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Mathien S, Tesnière C, Meloche S. Regulation of Mitogen-Activated Protein Kinase Signaling Pathways by the Ubiquitin-Proteasome System and Its Pharmacological Potential. Pharmacol Rev 2021; 73:263-296. [PMID: 34732541 DOI: 10.1124/pharmrev.120.000170] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. SIGNIFICANCE STATEMENT: Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics.
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Affiliation(s)
- Simon Mathien
- Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada (S.Ma., C.T., S.Me.); and Molecular Biology Program, Faculty of Medicine (C.T., S.Me.) and Department of Pharmacology and Physiology (S.Me.), Université de Montréal, Montreal, Quebec, Canada
| | - Chloé Tesnière
- Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada (S.Ma., C.T., S.Me.); and Molecular Biology Program, Faculty of Medicine (C.T., S.Me.) and Department of Pharmacology and Physiology (S.Me.), Université de Montréal, Montreal, Quebec, Canada
| | - Sylvain Meloche
- Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada (S.Ma., C.T., S.Me.); and Molecular Biology Program, Faculty of Medicine (C.T., S.Me.) and Department of Pharmacology and Physiology (S.Me.), Université de Montréal, Montreal, Quebec, Canada
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Kiwada T, Katakasu H, Okumura S, Odani A. Characterization of platinum(II) complexes exhibiting inhibitory activity against the 20S proteasome. ROYAL SOCIETY OPEN SCIENCE 2020; 7:200545. [PMID: 32968518 PMCID: PMC7481701 DOI: 10.1098/rsos.200545] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/08/2020] [Accepted: 07/23/2020] [Indexed: 06/11/2023]
Abstract
Proteasome inhibitors are useful for biochemical research and clinical treatment. In our previous study, we reported that the 4N-coordinated platinum complexes with anthracenyl ring and heterocycle exhibited proteasome-inhibitory activity. In the present study, the structure-activity relationships and characterization of these complexes were determined for the elucidation of the role of aromatic ligands. Lineweaver-Burk analysis revealed that the chemical structure of heterocycles affects the binding mode of platinum complexes. Platinum complexes with anthracenyl ring and pyridine showed competitive inhibition, although platinum complexes with anthracenyl ring and phenanthroline showed non-competitive inhibition. The structure-activity relationships demonstrated that anthracenyl moiety plays a crucial role in proteasome-inhibitory activity. The platinum complexes with naphthyl or phenyl rings exhibited lower inhibitory activities than the platinum complex with anthracenyl ring. The reactivity with N-acetylcysteine varied according to the chemical structure of complexes.
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Affiliation(s)
- Tatsuto Kiwada
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
| | - Hiromu Katakasu
- School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
| | - Serina Okumura
- School of Pharmacy, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
| | - Akira Odani
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan
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Kiwada T, Takayama H, Katakasu H, Ogawa K, Odani A. 20S Proteasome Inhibitory Activity of [ N-(9-Anthracenylmethyl)-1,3-propanediamine] (2,2′-Bipyridine) Palladium(II) Chloride. CHEM LETT 2019. [DOI: 10.1246/cl.190251] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Tatsuto Kiwada
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
| | - Hiroshi Takayama
- Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-8602, Japan
| | - Hiromu Katakasu
- School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
| | - Kazuma Ogawa
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
- Institute for Frontier Science Initiative, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
| | - Akira Odani
- Faculty of Pharmacy, Institute of Medical, Pharmaceutical, and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan
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Inhibition of Proteasome Activity Upregulates IL-6 Expression in RPE Cells through the Activation of P38 MAPKs. J Ophthalmol 2018; 2018:5392432. [PMID: 30116631 PMCID: PMC6079444 DOI: 10.1155/2018/5392432] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2018] [Accepted: 06/19/2018] [Indexed: 12/22/2022] Open
Abstract
Purpose As far as we know, during the development of age-related macular degeneration (AMD), the activity of proteasome in retinal pigment epithelium cells (RPE) gradually decreases. And a lot of research has shown that age-related macular degeneration is closely related to inflammation and autoimmune. Moreover, there are many cytokines (CKs) involved in the course of inflammation. In this study, we are going to investigate how the decrease of proteasome activity affects the production of interleukin-6 (IL-6) in human retinal pigment epithelium cells (ARPE-19). Methods Cultured ARPE-19 was treated with or without MG132, a proteasome inhibitor, and the levels of IL-6 mRNA (messenger ribonucleic acid) in RPE at 1 h, 4 h, 8 h, and IL-6 protein in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were measured by real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA). The protein levels of MCP-1 (monocyte chemoattractant protein-1) in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were also measured by ELISA. Then we tested which of cell signal pathways regulating the production of IL-6 were activated when we added MG132 into the medium by Western blot and electrophoretic mobility shift assays (EMSA). After that, we put the inhibitors of these activated cell signal pathways into the medium individually to see which inhibitor can counteract the effect of upregulating the levels of IL-6 in the culture medium of RPE. Results MG132 decreased the secretion of MCP-1 in the culture medium of RPE, but it increased the expression of IL-6 mRNA in RPE and IL-6 protein level in the culture medium of RPE. MG132 treatment was also found to enhance the level of phosphorylated p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal Kinase (JNK) by Western blotting. More importantly, the effect of MG132 on upregulating the levels of IL-6 was inhibited by SB203580, an inhibitor of P38 MAP kinases. But the JNK inhibitor, SP600125, cannot prevent the effect of upregulating the levels of IL-6 by MG132 in the RPE culture medium. Conclusions We concluded that the proteasome inhibitor, MG132, upregulates IL-6 production in RPE cells through the activation of P38 MAPKs.
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Farhan MA, Azad AK, Touret N, Murray AG. FGD5 Regulates VEGF Receptor-2 Coupling to PI3 Kinase and Receptor Recycling. Arterioscler Thromb Vasc Biol 2017; 37:2301-2310. [PMID: 29051140 DOI: 10.1161/atvbaha.117.309978] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Accepted: 10/10/2017] [Indexed: 11/16/2022]
Abstract
OBJECTIVE VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)-a Rho-family guanine nucleotide exchange factor-is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood. APPROACH AND RESULTS Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation. CONCLUSIONS FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling.
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Affiliation(s)
- Maikel A Farhan
- From the Department of Pediatrics (M.A.F.), Department of Medicine (A.K.A., A.G.M.), and Department of Biochemistry (N.T.), University of Alberta, Edmonton, Canada
| | - Abul K Azad
- From the Department of Pediatrics (M.A.F.), Department of Medicine (A.K.A., A.G.M.), and Department of Biochemistry (N.T.), University of Alberta, Edmonton, Canada
| | - Nicolas Touret
- From the Department of Pediatrics (M.A.F.), Department of Medicine (A.K.A., A.G.M.), and Department of Biochemistry (N.T.), University of Alberta, Edmonton, Canada
| | - Allan G Murray
- From the Department of Pediatrics (M.A.F.), Department of Medicine (A.K.A., A.G.M.), and Department of Biochemistry (N.T.), University of Alberta, Edmonton, Canada.
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Kimura T, Suzuki M, Akagi T. Age-dependent changes in synaptic plasticity enhance tau oligomerization in the mouse hippocampus. Acta Neuropathol Commun 2017; 5:67. [PMID: 28874186 PMCID: PMC5586024 DOI: 10.1186/s40478-017-0469-x] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2017] [Accepted: 08/22/2017] [Indexed: 01/09/2023] Open
Abstract
The aggregation mechanism of phosphorylated tau is an important therapeutic target for tauopathies, including Alzheimer’s disease, although the mechanism by which aggregation occurs is still unknown. Because the phosphorylation process of tau is involved in the trafficking of AMPA receptors, which accompanies the long-term depression (LTD) of synapses, we examined the effect of LTD-inducing low-frequency stimulation (LFS) on the formation of pathological tau aggregates in adult and aged wild-type mice. Our biochemical analysis demonstrated that LFS led to the formation of sarkosyl-insoluble (SI) tau oligomers in aged hippocampi but not in adult hippocampi in wild-type mice. In parallel, electrophysiological experiments showed an increased contribution of the autophagy-lysosomal pathway (ALP) to LTD during aging, although the other properties of LFS-induced LTD that we investigated were not altered. Thus, we anticipate that the increased contribution of the ALP to the LTD cascade is involved in the age-dependent formation of tau oligomers that results from LFS. Analysis of the LC3 ratio, an indicator of autophagosome formation, showed that LFS increased cleaved LC3 (type II) in the aged hippocampus relative to type I LC3, suggesting potentiation of the ALP accompanied by LTD. Pharmacological inhibition of autophagosome formation depressed LFS-induced oligomerization of tau. Prevention of lysosomal function in the ALP enhanced the formation of tau oligomers by LFS. These results suggest the importance of the autophagosome for the LFS-induced oligomerization of tau and suggest a reason for its age dependency. Interestingly, the lysosomal disturbance promoted the formation of the fibrillar form of aggregates consisting of hyper-phosphorylated tau. The LTD-ALP cascade potentially acts as one of the suppliers of pathological aggregates of tau in aged neurons.
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Vergara M, Berrios J, Martínez I, Díaz-Barrera A, Acevedo C, Reyes JG, Gonzalez R, Altamirano C. Endoplasmic Reticulum-Associated rht-PA Processing in CHO Cells: Influence of Mild Hypothermia and Specific Growth Rates in Batch and Chemostat Cultures. PLoS One 2015; 10:e0144224. [PMID: 26659083 PMCID: PMC4676689 DOI: 10.1371/journal.pone.0144224] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Accepted: 11/16/2015] [Indexed: 12/11/2022] Open
Abstract
Background Chinese hamster ovary (CHO) cells are the main host for producing recombinant proteins with human therapeutic applications mainly because of their capability to perform proper folding and glycosylation processes. In addition, mild hypothermia is one of the main strategies for maximising the productivity of these systems. However, little information is available on the effect of culture temperature on the folding and degradation processes of recombinant proteins that takes place in the endoplasmic reticulum. Methods In order to evaluate the effect of the mild hypothermia on processing/endoplasmatic reticulum-associated degradation (ERAD) processes, batch cultures of CHO cells producing recombinant human tissue plasminogen activator (rht-PA) were carried out at two temperatures (37°C and 33°C) and treated with specific inhibitors of glycosylation and ERAD I (Ubiquitin/Proteasome system) or ERAD II (Autophagosoma/Lisosomal system) pathways. The effect of mild hypothermia was analysed separately from its indirect effect on specific cell growth rate. To do this, chemostat cultures were carried out at the same incubation conditions as the batch cultures, controlling cell growth at high (0.017 h-1) and low (0.012 h-1) dilution rates. For a better understanding of the investigated phenomenon, cell behaviour was also analysed using principal component analysis (PCA). Results and Conclusion Results suggest that rht-PA is susceptible to degradation by both ERAD pathways studied, revealing that processing and/or ERAD processes are sensitive to temperature cultivation in batch culture. Moreover, by isolating the effect of culture temperature from the effect of cell growth rate verifyed by using chemostat cultures, we have found that processing and/or ERAD processes are more sensitive to reduction in specific growth rate than low temperature, and that temperature reduction may have a positive effect on protein processing. Interestingly, PCA indicated that the integrated performance displayed by CHO cells is modulated predominantly by specific growth rate, indicating that the culture temperature has a lower weighted effect within the range of conditions evaluated in this work.
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Affiliation(s)
- Mauricio Vergara
- Institute of Chemistry, Pontificia Universidad Católica de Valparaíso, Av. Universidad 330, Curauma, Chile
- School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, 4059, Chile
| | - Julio Berrios
- School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, 4059, Chile
| | - Irene Martínez
- School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, 4059, Chile
| | - Alvaro Díaz-Barrera
- School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, 4059, Chile
| | - Cristian Acevedo
- Biotechnology Center “Dr. Daniel Alkalay Lowitt”, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso, Chile
| | - Juan G. Reyes
- Institute of Chemistry, Pontificia Universidad Católica de Valparaíso, Av. Universidad 330, Curauma, Chile
| | - Ramon Gonzalez
- Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States of America
- Department of Bioengineering, Rice University, Houston, Texas, United States of America
| | - Claudia Altamirano
- School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Av. Brasil 2085, Valparaíso, 4059, Chile
- CREAS CONICYT Regional GORE Valparaíso R0GI1004. Av. Universidad, Curauma, Chile
- * E-mail:
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Skalniak L, Koj A, Jura J. Proteasome inhibitor MG-132 induces MCPIP1 expression. FEBS J 2013; 280:2665-74. [PMID: 23551903 PMCID: PMC3806276 DOI: 10.1111/febs.12264] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2013] [Revised: 03/19/2013] [Accepted: 03/22/2013] [Indexed: 01/14/2023]
Abstract
The proteasome is a protein complex responsible for the degradation of polyubiquitin-tagged proteins. Besides the removal of target proteins, the proteasome also participates in the regulation of gene transcription in both proteolytic and non-proteolytic fashion. In this study the effect of proteasome inhibition on the basal expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1) was examined. Treatment of HepG2 or HeLa cells with proteasome inhibitor MG-132 resulted in a significant increase of MCPIP1 expression, both at mRNA and protein level. Interestingly, MG-132 did not alter MCPIP1 stability. Instead, the observed protein increase was blocked by actinomycin D, suggesting the involvement of de novo mRNA synthesis in the increase of MCPIP1 protein following MG-132 treatment. Using several inhibitors we determined the participation of extracellular-signal-regulated kinase 1/2 and p38 kinases in MCPIP1 upregulation by MG-132. Our findings show for the first time the impact of proteasome inhibition on MCPIP1 protein expression by modulation of the activity of intracellular signaling pathways. Overexpression of MCPIP1-myc protein decreased the viability of HeLa cells but not HepG2 cells, which correlates with the increased susceptibility of HeLa cells to MG-132 toxicity. Notably, both MG-132 treatment and MCPIP1-myc overexpression led to the activation of apoptosis, as revealed by the induction of caspases 3/7 in both types of cell lines. This suggests the involvement of MCPIP1 upregulation in toxic properties of proteasome inhibition, which is an acknowledged approach to the treatment of several cancer types.
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Affiliation(s)
- Lukasz Skalniak
- Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland
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Fu YY, Nergard JC, Barnette NK, Wang YL, Chai KX, Chen LM. Proteasome inhibition augments cigarette smoke-induced GM-CSF expression in trophoblast cells via the epidermal growth factor receptor. PLoS One 2012; 7:e43042. [PMID: 22912784 PMCID: PMC3422336 DOI: 10.1371/journal.pone.0043042] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2012] [Accepted: 07/16/2012] [Indexed: 01/14/2023] Open
Abstract
Maternal cigarette smoking has adverse effects on pregnancy outcomes. The granulocyte-macrophage colony-stimulating factor (GM-CSF) is an essential cytokine for a normal pregnancy. We investigated the impact of cigarette smoke extract (CSE) on GM-CSF expression in human cytotrophoblast cells and suggested a cellular mechanism underlying the CSE-induced GM-CSF expression. An immortalized normal human trophoblast cell line (B6Tert-1) was treated with CSE. The viability and proliferation of the CSE-treated B6Tert-1 cells were evaluated, and the expression of GM-CSF in these cells was quantified at the mRNA and the protein levels by means of reverse-transcription and quantitative polymerase chain reaction (RT-qPCR); and enzyme-linked immunosorbent assay (ELISA), respectively. Human trophoblast cells treated with CSE had an increased expression of GM-CSF at both the mRNA and the protein levels. The CSE-induced GM-CSF expression was synergistically enhanced by the addition of the proteasome inhibitor MG-132, but inhibited by AG-1478, an inhibitor of the epidermal growth factor receptor (EGFR) kinase. Furthermore, CSE treatment increased the phosphorylation of the extracellular-signal regulated kinases (ERK1/2) in the trophoblast cells. The expression of other growth factors such as heparin-binding epidermal growth factor-like growth factor (HB-EGF) and vascular endothelial growth factor (VEGF) was also evaluated. Our data suggested that cigarette smoking and proteasome inhibition synergistically up-regulate GM-CSF cytokine expression by activating the EGFR signaling pathway.
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Affiliation(s)
- Ya-Yuan Fu
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China
| | - Jennifer C. Nergard
- Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America
| | - Nicole K. Barnette
- Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America
| | - Yan-Ling Wang
- State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People's Republic of China
| | - Karl X. Chai
- Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America
| | - Li-Mei Chen
- Burnett School of Biomedical Sciences, University of Central Florida College of Medicine, Orlando, Florida, United States of America
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Abdala-Valencia H, Berdnikovs S, Cook-Mills JM. Mechanisms for vascular cell adhesion molecule-1 activation of ERK1/2 during leukocyte transendothelial migration. PLoS One 2011; 6:e26706. [PMID: 22031842 PMCID: PMC3198778 DOI: 10.1371/journal.pone.0026706] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2011] [Accepted: 10/02/2011] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND During inflammation, adhesion molecules regulate recruitment of leukocytes to inflamed tissues. It is reported that vascular cell adhesion molecule-1 (VCAM-1) activates extracellular regulated kinases 1 and 2 (ERK1/2), but the mechanism for this activation is not known. Pharmacological inhibitors of ERK1/2 partially inhibit leukocyte transendothelial migration in a multi-receptor system but it is not known whether VCAM-1 activation of ERK1/2 is required for leukocyte transendothelial migration (TEM) on VCAM-1. METHODOLOGY/PRINCIPAL FINDINGS In this study, we identified a mechanism for VCAM-1 activation of ERK1/2 in human and mouse endothelial cells. VCAM-1 signaling, which occurs through endothelial cell NADPH oxidase, protein kinase Cα (PKCα), and protein tyrosine phosphatase 1B (PTP1B), activates endothelial cell ERK1/2. Inhibition of these signals blocked VCAM-1 activation of ERK1/2, indicating that ERK1/2 is activated downstream of PTP1B during VCAM-1 signaling. Furthermore, VCAM-1-specific leukocyte migration under physiological laminar flow of 2 dynes/cm(2) was blocked by pretreatment of endothelial cells with dominant-negative ERK2 K52R or the MEK/ERK inhibitors, PD98059 and U0126, indicating for the first time that ERK regulates VCAM-1-dependent leukocyte transendothelial migration. CONCLUSIONS/SIGNIFICANCE VCAM-1 activation of endothelial cell NADPH oxidase/PKCα/PTP1B induces transient ERK1/2 activation that is necessary for VCAM-1-dependent leukocyte TEM.
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Affiliation(s)
- Hiam Abdala-Valencia
- Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America
| | - Sergejs Berdnikovs
- Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America
| | - Joan M. Cook-Mills
- Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America
- * E-mail:
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Fujita H, Kato T, Watanabe N, Takahashi T, Kitagawa S. Calpain inhibitors stimulate phagocyte functions via activation of human formyl peptide receptors. Arch Biochem Biophys 2011; 513:51-60. [DOI: 10.1016/j.abb.2011.06.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2011] [Revised: 06/10/2011] [Accepted: 06/16/2011] [Indexed: 11/30/2022]
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Rehni AK, Singh TG, Behl N, Arora S. Possible involvement of ubiquitin proteasome system and other proteases in acute and delayed aspects of ischemic preconditioning of brain in mice. Biol Pharm Bull 2011; 33:1953-7. [PMID: 21139232 DOI: 10.1248/bpb.33.1953] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The present study has been designed to investigate the potential role of ubiquitin proteasome system and other proteases in acute as well as delayed aspects of ischemic preconditioning induced reversal of ischemia-reperfusion injury in mouse brain. Bilateral carotid artery occlusion of 17 min followed by reperfusion for 24 h was employed in present study to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was evaluated using elevated plus-maze test. Rota rod test was employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1 min and reperfusion of 1 min (ischemic preconditioning) both immediately before (for acute preconditioning) and 24 h before (for delayed preconditioning) global cerebral ischemia prevented markedly ischemia-reperfusion-induced cerebral injury as measured in terms of infarct size, loss of memory and motor coordination. Z-Leu-Leu-Phe-Chinese hamster ovary (CHO) (2 mg/kg, intraperitoneally (i.p.)), an inhibitor of ubiquitin proteasome system and other proteases attenuated the neuroprotective effect of both the acute as well as delayed ischemic preconditioning. It is concluded that the neuroprotective effect of both the acute as well as delayed phases of ischemic preconditioning may be due to the activation of ubiquitin proteasome system and other proteases.
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Sadanari H, Tanaka J, Li Z, Yamada R, Matsubara K, Murayama T. Proteasome inhibitor differentially regulates expression of the major immediate early genes of human cytomegalovirus in human central nervous system-derived cell lines. Virus Res 2009; 142:68-77. [PMID: 19201384 DOI: 10.1016/j.virusres.2009.01.010] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2008] [Revised: 01/13/2009] [Accepted: 01/13/2009] [Indexed: 10/21/2022]
Abstract
Proteasome inhibitor, which inhibits NF-kappaB activation, has been reported to activate c-Jun N-terminal kinase (JNK)-c-Jun pathway. In this study, we investigated the effects of proteasome inhibitor on the human cytomegalovirus (HCMV) major immediate early (MIE) gene expression in human central nervous system (CNS)-derived cell lines. Treatment of HCMV-infected 118MGC glioma and U373-MG astrocytoma cells with three proteasome inhibitors, MG132, clasto-lactacystin beta-lactone, and epoxomicin, suppressed MIE protein expression. In contrast, in HCMV-infected IMR-32 neuroblastoma cells, the proteasome inhibitors increased MIE protein expression, even in the presence of NF-kappaB inhibitor SN-50. A luciferase reporter assay demonstrated that MG132 markedly elevated the MIE promoter/enhancer (MIEP) activity in IMR-32 cells, but down-regulated it in 118MGC and U373-MG cells. Mutation in five cAMP response elements (CREs) within the MIEP resulted in a loss of the ability to respond to MG132 in IMR-32 cells. Moreover, Western blotting analysis revealed that MG132 induced c-Jun phosphorylation in all three CNS-derived cell lines, whereas a high level of activating transcription factor-2 (ATF-2) phosphorylation was observed only in IMR-32 cells. Finally, MG132-induced MIE protein expression was suppressed by JNK inhibitor that reduced the phosphorylation levels of both c-Jun and ATF-2. Taken together, these results suggest that the proteasome inhibitors activate CRE binding proteins consisting of c-Jun and ATF-2 through activating the JNK-c-Jun pathway, thereby inducing MIE protein synthesis in IMR-32 cells under the condition where NF-kappaB activity is inhibited.
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Affiliation(s)
- Hidetaka Sadanari
- Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa, Ishikawa 920-1181, Japan.
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14
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Kwak HB, Lee MS, Kim HS, Cho HJ, Kim JW, Lee ZH, Oh J. Proteasome inhibitors induce osteoclast survival by activating the Akt pathway. Biochem Biophys Res Commun 2008; 377:1-6. [PMID: 18492488 DOI: 10.1016/j.bbrc.2008.05.048] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2008] [Accepted: 05/07/2008] [Indexed: 12/19/2022]
Abstract
Osteoclasts rapidly undergo spontaneous apoptosis when deprived of survival factors. Regulation of osteoclast survival is important to treat bone-related diseases, such as osteoporosis. In this study, we found that the proteasome inhibitors, MG132 and ALLN, significantly inhibited osteoclast apoptosis induced by etoposide, as well as under conditions of survival factor deprivation. MG132 and ALLN inhibited the release of cytochrome c from mitochondria into the cytosol in the absence of survival factors and suppressed the cleavage of pro-caspase-9 and -3 to its active forms induced by etoposide. In addition, MG132 and ALLN enhanced the phosphorylation of Akt and ERK in osteoclasts. However, MG132 and ALLN did not inhibit the cleavage of caspase-9 and -3 in the presence of the phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002, while the inhibitory effect of MG132 and ALLN were intact in presence of the MEK1/2 inhibitor, U0126. LY294002 inhibited the survival of osteoclasts induced by MG132 and ALLN. Taken together, our results have demonstrated that proteasome inhibitors suppressed osteoclast apoptosis under conditions of survival factors deprivation through activation of the PI-3K/Akt pathway.
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Affiliation(s)
- Han Bok Kwak
- Department of Anatomy, School of Medicine, Wonkwang University College of Medicine, Iksan, Chonbuk, Republic of Korea
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15
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von Brandenstein MG, Ngum Abety A, Depping R, Roth T, Koehler M, Dienes HP, Fries JWU. A p38-p65 transcription complex induced by endothelin-1 mediates signal transduction in cancer cells. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2008; 1783:1613-22. [PMID: 18457675 DOI: 10.1016/j.bbamcr.2008.04.003] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2007] [Revised: 03/31/2008] [Accepted: 04/01/2008] [Indexed: 11/29/2022]
Abstract
Endothelin-1 is a powerful mitogen for various tumor and non-tumor cells. Its signaling cascade induces the inflammatory NF-kappaB complex, leading to expression of a number of target genes. In this context, MAPK p38 has been regarded as a potential phosphate donor for the p65 subunit of NF-kappaB. In the present study in HeLa cells, we have found that ET-1 induced signalling activates the NF-kappaB transcription complex (TC) in the nucleus at 6 h specifically via ET-A - but not ET-B receptor. The TC contains p65, p38 (alpha and beta) - binding to the NLS of p65 in the cytoplasm - as well as p50, but no IkappaBalpha. Specific p38 inhibition by SB203580 or by siRNA interferes markedly with gene expression of several target genes. Complex formation occurs in the cytoplasm, and both transcription factors transmigrate as a complex in the nucleus. Overexpression of p38, treatment with Chrysin, MG132, or dimethylformamide shows dependence of TC on p38 as partner. In other tumor cells lines studied, ET-1 activates TC, with p38 as an important complex partner of p65. TC-induction by ET-1 contains about twice the amount of p38 than by TNFalpha. Thus, p38 may be an additional therapeutic target to control inflammatory gene expression in tumor cells.
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16
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Sahni SK, Rydkina E, Sahni A. The proteasome inhibitor MG132 induces nuclear translocation of erythroid transcription factor Nrf2 and cyclooxygenase-2 expression in human vascular endothelial cells. Thromb Res 2008; 122:820-5. [PMID: 18377959 DOI: 10.1016/j.thromres.2008.01.011] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2007] [Revised: 01/21/2008] [Accepted: 01/22/2008] [Indexed: 11/27/2022]
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17
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Utama B, Shen YH, Mitchell BM, Makagiansar IT, Gan Y, Muthuswamy R, Duraisamy S, Martin D, Wang X, Zhang MX, Wang J, Wang J, Vercellotti GM, Gu W, Wang XL. Mechanisms for human cytomegalovirus-induced cytoplasmic p53 sequestration in endothelial cells. J Cell Sci 2006; 119:2457-67. [PMID: 16720642 DOI: 10.1242/jcs.02974] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Human cytomegalovirus (HCMV) infection results in endothelial dysfunction, typically known as dysregulated apoptosis, and aberrant expression and sub-cellular localization of p53, a tumor suppressor that accumulates at the late stage of infection. In this study, we examined three hypotheses that could be responsible for HCMV-induced cytoplasmic p53 accumulation at the later stage of infection: hyperactive nuclear export, cytoplasmic p53 tethering and delayed p53 degradation. Leptomycin B treatment, a nuclear export inhibitor, was unable to reduce cytoplasmic p53, thereby eliminating the hyperactive nuclear export mechanism. The findings that nascent p53 still entered nuclei after the nuclear export inhibition indicated that cytoplasmic tethering may play a minor role. Cytoplasmic p53 was still observed after the translation activities were blocked by cycloheximide. There was more than an eight-fold increase in the cytoplasmic p53 half-life with abnormal p53 ubiquitination. Taken together, these results suggest that delayed degradation could be responsible for the cytoplasmic p53 accumulation. The general slow-down of the proteasomal activity and the dysregulated p53 ubiquitination process at the later stage of infection could contribute to the reduced cytoplasmic p53 degradation and might be relevant to dysregulated endothelial apoptosis. The HCMV-induced changes in p53 dynamics could contribute to endothelial dysfunction.
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Affiliation(s)
- Budi Utama
- Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA
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18
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Holecek M, Muthny T, Kovarik M, Sispera L. Proteasome inhibitor MG-132 enhances whole-body protein turnover in rat. Biochem Biophys Res Commun 2006; 345:38-42. [PMID: 16674919 DOI: 10.1016/j.bbrc.2006.04.053] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2006] [Accepted: 04/13/2006] [Indexed: 11/18/2022]
Abstract
Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.
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Affiliation(s)
- Milan Holecek
- Department of Physiology, Charles University, Medical Faculty, Hradec Kralove, Czech Republic.
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19
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Woo KJ, Park JW, Kwon TK. Proteasome inhibitor-induced cyclooxygenase-2 expression in Raw264.7 cells is potentiated by inhibition of c-Jun N-terminal kinase activation. Biochem Biophys Res Commun 2006; 342:1334-40. [PMID: 16516846 DOI: 10.1016/j.bbrc.2006.02.105] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2006] [Accepted: 02/16/2006] [Indexed: 12/16/2022]
Abstract
Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. MG132, proteasome inhibitor, is known to anti-tumor agent activity and anti-inflammation with inhibitory property of NF-kappaB. We investigated the effect of MG132 on the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGE(2), using macrophage cell line, Raw264.7. Our results showed that COX-2 expression is up-regulated by MG132 treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in MG132-induced COX-2 expression. The p38 MAPK inhibitor (SB 203580) prevented MG132-induced COX-2 expression, whereas c-Jun N-terminal kinase (JNK) inhibitor (SP 600125) and MAPK kinase 4 (MKK4)-DN (dominant negative mutant) and MKK7-DN significantly enhanced COX-2 expression. These results suggest that MG132-induced COX-2 expression is associated with the activation of p38 MAPK and the inhibition of JNK signaling pathways.
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Affiliation(s)
- Kyung Jin Woo
- Department of Immunology, School of Medicine, Keimyung University, 194 DongSan-Dong Jung-Gu, Taegu 700-712, Republic of Korea
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20
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Gunn WG, Conley A, Deininger L, Olson SD, Prockop DJ, Gregory CA. A crosstalk between myeloma cells and marrow stromal cells stimulates production of DKK1 and interleukin-6: a potential role in the development of lytic bone disease and tumor progression in multiple myeloma. Stem Cells 2005; 24:986-91. [PMID: 16293576 DOI: 10.1634/stemcells.2005-0220] [Citation(s) in RCA: 204] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells. B-cell plasmacytomas stimulate bone resorption and angiogenesis, resulting in osteolytic lesions in the skeleton which persist upon successful treatment of the malignancy with chemotherapy. We found that an interaction between MM cells and mesenchymal stem cells (MSCs) from bone marrow stroma results in the formation and persistence of osteolytic bone lesions. It is known that MM cells activate osteoclast activity and secrete high levels of the Wnt inhibitor, Dickkopf-1, which prevents MSCs from differentiating into osteoblasts. We show that the Wnt signaling activator 6-bromoindirubin-3'-monoxime (BIO) releases MSCs from the osteoinhibitory effects of Dickkopf-1, whereas LiCl treatment does not. Additionally, we show that the >5-kDa fraction of MSC-conditioned medium promotes the proliferation of Dickkopf-1-secreting MM cells and that an interleukin-6 (IL-6)-neutralizing antibody blocks this effect. IL-6 expression levels were higher in undifferentiated MSCs than in MSCs treated with osteogenic medium, remained high in the presence of Dkk1, and were reduced by BIO treatment. Therefore, BIO treatment reduces the MSC-stimulated proliferation of MM cells and may enable MSCs to repair existing osteolytic lesions.
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Affiliation(s)
- William G Gunn
- Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana, USA
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21
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Chen JJ, Huang WC, Chen CC. Transcriptional regulation of cyclooxygenase-2 in response to proteasome inhibitors involves reactive oxygen species-mediated signaling pathway and recruitment of CCAAT/enhancer-binding protein delta and CREB-binding protein. Mol Biol Cell 2005; 16:5579-91. [PMID: 16195339 PMCID: PMC1289404 DOI: 10.1091/mbc.e05-08-0778] [Citation(s) in RCA: 70] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Inhibition of ubiquitin-proteasome pathway has been shown to be a promising strategy for the treatment of inflammation and cancer. Here, we show that proteasome inhibitors MG132, PSI-1, and lactacystin induce COX-2 expression via enhancing gene transcription rather than preventing protein degradation in the human alveolar NCI-H292 and A549, and gastric AGS epithelial cells. NF-IL6 and CRE, but not NF-kappaB elements on the COX-2 promoter were involved in the gene transcription event. The binding of CCAAT/enhancer binding protein (C/EBP)beta and C/EBPdelta to the CRE and NF-IL6 elements, as well as the recruitment of CBP and the enhancement of histone H3 and H4 acetylation on the COX-2 promoter was enhanced by MG132. However, it did not affect the total protein levels of C/EBPbeta and C/EBPdelta. MG132-induced DNA-binding activity of C/EBPdelta, but not C/EBPbeta was regulated by p38, PI3K, Src, and protein kinase C. Small interfering RNA of C/EBPdelta suppressed COX-2 expression, further strengthening the role of C/EBPdelta in COX-2 gene transcription. In addition, the generation of intracellular reactive oxygen species (ROS) in response to MG132 contributed to the activation of MAPKs and Akt. These findings reveal that the induction of COX-2 transcription induced by proteasome inhibitors requires ROS-dependent protein kinases activation and the subsequent recruitments of C/EBPdelta and CBP.
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Affiliation(s)
- Jun-Jie Chen
- Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan
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22
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MacInnis BL, Campenot RB. Regulation of Wallerian degeneration and nerve growth factor withdrawal-induced pruning of axons of sympathetic neurons by the proteasome and the MEK/Erk pathway. Mol Cell Neurosci 2005; 28:430-9. [PMID: 15737734 DOI: 10.1016/j.mcn.2004.10.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2004] [Revised: 09/23/2004] [Accepted: 10/06/2004] [Indexed: 10/26/2022] Open
Abstract
Treatment of transected distal axons of rat sympathetic neurons in compartmented cultures with MG132 (5 microM) and other inhibitors of proteasome activity, preserved axonal mitochondrial function, assessed by Mitotracker-Orange and MTT staining, for at least 24 h. MG132 similarly protected axons from undergoing branch elimination (pruning) in response to local NGF deprivation. Axons protected by MG132 displayed persistent phosphorylation of Erk1/2, and pharmacological inhibition of MEK activity with U0126 (50 microM) restored rapid axonal degeneration. Therefore, the proteasome does not appear to be necessary as a general effector of protein degradation during axonal degeneration. Rather, the proteasome functions in the regulation of signaling pathways that control axonal survival and degeneration. Specifically, the down-regulation of the MEK/Erk pathway by the proteasome plays roles in Wallerian degeneration of severed axons and axonal pruning in response to local NGF deprivation. Identification of the pathways that regulate axonal survival and degeneration will provide possible target sites for pharmacological treatments of neurodegenerative diseases and traumatic injury.
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Affiliation(s)
- Bronwyn L MacInnis
- Department of Cell Biology, University of Alberta, 6-22 Medical Sciences Building, Edmonton, AB, Canada T6G 2H7
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Lu H, Wu JY, Kudo T, Ohno T, Graham DY, Yamaoka Y. Regulation of interleukin-6 promoter activation in gastric epithelial cells infected with Helicobacter pylori. Mol Biol Cell 2005; 16:4954-66. [PMID: 16030249 PMCID: PMC1237095 DOI: 10.1091/mbc.e05-05-0426] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The regulation of Helicobacter pylori induced interleukin (IL)-6 in the gastric epithelium remains unclear. Primary gastric epithelial cells and MKN28 cells were cocultured with H. pylori and its isogenic cag pathogenicity island (PAI) mutant and/or oipA mutants. H. pylori infection-induced IL-6 mRNA expression and IL-6 protein production, which was further enhanced by the cag PAI and OipA. Luciferase reporter gene assays and electrophoretic mobility shift assays showed that full IL-6 transcription required binding sites for nuclear factor-kappaB (NF-kappaB), cAMP response element (CRE), CCAAT/enhancer binding protein (C/EBP), and activator protein (AP)-1. The cag PAI and OipA were involved in binding to NF-kappaB, AP-1, CRE, and C/EBP sites. The cag PAI activated the extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK) pathways; OipA activated the p38 pathway. Transfection of dominant negative G-protein confirmed roles for Raf, Rac1, and RhoA in IL-6 induction. Overall, the cag PAI-related IL-6 signal transduction pathway involved the Ras/Raf/MEK1/2/ERK/AP-1/CRE pathway and the JNK/AP-1/CRE pathway; the OipA-related pathway is p38/AP-1/CRE and both the cag PAI and OipA appear to be involved in the RhoA/Rac1/NF-kappaB pathway. Combination of different pathways by the cag PAI and OipA will lead to the maximum IL-6 induction.
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Affiliation(s)
- Hong Lu
- Department of Medicine, Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX 77030, USA
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24
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Zhang WG, Yu JP, Wu QM, Tong Q, Li SB, Wang XH, Xie GJ. Inhibitory effect of ubiquitin-proteasome pathway on proliferation of esophageal carcinoma cells. World J Gastroenterol 2004; 10:2779-84. [PMID: 15334669 PMCID: PMC4572101 DOI: 10.3748/wjg.v10.i19.2779] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To investigate the inhibitory effect of ubiquitin-proteasome pathway (UPP) on proliferation of esophageal carcinoma cells.
METHODS: Esophageal carcinoma cell strain EC9706 was treated with MG-132 to inhibit its UPP specificity. Cell growth suppression was evaluated with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. DNA synthesis was evaluated by 3H-thymidine (3H-TdR) incorporation. Morphologic changes of cells were observed under microscope. Activity of telomerase was examined by telomeric repeat amplification protocol (TRAP) of PCR-ELISA. Cell cycle and apoptosis were detected by flow cytometry (FCM). DNA fragment analysis was used to confirm the presence of apoptosis. Expression of p27kip1 was detected by immunocytochemical technique.
RESULTS: After exposed to MG-132, the growth and value of 3H-TdR incorporation of EC9706 cells were obviously inhibited. Cells became round, small and exfoliative under microscope. TRAP PCR-ELISA showed that light absorption of cells gradually decreased after exposed to 5 μmol/L of MG-132 for 24, 48, 72 and 96 h (P < 0.01). The percentage of cells at G0/G1 phase was increased and that at S and G2/M was decreased (P < 0.01). The rate of apoptotic cells treated with 5 μmol/L of MG-132 for 48 and 96 h was 31.7% and 66.4%, respectively. Agarose electrophoresis showed marked ladders. In addition, the positive signals of p27kip1 were located in cytoplasm and nuclei in MG-132 group in contrast to cytoplasm staining in control group.
CONCLUSION: MG-132 can obviously inhibit proliferation of EC9706 cells and induce apoptosis. The mechanisms include upregulation of p27kip1 expression, G1 arrest and depression of telomerase activity. The results indicate that inhibiting UPP is a novel strategy for esophageal carcinoma therapy.
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Affiliation(s)
- Wei-Guo Zhang
- Digestive Department, Taihe Hospital, Yunyang Medical College, Shiyan 442000, Hubei Province, China.
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25
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Imaizumi T, Yoshida H, Satoh K. Regulation of CX3CL1/fractalkine expression in endothelial cells. J Atheroscler Thromb 2004; 11:15-21. [PMID: 15067194 DOI: 10.5551/jat.11.15] [Citation(s) in RCA: 127] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
CX3CL1/fractalkine is a chemokine with a unique CX3C motif. Fractalkine is synthesized in endothelial cells as a membrane protein, and the N-terminal domain containing a CX3C motif is cleaved and secreted. CX3CR1, the specific receptor for fractalkine, is expressed in monocytes and lymphocytes. Membrane-bound fractalkine works as an adhesion molecule for these leukocytes and the secreted form as a chemotactic factor. Fractalkine is produced by endothelial cells stimulated with tumor necrosis factor-alpha, interleukin-1 (IL-1), lipopolysaccharide and interferon-gamma. Expression of fractalkine in endothelial cells is inhibited by the soluble form of IL-6 receptor-alpha, 15-deoxy-Delta(12,14)-prostaglandin J(2), and hypoxia. The expression of fractalkine is tightly regulated and fractalkine plays an important role in the interaction between leukocytes and endothelial cells.
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Affiliation(s)
- Tadaatsu Imaizumi
- Department of Vascular Biology, Hirosaki University School of Medicine, Aomori, Japan.
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26
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Lim JH, Chang YC, Park YB, Park JW, Kwon TK. Transcriptional repression of E2F gene by proteasome inhibitors in human osteosarcoma cells. Biochem Biophys Res Commun 2004; 318:868-72. [PMID: 15147952 DOI: 10.1016/j.bbrc.2004.04.103] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2004] [Indexed: 11/26/2022]
Abstract
E2F family of transcription factors regulates the transcription of genes required for DNA synthesis. E2F is itself controlled by a series of transcriptional and post-transcriptional pathways. Here we provide evidence that proteasome inhibitor-mediated E2F1 gene down-regulation is regulated by transcriptional events. Using the proteasome-specific inhibitors, MG132 and lactacystin, we show that the p53, the cdk inhibitors p21 and p27, and cyclin A are degraded by the ubiquitin-proteasome pathway in human osteosarcoma cells. Interestingly, the expression levels of E2F1 and E2F2 are down-regulated by proteasome inhibitors. E2F promoter and RT-PCR assay clearly demonstrated that proteasome inhibitors could reduce E2F transcriptional activation. However, MG132-induced repression of E2F1 and E2F2 is not associated with ROS generation.
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Affiliation(s)
- Jun Hee Lim
- Kidney Institute, School of Medicine, Keimyung University, 194 DongSan-Dong, Taegu 700-712, Republic of Korea
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Hiramatsu N, Kasai A, Yao J, Meng Y, Takeda M, Maeda S, Kitamura M. AP-1-independent sensitization to oxidative stress-induced apoptosis by proteasome inhibitors. Biochem Biophys Res Commun 2004; 316:545-52. [PMID: 15020252 DOI: 10.1016/j.bbrc.2004.02.081] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2004] [Indexed: 10/26/2022]
Abstract
Hydrogen peroxide (H(2)O(2)) induces apoptosis of mesangial cells via c-Jun N-terminal kinase (JNK)-activator protein-1 (AP-1) and extracellular signal-regulated kinase (ERK)-AP-1 pathways. We recently found that subtoxic doses of proteasome inhibitors, MG132 and lactacystin, dramatically enhanced H(2)O(2)-induced apoptosis in mesangial cells. In this report, we examined molecular mechanisms involved in this phenomenon, especially focusing on AP-1 pathways. Reporter assays showed that MG132 induced activation of AP-1. However, pharmacological inhibitors of AP-1, retinoic acid, and curcumin, did not suppress the proapoptotic effect of MG132. Suppression of JNK-AP-1 by transfection with either a dominant-negative mutant of JNK or a dominant-negative mutant of c-Jun did not attenuate the apoptosis enhancement by MG132. Similarly, suppression of ERK-AP-1 by PD98059 or dominant-negative mutants of ERK did not affect the apoptosis-promoting effect of MG132. Interestingly, pretreatment with MG132 did not enhance activation of AP-1 by H(2)O(2). These data suggested a novel, AP-1-independent promotion of apoptosis by proteasome inhibitors.
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Affiliation(s)
- Nobuhiko Hiramatsu
- Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Tamaho, Yamanashi 409-3898, Japan
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28
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Shibata T, Imaizumi T, Matsumiya T, Tamo W, Hatakeyama M, Yoshida H, Munakata H, Fukuda I, Satoh K. Effect of MG132, a proteasome inhibitor, on the expression of growth related oncogene protein-α in human umbilical vein endothelial cells. Cytokine 2003; 24:67-73. [PMID: 14581000 DOI: 10.1016/s1043-4666(03)00271-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Growth related oncogene protein-alpha (GRO-alpha) is a member of C-X-C chemokine and plays an important role in inflammatory responses. Expression of GRO gene family is regulated by a number of factors at both transcriptional and posttranscriptional levels. In the present study, we have addressed the possible regulation of GRO-alpha expression by ubiquitin-proteasome system. Cultures of human umbilical vein endothelial cells were treated with a proteasome inhibitor, MG132, and the levels of GRO-alpha mRNA were analyzed by reverse transcription-polymerase chain reaction or northern blotting. Levels of GRO-alpha protein in the cell-conditioned medium were determined by enzyme-linked immunosorbent assay. MG132 alone increased the levels of GRO-alpha mRNA and protein; however, it did not affect the GRO-alpha mRNA induced by lipopolysaccharide (LPS) and inhibited the LPS-induced decrease in IkappaB levels. Other proteasome inhibitors, MG115 and lactacystin, also induced the expression of GRO-alpha mRNA. MG132 induced the phosphorylation of p38 MAPK, MEK and JNK. Pretreatment of the cells with SB203580, an inhibitor of p38 MAPK, suppressed the MG132-induced GRO-alpha expression, but pretreatment of the cells with U0126, PD98059 or SP600125, inhibitors of MEK1/2 or JNK, did not influence the effect of MG132. We conclude that MG132 upregulates GRO-alpha expression in vascular endothelial cells, at least in part, through the activation of p38 MAPK.
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Affiliation(s)
- Takeo Shibata
- Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
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