complementary to fluorescence microscopy.
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Affiliation(s)
- Liam T Wilson
- Department of Pure and Applied Chemistry, WestCHEM, Thomas Graham Building, University of Strathclyde, 295 Cathedral Street, Glasgow, G1 1XL, UK.
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Schotthöfer SK, Bohrmann J. Analysing bioelectrical phenomena in the Drosophila ovary with genetic tools: tissue-specific expression of sensors for membrane potential and intracellular pH, and RNAi-knockdown of mechanisms involved in ion exchange. BMC DEVELOPMENTAL BIOLOGY 2020; 20:15. [PMID: 32635900 PMCID: PMC7341674 DOI: 10.1186/s12861-020-00220-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Accepted: 06/10/2020] [Indexed: 01/16/2023]
Abstract
Background Changes in transcellular bioelectrical patterns are known to play important roles during developmental and regenerative processes. The Drosophila follicular epithelium has proven to be an appropriate model system for studying the mechanisms by which bioelectrical signals emerge and act. Fluorescent indicator dyes in combination with various inhibitors of ion-transport mechanisms have been used to investigate the generation of membrane potentials (Vmem) and intracellular pH (pHi). Both parameters as well as their anteroposterior and dorsoventral gradients were affected by the inhibitors which, in addition, led to alterations of microfilament and microtubule patterns equivalent to those observed during follicle-cell differentiation. Results We expressed two genetically-encoded fluorescent sensors for Vmem and pHi, ArcLight and pHluorin-Moesin, in the follicular epithelium of Drosophila. By means of the respective inhibitors, we obtained comparable effects on Vmem and/or pHi as previously described for Vmem- and pHi-sensitive fluorescent dyes. In a RNAi-knockdown screen, five genes of ion-transport mechanisms and gap-junction subunits were identified exerting influence on ovary development and/or oogenesis. Loss of ovaries or small ovaries were the results of soma knockdowns of the innexins inx1 and inx3, and of the DEG/ENaC family member ripped pocket (rpk). Germline knockdown of rpk also resulted in smaller ovaries. Soma knockdown of the V-ATPase-subunit vha55 caused size-reduced ovaries with degenerating follicles from stage 10A onward. In addition, soma knockdown of the open rectifier K+channel 1 (ork1) resulted in a characteristic round-egg phenotype with altered microfilament and microtubule organisation in the follicular epithelium. Conclusions The genetic tool box of Drosophila provides means for a refined and extended analysis of bioelectrical phenomena. Tissue-specifically expressed Vmem- and pHi-sensors exhibit some practical advantages compared to fluorescent indicator dyes. Their use confirms that the ion-transport mechanisms targeted by inhibitors play important roles in the generation of bioelectrical signals. Moreover, modulation of bioelectrical signals via RNAi-knockdown of genes coding for ion-transport mechanisms and gap-junction subunits exerts influence on crucial processes during ovary development and results in cytoskeletal changes and altered follicle shape. Thus, further evidence amounts for bioelectrical regulation of developmental processes via the control of both signalling pathways and cytoskeletal organisation.
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Affiliation(s)
- Susanne Katharina Schotthöfer
- RWTH Aachen University, Institut für Biologie II, Abt. Zoologie und Humanbiologie, Worringerweg 3, 52056, Aachen, Germany
| | - Johannes Bohrmann
- RWTH Aachen University, Institut für Biologie II, Abt. Zoologie und Humanbiologie, Worringerweg 3, 52056, Aachen, Germany.
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tpHusion: An efficient tool for clonal pH determination in Drosophila. PLoS One 2020; 15:e0228995. [PMID: 32059043 PMCID: PMC7021318 DOI: 10.1371/journal.pone.0228995] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Accepted: 01/27/2020] [Indexed: 02/04/2023] Open
Abstract
Genetically encoded pH indicators (GEpHI) have emerged as important tools for investigating intracellular pH (pHi) dynamics in Drosophila. However, most of the indicators are based on the Gal4/UAS binary expression system. Here, we report the generation of a ubiquitously-expressed GEpHI. The fusion protein of super ecliptic pHluorin and FusionRed was cloned under the tubulin promoter (tpHusion) to drive it independently of the Gal4/UAS system. The function of tpHusion was validated in various tissues from different developmental stages of Drosophila. Differences in pHi were also indicated correctly in fixed tissues. Finally, we describe the use of tpHusion for comparative analysis of pHi in manipulated clones and the surrounding cells in epithelial tissues. Our findings establish tpHusion as a robust tool for studying pHi in Drosophila.
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Abstract
Acidic metabolic waste products accumulate in the tumor microenvironment because of high metabolic activity and insufficient perfusion. In tumors, the acidity of the interstitial space and the relatively well-maintained intracellular pH influence cancer and stromal cell function, their mutual interplay, and their interactions with the extracellular matrix. Tumor pH is spatially and temporally heterogeneous, and the fitness advantage of cancer cells adapted to extracellular acidity is likely particularly evident when they encounter less acidic tumor regions, for instance, during invasion. Through complex effects on genetic stability, epigenetics, cellular metabolism, proliferation, and survival, the compartmentalized pH microenvironment favors cancer development. Cellular selection exacerbates the malignant phenotype, which is further enhanced by acid-induced cell motility, extracellular matrix degradation, attenuated immune responses, and modified cellular and intercellular signaling. In this review, we discuss how the acidity of the tumor microenvironment influences each stage in cancer development, from dysplasia to full-blown metastatic disease.
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Affiliation(s)
- Ebbe Boedtkjer
- Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark
| | - Stine F. Pedersen
- Department of Biology, University of Copenhagen, DK-2100 Copenhagen, Denmark
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Loh SH, Tsai YT, Huang SF, Yu TC, Kuo PC, Chao SC, Chou MF, Tsai CS, Lee SP. Effects of Andrographolide on Intracellular pH Regulation, Cellular Migration, and Apoptosis in Human Cervical Cancer Cells †. Cancers (Basel) 2020; 12:cancers12020387. [PMID: 32046125 PMCID: PMC7072207 DOI: 10.3390/cancers12020387] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2019] [Revised: 02/03/2020] [Accepted: 02/04/2020] [Indexed: 12/15/2022] Open
Abstract
Cancer cells have been characterized with alkaline intracellular pH (pHi) values (≥7.2) to enable cancer proliferation, migration, and progression. The aim of the present study was to explore the concentration-dependent effects of Andrographolide, an active diterpenoid compound of herb Andrographis paniculata, on Na+/H+ exchanger isoform 1 (NHE1), cellular migration and apoptosis in human cervical cancer cells (HeLa). The pHi was detected by microspectrofluorometry method, and intracellular acidification was induced by NH4Cl prepulse technique. Viability and protein expression were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and Western blot, respectively. Human normal endocervical cells (End1), ectocervical cells (Ect1), and HeLa were bought commercially. The resting pHi value of HeLa (≈7.47) was significantly higher than that of End1 and Ect1 (≈7.30), and shifted from alkaline to acidic following acid/base impacts. In HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid | N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) -buffered superfusate, NHE1 and V-ATPase co-existed functionally for acid extrusion in HeLa, while only NHE1 existed functionally in End/Ect1. Andrographolide (3–1000 μM) concentration-dependently inhibited NHE1 activity. Cell-migration and expressions of NHE1, V-ATPase, PARP (poly-ADP-ribose-polymerase), pro-Caspase-3, and Bcl-2 were significantly reduced by pretreating with Andrographolide (≥100 μM) for 24–48 h in HeLa. Andrographolide inhibited cell viability of End1-cells/Ect1 and HeLa (≥100 and ≥30 μM, respectively). The present findings implicate the promising clinical applications of Andrographolide on cervical cancer treatment.
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Affiliation(s)
- Shih-Hurng Loh
- Department of Clinical Pharmacy, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan; (S.-H.L.); (S.-F.H.); (M.-F.C.)
- Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan; (T.-C.Y.); (P.-C.K.)
| | - Yi-Ting Tsai
- Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan; (Y.-T.T.); (C.-S.T.)
| | - Shu-Fu Huang
- Department of Clinical Pharmacy, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan; (S.-H.L.); (S.-F.H.); (M.-F.C.)
| | - Tien-Chieh Yu
- Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan; (T.-C.Y.); (P.-C.K.)
| | - Pei-Chun Kuo
- Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan; (T.-C.Y.); (P.-C.K.)
| | - Shih-Chi Chao
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan;
| | - Mei-Fang Chou
- Department of Clinical Pharmacy, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan; (S.-H.L.); (S.-F.H.); (M.-F.C.)
| | - Chien-Sung Tsai
- Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan; (Y.-T.T.); (C.-S.T.)
| | - Shiao-Pieng Lee
- Department of Biomedical Engineering, National Defense Medical Center, Taipei 11490, Taiwan
- Department of Oral and Maxillofacial Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
- Correspondence:
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Rust K, Nystul T. Signal transduction in the early Drosophila follicle stem cell lineage. CURRENT OPINION IN INSECT SCIENCE 2020; 37:39-48. [PMID: 32087562 PMCID: PMC7155752 DOI: 10.1016/j.cois.2019.11.005] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 11/13/2019] [Accepted: 11/16/2019] [Indexed: 05/08/2023]
Abstract
The follicle stem cell (FSC) lineage in the Drosophila ovary is a highly informative model of in vivo epithelial stem cell biology. Studies over the past 30 years have identified roles for every major signaling pathway in the early FSC lineage. These pathways regulate a wide variety of cell behaviors, including self-renewal, proliferation, survival and differentiation. Studies of cell signaling in the follicle epithelium have provided new insights into how these cell behaviors are coordinated within an epithelial stem cell lineage and how signaling pathways interact with each other in the native, in vivo context of a living tissue. Here, we review these studies, with a particular focus on how these pathways specify differences between the FSCs and their daughter cells. We also describe common themes that have emerged from these studies, and highlight new research directions that have been made possible by the detailed understanding of the follicle epithelium.
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Raja DA, Gotherwal V, Burse SA, Subramaniam YJ, Sultan F, Vats A, Gautam H, Sharma B, Sharma S, Singh A, Sivasubbu S, Gokhale RS, Natarajan VT. pH-controlled histone acetylation amplifies melanocyte differentiation downstream of MITF. EMBO Rep 2020; 21:e48333. [PMID: 31709752 PMCID: PMC6945066 DOI: 10.15252/embr.201948333] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2019] [Revised: 10/04/2019] [Accepted: 10/15/2019] [Indexed: 12/20/2022] Open
Abstract
Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. This involves the rapid and selective induction of melanocyte maturation genes, while concomitantly the expression of other effector genes is maintained. In this study, using cell-based and zebrafish model systems, we report on a pH-mediated feed-forward mechanism of epigenetic regulation that enables selective amplification of the melanocyte maturation program. We demonstrate that MITF activation directly elevates the expression of the enzyme carbonic anhydrase 14 (CA14). Nuclear localization of CA14 leads to an increase of the intracellular pH, resulting in the activation of the histone acetyl transferase p300/CBP. In turn, enhanced H3K27 histone acetylation at selected differentiation genes facilitates their amplified expression via MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in the formation of immature acidic melanocytes with decreased pigmentation, establishing a central role for this mechanism during melanocyte differentiation in vivo. Thus, we describe an epigenetic control system via pH modulation that reinforces cell fate determination by altering chromatin dynamics.
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Affiliation(s)
- Desingu Ayyappa Raja
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Vishvabandhu Gotherwal
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Shaunak A Burse
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Yogaspoorthi J Subramaniam
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Farina Sultan
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Archana Vats
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
| | - Hemlata Gautam
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
| | - Babita Sharma
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | - Sachin Sharma
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
- Present address:
National Institute of ImmunologyNew DelhiIndia
| | - Archana Singh
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
| | | | - Rajesh S Gokhale
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Present address:
National Institute of ImmunologyNew DelhiIndia
| | - Vivek T Natarajan
- CSIR‐Institute of Genomics and Integrative BiologyNew DelhiIndia
- Academy of Scientific and Innovative ResearchTaramani, Chennai
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Weiß I, Bohrmann J. Electrochemical gradients are involved in regulating cytoskeletal patterns during epithelial morphogenesis in the Drosophila ovary. BMC DEVELOPMENTAL BIOLOGY 2019; 19:22. [PMID: 31718540 PMCID: PMC6852995 DOI: 10.1186/s12861-019-0203-y] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/13/2019] [Accepted: 10/24/2019] [Indexed: 12/14/2022]
Abstract
BACKGROUND During Drosophila oogenesis, the follicular epithelium differentiates into several morphologically distinct follicle-cell populations. Characteristic bioelectrical properties make this tissue a suitable model system for studying connections between electrochemical signals and the organisation of the cytoskeleton. Recently, we have described stage-specific transcellular antero-posterior and dorso-ventral gradients of intracellular pH (pHi) and membrane potential (Vmem) depending on the asymmetrical distribution and/or activity of various ion-transport mechanisms. In the present study, we analysed the patterns of basal microfilaments (bMF) and microtubules (MT) in relation to electrochemical signals. RESULTS The bMF- and MT-patterns in developmental stages 8 to 12 were visualised using labelled phalloidin and an antibody against acetylated α-tubulin as well as follicle-cell specific expression of GFP-actin and GFP-α-tubulin. Obviously, stage-specific changes of the pHi- and Vmem-gradients correlate with modifications of the bMF- and MT-organisation. In order to test whether cytoskeletal modifications depend directly on bioelectrical changes, we used inhibitors of ion-transport mechanisms that have previously been shown to modify pHi and Vmem as well as the respective gradients. We inhibited, in stage 10b, Na+/H+-exchangers and Na+-channels with amiloride, V-ATPases with bafilomycin, ATP-sensitive K+-channels with glibenclamide, voltage-dependent L-type Ca2+-channels with verapamil, Cl--channels with 9-anthroic acid and Na+/K+/2Cl--cotransporters with furosemide, respectively. The correlations between pHi, Vmem, bMF and MT observed in different follicle-cell types are in line with the correlations resulting from the inhibition experiments. While relative alkalisation and/or hyperpolarisation stabilised the parallel transversal alignment of bMF, acidification led to increasing disorder and to condensations of bMF. On the other hand, relative acidification as well as hyperpolarisation stabilised the longitudinal orientation of MT, whereas alkalisation led to loss of this arrangement and to partial disintegration of MT. CONCLUSIONS We conclude that the pHi- and Vmem-changes induced by inhibitors of ion-transport mechanisms simulate bioelectrical changes occurring naturally and leading to the cytoskeletal changes observed during differentiation of the follicle-cell epithelium. Therefore, gradual modifications of electrochemical signals can serve as physiological means to regulate cell and tissue architecture by modifying cytoskeletal patterns.
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Affiliation(s)
- Isabel Weiß
- Institut für Biologie II, Abt. Zoologie und Humanbiologie, RWTH Aachen University, Worringerweg 3, 52056, Aachen, Germany
| | - Johannes Bohrmann
- Institut für Biologie II, Abt. Zoologie und Humanbiologie, RWTH Aachen University, Worringerweg 3, 52056, Aachen, Germany.
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Vélez JI, Lopera F, Silva CT, Villegas A, Espinosa LG, Vidal OM, Mastronardi CA, Arcos-Burgos M. Familial Alzheimer's Disease and Recessive Modifiers. Mol Neurobiol 2019; 57:1035-1043. [PMID: 31664702 PMCID: PMC7031188 DOI: 10.1007/s12035-019-01798-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2019] [Accepted: 09/22/2019] [Indexed: 12/15/2022]
Abstract
Alzheimer’s disease (AD) is progressive brain disorder that affects ~ 50 million people worldwide and has no current effective treatment. AD age of onset (ADAOO) has shown to be critical for the identification of genes that modify the appearance of AD signs and symptoms in a specific population. We clinically characterized and whole-exome genotyped 71 individuals with AD from the Paisa genetic isolate, segregating the (PSEN1) E280A dominant fully penetrant mutation, and analyzed the potential recessive effects of ~ 50,000 common functional genomic variants to the ADAOO. Standard quality control and filtering procedures were applied, and recessive single- and multi-locus linear mixed-effects models were used. We identified genetic variants in the SLC9C1, CSN1S1, and LOXL4 acting recessively to delay ADAOO up to ~ 11, ~ 6, and ~ 9 years on average, respectively. In contrast, the CC recessive genotype in marker DHRS4L2-rs2273946 accelerates ADAOO by ~ 8 years. This study, reports new recessive variants modifying ADAOO in PSEN1 E280A mutation carriers. This set of genes are implicated in important biological processes and molecular functions commonly affected by genes associated with the etiology of AD such as APP, APOE, and CLU. Future functional studies using modern techniques such as induced pluripotent stem cells will allow a better understanding of the over expression and down regulation of these recessive modifier variants and hence the pathogenesis of AD. These results are important for prediction of AD and ultimately, substantial to develop new therapeutic strategies for individuals at risk or affected by AD.
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Affiliation(s)
| | - Francisco Lopera
- Neuroscience Research Group, University of Antioquia, Medellín, Colombia
| | - Claudia T Silva
- Neuroscience Research Group, University of Antioquia, Medellín, Colombia
| | - Andrés Villegas
- Neuroscience Research Group, University of Antioquia, Medellín, Colombia
| | - Lady G Espinosa
- INPAC Research Group, Fundación Universitaria Sanitas, Bogotá, Colombia
| | | | | | - Mauricio Arcos-Burgos
- Grupo de Investigación en Psiquiatría (GIPSI), Departamento de Psiquiatría, Instituto de Investigaciones Médicas (IIM), Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.
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Pedersen SF, Counillon L. The SLC9A-C Mammalian Na +/H + Exchanger Family: Molecules, Mechanisms, and Physiology. Physiol Rev 2019; 99:2015-2113. [PMID: 31507243 DOI: 10.1152/physrev.00028.2018] [Citation(s) in RCA: 112] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Na+/H+ exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na+ and H+ across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na+/H+ exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.
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Affiliation(s)
- S F Pedersen
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; and Université Côte d'Azur, CNRS, Laboratoire de Physiomédecine Moléculaire, LP2M, France, and Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France
| | - L Counillon
- Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen, Copenhagen, Denmark; and Université Côte d'Azur, CNRS, Laboratoire de Physiomédecine Moléculaire, LP2M, France, and Laboratories of Excellence Ion Channel Science and Therapeutics, Nice, France
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Rodimova SA, Meleshina AV, Kalabusheva EP, Dashinimaev EB, Reunov DG, Torgomyan HG, Vorotelyak EA, Zagaynova EV. Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions. Methods Appl Fluoresc 2019; 7:044002. [PMID: 31412329 DOI: 10.1088/2050-6120/ab3b3d] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.
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Affiliation(s)
- Svetlana A Rodimova
- Privolzhsky Research Medical University, 10/1 Minin and Pozharsky sq., Nizhny Novgorod, 603950, Russia. Lobachevsky State University of Nizhny Novgorod, 23 Gagarin Avenue, Novgorod, Nizhny Novgorod, 603950, Russia
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Identification of alkaline pH optimum of human glucokinase because of ATP-mediated bias correction in outcomes of enzyme assays. Sci Rep 2019; 9:11422. [PMID: 31388064 PMCID: PMC6684659 DOI: 10.1038/s41598-019-47883-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2018] [Accepted: 07/08/2019] [Indexed: 12/16/2022] Open
Abstract
Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected KM of human hexokinase 2 and S0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5-8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.
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Electrochemical patterns during Drosophila oogenesis: ion-transport mechanisms generate stage-specific gradients of pH and membrane potential in the follicle-cell epithelium. BMC DEVELOPMENTAL BIOLOGY 2019; 19:12. [PMID: 31226923 PMCID: PMC6588877 DOI: 10.1186/s12861-019-0192-x] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2019] [Accepted: 06/06/2019] [Indexed: 12/14/2022]
Abstract
Background Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. Results Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl−-channels (9-anthroic acid) and Na+/K+/2Cl−-cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl−-cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl−-cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. Conclusions Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation. Electronic supplementary material The online version of this article (10.1186/s12861-019-0192-x) contains supplementary material, which is available to authorized users.
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Benitez M, Tatapudy S, Liu Y, Barber DL, Nystul TG. Drosophila anion exchanger 2 is required for proper ovary development and oogenesis. Dev Biol 2019; 452:127-133. [PMID: 31071312 DOI: 10.1016/j.ydbio.2019.04.018] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2018] [Revised: 03/22/2019] [Accepted: 04/26/2019] [Indexed: 02/06/2023]
Abstract
Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene anion exchanger 2 (ae2). Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.
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Affiliation(s)
- Marimar Benitez
- Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, USA
| | - Sumitra Tatapudy
- Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, USA
| | - Yi Liu
- Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, USA
| | - Diane L Barber
- Department of Cell and Tissue Biology, University of California, San Francisco, USA
| | - Todd G Nystul
- Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, USA.
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Grillo-Hill BK, White KA. Oncogenic β-catenin mutations evade pH-regulated degradation. Mol Cell Oncol 2019; 6:1554470. [PMID: 30788422 PMCID: PMC6370367 DOI: 10.1080/23723556.2018.1554470] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2018] [Revised: 11/25/2018] [Accepted: 11/28/2018] [Indexed: 02/04/2023]
Abstract
β-catenin has roles in cell-cell adhesion and Wnt signaling. We recently showed that β-catenin protein abundance is decreased at higher intracellular pH (pHi), mediated by pH-sensitive interaction with the beta-transducin repeat containing E3 ubiquitin protein ligase (β-TrCP). Increased pHi facilitates β-TrCP binding and degradation of β-catenin. β-catenin mutations that abrogate the pH-sensitive interaction induce significant tumors not seen with other β-catenin stabilizing mutants.
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Affiliation(s)
- Bree K Grillo-Hill
- Department of Biological Sciences, San Jose State University, San Jose, CA, USA
| | - Katharine A White
- Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA.,Department of Chemistry and Biochemistry, Harper Cancer Research Institute, University of Notre Dame, South Bend, IN, USA
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Chao SC, Wu GJ, Huang SF, Dai NT, Huang HK, Chou MF, Tsai YT, Lee SP, Loh SH. Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells. World J Stem Cells 2018; 10:196-211. [PMID: 30613313 PMCID: PMC6306555 DOI: 10.4252/wjsc.v10.i12.196] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Revised: 11/14/2018] [Accepted: 12/05/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To establish a functional and molecular model of the intracellular pH (pHi) regulatory mechanism in human induced pluripotent stem cells (hiPSCs).
METHODS hiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pHi were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K+/nigericin method. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (β) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers.
RESULTS In this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3--buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3--buffered system, the values of total intracellular buffering power (β) can be described by the following equation: βtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3- cotransporter (NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1; (5) the Cl-/ OH- exchanger (CHE) and the Cl-/HCO3- anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl--independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pHi value and diminished the functional activity and protein expression of the NHE and the NBC.
CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.
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Affiliation(s)
- Shih-Chi Chao
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan
| | - Gwo-Jang Wu
- Department of Obstetrics and Gynecology, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Shu-Fu Huang
- Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Niann-Tzyy Dai
- Division of Plastic and Reconstructive Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Hsu-Kai Huang
- Division of Chest Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Mei-Fang Chou
- Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Yi-Ting Tsai
- Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei 11490, Taiwan
- Division of Cardiovascular Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
| | - Shiao-Pieng Lee
- Division of Oral and Maxillofacial Surgery, Department of Dentistry, School of Dentistry, Tri-Service General Hospital and National Defense Medical Center, Taipei 11490, Taiwan
| | - Shih-Hurng Loh
- Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan
- Department of Pharmacy Practice, Tri-Service General Hospital, National Defense Medical Center, Taipei 11490, Taiwan
- Department of Pharmacology, National Defense Medical Center, Taipei 11490, Taiwan
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