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Hashmi H, Matsumoto R, Corcoran D, Kawakami Y, Araki T. Genetic models of Cushing's disease : From cells, in vivo transgenic models to human pituitary organoids. Pituitary 2025; 28:47. [PMID: 40186634 DOI: 10.1007/s11102-025-01516-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 03/10/2025] [Indexed: 04/07/2025]
Abstract
Cushing's disease (CD) is caused by pituitary tumors that overproduce adrenocorticotropic hormone (ACTH); however, effective medical treatments remain limited, significantly impairing patients' quality of life and prognosis. Despite extensive molecular analyses, the pathogenesis of CD remains unclear. Although previous molecular studies have relied heavily on rodent-derived cells and rodent transgenic models, significant species differences exist in the tumorigenesis of CD between humans and rodents. To date, an established human CD cell model is lacking, as human CD cells are limited in availability and sustainability over time. Additionally, the gene modifications used in transgenic models do not necessarily reflect the causative genes in CD. CD tumors exhibit wide phenotypic heterogeneity, which further complicates the development of an ideal genetic model. In this review, we provide an analysis of 11 genetic models used to study CD, outlining their historical development, strengths, and limitations. Additionally, we discuss the ongoing development of human induced pluripotent stem cell (iPSC)-derived pituitary organoids and further describe various models of pituitary organoids as an emerging novel approach to studying CD. By comparing all these models, we highlight the necessity of advancing genetic models to improve our understanding and treatment of CD.
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Affiliation(s)
- Hiba Hashmi
- Division of Endocrinology & Metabolism, Department of Medicine, Western University, London, ON, Canada
- Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, University of Minnesota, 420 Delaware SE, Minneapolis, MN, USA
| | - Ryusaku Matsumoto
- Center for Ips Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Dylan Corcoran
- Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA
| | - Yasuhiko Kawakami
- Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA
- Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
| | - Takako Araki
- Division of Endocrinology, Diabetes & Metabolism, Department of Medicine, University of Minnesota, 420 Delaware SE, Minneapolis, MN, USA.
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Ear PH, Marinoni I, Dayton T, Guenter R, Quelle DE, Battistella A, Buishand FO, Chittaranjan S, Nancy Du YC, Marques I, Pellegata NS, Sadowski SM, Tirosh A, April-Monn S, Aurilia C, Jaskula-Sztul R, Baena Moreno MJ, Donati S, English KA, Hernandez Llorens MA, Hodgetts H, Marini F, Martins M, Palmini G, Soldevilla B, Schrader J, Thakker RV, Lines KE. NET Models Meeting 2024 white paper: the current state of neuroendocrine tumour research models and our future aspirations. ENDOCRINE ONCOLOGY (BRISTOL, ENGLAND) 2024; 4:e240055. [PMID: 39822778 PMCID: PMC11737514 DOI: 10.1530/eo-24-0055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/10/2024] [Revised: 11/12/2024] [Accepted: 11/19/2024] [Indexed: 01/19/2025]
Abstract
Current models for the study of neuroendocrine tumours (NETs) are severely limited. While in vitro (e.g. cell lines), ex vivo (e.g. organoids) and in vivo (e.g. mice) models all exist, each has limitations. To address these limitations and collectively identify strategies to move the NET models field forward, we held an inaugural NET models meeting, hosted by our founding group: Dr Lines (Oxford), Prof. Quelle (Iowa), Dr Dayton (Barcelona), Dr Ear (Iowa), Dr Marinoni (Bern) and Dr Guenter (Alabama). This two-day meeting in Oxford (UK) was organised and supported by Bioscientifica Ltd and was solely dedicated to the discussion of NET models. The meeting was attended by ∼30 international researchers (from the UK, EU, Israel, USA and Canada). Plenary talks were given by Prof. Thakker, who summarised NET research over the past few decades, and Dr Schrader, who described the process and pitfalls of generating new cell lines. Eight researchers also presented their work on topics ranging from human cell 3D bioprinting to zebrafish models and included novel ideas and improvements on current concepts. This was followed by an interactive workshop, where discussion topics included a summary of currently available NET models, limitations of these models, barriers to developing new models, and how we can address these issues going forward. This white paper summarises the key points raised in these discussions and the future aspirations of the NET Models Consortium. The next meeting will take place in Oxford (UK) in 2025; contact contact@netcancerfoundation.com for more information.
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Affiliation(s)
- Po Hien Ear
- Department of Surgery, University
of Iowa, Iowa City, Iowa,
USA
| | - Ilaria Marinoni
- Institute of Tissue Medicine and
Pathology, University of Bern, Bern,
Switzerland
| | - Talya Dayton
- European Molecular Biology
Laboratory (EMBL) Barcelona, Tissue Biology and Disease Modelling,
Barcelona, Spain
| | - Rachael Guenter
- Department of Surgery, School of
Medicine, University of Alabama at Birmingham,
Birmingham, Alabama, USA
| | - Dawn E Quelle
- Department of Neuroscience and
Pharmacology, University of Iowa, Iowa City,
Iowa, USA
| | - Anna Battistella
- Pancreatic Surgery Unit, Pancreas
Translational and Clinical Research Center, IRCCS San Raffaele Scientific
Institute, Vita-Salute San Raffaele University,
Milan, Italy
- Malignant B Cells Biology and 3D
Modelling Unit, Experimental Oncology Department, Vita-Salute San Raffaele
University, Milan, Italy
| | - Floryne O Buishand
- Department of Clinical Science
and Services, Royal Veterinary College, Hatfield,
UK
| | - Suganthi Chittaranjan
- Department of Genome Sciences
Centre, BC Cancer, Vancouver, British
Columbia, Canada
| | - Yi-Cheih Nancy Du
- Department of Pathology and
Laboratory Medicine, Weill Cornell Medicine, New
York, New York, USA
| | - Ines Marques
- Institute of Anatomy,
University of Bern, Bern,
Switzerland
| | - Natalia S Pellegata
- Department of Biology and
Biotechnology, University of Pavia, Pavia,
Italy
- Institute for Diabetes and
Cancer, Helmholtz Munich, Munich,
Germany
| | - Samira M Sadowski
- Endocrine Surgery Section,
Surgical Oncology Program, Center for Cancer Research, NCI, NIH,
Bethesda, Maryland, USA
| | - Amit Tirosh
- ENTIRE – Endocrine
Neoplasia Translational Research Center, Sheba Medical Center, and Tel Aviv
University Faculty of Medicine, Tel Aviv,
Israel
| | - Simon April-Monn
- Department of Pathology and
Molecular Pathology, University Hospital Zurich,
Zurich, Switzerland
| | - Cinzia Aurilia
- Associazione Italiana Neoplasie
Endocrine Multiple di tipo 1 e di tipo 2 (AIMEN 1 e 2),
Sondrio, Italy
| | - Renata Jaskula-Sztul
- Department of Surgery, School of
Medicine, University of Alabama at Birmingham,
Birmingham, Alabama, USA
| | - Maria Jesús Baena Moreno
- Center of Experimental
Oncology, Gastrointestinal and Neuroendocrine Tumors Research Group, Research
Institute Hospital 12 de Octubre (i+12),
Madrid, Spain
| | - Simone Donati
- Associazione Italiana Neoplasie
Endocrine Multiple di tipo 1 e di tipo 2 (AIMEN 1 e 2),
Sondrio, Italy
| | - Katherine A English
- OCDEM, Radcliffe Department of
Medicine, University of Oxford, Churchill Hospital,
Oxford, UK
| | - Maria Almudena Hernandez Llorens
- Center of Experimental
Oncology, Gastrointestinal and Neuroendocrine Tumors Research Group, Research
Institute Hospital 12 de Octubre (i+12),
Madrid, Spain
| | - Harry Hodgetts
- Regenerative Medicine and
Fibrosis Group, Institute for Liver and Digestive Health, University College
London, Royal Free Campus, London,
UK
| | - Francesca Marini
- FIRMO Foundation (Italian
Foundation for the Research on Bone Diseases),
Florence, Italy
| | - Maria Martins
- Regenerative Medicine and
Fibrosis Group, Institute for Liver and Digestive Health, University College
London, Royal Free Campus, London,
UK
| | - Gaia Palmini
- FIRMO Foundation (Italian
Foundation for the Research on Bone Diseases),
Florence, Italy
| | - Beatriz Soldevilla
- Center of Experimental
Oncology, Gastrointestinal and Neuroendocrine Tumors Research Group, Research
Institute Hospital 12 de Octubre (i+12),
Madrid, Spain
- Department of Genetics,
Physiology and Microbiology, Faculty of Biology, Universidad Complutense de
Madrid (UCM)Madrid, Spain
| | - Jörg Schrader
- Department of Medicine,
University Medical Center Hamburg-Eppendorf, Hamburg,
Germany
| | - Rajesh V Thakker
- OCDEM, Radcliffe Department of
Medicine, University of Oxford, Churchill Hospital,
Oxford, UK
- Oxford NIHR Biomedical Research
Centre, Oxford University Hospitals Trust, Oxford,
UK
- Centre for Endocrinology,
William Harvey Research Institute, Barts and the London School of Medicine,
Queen Mary University of London, London,
UK
| | - Kate E Lines
- OCDEM, Radcliffe Department of
Medicine, University of Oxford, Churchill Hospital,
Oxford, UK
- School of Biological and
Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes
University, Oxford, UK
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Miller WL, White PC. History of Adrenal Research: From Ancient Anatomy to Contemporary Molecular Biology. Endocr Rev 2023; 44:70-116. [PMID: 35947694 PMCID: PMC9835964 DOI: 10.1210/endrev/bnac019] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Indexed: 01/20/2023]
Abstract
The adrenal is a small, anatomically unimposing structure that escaped scientific notice until 1564 and whose existence was doubted by many until the 18th century. Adrenal functions were inferred from the adrenal insufficiency syndrome described by Addison and from the obesity and virilization that accompanied many adrenal malignancies, but early physiologists sometimes confused the roles of the cortex and medulla. Medullary epinephrine was the first hormone to be isolated (in 1901), and numerous cortical steroids were isolated between 1930 and 1949. The treatment of arthritis, Addison's disease, and congenital adrenal hyperplasia (CAH) with cortisone in the 1950s revolutionized clinical endocrinology and steroid research. Cases of CAH had been reported in the 19th century, but a defect in 21-hydroxylation in CAH was not identified until 1957. Other forms of CAH, including deficiencies of 3β-hydroxysteroid dehydrogenase, 11β-hydroxylase, and 17α-hydroxylase were defined hormonally in the 1960s. Cytochrome P450 enzymes were described in 1962-1964, and steroid 21-hydroxylation was the first biosynthetic activity associated with a P450. Understanding of the genetic and biochemical bases of these disorders advanced rapidly from 1984 to 2004. The cloning of genes for steroidogenic enzymes and related factors revealed many mutations causing known diseases and facilitated the discovery of new disorders. Genetics and cell biology have replaced steroid chemistry as the key disciplines for understanding and teaching steroidogenesis and its disorders.
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Affiliation(s)
- Walter L Miller
- Department of Pediatrics, Center for Reproductive Sciences, and Institute for Human Genetics, University of California, San Francisco, CA, USA
| | - Perrin C White
- Division of Pediatric Endocrinology, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Koganti PP, Zhao AH, Selvaraj V. Exogenous cholesterol acquisition signaling in LH-responsive MA-10 Leydig cells and in adult mice. J Endocrinol 2022; 254:187-199. [PMID: 35900012 PMCID: PMC9840751 DOI: 10.1530/joe-22-0043] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/19/2022] [Accepted: 07/27/2022] [Indexed: 01/17/2023]
Abstract
MA-10 cells, established 4 decades ago from a murine Leydig cell tumor, has served as a key model system for studying steroidogenesis. Despite a precipitous loss in their innate ability to respond to luteinizing hormone (LH), the use of a cell-permeable cAMP analog for induction ensured their continued use. In parallel, a paradigm that serum-free conditions are essential for trophic steroidogenic stimulation was rationalized. Through the selection of LH-responsive single-cell MA-10Slip clones, we uncovered that Leydig cells remain responsive in the presence of serum in vitro and that exogenous cholesterol delivery by lipoproteins provided a significantly elevated steroid biosynthetic response (>2-fold). In scrutinizing the underlying regulation, systems biology of the MA-10 cell proteome identified multiple Rho-GTPase signaling pathways as highly enriched. Testing Rho function in steroidogenesis revealed that its modulation can negate the specific elevation in steroid biosynthesis observed in the presence of lipoproteins/serum. This signaling modality primarily linked to the regulation of endocytic traffic is evident only in the presence of exogenous cholesterol. Inhibiting Rho function in vivo also decreased hCG-induced testosterone production in mice. Collectively, our findings dispel a long-held view that the use of serum could confound or interfere with trophic stimulation and underscore the need for exogenous lipoproteins when dissecting physiological signaling and cholesterol trafficking for steroid biosynthesis in vitro. The LH-responsive MA-10Slip clones derived in this study present a reformed platform enabling biomimicry to study the cellular and molecular basis of mammalian steroidogenesis.
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Affiliation(s)
- Prasanthi P. Koganti
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853, USA
| | - Amy H. Zhao
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853, USA
| | - Vimal Selvaraj
- Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853, USA
- Correspondence should be addressed to: Vimal Selvaraj, Department of Animal Science, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853; ; Tel. 607-255-6138; Fax. 607-255-9829
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5
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Liu J, Wang J, Tian W, Xu Y, Li R, Zhao K, You C, Zhu Y, Bartsch JW, Niu H, Zhang H, Shu K, Lei T. PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling. Aging (Albany NY) 2022; 14:6066-6080. [PMID: 35963638 PMCID: PMC9417224 DOI: 10.18632/aging.204206] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2022] [Accepted: 07/27/2022] [Indexed: 12/05/2022]
Abstract
As the second most common primary intracranial neoplasms, about 40% of pituitary adenomas (PAs) exhibit aggressive behaviors and resulting in poor patient prognosis. The molecular mechanisms underlying the aggressive behaviors of PAs are not yet fully understood. Biochemical studies have reported that programmed cell death 10 (PDCD10) is a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex and plays a dual role in cancers in a tissue- or disease-specific manner. In the present study, we report for the first time that the role of PDCD10 in PAs. Cell proliferation, migration and invasion were either enhanced by overexpressing or inhibited by silencing PDCD10 in PA cells. Moreover, PDCD10 significantly promoted epithelial–mesenchymal transition (EMT) of pituitary adenoma cells. Mechanistically, we showed that the expression of CXCR2, together with phosphorylation levels of AKT and ERK1/2 were regulated by PDCD10. Activation of CXCR2 inversed inactivation of AKT/ERK signal pathways and the tumor-suppressive effects induced by PDCD10 silencing. Finally, the pro-oncogenic effect of PDCD10 was confirmed by in vivo tumor grafting. Taken together, we demonstrate for the first time that PDCD10 can induce aggressive behaviors of PAs by promoting cellular proliferation, migration, invasion and EMT through CXCR2-AKT/ERK signaling axis.
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Affiliation(s)
- Jingdian Liu
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Junwen Wang
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Weidong Tian
- Department of Neurosurgery, The First Affiliated Hospital, School of Medicine, Shihezi University, Shihezi, China
| | - Yu Xu
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ran Li
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Kai Zhao
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Chao You
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Yuan Zhu
- Department of Neurosurgery, University of Duisburg-Essen, Essen, Germany
| | | | - Hongquan Niu
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Huaqiu Zhang
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Kai Shu
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ting Lei
- Department of Neurosurgery, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Nunes B, Pópulo H, Lopes JM, Reis M, Nascimento G, Nascimento AG, Fernandes J, Faria M, de Carvalho DP, Soares P, Miranda-Alves L. Connexin Expression in Pituitary Adenomas and the Effects of Overexpression of Connexin 43 in Pituitary Tumor Cell Lines. Genes (Basel) 2022; 13:genes13040674. [PMID: 35456480 PMCID: PMC9032236 DOI: 10.3390/genes13040674] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/22/2022] [Accepted: 04/06/2022] [Indexed: 01/27/2023] Open
Abstract
Gap junction intercellular communication (GJIC) is considered a key mechanism in the regulation of tissue homeostasis. GJIC structures are organized in two transmembrane channels, with each channel formed by connexins (Cxs). GJIC and Cxs expression alterations are related to the process of tumorigenesis in different cell types. Pituitary neuroendocrine tumors (PitNETs) represent 15–20% of intracranial neoplasms, and usually display benign behavior. Nevertheless, some may have aggressive behavior, invading adjacent tissues, and featuring a high proliferation rate. We aimed to assess the expression and relevance of GJIC and Cxs proteins in PitNETs. We evaluated the mRNA expression levels of Cx26, 32, and 43, and the protein expression of Cx43 in a series of PitNETs. In addition, we overexpressed Cx43 in pituitary tumor cell lines. At the mRNA level, we observed variable expression of all the connexins in the tumor samples. Cx43 protein expression was absent in most of the pituitary tumor samples that were studied. Moreover, in vitro studies revealed that the overexpression of Cx43 decreases cell growth and induces apoptosis in pituitary tumor cell lines. Our results indicate that the downregulation of Cx43 protein might be involved in the tumorigenesis of most pituitary adenomas and have a potential therapeutic value for pituitary tumor therapy.
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Affiliation(s)
- Bruno Nunes
- Laboratory of Experimental Endocrinology—LEEx, Institute of Biomedical Science, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil; (B.N.); (D.P.d.C.); (L.M.-A.)
- Postgraduate Program in Endocrinology, Faculty of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
- Laboratory of Endocrine Physiology, Doris Rosenthal, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
| | - Helena Pópulo
- Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal; (H.P.); (J.M.L.); (M.R.)
- Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)—Cancer Signalling & Metabolism, 4200-135 Porto, Portugal
- Department of Pathology, Medical Faculty of the University of Porto, 4200-319 Porto, Portugal
| | - José Manuel Lopes
- Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal; (H.P.); (J.M.L.); (M.R.)
- Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)—Cancer Signalling & Metabolism, 4200-135 Porto, Portugal
- Department of Pathology, Medical Faculty of the University of Porto, 4200-319 Porto, Portugal
| | - Marta Reis
- Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal; (H.P.); (J.M.L.); (M.R.)
- Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)—Cancer Signalling & Metabolism, 4200-135 Porto, Portugal
| | - Gilvan Nascimento
- Centre of Clinical Research (CEPEC), President Dutra Hospital of Federal University of Maranhão (UFMA), São Luís 65020-600, Brazil; (G.N.); (M.F.)
- Endocrinology Service, President Dutra Hospital of Federal University of Maranhão (UFMA), São Luís 65060-600, Brazil
| | - Ana Giselia Nascimento
- Pathology Service, President Dutra Hospital of Federal University of Maranhão (UFMA), São Luís 65020-070, Brazil;
| | - Janaína Fernandes
- NUPEX, Polo Duque de Caxias, Universidade Federal do Rio de Janeiro, Rio de Janeiro 25240-005, Brazil;
| | - Manuel Faria
- Centre of Clinical Research (CEPEC), President Dutra Hospital of Federal University of Maranhão (UFMA), São Luís 65020-600, Brazil; (G.N.); (M.F.)
- Endocrinology Service, President Dutra Hospital of Federal University of Maranhão (UFMA), São Luís 65060-600, Brazil
| | - Denise Pires de Carvalho
- Laboratory of Experimental Endocrinology—LEEx, Institute of Biomedical Science, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil; (B.N.); (D.P.d.C.); (L.M.-A.)
- Postgraduate Program in Endocrinology, Faculty of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
- Laboratory of Endocrine Physiology, Doris Rosenthal, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
| | - Paula Soares
- Institute for Research and Innovation in Health, University of Porto, 4200-135 Porto, Portugal; (H.P.); (J.M.L.); (M.R.)
- Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)—Cancer Signalling & Metabolism, 4200-135 Porto, Portugal
- Department of Pathology, Medical Faculty of the University of Porto, 4200-319 Porto, Portugal
- Correspondence:
| | - Leandro Miranda-Alves
- Laboratory of Experimental Endocrinology—LEEx, Institute of Biomedical Science, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil; (B.N.); (D.P.d.C.); (L.M.-A.)
- Postgraduate Program in Endocrinology, Faculty of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
- Laboratory of Endocrine Physiology, Doris Rosenthal, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
- Postgraduate Program in Pharmacology and Medicinal Chemistry, Institute of Biomedical Science, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil
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Liu C, Nakano-Tateno T, Satou M, Chik C, Tateno T. Emerging role of signal transducer and activator of transcription 3 (STAT3) in pituitary adenomas. Endocr J 2021; 68:1143-1153. [PMID: 34248112 DOI: 10.1507/endocrj.ej21-0106] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Pituitary adenomas are benign tumours that can cause an individual various clinical manifestations including tumour mass effects and/or the diverse effects of abnormal pituitary hormone secretion. Given the morbidity and limited treatment options for pituitary adenomas, there is a need for better biomarkers and treatment options. One molecule that is of specific interest is the signal transducer and activator of transcription 3 (STAT3), a transcription factor that plays a critical role in mediating cytokine-induced changes in gene expression. In addition, STAT3 controls cell proliferation by regulating mitochondrial activity. Not only does activation of STAT3 play a crucial role in tumorigenesis, including pituitary tumorigenesis, but a number of studies also demonstrate pharmacological STAT3 inhibition as a promising treatment approach for many types of tumours, including pituitary tumours. This review will focus on the role of STAT3 in different pituitary adenomas, in particular, growth hormone-producing adenomas and null cell adenomas. Furthermore, how STAT3 is involved in the cell proliferation and hormone regulation in pituitary adenomas and its potential role as a molecular therapeutic target in pituitary adenomas will be summarized.
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Affiliation(s)
- Cyndy Liu
- Division of Endocrinology and Metabolism, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Tae Nakano-Tateno
- Division of Endocrinology and Metabolism, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Motoyasu Satou
- Division of Endocrinology and Metabolism, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
- Department of Biochemistry, Dokkyo Medical University School of Medicine, Mibu, Tochigi, Japan
| | - Constance Chik
- Division of Endocrinology and Metabolism, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
| | - Toru Tateno
- Division of Endocrinology and Metabolism, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada
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8
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Ma CIJ, Burgess J, Brill JA. Maturing secretory granules: Where secretory and endocytic pathways converge. Adv Biol Regul 2021; 80:100807. [PMID: 33866198 DOI: 10.1016/j.jbior.2021.100807] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2021] [Revised: 03/10/2021] [Accepted: 03/18/2021] [Indexed: 10/21/2022]
Abstract
Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.
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Affiliation(s)
- Cheng-I Jonathan Ma
- Cell Biology Program, The Hospital for Sick Children, PGCRL Building, Room 15.9716, 686 Bay Street, Toronto, Ontario, M5G 0A4, Canada; Institute of Medical Science, University of Toronto, Medical Sciences Building, Room 2374, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada
| | - Jason Burgess
- Cell Biology Program, The Hospital for Sick Children, PGCRL Building, Room 15.9716, 686 Bay Street, Toronto, Ontario, M5G 0A4, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, Room 4396, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada
| | - Julie A Brill
- Cell Biology Program, The Hospital for Sick Children, PGCRL Building, Room 15.9716, 686 Bay Street, Toronto, Ontario, M5G 0A4, Canada; Institute of Medical Science, University of Toronto, Medical Sciences Building, Room 2374, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada; Department of Molecular Genetics, University of Toronto, Medical Sciences Building, Room 4396, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada.
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9
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Serrero G. Progranulin/GP88, A Complex and Multifaceted Player of Tumor Growth by Direct Action and via the Tumor Microenvironment. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1329:475-498. [PMID: 34664252 DOI: 10.1007/978-3-030-73119-9_22] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Investigation of the role of progranulin/GP88 on the proliferation and survival of a wide variety of cells has been steadily increasing. Several human diseases stem from progranulin dysregulation either through its overexpression in cancer or its absence as in the case of null mutations in some form of frontotemporal dementia. The present review focuses on the role of progranulin/GP88 in cancer development, progression, and drug resistance. Various aspects of progranulin identification, biology, and signaling pathways will be described. Information will be provided about its direct role as an autocrine growth and survival factor and its paracrine effect as a systemic factor as well as via interaction with extracellular matrix proteins and with components of the tumor microenvironment to influence drug resistance, migration, angiogenesis, inflammation, and immune modulation. This chapter will also describe studies examining progranulin/GP88 tumor tissue expression as well as circulating level as a prognostic factor for several cancers. Due to the wealth of publications in progranulin, this review does not attempt to be exhaustive but rather provide a thread to lead the readers toward more in-depth exploration of this fascinating and unique protein.
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Lines KE, Filippakopoulos P, Stevenson M, Müller S, Lockstone HE, Wright B, Knapp S, Buck D, Bountra C, Thakker RV. Effects of epigenetic pathway inhibitors on corticotroph tumour AtT20 cells. Endocr Relat Cancer 2020; 27:163-174. [PMID: 31935194 PMCID: PMC7040567 DOI: 10.1530/erc-19-0448] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2019] [Accepted: 01/13/2020] [Indexed: 12/13/2022]
Abstract
Medical treatments for corticotrophinomas are limited, and we therefore investigated the effects of epigenetic modulators, a new class of anti-tumour drugs, on the murine adrenocorticotropic hormone (ACTH)-secreting corticotrophinoma cell line AtT20. We found that AtT20 cells express members of the bromo and extra-terminal (BET) protein family, which bind acetylated histones, and therefore, studied the anti-proliferative and pro-apoptotic effects of two BET inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, using CellTiter Blue and Caspase Glo assays, respectively. JQ1 and PFI-1 significantly decreased proliferation by 95% (P < 0.0005) and 43% (P < 0.0005), respectively, but only JQ1 significantly increased apoptosis by >50-fold (P < 0.0005), when compared to untreated control cells. The anti-proliferative effects of JQ1 and PFI-1 remained for 96 h after removal of the respective compound. JQ1, but not PFI-1, affected the cell cycle, as assessed by propidium iodide staining and flow cytometry, and resulted in a higher number of AtT20 cells in the sub G1 phase. RNA-sequence analysis, which was confirmed by qRT-PCR and Western blot analyses, revealed that JQ1 treatment significantly altered expression of genes involved in apoptosis, such as NFκB, and the somatostatin receptor 2 (SSTR2) anti-proliferative signalling pathway, including SSTR2. JQ1 treatment also significantly reduced transcription and protein expression of the ACTH precursor pro-opiomelanocortin (POMC) and ACTH secretion by AtT20 cells. Thus, JQ1 treatment has anti-proliferative and pro-apoptotic effects on AtT20 cells and reduces ACTH secretion, thereby indicating that BET inhibition may provide a novel approach for treatment of corticotrophinomas.
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Affiliation(s)
- K E Lines
- OCDEM, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford, UK
| | | | - M Stevenson
- OCDEM, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford, UK
| | - S Müller
- Structural Genomics Consortium, Buchmann Institute for Life Sciences, Goethe-University Frankfurt, Frankfurt, Germany
| | - H E Lockstone
- Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - B Wright
- Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - S Knapp
- Structural Genomics Consortium, Buchmann Institute for Life Sciences, Goethe-University Frankfurt, Frankfurt, Germany
- Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Frankfurt, Germany
| | - D Buck
- Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
| | - C Bountra
- Structural Genomics Consortium, University of Oxford, Oxford, UK
| | - R V Thakker
- OCDEM, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford, UK
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Shida A, Ikeda T, Tani N, Morioka F, Aoki Y, Ikeda K, Watanabe M, Ishikawa T. Cortisol levels after cold exposure are independent of adrenocorticotropic hormone stimulation. PLoS One 2020; 15:e0218910. [PMID: 32069307 PMCID: PMC7028257 DOI: 10.1371/journal.pone.0218910] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Accepted: 01/27/2020] [Indexed: 02/08/2023] Open
Abstract
We previously showed that postmortem serum levels of adrenocorticotropic hormone (ACTH) were significantly higher in cases of hypothermia (cold exposure) than other causes of death. This study examined how the human hypothalamic-pituitary-adrenal axis, and specifically cortisol, responds to hypothermia. Human samples: Autopsies on 205 subjects (147 men and 58 women; age 15-98 years, median 60 years) were performed within 3 days of death. Cause of death was classified as either hypothermia (cold exposure, n = 14) or non-cold exposure (controls; n = 191). Cortisol levels were determined in blood samples obtained from the left and right cardiac chambers and common iliac veins using a chemiluminescent enzyme immunoassay. Adrenal gland tissues samples were stained for cortisol using a rabbit anti-human polyclonal antibody. Cell culture: AtT20, a mouse ACTH secretory cell line, and Y-1, a corticosterone secretory cell line derived from a mouse adrenal tumor, were analyzed in mono-and co-culture, and times courses of ACTH (in AtT20) and corticosterone (in Y-1) secretion were assessed after low temperature exposure mimicking hypothermia and compared with data for samples collected postmortem for other cases of death. However, no correlation between ACTH concentration and cortisol levels was observed in hypothermia cases. Immunohistologic analyses of samples from hypothermia cases showed that cortisol staining was localized primarily to the nucleus rather than the cytoplasm of cells in the zona fasciculata of the adrenal gland. During both mono-culture and co-culture, AtT20 cells secreted high levels of ACTH after 10-15 minutes of cold exposure, whereas corticosterone secretion by Y-1 cells increased slowly during the first 15-20 minutes of cold exposure. Similar to autopsy results, no correlation was detected between ACTH levels and corticosterone secretion, either in mono-culture or co-culture experiments. These results suggested that ACTH-independent cortisol secretion may function as a stress response during cold exposure.
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Affiliation(s)
- Alissa Shida
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
- * E-mail:
| | - Tomoya Ikeda
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan
| | - Naoto Tani
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan
| | - Fumiya Morioka
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - Yayoi Aoki
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - Kei Ikeda
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
| | - Miho Watanabe
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
- Laboratory of Clinical Regenerative Medicine Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, Health and Medical Science Innovation laboratory, Tsukuba City, Ibaraki, Japan
| | - Takaki Ishikawa
- Department of Legal Medicine, Osaka City University Medical School, Osaka, Japan
- Forensic Autopsy Section, Medico-legal Consultation and Postmortem Investigation Support Center (MLCPI-SC), Osaka, Japan
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Zemková H, Stojilkovic SS. Neurotransmitter receptors as signaling platforms in anterior pituitary cells. Mol Cell Endocrinol 2018; 463:49-64. [PMID: 28684290 PMCID: PMC5752632 DOI: 10.1016/j.mce.2017.07.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Revised: 06/29/2017] [Accepted: 07/02/2017] [Indexed: 02/07/2023]
Abstract
The functions of anterior pituitary cells are controlled by two major groups of hypothalamic and intrapituitary ligands: one exclusively acts on G protein-coupled receptors and the other activates both G protein-coupled receptors and ligand-gated receptor channels. The second group of ligands operates as neurotransmitters in neuronal cells and their receptors are termed as neurotransmitter receptors. Most information about pituitary neurotransmitter receptors was obtained from secretory studies, RT-PCR analyses of mRNA expression and immunohistochemical and biochemical analyses, all of which were performed using a mixed population of pituitary cells. However, recent electrophysiological and imaging experiments have characterized γ-aminobutyric acid-, acetylcholine-, and ATP-activated receptors and channels in single pituitary cell types, expanding this picture and revealing surprising differences in their expression between subtypes of secretory cells and between native and immortalized pituitary cells. The main focus of this review is on the electrophysiological and pharmacological properties of these receptors and their roles in calcium signaling and calcium-controlled hormone secretion.
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Affiliation(s)
- Hana Zemková
- Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology, ASCR, Prague, Czech Republic.
| | - Stanko S Stojilkovic
- Sections on Cellular Signaling, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA
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Sato JD, Okamoto T, Barnes D, Hayashi J, Serrero G, McKeehan WL. A tribute to Dr. Gordon Hisashi Sato (December 24, 1927-March 31, 2017). In Vitro Cell Dev Biol Anim 2018; 54:177-193. [PMID: 29435725 DOI: 10.1007/s11626-018-0230-1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2017] [Accepted: 01/02/2018] [Indexed: 11/26/2022]
Abstract
Gordon H. Sato, an innovator in mammalian tissue culture and integrated cellular physiology, passed away in 2017. In tribute to Dr. Sato, In Vitro Cellular and Developmental Biology-Animal presents a collection of invited remembrances from six colleagues whose associations with Dr. Sato spanned more than 40 years. Dr. Sato was a past president of the Tissue Culture Association (now the Society for In Vitro Biology), editor-in-chief of In Vitro Cellular and Developmental Biology (1987-1991), and the recipient of the lifetime achievement award from the Society for In Vitro Biology (2002). He was elected to the US National Academy of Sciences in 1984.
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Affiliation(s)
- J Denry Sato
- Manzanar Project Foundation, 27 Cedar St., Wenham, MA, 01984, USA
| | - Tetsuji Okamoto
- Department of Molecular Oral Medicine & Maxillofacial Surgery, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.
| | - David Barnes
- School of Science and Technology, Georgia Gwinnett College, Lawrenceville, GA, 30043, USA
| | - Jun Hayashi
- Precision Antibody Inc., Columbia, MD, 21045, USA
| | | | - Wallace L McKeehan
- Center for Translational Cancer Research, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, 77030, USA
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Ohsaki Y, Kawai T, Yoshikawa Y, Cheng J, Jokitalo E, Fujimoto T. PML isoform II plays a critical role in nuclear lipid droplet formation. J Cell Biol 2016; 212:29-38. [PMID: 26728854 PMCID: PMC4700481 DOI: 10.1083/jcb.201507122] [Citation(s) in RCA: 136] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
PML-II plays a critical role in generating nuclear lipid droplets, which are associated with promyelocytic leukemia nuclear bodies as well as with the extension of the inner nuclear membrane. Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types.
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Affiliation(s)
- Yuki Ohsaki
- Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Takeshi Kawai
- Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Yukichika Yoshikawa
- Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Jinglei Cheng
- Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Eija Jokitalo
- Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland
| | - Toyoshi Fujimoto
- Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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Basham KJ, Hung HA, Lerario AM, Hammer GD. Mouse models of adrenocortical tumors. Mol Cell Endocrinol 2016; 421:82-97. [PMID: 26678830 PMCID: PMC4720156 DOI: 10.1016/j.mce.2015.11.031] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/07/2015] [Revised: 11/23/2015] [Accepted: 11/24/2015] [Indexed: 12/17/2022]
Abstract
The molecular basis of the organogenesis, homeostasis, and tumorigenesis of the adrenal cortex has been the subject of intense study for many decades. Specifically, characterization of tumor predisposition syndromes with adrenocortical manifestations and molecular profiling of sporadic adrenocortical tumors have led to the discovery of key molecular pathways that promote pathological adrenal growth. However, given the observational nature of such studies, several important questions regarding the molecular pathogenesis of adrenocortical tumors have remained. This review will summarize naturally occurring and genetically engineered mouse models that have provided novel tools to explore the molecular and cellular underpinnings of adrenocortical tumors. New paradigms of cancer initiation, maintenance, and progression that have emerged from this work will be discussed.
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Affiliation(s)
- Kaitlin J Basham
- Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, USA; Endocrine Oncology Program, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Holly A Hung
- Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, USA; Endocrine Oncology Program, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Antonio M Lerario
- Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, USA; Endocrine Oncology Program, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA
| | - Gary D Hammer
- Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, USA; Endocrine Oncology Program, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI, 48109, USA.
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HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity. PLoS One 2015; 10:e0130189. [PMID: 26066624 PMCID: PMC4465743 DOI: 10.1371/journal.pone.0130189] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2013] [Accepted: 05/17/2015] [Indexed: 12/21/2022] Open
Abstract
Background The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors. Methodology/Principal Findings We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells. Conclusions These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.
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Isovaline does not activate GABA(B) receptor-coupled potassium currents in GABA(B) expressing AtT-20 cells and cultured rat hippocampal neurons. PLoS One 2015; 10:e0118497. [PMID: 25706125 PMCID: PMC4337901 DOI: 10.1371/journal.pone.0118497] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2014] [Accepted: 01/19/2015] [Indexed: 01/13/2023] Open
Abstract
Isovaline is a non-proteinogenic amino acid that has analgesic properties. R-isovaline is a proposed agonist of the γ-aminobutyric acid type B (GABAB) receptor in the thalamus and peripheral tissue. Interestingly, the responses to R-isovaline differ from those of the canonical GABAB receptor agonist R-baclofen, warranting further investigation. Using whole cell recording techniques we explored isovaline actions on GABAB receptors coupled to rectifying K+ channels in cells of recombinant and native receptor preparations. In AtT-20 cells transfected with GABAB receptor subunits, bath application of the GABAB receptor agonists, GABA (1 μM) and R-baclofen (5 μM) produced inwardly rectifying currents that reversed approximately at the calculated reversal potential for K+ R- isovaline (50 μM to 1 mM) and S-isovaline (500 μM) did not evoke a current. R-isovaline applied either extracellularly (250 μM) or intracellularly (10 μM) did not alter responses to GABA at 1 μM. Co-administration of R-isovaline (250 μM) with a low concentration (10 nM) of GABA did not result in a response. In cultured rat hippocampal neurons that natively express GABAB receptors, R-baclofen (5 μM) induced GABAB receptor-dependent inward currents. Under the same conditions R-isovaline (1 or 50 μM) did not evoke a current or significantly alter R-baclofen-induced effects. Therefore, R-isovaline does not interact with recombinant or native GABAB receptors to open K+ channels in these preparations.
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Paul SD, Shrestha R, Palat A, Anbarasan S. Variation in growth pattern and morphological appearance of primary monolayer cultures of chondrocytes and neural cells isolated from the chick embryo at different stages. ACTA MEDICA INTERNATIONAL 2015; 2:61. [DOI: 10.5530/ami.2015.3.3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/22/2025] Open
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Hoshino S, Kurotani R, Miyano Y, Sakahara S, Koike K, Maruyama M, Ishikawa F, Sakatai I, Abe H, Sakai T. Macrophage Colony-Stimulating Factor Induces Prolactin Expression in Rat Pituitary Gland. Zoolog Sci 2014; 31:390-7. [DOI: 10.2108/zs130226] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Affiliation(s)
- Satoya Hoshino
- Department of Regulation Biology, Faculty of Science, Saitama University,Sakuraku Saitama 338-8570, Japan
| | - Reiko Kurotani
- Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan
| | - Yuki Miyano
- Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan
| | - Satoshi Sakahara
- Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan
| | - Kanako Koike
- Department of Regulation Biology, Faculty of Science, Saitama University,Sakuraku Saitama 338-8570, Japan
| | - Minoru Maruyama
- Department of Regulation Biology, Faculty of Science, Saitama University,Sakuraku Saitama 338-8570, Japan
| | - Fumio Ishikawa
- Department of Immunology, School of Medicine, Toho University, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan
| | - Ichiro Sakatai
- Department of Regulation Biology, Faculty of Science, Saitama University,Sakuraku Saitama 338-8570, Japan
| | - Hiroyuki Abe
- Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992-8510, Japan
| | - Takafumi Sakai
- Department of Regulation Biology, Faculty of Science, Saitama University,Sakuraku Saitama 338-8570, Japan
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Martin TJ. Historically significant events in the discovery of RANK/RANKL/OPG. World J Orthop 2013; 4:186-197. [PMID: 24147254 PMCID: PMC3801238 DOI: 10.5312/wjo.v4.i4.186] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/04/2012] [Revised: 01/07/2013] [Accepted: 03/23/2013] [Indexed: 02/06/2023] Open
Abstract
After it was suggested 30 years ago that the osteoblast lineage controlled the formation of osteoclasts, methods were developed that established this to be the case, but the molecular controls were elusive. Over more than a decade much evidence was obtained for signaling mechanisms that regulated the production of a membrane - bound regulator of osteoclastogenesis, in the course of which intercellular communication in bone was revealed in its complexity. The discovery of regulation by tumor necrosis factor ligand and receptor families was made in the last few years of the twentieth century, leading since then to a new physiology of bone, and to exciting drug development.
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The CpG island in the murine foxl2 proximal promoter is differentially methylated in primary and immortalized cells. PLoS One 2013; 8:e76642. [PMID: 24098544 PMCID: PMC3788739 DOI: 10.1371/journal.pone.0076642] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2013] [Accepted: 08/30/2013] [Indexed: 11/23/2022] Open
Abstract
Forkhead box L2 (Foxl2), a member of the forkhead transcription factor family, plays important roles in pituitary follicle-stimulating hormone synthesis and in ovarian maintenance and function. Mutations in the human FOXL2 gene cause eyelid malformations and premature ovarian failure. FOXL2/Foxl2 is expressed in pituitary gonadotrope and thyrotrope cells, the perioptic mesenchyme of the developing eyelid, and ovarian granulosa cells. The mechanisms governing this cell-restricted expression have not been described. We mapped the Foxl2 transcriptional start site in immortalized murine gonadotrope-like cells, LβT2, by 5’ rapid amplification of cDNA ends and then PCR amplified approximately 1 kb of 5’ flanking sequence from murine genomic DNA. When ligated into a reporter plasmid, the proximal promoter conferred luciferase activity in both homologous (LβT2) and, unexpectedly, heterologous (NIH3T3) cells. In silico analyses identified a CpG island in the proximal promoter and 5’ untranslated region, suggesting that Foxl2 transcription might be regulated epigenetically. Indeed, pyrosequencing and quantitative analysis of DNA methylation using real-time PCR revealed Foxl2 proximal promoter hypomethylation in homologous compared to some, though not all, heterologous cell lines. The promoter was also hypomethylated in purified murine gonadotropes. In vitro promoter methylation completely silenced reporter activity in heterologous and homologous cells. Collectively, the data suggest that differential proximal promoter DNA methylation may contribute to cell-specific Foxl2 expression in some cellular contexts. However, gonadotrope-specific expression of the gene cannot be explained by promoter hypomethylation alone.
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Okamoto T, Sato JD, Barnes DW, Sato GH. Biomedical advances from tissue culture. Cytotechnology 2013; 65:967-71. [PMID: 23828098 DOI: 10.1007/s10616-013-9591-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Accepted: 05/29/2013] [Indexed: 12/13/2022] Open
Abstract
The demonstration that the "dedifferentiation" of cells commonly observed in the early days of tissue culture was due to selective overgrowth of fibroblasts led to enrichment culture techniques (alternate animal and culture passage) designed to give a selective advantage to functionally differentiated tumor cells. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types. These results gave rise to the hypothesis that cells in culture accurately represent cells in vivo but without the complex in vivo environment. This concept has been strengthened with the development of hormonally defined culture media in combination with functionally differentiated clonal cell lines, which have augmented the potential of tissue culture studies. The use of hormonally defined media in place of serum-supplemented media demonstrates that hormonal responses and dependencies can be discovered in culture. Discoveries of hormonal dependencies of cancer cells has led to therapies targeting intracellular signaling pathways while discoveries of hormonal responses of pluripotent cells are helping to identify the potential application of stem cells. In these and other ways tissue culture technology will continue to contribute to solving problems of human health.
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Affiliation(s)
- Tetsuji Okamoto
- The Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, 734-8553, Japan,
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Haddad NF, Teodoro AJ, Leite de Oliveira F, Soares N, de Mattos RM, Hecht F, Dezonne RS, Vairo L, Goldenberg RCDS, Gomes FCA, de Carvalho DP, Gadelha MR, Nasciutti LE, Miranda-Alves L. Lycopene and beta-carotene induce growth inhibition and proapoptotic effects on ACTH-secreting pituitary adenoma cells. PLoS One 2013; 8:e62773. [PMID: 23667519 PMCID: PMC3647049 DOI: 10.1371/journal.pone.0062773] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2012] [Accepted: 03/25/2013] [Indexed: 12/21/2022] Open
Abstract
Pituitary adenomas comprise approximately 10-15% of intracranial tumors and result in morbidity associated with altered hormonal patterns, therapy and compression of adjacent sella turcica structures. The use of functional foods containing carotenoids contributes to reduce the risk of chronic diseases such as cancer and vascular disorders. In this study, we evaluated the influence of different concentrations of beta-carotene and lycopene on cell viability, colony formation, cell cycle, apoptosis, hormone secretion, intercellular communication and expression of connexin 43, Skp2 and p27(kip1) in ACTH-secreting pituitary adenoma cells, the AtT20 cells, incubated for 48 and 96 h with these carotenoids. We observed a decrease in cell viability caused by the lycopene and beta-carotene treatments; in these conditions, the clonogenic ability of the cells was also significantly decreased. Cell cycle analysis revealed that beta-carotene induced an increase of the cells in S and G2/M phases; furthermore, lycopene increased the proportion of these cells in G0/G1 while decreasing the S and G2/M phases. Also, carotenoids induced apoptosis after 96 h. Lycopene and beta-carotene decreased the secretion of ACTH in AtT20 cells in a dose-dependent manner. Carotenoids blocked the gap junction intercellular communication. In addition, the treatments increased the expression of phosphorylated connexin43. Finally, we also demonstrate decreased expression of S-phase kinase-associated protein 2 (Skp2) and increased expression of p27(kip1) in carotenoid-treated cells. These results show that lycopene and beta-carotene were able to negatively modulate events related to the malignant phenotype of AtT-20 cells, through a mechanism that could involve changes in the expression of connexin 43, Skp2 and p27(kip1); and suggest that these compounds might provide a novel pharmacological approach to the treatment of Cushing's disease.
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Affiliation(s)
- Natália F. Haddad
- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil
- Serviço de Endocrinologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil
| | - Anderson J. Teodoro
- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil
- Programa de Nutrição e Alimentos, Universidade do Estado do Rio de Janeiro, Brazil
| | | | - Nathália Soares
- Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Brazil
| | | | - Fábio Hecht
- Serviço de Endocrinologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil
| | | | - Leandro Vairo
- Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil
| | | | | | - Denise Pires de Carvalho
- Serviço de Endocrinologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil
| | - Mônica R. Gadelha
- Serviço de Endocrinologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil
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24
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Application of a new wall-less plate technology to complex multistep cell-based investigations using suspension cells. Blood 2013; 121:e25-33. [DOI: 10.1182/blood-2012-07-446294] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Key PointsThe DropArray technology is compatible with the retention of suspension cells in multistep procedures thus enabling novel assay methods. This technology enabled visualization and quantification of specific killing events triggered by bispecific antibodies engaging T cells.
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25
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Kim GL, Wang X, Chalmers JA, Thompson DR, Dhillon SS, Koletar MM, Belsham DD. Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes. PLoS One 2011; 6:e27799. [PMID: 22132145 PMCID: PMC3221660 DOI: 10.1371/journal.pone.0027799] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2011] [Accepted: 10/25/2011] [Indexed: 11/21/2022] Open
Abstract
The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.
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Affiliation(s)
- Ginah L. Kim
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Xiaomei Wang
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | | | - David R. Thompson
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | - Sandeep S. Dhillon
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
| | | | - Denise D. Belsham
- Department of Physiology, University of Toronto, Toronto, Ontario, Canada
- Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, Canada
- Department of Medicine, University of Toronto, Toronto, Ontario, Canada
- Division of Cellular and Molecular Biology, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada
- * E-mail:
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26
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Maixnerová J, Špolcová A, Pýchová M, Blechová M, Elbert T, Rezáčová M, Zelezná B, Maletínská L. Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor. Peptides 2011; 32:811-7. [PMID: 21185342 DOI: 10.1016/j.peptides.2010.12.011] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/28/2010] [Revised: 12/06/2010] [Accepted: 12/14/2010] [Indexed: 11/24/2022]
Abstract
The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K(d) in the 10(-9)M range and a B(max) in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with (125)I-PrRP31 with a similar K(i). The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.
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Affiliation(s)
- Jana Maixnerová
- Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
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28
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Sato GH, Sato JD, Okamoto T, McKeehan WL, Barnes DW. Tissue culture: the unlimited potential. In Vitro Cell Dev Biol Anim 2010; 46:590-4. [PMID: 20512426 DOI: 10.1007/s11626-010-9315-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2009] [Accepted: 03/31/2010] [Indexed: 12/31/2022]
Abstract
Lack of tissue-specific differentiated functions of cells in tissue culture, once thought to be due to "dedifferentiation", was shown to be due to selective overgrowth of fibroblasts by a series of simple experiments that challenged the prevailing dogma. Following this insight, enrichment culture techniques (alternate animal and culture passage) were designed to give functionally differentiated tumor cells selective advantage over the fibroblasts. These experiments resulted in the derivation of a large number of functionally differentiated clonal strains of a range of cell types, providing the final point of destruction of the dogma of "dedifferentiation." Instead, the hypothesis was proposed that cells in culture accurately represent cells in vivo, but without the complex in vivo environment. With the development of hormonally defined media and its combination with functionally differentiated clonal cell lines, this concept has been strengthened and the potential of tissue culture studies has been greatly augmented. Hormonally defined media allow the culture of cell types that cannot be grown in conventional, serum-supplemented media. These approaches demonstrate that hormonal responses and dependencies can be discovered in culture. Following this thinking and the discovery of hormonal dependencies of cancer cells has led to a new rationale for therapy. Tissue culture and cell technology continue to play an important role in solving human health problems.
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Affiliation(s)
- Gordon H Sato
- The Manzanar Project Foundation, 27 Cedar St., Wenham, MA 01984, USA.
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29
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Kowal J, Srinivasan S, Saito T. Calcium Modulation of Acth and Cholera Toxin Stimulated Adrenal Steroid and Cyclic-AMP Biosynthesis. ACTA ACUST UNITED AC 2009. [DOI: 10.3109/07435807409088996] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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30
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Sato G. Tissue culture: the unrealized potential. Cytotechnology 2007; 57:111-4. [PMID: 19003154 PMCID: PMC2553672 DOI: 10.1007/s10616-007-9109-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2007] [Accepted: 10/22/2007] [Indexed: 11/08/2022] Open
Abstract
Lack of differentiated functions of the tissue of origin in tissue culture thought to be due to dedifferentiation was shown to be due to selective overgrowth of fibroblasts. Enrichment culture techniques, (alternate animal and culture passage), designed to give the functionally differentiated cells selective advantage over the fibroblasts resulted in a large number of functionally differentiated clonal strains. Thus the dogma of dedifferentiation was destroyed. It is proposed to substitute the dedifferentiation hypothesis with the hypothesis that cells in culture accurately represent cells in vivo without the complex in vivo environment. With the development of hormonally defined media, combined with functionally differentiated clonal cell lines, the potential of tissue culture studies is greatly augmented. Hormonal responses and dependencies can be discovered in culture and the discovery of dependencies of cancer cells has led to a new rationale for therapy.
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Affiliation(s)
- Gordon Sato
- A and G Pharmaceutical Corp., 27 Cedar St., Wenham, MA, 01984, USA,
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31
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Ascoli M. Immortalized Leydig Cell Lines as Models for Studying Leydig Cell Physiology. CONTEMPORARY ENDOCRINOLOGY 2007:373-381. [DOI: 10.1007/978-1-59745-453-7_26] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/02/2023]
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32
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Aikawa S, Sato T, Ono T, Kato T, Kato Y. High level expression of Prop-1 gene in gonadotropic cell lines. J Reprod Dev 2005; 52:195-201. [PMID: 16394624 DOI: 10.1262/jrd.17050] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Prop-1 acts as an upstream regulator for the Pit-1 gene to induce development of Pit-1 lineage pituitary cell lines, GH-, PRL-, and TSH-producing cells, in the early stage of pituitary organogenesis. Furthermore, Prop-1 is presumed to be involved in the function of FSH/LH-producing cells, gonadotropes, since the defective Prop-1 gene shows hypogonadism. Recently, we reported evidence that Prop-1 directly regulates expression of the porcine FSHbeta gene, thus providing a novel advance in understanding the function of Prop-1 in FSH/LH production and hypogonadism. This study was intended to demonstrate the expressions of Prop-1 gene in pituitary tumor-derived cell lines. RT-PCR analyses were conducted of Pit-1, glycoprotein alpha subunit (alphaGSU), GnRH receptor, and cyclophilin A (a ubiquitously expressing gene). We observed expression of the Pit-1 gene in alphaT1-1, TalphaT1, MtT/S, GH3, and TtT/GF cells, expression of the alphaGSU gene in alphaT1-1, alphaT3-1, LbetaT2, LbetaT4, TalphaT1, and GH3 cells, and expression of GnRH receptor gene in alphaT3-1, LbetaT2, LbetaT4, and GH3 cells, respectively. These expression profiles were in accord with their cell lineages, with only a few exceptions. To accurately measure the expression level of the Prop-1 gene, a quantitative analysis was performed using the real-time PCR method. This analysis demonstrated that the LbetaT2 and LbetaT4 gonadotrope cell lines, which express the FSHbeta gene, express the Prop-1 gene. Taken together with our previous observation that Prop-1 is present in the adult porcine pituitary gonadotropes, Prop-1 might also be involved in development of gonadotropes and hormone production.
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Affiliation(s)
- Satoko Aikawa
- Laboratory of Molecular Biology and Gene Regulation, Department of Life Science, School of Agriculture, Meiji University, Kawasaki, Kanagawa, Japan
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33
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Abstract
The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones can make use of cells isolated from the adrenal gland but this requires animal sacrifice and the need for continued isolation for long-term studies. In addition primary cultures of adrenal cells have a limited life-span in culture and the cultured cells are often contaminated by the presence of non-steroidogenic cells. For that reason in vitro cell culture models have several benefits for research on adrenocortical function. Herein we discuss the available adrenocortical cell lines and their uses as model systems for adrenal studies. Focus is placed on the human NCI-H295 and mouse Y-1 adrenal cell lines, which have been used extensively as adrenocortical model systems. These cell lines have proven to be of considerable value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis. The current review will discuss the attributes and limitations of the currently available adrenocortical cell lines as models for adrenal studies.
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Affiliation(s)
- William E Rainey
- Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9032, USA.
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34
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Ooi GT, Tawadros N, Escalona RM. Pituitary cell lines and their endocrine applications. Mol Cell Endocrinol 2004; 228:1-21. [PMID: 15541569 DOI: 10.1016/j.mce.2004.07.018] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/17/2004] [Accepted: 07/15/2004] [Indexed: 10/26/2022]
Abstract
The pituitary gland is an important component of the endocrine system, and together with the hypothalamus, exerts considerable influence over the functions of other endocrine glands. The hypothalamus either positively or negatively regulates hormonal productions in the pituitary through its release of various trophic hormones which act on specific cell types in the pituitary to secrete a variety of pituitary hormones that are important for growth and development, metabolism, reproductive and nervous system functions. The pituitary is divided into three sections-the anterior lobe which constitute the majority of the pituitary mass and is composed primarily of five hormone-producing cell types (thyrotropes, lactotropes, corticotropes, somatotropes and gonadotropes) each secreting thyrotropin, prolactin, ACTH, growth hormone and gonadotropins (FSH and LH) respectively. There is also a sixth cell type in the anterior lobe-the non-endocrine, agranular, folliculostellate cells. The intermediate lobe produces melanocyte-stimulating hormone and endorphins, whereas the posterior lobe secretes anti-diuretic hormone (vasopressin) and oxytocin. Representative cell lines of all the six cell types of the anterior pituitary have been established and have provided valuable information on genealogy of the various cell lineages, endocrine feedback control of hormone synthesis and secretions, intrapituitary interactions between the various cell types, as well as the role of specific transcription factors that determine each differentiated cell phenotype. In this review, we will discuss the morphology and function of the cell types that make up the anterior pituitary, and the characteristics of the various functional anterior pituitary cell systems that have been established to be representative of each anterior pituitary cell lineage.
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Affiliation(s)
- Guck T Ooi
- Prince Henry's Institute of Medical Research, Monash Medical Centre, Block E, Level 4, 246 Clayton Road, Clayton, Victoria 3168, Australia.
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35
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Clipsham R, Zhang YH, Huang BL, McCabe ERB. Genetic network identification by high density, multiplexed reversed transcriptional (HD-MRT) analysis in steroidogenic axis model cell lines. Mol Genet Metab 2002; 77:159-78. [PMID: 12359144 DOI: 10.1016/s1096-7192(02)00119-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Transcriptional network analysis in steroidogenic axis cell lines requires an understanding of cellular network composition and complexity. Previous studies have shown that absence of transcriptional network components in a cell line compromises that cell line's functional capacity for transcriptional regulation. Our goal was to analyze qualitatively steroidogenic axis-derived cell lines' expression of a putative transcriptional network involved in human and mouse development. To pursue this analysis we used Northern blots and a high density-multiplexed reverse transcription-polymerase chain reaction (HD-MRT-PCR) approach. Our results revealed that, while some members of this putative network were universally expressed, only a minority of the non-constitutive targeted transcripts were present in any single line. Based on our data and previously published results for contextual expression of these transcription factors, a model was constructed possessing the topology suggestive of a scale-free network: certain network members were highly connected nodes and would represent critical sites of vulnerability. The importance of these highly connected nodes for network function is supported by the severe phenotypes exhibited by human patients and animal models when these genes are mutated. We conclude that knowledge of network composition in specific cell lines is essential for their use as models to investigate functional interactions within selected subnetworks.
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Affiliation(s)
- R Clipsham
- UCLA Molecular Biology Institute, Los Angeles, CA, USA
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36
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Affiliation(s)
- J. Denry Sato
- Adirondack Biomedical Research Institute Lake Placid New York
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37
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Abstract
The pituitary corticotrope AtT-20 stable cell line has been used as a model system to study peptide secretion, glucocorticoid regulation, and several other processes. In order to better understand this model cell line, a phage cDNA library was generated from AtT-20/D-16v cell mRNA and cDNA sequences were obtained for 317 clones representing 203 known genes and 48 novel cDNAs. The sequencing results revealed the prevalence of the mouse leukemia virus in this cell line and also identified a number of putatively secreted molecules that were not previously recognized as being secreted from AtT-20/D-16v cells or pituitary corticotropes. Nine completely novel cDNAs and 39 cDNAs homologous to known ESTs were also identified. A listing of other genes known to be expressed in AtT-20/D-16v cells is also provided.
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Affiliation(s)
- M R Schiller
- Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Ave., Farmington, Connecticut, USA.
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38
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Kurotani R, Osamura R, Tahara S, Sanno N, Teramoto A, Mellon P, Inoue K, Yoshimura S. Expression of Ptx1 in the adult rat pituitary glands and pituitary cell lines: hormone-secreting cells and folliculo-stellate cells. Cell Tissue Res 1999; 298:55-61. [PMID: 10555539 PMCID: PMC6095709 DOI: 10.1007/pl00008809] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
The pituitary homeobox1 gene (Ptx1) was initially identified as encoding a pituitary-restricted transcription factor for the proopiomelanocortin (POMC) gene. In order to elucidate the expression pattern of the Ptx1 protein, we investigated the localization of the protein in adult rat pituitary gland and in various pituitary cell lines. We produced an antibody specific for Ptxl protein, and confirmed its specificity by Western blot analysis. Immunohistochemically, many nuclei in the anterior pituitary cells as well as in the intermediate cells were positive for Ptxl staining with this specific antibody. Immunohistochemical double staining revealed the presence of Ptx1 not only in all types of hormone-secreting cells but also in some folliculo-stellate (FS) cells. Furthermore, the expression of Ptx1 mRNA was confirmed in various pituitary cell lines and in the FS cell line by using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Our studies indicated that Ptxl may not only play a role as a basic transcriptional factor for production of various hormones, but may also play some important role(s) in FS cells. Possible synergistic actions with other factors remain to be investigated. The novel finding of Ptx1 in FS cells is of particular interest, and may suggest that FS cells and hormone-secreting cells are derived from a common cellular ancestor.
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Affiliation(s)
| | - R.Y. Osamura
- Department of Pathology, Tokai University School of Medicine, Boseidai Isehara City, Kanagawa 259-1193, Japan, ; Tel: +81 463 93 1121; Fax: +81 463 91 1370
| | | | | | - A. Teramoto
- Department of Neurosurgery, Nippon Medical School, Tokyo
| | - P.L. Mellon
- Department of Reproductive Medicine and Neuroscience, School of Medicine, University of California, San Diego, USA
| | - K. Inoue
- Department of Regulation Biology, Faculty of Science, Saitama University, Saitama, Japan
| | - S. Yoshimura
- Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan
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Zhang H, Serrero G. Inhibition of tumorigenicity of the teratoma PC cell line by transfection with antisense cDNA for PC cell-derived growth factor (PCDGF, epithelin/granulin precursor). Proc Natl Acad Sci U S A 1998; 95:14202-7. [PMID: 9826678 PMCID: PMC24351 DOI: 10.1073/pnas.95.24.14202] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The PC cell line is a highly tumorigenic, insulin-independent, teratoma-derived cell line isolated from the nontumorigenic, insulin-dependent 1246 cell line. Studies of the PC cell growth properties have led to the purification of an 88-kDa secreted glycoprotein called PC cell-derived growth factor (PCDGF), which has been shown to stimulate the growth of PC cells as well as 3T3 fibroblasts. Sequencing of PCDGF cDNA demonstrated its identity to the precursor of a family of 6-kDa double-cysteine-rich polypeptides called epithelins or granulins (epithelin/granulin precursor). Since PCDGF was isolated from highly tumorigenic cells, its level of expression was examined in PC cells as well as in nontumorigenic and moderately tumorigenic cells from which PC cells were derived. Northern blot and Western blot analyses indicate that the levels of PCDGF mRNA and protein were very low in the nontumorigenic cells and increased in tumorigenic cell lines in a positive correlation with their tumorigenic properties. Experiments were performed to determine whether the autocrine production of PCDGF was involved in the tumorigenicity of PC cells. For this purpose, we examined the in vivo growth properties in syngeneic C3H mice of PC cells where PCDGF expression had been inhibited by transfection of antisense PCDGF cDNA. The results show that inhibition of PCDGF expression resulted in a dramatic inhibition of tumorigenicity of the transfected cells when compared with empty-vector control cells. These data demonstrate the importance in tumor formation of overexpression of the novel growth factor PCDGF.
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Affiliation(s)
- H Zhang
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy and Program of Oncology, Marlene and Stewart Greenebaum Cancer Center of the University of Maryland, Baltimore, MD 21201-1180, USA
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40
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Ham J, Webster J, Bond JA, Jasani B, Lewis MD, Hepburn PJ, Davies JS, Lewis BM, Thomas DW, Scanlon MF. Immortalized human pituitary cells express glycoprotein alpha-subunit and thyrotropin beta (TSH beta). J Clin Endocrinol Metab 1998; 83:1598-603. [PMID: 9589662 DOI: 10.1210/jcem.83.5.4803] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
A major problem in the study of human pituitary cells is their lack of proliferative capacity in vitro. To address this issue, we have infected normal human, postmortem pituitary cells in monolayer culture with a temperature-sensitive (tsA58) mutant of SV40 large T antigen. Several epithelial-like colonies were isolated; and one, designated CHP2, has been studied in detail to identify its functional characteristics. CHP2 cells have undergone more than 150 culture passages and retain an epithelial morphology. They exhibit tight temperature-dependent growth, in the presence and absence of serum, with cell division at 33 C and growth inhibition at 39 C. CHP2 cells, at both temperatures, showed diffuse immunostaining for human alpha-subunit and focal staining for TSH beta. Gene expression was confirmed by RT-PCR and sequencing. TRH and GnRH receptors were not detectable, and their absence was confirmed by their lack of effects on intracellular calcium and inositol phospholipids. Cytogenetic analysis showed that the cells had a modal peak in the diploid range and a smaller peak in the tetraploid range. There was also a consistent loss of chromosome 22 and a normal chromosome 2 homologue, the latter being replaced by one of two chromosome 2 markers, M2A or M2B. In conclusion, we have immortalized human pituitary cells using SV40 tsT, from which we have cloned a cell line expressing alpha-subunit and TSH beta.
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Affiliation(s)
- J Ham
- Department of Medicine, University of Wales College of Medicine, Cardiff, United Kingdom
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Beinfeld MC. Prohormone and proneuropeptide processing. Recent progress and future challenges. Endocrine 1998; 8:1-5. [PMID: 9666338 DOI: 10.1385/endo:8:1:1] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/1997] [Accepted: 09/18/1997] [Indexed: 02/08/2023]
Abstract
Our knowledge of prohormone and proneuropeptide processing and its relationship to the secretory pathway has advanced significantly in the last five years. The recent discovery of the prohormone convertase family of proteolytic enzymes has provided new candidates for the prohormone and proneuropeptide convertases. The increasing appreciation of the role of proteolysis in diverse cellular processes has also brought the prohormone processing field closer to the fields of growth factor processing, the role of host proteases in viral and bacterial pathogenesis and toxicity, control of the cell cycle, inflammation, and apoptosis. The last five years have been very productive, but the most interesting questions are still unanswered. Which enzymes are actually responsible for prohormone cleavages in specific tissues? What structural features of the prohormones determine where it will be processed or how it is recognized as secretory material by the sorting machinery? How is tissue-specific processing determined and regulated? The availability of protease knockout mice and and a more detailed understanding of the complex biosynthetic activation of these enzymes will provide at least some of the answers.
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Affiliation(s)
- M C Beinfeld
- Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, MA 02111, USA
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Brasaemle DL, Barber T, Kimmel AR, Londos C. Post-translational regulation of perilipin expression. Stabilization by stored intracellular neutral lipids. J Biol Chem 1997; 272:9378-87. [PMID: 9083075 DOI: 10.1074/jbc.272.14.9378] [Citation(s) in RCA: 118] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023] Open
Abstract
The perilipins are a family of polyphosphorylated proteins found exclusively surrounding neutral lipid storage droplets in adipocytes and steroidogenic cells. In steroidogenic cells, the cholesterol ester-rich lipid storage droplets are encoated with perilipins A and C. This study describes the dependence of perilipin levels on neutral lipid storage in cultured Y-1 adrenal cortical cells. The addition of fatty acids and cholesterol to the culture medium of Y-1 adrenal cortical cells greatly increased the storage of cholesterol esters and triacylglycerols concomitant with the formation of many new lipid storage droplets. The addition of fatty acids to the culture medium also produced a transient 6-fold increase in levels of perilipin A, but not C, mRNA, while much larger and stable increases in both perilipin A and C proteins were observed. The increases in perilipin protein levels were dependent upon the metabolism of fatty acids to triacylglycerol or cholesterol esters, since the incubation of cells with bromopalmitate, a poorly metabolized fatty acid, failed to yield large increases in lipid content or perilipin levels. Constitutive expression of epitope-tagged perilipins in transfected Y-1 adrenal cortical cells was regulated by lipid similarly to expression of the endogenous perilipins despite an absence of untranslated perilipin mRNA sequences in the expression constructs. Epitope-tagged perilipin A mRNAs were efficiently loaded with polyribosomes whether or not fatty acids were added to the culture medium; therefore, the increase in perilipin levels in the presence of fatty acids is likely due to factors other than increased translational efficiency. We suggest that the large increase in cellular perilipin levels upon lipid loading of cells is the result of post-translational stabilization of newly synthesized perilipins by stored neutral lipids.
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Affiliation(s)
- D L Brasaemle
- Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-2715, USA
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Abstract
A number of apparently unrelated factors are known to have a profound effect on the metabolism of cultured mammalian cells; and some of these may be operative as metabolic controls in the whole animal as well. The more complete exploration of (i) homotypic and heterotypic cellular interactions, (ii) the spontaneous transformations sometimes observed in cultured cells, (iii) the mode of action of cytotoxic agents, (iv) the multiple metabolic effects of viral infection, and (v) the conditions necessary for the maintenance of specialized function in cultured cells, can be expected to throw light on the basic mechanisms underlying such complex processes as differentiation, senescence, and cancer.
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44
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Gamby C, Waage MC, Allen RG, Baizer L. Growth-associated protein-43 (GAP-43) facilitates peptide hormone secretion in mouse anterior pituitary AtT-20 cells. J Biol Chem 1996; 271:10023-8. [PMID: 8626556 DOI: 10.1074/jbc.271.17.10023] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The neuronal growth-associated protein (GAP)-43 (neuromodulin, B-50, F1), which is concentrated in the growth cones of elongating axons during neuronal development and in nerve terminals in restricted regions of the adult nervous system, has been implicated in the release of neurotransmitter. To study the role of GAP-43 in evoked secretion, we transfected mouse anterior pituitary AtT-20 cells with the rat GAP-43 cDNA and derived stably transfected cell lines. Depolarization-mediated beta-endorphin secretion was greatly enhanced in the GAP-43-expressing AtT-20 cells without a significant change in Ca2+ influx; in contrast, expression of GAP-43 did not alter corticotropin-releasing factor-evoked hormone secretion. The transfected cells also displayed a flattened morphology and extended processes when plated on laminin-coated substrates. These results suggest that AtT-20 cells are a useful model system for further investigations on the precise biological function(s) of GAP-43.
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Affiliation(s)
- C Gamby
- R. S. DOW Neurological Sciences Institute, Good Samaritan Hospital and Medical Center, Portland, Oregon 97209, USA
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46
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Servetnick DA, Brasaemle DL, Gruia-Gray J, Kimmel AR, Wolff J, Londos C. Perilipins are associated with cholesteryl ester droplets in steroidogenic adrenal cortical and Leydig cells. J Biol Chem 1995; 270:16970-3. [PMID: 7622516 DOI: 10.1074/jbc.270.28.16970] [Citation(s) in RCA: 148] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Steroidogenic cells store cholesteryl esters, precursors for steroid hormone synthesis, in intracellular lipid droplets. Cholesteryl ester hydrolysis is activated by protein kinase A and catalyzed by cholesteryl esterase. The esterase is similar, if not identical, to hormone-sensitive lipase in adipocytes where an analogous lipolytic mechanism occurs. Perilipins, proteins located exclusively at lipid droplet surfaces in adipocytes, are polyphosphorylated by protein kinase A in response to lipolytic stimuli, suggesting a role for these proteins in mediating lipid metabolism. The present study reveals that perilipins are associated with cholesteryl ester droplets in two steroidogenic cell lines: Y-1 adrenal cortical cells and MA-10 Leydig cells. The relative abundance of perilipin mRNAs and protein is much less in steroidogenic cells than in adipocytes. Like adipocytes, steroidogenic cells express perilipin A; additionally, the latter cells contain relatively abundant amounts of perilipin C, a protein that is not detectable in adipocytes by Western analysis. The data suggest a strong link between perilipins and lipid hydrolysis that is mediated by the hormone-sensitive lipase/cholesteryl esterase class of enzymes.
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Affiliation(s)
- D A Servetnick
- Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA
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Saitoh Y, Taki T, Arita N, Ohnishi T, Hayakawa T. Analgesia induced by transplantation of encapsulated tumor cells secreting beta-endorphin. J Neurosurg 1995; 82:630-4. [PMID: 7897526 DOI: 10.3171/jns.1995.82.4.0630] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The purpose of this study was to assess whether xenogeneic tumor cells immunologically isolated in polymer capsules could survive and continue to reduce pain when transplanted into the cerebrospinal fluid (CSF) of rats. The mouse tumor cell lines AtT-20 and gene-transfected Neuro2A, which secrete beta-endorphin, were enclosed in polymer capsules at a density of 5 x 10(6) cells/ml and transplanted into the spinal CSF space of the occipitoatlantal junction in male Sprague-Dawley rats. The analgesiometric tests (tail pinch, hot plate, and electrical stimulation) showed that the five rats with encapsulated AtT-20 or Neuro2A (eight rats) were significantly less sensitive to pain after transplantation than the eight control animals (analysis of variance; p < 0.05). The analgesia induced by encapsulated cells secreting beta-endorphin could be attenuated by the opiate antagonist naloxone, which suggested the involvement of opiate in mediating this response. Morphological study revealed that the cells in polymer capsules survived 1 month after transplantation in the CSF space. In vitro experiments with cultured capsules showed that both encapsulated AtT-20 and Neuro2A secrete peptide for 1 month. The results of this study suggest that immunologically isolated xenogeneic tumor cells can secrete opiate in the CSF space, and this method may be applied to the treatment of cancer pain.
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Affiliation(s)
- Y Saitoh
- Department of Neurosurgery, Osaka University Medical School, Japan
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Rudnick A, Ling TY, Odagiri H, Rutter WJ, German MS. Pancreatic beta cells express a diverse set of homeobox genes. Proc Natl Acad Sci U S A 1994; 91:12203-7. [PMID: 7991607 PMCID: PMC45405 DOI: 10.1073/pnas.91.25.12203] [Citation(s) in RCA: 110] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Homeobox genes, which are found in all eukaryotic organisms, encode transcriptional regulators involved in cell-type differentiation and development. Several homeobox genes encoding homeodomain proteins that bind and activate the insulin gene promoter have been described. In an attempt to identify additional beta-cell homeodomain proteins, we designed primers based on the sequences of beta-cell homeobox genes cdx3 and lmx1 and the Drosophila homeodomain protein Antennapedia and used these primers to amplify inserts by PCR from an insulinoma cDNA library. The resulting amplification products include sequences encoding 10 distinct homeodomain proteins; 3 of these proteins have not been described previously. In addition, an insert was obtained encoding a splice variant of engrailed-2, a homeodomain protein previously identified in the central nervous system. Northern analysis revealed a distinct pattern of expression for each homeobox gene. Interestingly, the PCR-derived clones do not represent a complete sampling of the beta-cell library because no inserts encoding cdx3 or lmx1 protein were obtained. Beta cells probably express additional homeobox genes. The abundance and diversity of homeodomain proteins found in beta cells illustrate the remarkable complexity and redundancy of the machinery controlling beta-cell development and differentiation.
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Affiliation(s)
- A Rudnick
- Hormone Research Institute, University of California, San Francisco 94143-0534
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Tramontano D, Villone G. About thyroid cells in culture. J Endocrinol Invest 1994; 17:875-90. [PMID: 7745236 DOI: 10.1007/bf03347795] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Affiliation(s)
- D Tramontano
- Dipartimento di Medicina Sperimentale e Clinica, Università degli Studi di Reggio Calabria, Catanzaro, Italy
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Blass-Kampmann S, Reinhardt-Maelicke S, Kindler-Röhrborn A, Cleeves V, Rajewsky MF. In vitro differentiation of E-N-CAM expressing rat neural precursor cells isolated by FACS during prenatal development. J Neurosci Res 1994; 37:359-73. [PMID: 8176758 DOI: 10.1002/jnr.490370308] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Most fetal rat brain cells expressing the embryonal, highly sialylated form of the cell adhesion molecule N-CAM (E-N-CAM) are precursor cells, as judged from the absence of marker molecules specific for mature neural cell types. However, the detection of E-N-CAM+ cells in frozen sections does not provide information on the lineage-specific differentiation of these cells during development. To investigate their differentiation behaviour in vitro, E-N-CAM+ cells were isolated at different times of brain development by fluorescence-activated cell sorting (FACS), using a monoclonal antibody (Mab RB21-7) which specifically recognizes polysialic acid (PSA) residues on E-N-CAM. Double-immunofluorescence analyses showed that the majority of E-N-CAM+ cells isolated on prenatal days 15 to 18 differentiated into neurons while a small subset of Mab RB21-7 binding cells proved to be astrocytic precursors and/or bipotential. The proportion of E-N-CAM+ astrocytic precursors increased during later development (prenatal day 22) concomitantly with the onset of gliogenesis. While conversion of E-N-CAM to mature forms of N-CAM was never observed in neurons during cultivation, E-N-CAM+ cells of the astrocyte lineage switched to N-CAM soon after the onset of GFAP expression. A lineage-specific transition of E-N-CAM to mature N-CAM expression is, therefore, suggested for these astrocytic progenitor cells during rat brain development.
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Affiliation(s)
- S Blass-Kampmann
- Institute of Cell Biology (Cancer Research), University of Essen Medical School, Germany
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