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Danieli A, Vucak G, Baccarini M, Martens S. Sequestration of translation initiation factors in p62 condensates. Cell Rep 2023; 42:113583. [PMID: 38096057 DOI: 10.1016/j.celrep.2023.113583] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2023] [Revised: 10/20/2023] [Accepted: 11/29/2023] [Indexed: 12/30/2023] Open
Abstract
Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.
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Affiliation(s)
- Alberto Danieli
- Max Perutz Labs, Vienna Biocenter Campus (VBC), Dr.-Bohr-Gasse 9, 1030 Vienna, Austria; University of Vienna, Center for Molecular Biology, Department of Biochemistry and Cell Biology, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria; Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Campus-Vienna-Biocenter 1, 1030 Vienna, Austria.
| | - Georg Vucak
- Max Perutz Labs, Vienna Biocenter Campus (VBC), Dr.-Bohr-Gasse 9, 1030 Vienna, Austria; Vienna BioCenter PhD Program, Doctoral School of the University of Vienna and Medical University of Vienna, Campus-Vienna-Biocenter 1, 1030 Vienna, Austria; University of Vienna, Center for Molecular Biology, Department of Microbiology, Immunobiology and Genetics, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
| | - Manuela Baccarini
- Max Perutz Labs, Vienna Biocenter Campus (VBC), Dr.-Bohr-Gasse 9, 1030 Vienna, Austria; University of Vienna, Center for Molecular Biology, Department of Microbiology, Immunobiology and Genetics, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
| | - Sascha Martens
- Max Perutz Labs, Vienna Biocenter Campus (VBC), Dr.-Bohr-Gasse 9, 1030 Vienna, Austria; University of Vienna, Center for Molecular Biology, Department of Biochemistry and Cell Biology, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria.
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2
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Wang Y, Lei J, Zhang S, Wang X, Jin J, Liu Y, Gan M, Yuan Y, Sun L, Li X, Han T, Wang JB. 4EBP1 senses extracellular glucose deprivation and initiates cell death signaling in lung cancer. Cell Death Dis 2022; 13:1075. [PMID: 36575176 PMCID: PMC9794714 DOI: 10.1038/s41419-022-05466-5] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2022] [Revised: 11/22/2022] [Accepted: 11/24/2022] [Indexed: 12/28/2022]
Abstract
Nutrient-limiting conditions are common during cancer development. The coordination of cellular glucose levels and cell survival is a fundamental question in cell biology and has not been completely understood. 4EBP1 is known as a translational repressor to regulate cell proliferation and survival by controlling translation initiation, however, whether 4EBP1 could participate in tumor survival by other mechanism except for translational repression function, especially under glucose starvation conditions remains unknown. Here, we found that protein levels of 4EBP1 was up-regulated in the central region of the tumor which always suffered nutrient deprivation compared with the peripheral region. We further discovered that 4EBP1 was dephosphorylated by PTPMT1 under glucose starvation conditions, which prevented 4EBP1 from being targeted for ubiquitin-mediated proteasomal degradation by HERC5. After that, 4EBP1 translocated to cytoplasm and interacted with STAT3 by competing with JAK and ERK, leading to the inactivation of STAT3 in the cytoplasm, resulting in apoptosis under glucose withdrawal conditions. Moreover, 4EBP1 knockdown increased the tumor volume and weight in xenograft models by inhibiting apoptosis in the central region of tumor. These findings highlight a novel mechanism for 4EBP1 as a new cellular glucose sensor in regulating cancer cell death under glucose deprivation conditions, which was different from its classical function as a translational repressor.
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Affiliation(s)
- Yanan Wang
- grid.412604.50000 0004 1758 4073Jiangxi Institute of Respiratory Disease, The First Affiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China ,Jiangxi Hospital of China-Japan Friendship Hospital, Nanchang City, 330052 Jiangxi China ,Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City, 330006 Jiangxi China
| | - Jiapeng Lei
- School of Basic Medical Sciences, Nanchang Medical College, Nanchang City, 330006 Jiangxi China
| | - Song Zhang
- grid.412465.0Department of Hepatobiliary and Pancreatic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou City, 310009 Zhejiang China
| | - Xiaomei Wang
- grid.415912.a0000 0004 4903 149XDepartment of Pharmacy, Liaocheng People’s Hospital, Liaocheng City, 252000 Shandong China
| | - Jiangbo Jin
- grid.260463.50000 0001 2182 8825Department of Thoracic Surgery, The First Affifiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China
| | - Yufeng Liu
- grid.260463.50000 0001 2182 8825School of Basic Medical Sciences, Nanchang University, Nanchang City, 330031 Jiangxi China
| | - Mingxi Gan
- grid.260463.50000 0001 2182 8825School of Basic Medical Sciences, Nanchang University, Nanchang City, 330031 Jiangxi China
| | - Yi Yuan
- grid.260463.50000 0001 2182 8825Huankui Academy, Nanchang University, Nanchang City, 330031 Jiangxi China
| | - Longhua Sun
- grid.412604.50000 0004 1758 4073Departments of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China
| | - Xiaolei Li
- grid.412604.50000 0004 1758 4073Jiangxi Institute of Respiratory Disease, The First Affiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China
| | - Tianyu Han
- grid.412604.50000 0004 1758 4073Jiangxi Institute of Respiratory Disease, The First Affiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China ,Jiangxi Hospital of China-Japan Friendship Hospital, Nanchang City, 330052 Jiangxi China ,Jiangxi Clinical Research Center for Respiratory Diseases, Nanchang City, 330006 Jiangxi China
| | - Jian-Bin Wang
- grid.260463.50000 0001 2182 8825Department of Thoracic Surgery, The First Affifiliated Hospital of Nanchang University, Nanchang City, 330006 Jiangxi China ,grid.260463.50000 0001 2182 8825School of Basic Medical Sciences, Nanchang University, Nanchang City, 330031 Jiangxi China
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3
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O'Reilly CL, Uranga S, Fluckey JD. Culprits or consequences: Understanding the metabolic dysregulation of muscle in diabetes. World J Biol Chem 2021; 12:70-86. [PMID: 34630911 PMCID: PMC8473417 DOI: 10.4331/wjbc.v12.i5.70] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2021] [Revised: 06/21/2021] [Accepted: 08/03/2021] [Indexed: 02/06/2023] Open
Abstract
The prevalence of type 2 diabetes (T2D) continues to rise despite the amount of research dedicated to finding the culprits of this debilitating disease. Skeletal muscle is arguably the most important contributor to glucose disposal making it a clear target in insulin resistance and T2D research. Within skeletal muscle there is a clear link to metabolic dysregulation during the progression of T2D but the determination of culprits vs consequences of the disease has been elusive. Emerging evidence in skeletal muscle implicates influential cross talk between a key anabolic regulatory protein, the mammalian target of rapamycin (mTOR) and its associated complexes (mTORC1 and mTORC2), and the well-described canonical signaling for insulin-stimulated glucose uptake. This new understanding of cellular signaling crosstalk has blurred the lines of what is a culprit and what is a consequence with regard to insulin resistance. Here, we briefly review the most recent understanding of insulin signaling in skeletal muscle, and how anabolic responses favoring anabolism directly impact cellular glucose disposal. This review highlights key cross-over interactions between protein and glucose regulatory pathways and the implications this may have for the design of new therapeutic targets for the control of glucoregulatory function in skeletal muscle.
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Affiliation(s)
| | - Selina Uranga
- Health and Kinesiology, Texas A&M University, TX 77843, United States
| | - James D Fluckey
- Health and Kinesiology, Texas A&M University, TX 77843, United States
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Abstract
Cells metabolize nutrients for biosynthetic and bioenergetic needs to fuel growth and proliferation. The uptake of nutrients from the environment and their intracellular metabolism is a highly controlled process that involves cross talk between growth signaling and metabolic pathways. Despite constant fluctuations in nutrient availability and environmental signals, normal cells restore metabolic homeostasis to maintain cellular functions and prevent disease. A central signaling molecule that integrates growth with metabolism is the mechanistic target of rapamycin (mTOR). mTOR is a protein kinase that responds to levels of nutrients and growth signals. mTOR forms two protein complexes, mTORC1, which is sensitive to rapamycin, and mTORC2, which is not directly inhibited by this drug. Rapamycin has facilitated the discovery of the various functions of mTORC1 in metabolism. Genetic models that disrupt either mTORC1 or mTORC2 have expanded our knowledge of their cellular, tissue, as well as systemic functions in metabolism. Nevertheless, our knowledge of the regulation and functions of mTORC2, particularly in metabolism, has lagged behind. Since mTOR is an important target for cancer, aging, and other metabolism-related pathologies, understanding the distinct and overlapping regulation and functions of the two mTOR complexes is vital for the development of more effective therapeutic strategies. This review discusses the key discoveries and recent findings on the regulation and metabolic functions of the mTOR complexes. We highlight findings from cancer models but also discuss other examples of the mTOR-mediated metabolic reprogramming occurring in stem and immune cells, type 2 diabetes/obesity, neurodegenerative disorders, and aging.
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Affiliation(s)
- Angelia Szwed
- Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey
| | - Eugene Kim
- Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey
| | - Estela Jacinto
- Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey
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Yang W, Huang J, Wu H, Wang Y, Du Z, Ling Y, Wang W, Wu Q, Gao W. Molecular mechanisms of cancer cachexia‑induced muscle atrophy (Review). Mol Med Rep 2020; 22:4967-4980. [PMID: 33174001 PMCID: PMC7646947 DOI: 10.3892/mmr.2020.11608] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2020] [Accepted: 09/09/2020] [Indexed: 12/20/2022] Open
Abstract
Muscle atrophy is a severe clinical problem involving the loss of muscle mass and strength that frequently accompanies the development of numerous types of cancer, including pancreatic, lung and gastric cancers. Cancer cachexia is a multifactorial syndrome characterized by a continuous decline in skeletal muscle mass that cannot be reversed by conventional nutritional therapy. The pathophysiological characteristic of cancer cachexia is a negative protein and energy balance caused by a combination of factors, including reduced food intake and metabolic abnormalities. Numerous necessary cellular processes are disrupted by the presence of abnormal metabolites, which mediate several intracellular signaling pathways and result in the net loss of cytoplasm and organelles in atrophic skeletal muscle during various states of cancer cachexia. Currently, the clinical morbidity and mortality rates of patients with cancer cachexia are high. Once a patient enters the cachexia phase, the consequences are difficult to reverse and the treatment methods for cancer cachexia are very limited. The present review aimed to summarize the recent discoveries regarding the pathogenesis of cancer cachexia-induced muscle atrophy and provided novel ideas for the comprehensive treatment to improve the prognosis of affected patients.
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Affiliation(s)
- Wei Yang
- Department of Oncology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China
| | - Jianhui Huang
- Department of Oncology, Lishui Municipal Central Hospital, Lishui, Zhejiang 323000, P.R. China
| | - Hui Wu
- Department of Clinical Medicine, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
| | - Yuqing Wang
- Department of Clinical Medicine, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
| | - Zhiyin Du
- Department of Clinical Medicine, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
| | - Yuanbo Ling
- Department of Clinical Medicine, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
| | - Weizhuo Wang
- Department of Clinical Medicine, Anhui University of Science and Technology, Huainan, Anhui 232001, P.R. China
| | - Qian Wu
- Department of Oncology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China
| | - Wenbin Gao
- Department of Oncology, The Third Affiliated Hospital of Shenzhen University, Shenzhen, Guangdong 518000, P.R. China
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Wang XX, Geng SL, Zhang XS, Xu WH. P-S6K is associated with insect diapause via the ROS/AKT/ S6K/CREB/HIF-1 pathway in the cotton bollworm, Helicoverpa armigera. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2020; 120:103262. [PMID: 32088323 DOI: 10.1016/j.ibmb.2019.103262] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2019] [Revised: 10/17/2019] [Accepted: 10/29/2019] [Indexed: 06/10/2023]
Abstract
Diapause is a complex physiological response that allows insects to survive unfavorable environmental conditions, and many signaling pathways participate in regulating this process. However, little is known about TOR signaling in the regulation of diapause. In this study, we found that the TOR pathway-related proteins TOR and Raptor are expressed at low levels in the brains of diapause-destined pupae of Helicoverpa armigera, consistent with a previous report that TOR signaling is associated with development. Interestingly, another TOR signaling-related protein, p-S6K, was increased in the brains of diapause-destined pupae. Our results showed that p-S6K in the brains of diapause-destined pupae can respond to the upstream signals reactive oxygen species (ROS) and AKT and that S6K activates the level of CREB, which binds to the HIF-1α promoter and increases its expression. Previous study has shown that HIF-1α levels elevated by ROS in the brains of diapause-destined pupae cause low mitochondrial activity for insect diapause. Thus, p-S6K in response to ROS/AKT regulates HIF-1α via activating transcription factor CREB for diapause initiation.
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Affiliation(s)
- Xiao-Xue Wang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, China
| | - Shao-Lei Geng
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, China
| | - Xiao-Shuai Zhang
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, China
| | - Wei-Hua Xu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510006, China.
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7
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Gioldasi S, Karvela A, Rojas-Gil AP, Rodi M, de Lastic AL, Thomas I, Spiliotis BE, Mouzaki A. Metabolic Association between Leptin and the Corticotropin Releasing Hormone. Endocr Metab Immune Disord Drug Targets 2020; 19:458-466. [PMID: 30727936 PMCID: PMC7360915 DOI: 10.2174/1871530319666190206165626] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/13/2018] [Revised: 10/31/2018] [Accepted: 12/27/2018] [Indexed: 01/29/2023]
Abstract
Objective In healthy individuals, leptin is produced from adipose tissue and is secreted into the circulation to communicate energy balance status to the brain and control fat metabolism. Corticotropin-Releasing Hormone (CRH) is synthesized in the hypothalamus and regulates stress responses. Among the many adipokines and hormones that control fat metabolism, leptin and CRH both curb appetite and inhibit food intake. Despite numerous reports on leptin and CRH properties and function, little has been actually shown about their association in the adipose tissue environment. Methods In this article, we summarized the salient information on leptin and CRH in relation to metabolism. We also investigated the direct effect of recombinant CRH on leptin secretion by primary cultures of human adipocytes isolated from subcutaneous abdominal adipose tissue of 7 healthy children and adolescents, and measured CRH and leptin levels in plasma collected from peripheral blood of 24 healthy children and adolescents to assess whether a correlation exists between CRH and leptin levels in the periphery. Results and Conclusion The available data indicate that CRH exerts a role in the regulation of leptin in human adipocytes. We show that CRH downregulates leptin production by mature adipocytes and that a strong negative correlation exists between CRH and leptin levels in the periphery, and suggest the possible mechanisms of CRH control of leptin. Delineation of CRH control of leptin production by adipocytes may explain unknown pathogenic mechanisms linking stress and metabolism.
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Affiliation(s)
- Sofia Gioldasi
- Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece
| | - Alexia Karvela
- Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Medical School, University of Patras, Patras, Greece
| | | | - Maria Rodi
- Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece
| | - Anne-Lise de Lastic
- Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece
| | - Iason Thomas
- Department of Allergy, Guy's and St Thomas' NHS Foundation Trust, London, United Kingdom
| | - Bessie E Spiliotis
- Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics, Medical School, University of Patras, Patras, Greece
| | - Athanasia Mouzaki
- Division of Hematology, Department of Internal Medicine, Medical School, University of Patras, Patras, Greece
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8
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Rice KM, Manne ND, Arvapalli R, Ginjupalli GK, Blough ER. Diabetes alters vascular mechanotransduction data: Pressure-induced regulation of mTor and associated signaling in the rat inferior vena cava. Data Brief 2017; 15:63-71. [PMID: 28971124 PMCID: PMC5612793 DOI: 10.1016/j.dib.2017.09.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2017] [Revised: 08/29/2017] [Accepted: 09/01/2017] [Indexed: 11/30/2022] Open
Abstract
Diabetes is a multifaceted disease with various etiologies. The complexity of this pathology creates a myriad of factors that must be considered when addressing surgical outcomes and prognosis. Of vital importance to cardiovascular surgery is the viability of homographic vein grafts. Due to the fact, diabetic patients have a higher rate of vein graph failure, a greater understanding of the effect diabetes has on vascular mechano-transductive response is critical to improving patient prognosis. This article represents data regarding a study published in Cardiovascular Diabetology (Rice et al., 2006) [1] and Open Journal of Endocrine and Metabolic Diseases (Rice et al., 2015) [2] with the purpose of evaluating the effect of pressurization on rat inferior venae cavae (IVC). Here we provide the information about the method and processing of raw data related to our prior publish work and Data in Brief articles (Rice et al., Submitted for publication) [3,4]. The data contained in this article evaluates the contribution of mTor signaling and associated proteins. IVC from lean and obese animals were exposed to a 30 min perfusion of 120 mm Hg pressure and evaluated for changes in expression and phosphorylation of mTor, p70s6k, GSK3β, and 4EBP-1.
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Affiliation(s)
- Kevin M. Rice
- Center for Diagnostic Nanosystems, Marshall University, Huntington, WV, United States
- Department of Internal Medicine, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States
- Biotechnology Graduate Program West Virginia State University, Institute, WV, United States
- Department of Health and Human Service, School of Kinesiology, Marshall University, Huntington, WV, United States
| | | | - Ravikumar Arvapalli
- Center for Diagnostic Nanosystems, Marshall University, Huntington, WV, United States
| | - Gautam K. Ginjupalli
- Center for Diagnostic Nanosystems, Marshall University, Huntington, WV, United States
| | - Eric R. Blough
- Center for Diagnostic Nanosystems, Marshall University, Huntington, WV, United States
- Biotechnology Graduate Program West Virginia State University, Institute, WV, United States
- Department of Pharmaceutical Sciences and Research, School of Pharmacy, Marshall University, Huntington, WV, United States
- Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, United States
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9
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Liu D, Bordicchia M, Zhang C, Fang H, Wei W, Li JL, Guilherme A, Guntur K, Czech MP, Collins S. Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning. J Clin Invest 2016; 126:1704-16. [PMID: 27018708 DOI: 10.1172/jci83532] [Citation(s) in RCA: 158] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Accepted: 02/19/2016] [Indexed: 12/27/2022] Open
Abstract
A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.
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MESH Headings
- 3T3-L1 Cells
- Adaptor Proteins, Signal Transducing/genetics
- Adaptor Proteins, Signal Transducing/metabolism
- Adipose Tissue, Brown/cytology
- Adipose Tissue, Brown/metabolism
- Adipose Tissue, White/cytology
- Adipose Tissue, White/metabolism
- Animals
- Cyclic AMP-Dependent Protein Kinases/genetics
- Cyclic AMP-Dependent Protein Kinases/metabolism
- HEK293 Cells
- Humans
- Insulin/genetics
- Insulin/metabolism
- Mechanistic Target of Rapamycin Complex 1
- Mice
- Mice, Knockout
- Multiprotein Complexes/genetics
- Multiprotein Complexes/metabolism
- Receptors, Adrenergic, beta/genetics
- Regulatory-Associated Protein of mTOR
- Ribosomal Protein S6 Kinases, 70-kDa/genetics
- Ribosomal Protein S6 Kinases, 70-kDa/metabolism
- Ribosomal Protein S6 Kinases, 90-kDa/genetics
- Ribosomal Protein S6 Kinases, 90-kDa/metabolism
- Signal Transduction/physiology
- TOR Serine-Threonine Kinases/genetics
- TOR Serine-Threonine Kinases/metabolism
- Uncoupling Protein 1/biosynthesis
- Uncoupling Protein 1/genetics
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11
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Shaheen ZR, Naatz A, Corbett JA. CCR5-Dependent Activation of mTORC1 Regulates Translation of Inducible NO Synthase and COX-2 during Encephalomyocarditis Virus Infection. THE JOURNAL OF IMMUNOLOGY 2015; 195:4406-14. [PMID: 26408666 DOI: 10.4049/jimmunol.1500704] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/25/2015] [Accepted: 08/27/2015] [Indexed: 11/19/2022]
Abstract
Encephalomyocarditis virus (EMCV) infection of macrophages results in the expression of a number of inflammatory and antiviral genes, including inducible NO synthase (iNOS) and cyclooxygenase (COX)-2. EMCV-induced macrophage activation has been shown to require the presence of CCR5 and the activation of PI3K-dependent signaling cascades. The purpose of this study was to determine the role of PI3K in regulating the macrophage responses to EMCV. We show that PI3K regulates EMCV-stimulated iNOS and COX-2 expression by two independent mechanisms. In response to EMCV infection, Akt is activated and regulates the translation of iNOS and COX-2 through the mammalian target of rapamycin complex (mTORC)1. The activation of mTORC1 during EMCV infection is CCR5-dependent and appears to function in a manner that promotes the translation of iNOS and COX-2. CCR5-dependent mTORC1 activation functions as an antiviral response, as mTORC1 inhibition increases the expression of EMCV polymerase. PI3K also regulates the transcriptional induction of iNOS and COX-2 in response to EMCV infection by a mechanism that is independent of Akt and mTORC1 regulation. These findings indicate that macrophage expression of the inflammatory genes iNOS and COX-2 occurs via PI3K- and Akt-dependent translational control of mTORC1 and PI3K-dependent, Akt-independent transcriptional control.
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Affiliation(s)
- Zachary R Shaheen
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee WI 53226
| | - Aaron Naatz
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee WI 53226
| | - John A Corbett
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee WI 53226
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12
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Rodriguez P, Rojas J. cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation. J Cell Biochem 2015; 117:741-50. [PMID: 26335579 DOI: 10.1002/jcb.25359] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2015] [Accepted: 09/01/2015] [Indexed: 01/28/2023]
Abstract
cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.
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Affiliation(s)
- Pedro Rodriguez
- Facultad de Ciencias M, é, dicas, Escuela de Medicina, Universidad de Santiago de Chile (USACH), el Belloto 3530, segundo piso. Avenida Libertador Bernardo O'Higgins n°3363, Estación Central, Santiago, Chile
| | - Juan Rojas
- Facultad de Ciencias M, é, dicas, Escuela de Medicina, Universidad de Santiago de Chile (USACH), el Belloto 3530, segundo piso. Avenida Libertador Bernardo O'Higgins n°3363, Estación Central, Santiago, Chile
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13
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Fouladi M, Perentesis JP, Wagner LM, Vinks AA, Reid JM, Ahern C, Thomas G, Mercer CA, Krueger DA, Houghton PJ, Doyle LA, Chen H, Weigel B, Blaney SM. A Phase I Study of Cixutumumab (IMC-A12) in Combination with Temsirolimus (CCI-779) in Children with Recurrent Solid Tumors: A Children's Oncology Group Phase I Consortium Report. Clin Cancer Res 2014; 21:1558-65. [PMID: 25467181 DOI: 10.1158/1078-0432.ccr-14-0595] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Accepted: 11/03/2014] [Indexed: 11/16/2022]
Abstract
PURPOSE To determine the MTD, dose-limiting toxicities (DLT), pharmacokinetics, and biologic effects of cixutumumab administered in combination with temsirolimus to children with refractory solid tumors. EXPERIMENTAL DESIGN Cixutumumab and temsirolimus were administered intravenously once every 7 days in 28-day cycles. Pharmacokinetic and biology studies, including assessment of mTOR downstream targets in peripheral blood mononuclear cells, were performed during the first cycle. RESULTS Thirty-nine patients, median age 11.8 years (range, 1-21.5), with recurrent solid or central nervous system tumors were enrolled, of whom 33 were fully assessable for toxicity. There were four dose levels, which included two dose reductions and a subsequent intermediated dose escalation: (i) IMC-A12 6 mg/kg, temsirolimus 15 mg/m(2); (ii) IMC-A12 6 mg/kg, temsirolimus 10 mg/m(2); (iii) IMC-A12 4 mg/kg, temsirolimus 8 mg/m(2); and (iv) IMC-A12 6 mg/kg, temsirolimus 8 mg/m(2). Mucositis was the predominant DLT. Other DLTs included hypercholesterolemia, fatigue, thrombocytopenia, and increased alanine aminotransferase. Target inhibition (decreased S6K1 and PAkt) in peripheral blood mononuclear cells was noted at all dose levels. Marked interpatient variability in temsirolimus pharmacokinetic parameters was noted. At 8 mg/m(2), the median temsirolimus AUC was 2,946 ng • h/mL (range, 937-5,536) with a median sirolimus AUC of 767 ng • h/mL (range, 245-3,675). CONCLUSIONS The recommended pediatric phase II doses for the combination of cixutumumab and temsirolimus are 6 mg/kg and 8 mg/m(2), respectively.
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Affiliation(s)
- Maryam Fouladi
- Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
| | | | - Lars M Wagner
- Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | | | - Joel M Reid
- Mayo Clinic College of Medicine, Rochester, Minnesota
| | - Charlotte Ahern
- Children's Oncology Group Operations Center, Arcadia, California
| | | | | | - Darcy A Krueger
- Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | | | - L Austin Doyle
- Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, Maryland
| | - Helen Chen
- Cancer Therapy Evaluation Program, National Cancer Institute, Bethesda, Maryland
| | | | - Susan M Blaney
- Texas Children's Cancer Center/Baylor College of Medicine, Houston, Texas
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14
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Chen LZ, Li XY, Huang H, Xing W, Guo W, He J, Sun ZY, Luo AX, Liang HP, Hu J, Xu X, Xu YS, Wang ZG. SUMO-2 promotes mRNA translation by enhancing interaction between eIF4E and eIF4G. PLoS One 2014; 9:e100457. [PMID: 24971752 PMCID: PMC4074059 DOI: 10.1371/journal.pone.0100457] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2014] [Accepted: 05/25/2014] [Indexed: 01/02/2023] Open
Abstract
Small ubiquitin-like modifier (SUMO) proteins regulate many important eukaryotic cellular processes through reversible covalent conjugation to target proteins. In addition to its many well-known biological consequences, like subcellular translocation of protein, subnuclear structure formation, and modulation of transcriptional activity, we show here that SUMO-2 also plays a role in mRNA translation. SUMO-2 promoted formation of the active eukaryotic initiation factor 4F (eIF4F) complex by enhancing interaction between Eukaryotic Initiation Factor 4E (eIF4E) and Eukaryotic Initiation Factor 4G (eIF4G), and induced translation of a subset of proteins, such as cyclinD1 and c-myc, which essential for cell proliferation and apoptosis. As expected, overexpression of SUMO-2 can partially cancel out the disrupting effect of 4EGI-1, a small molecule inhibitor of eIF4E/eIF4G interaction, on formation of the eIF4F complex, translation of the cap-dependent protein, cell proliferation and apoptosis. On the other hand, SUMO-2 knockdown via shRNA partially impaired cap-dependent translation and cell proliferation and promoted apoptosis. These results collectively suggest that SUMO-2 conjugation plays a crucial regulatory role in protein synthesis. Thus, this report might contribute to the basic understanding of mammalian protein translation and sheds some new light on the role of SUMO in this process.
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Affiliation(s)
- Li-zhao Chen
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
- Department of Neurosurgery, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Xiang-yun Li
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
- Cell-based Biotherapy Center, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Hong Huang
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Wei Xing
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Wei Guo
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Jing He
- Cell-based Biotherapy Center, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Zhi-ya Sun
- Cell-based Biotherapy Center, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - An-xiong Luo
- Cell-based Biotherapy Center, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Hua-ping Liang
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Jing Hu
- Department of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
| | - Xiang Xu
- First department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
- Cell-based Biotherapy Center, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
| | - Yun-sheng Xu
- Department of Dermatology, First Affiliated Hospital of Wenzhou Medical College, Wenzhou Zhejiang, China
| | - Zheng-guo Wang
- Fourth department, State Key Laboratory of Trauma, Burn and Combined Injury, Research Institute of Surgery and Daping Hospital, Third Military Medical University, Chongqing, China
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15
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Xue M, Masand P, Thompson P, Finegold M, Leung DH. Angiosarcoma successfully treated with liver transplantation and sirolimus. Pediatr Transplant 2014; 18:E114-9. [PMID: 24641525 DOI: 10.1111/petr.12245] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 01/29/2014] [Indexed: 12/14/2022]
Abstract
Malignant liver tumors represent approximately 1% of malignancies in children. HA is a high-grade tumor of endothelial cells that is even more rare in the pediatric population. HA has a limited response to chemotherapy, radiation and resection with universal tumor recurrence with LT and nearly 100% mortality by 18 months. This is the first reported successful case of hepatic angiosarcoma in a child who was treated by LT in combination with sirolimus. Sirolimus antagonizes the mTOR pathway, which regulates cell proliferation, differentiation, and migration, and is being studied as an anti-neoplastic agent for solid tumors.
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Affiliation(s)
- Megan Xue
- Pediatrics Baylor College of Medicine, Houston, TX, USA
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16
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Raina K, Agarwal C, Wadhwa R, Serkova NJ, Agarwal R. Energy deprivation by silibinin in colorectal cancer cells: a double-edged sword targeting both apoptotic and autophagic machineries. Autophagy 2013; 9:697-713. [PMID: 23445752 DOI: 10.4161/auto.23960] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Small molecules with the potential to initiate different types of programmed cell death could be useful 'adjunct therapy' where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨm) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT-MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly 'double-edged sword' against CRC cells thereby further advocating its clinical effectiveness against this malignancy.
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Affiliation(s)
- Komal Raina
- Department of Pharmaceutical Sciences, Skaggs School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
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17
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Sayano T, Kawakami Y, Kusada W, Suzuki T, Kawano Y, Watanabe A, Takashima K, Arimoto Y, Esaki K, Wada A, Yoshizawa F, Watanabe M, Okamoto M, Hirabayashi Y, Furuya S. L-serine deficiency caused by genetic Phgdh deletion leads to robust induction of 4E-BP1 and subsequent repression of translation initiation in the developing central nervous system. FEBS J 2013; 280:1502-17. [PMID: 23350942 DOI: 10.1111/febs.12146] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2012] [Revised: 01/09/2013] [Accepted: 01/21/2013] [Indexed: 12/23/2022]
Abstract
Targeted disruption in mice of the gene encoding D-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free L-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that L-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of L-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when L-serine was supplemented in the culture medium, indicating that reduced L-serine availability regulates the induction of Eif4ebp1/4E-BP1. These data suggest that elevated levels of 4E-BP1 may be involved in a mechanism to arrest brain development in Phgdh KO embryos.
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Affiliation(s)
- Tomoko Sayano
- Division of Systems Biology, Department of Bioscience and Biotechnology, Kyushu University, Fukuoka, Japan
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18
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The role of mTORC1 in regulating protein synthesis and skeletal muscle mass in response to various mechanical stimuli. Rev Physiol Biochem Pharmacol 2013; 166:43-95. [PMID: 24442322 DOI: 10.1007/112_2013_17] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Skeletal muscle plays a fundamental role in mobility, disease prevention, and quality of life. Skeletal muscle mass is, in part, determined by the rates of protein synthesis, and mechanical loading is a major regulator of protein synthesis and skeletal muscle mass. The mammalian/mechanistic target of rapamycin (mTOR), found in the multi-protein complex, mTORC1, is proposed to play an essential role in the regulation of protein synthesis and skeletal muscle mass. The purpose of this review is to examine the function of mTORC1 in relation to protein synthesis and cell growth, the current evidence from rodent and human studies for the activation of mTORC1 signaling by different types of mechanical stimuli, whether mTORC1 signaling is necessary for changes in protein synthesis and skeletal muscle mass that occur in response to different types of mechanical stimuli, and the proposed molecular signaling mechanisms that may be responsible for the mechanical activation of mTORC1 signaling.
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19
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Rai P, Plagov A, Kumar D, Pathak S, Ayasolla KR, Chawla AK, Mathieson PW, Saleem MA, Husain M, Malhotra A, Singhal PC. Rapamycin-induced modulation of HIV gene transcription attenuates progression of HIVAN. Exp Mol Pathol 2012; 94:255-61. [PMID: 23010541 DOI: 10.1016/j.yexmp.2012.09.009] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2012] [Accepted: 09/15/2012] [Indexed: 11/15/2022]
Abstract
HIV-associated nephropathy (HIVAN) is the manifestation of HIV gene expression by kidney cells in the presence of specific host factors. Recently, rapamycin (sirolimus) has been demonstrated to modulate the progression of HIVAN. We hypothesized that rapamycin would modulate the progression of HIVAN by attenuating HIV gene expression. To test our hypothesis, three weeks old Tg26 mice (n=6) were administered either vehicle or rapamycin (5 mg/kg, every other day, intraperitoneal) for eight weeks. At the end of the experimental period, the kidneys were harvested. In in vitro studies, human podocytes were transduced with either HIV-1 (NL4-3) or empty vector (EV), followed by treatment with either vehicle or rapamycin. Total RNA and proteins were extracted from renal tissues/cellular lysates and HIV gene transcription/translation was measured by real time PCR and Western blotting studies. Renal histological slides were graded for glomerular sclerosis and tubular dilatation with microcyst formation. Rapamycin attenuated both glomerular and tubular lesions in Tg26 mice. Rapamycin decreased transcription of HIV genes both in renal tissues as well as in HIV-1 transduced podocytes. Our data strongly indicate that HIV-1 long terminal repeat-mediated transcriptional activity was targeted by rapamycin. Rapamycin enhanced podocyte NF-κB and CREB activities but then it decreased AP-1 binding activity. Since expression of HIV genes by kidney cells has been demonstrated to be the key factor in the development HIVAN, it appears that rapamycin-induced altered transcription of HIV genes might have partly contributed to its disease modulating effects.
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Affiliation(s)
- Partab Rai
- Department of Medicine, Feinstein Institute for Medical Research, Hofstra North Shore LIJ Medical School, NY, USA
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20
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Abstract
In metazoans, TOR is an essential protein that functions as a master regulator of cellular growth and proliferation. Over the past decade, there has been an explosion of information about this critical master kinase, ranging from the composition of the TOR protein complex to its ability to act as an integrator of numerous extracellular signals. Unfortunately, this plethora of information has also raised numerous questions regarding TOR function. Currently, the prevailing view is that mammalian TOR (mTOR) exists in at least two molecular complexes, mTORC1 and mTORC2, which are largely defined by the presence of either RAPTOR or RICTOR. However, additional co-factors have been identified for each complex, and their importance in mediating mTOR signals has been incompletely elucidated. Similarly, there are differences in mTOR function that reflect the tissue of origin. In this review, we present an alternative view to mTOR complex formation and function, which envisions mTOR regulation and signal propagation as a reflection of cell type- and basal state-dependent conditions. The re-interpretation of mTOR biology in this framework may facilitate the design of therapies most likely to effectively inhibit this central regulator of cell behavior.
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Affiliation(s)
- Jason D Weber
- Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA
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21
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Hewer RC, Sala-Newby GB, Wu YJ, Newby AC, Bond M. PKA and Epac synergistically inhibit smooth muscle cell proliferation. J Mol Cell Cardiol 2010; 50:87-98. [PMID: 20971121 PMCID: PMC3093616 DOI: 10.1016/j.yjmcc.2010.10.010] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/01/2010] [Revised: 10/08/2010] [Accepted: 10/11/2010] [Indexed: 12/14/2022]
Abstract
Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. Cyclic AMP inhibits VSMC proliferation via mechanisms that are not fully understood. We investigated the role of PKA and Epac signalling on cAMP-induced inhibition of VSMC proliferation. cAMP-mediated growth arrest was PKA-dependent. However, selective PKA activation with 6-Benzoyl-cAMP did not inhibit VSMC proliferation, indicating a requirement for additional pathways. Epac activation using the selective cAMP analogue 8-CPT-2′-O-Me-cAMP, did not affect levels of hyperphosphorylated Retinoblastoma (Rb) protein, a marker of G1-S phase transition, or BrdU incorporation, despite activation of the Epac-effector Rap1. However, 6-Benzoyl-cAMP and 8-CPT-2′-O-Me-cAMP acted synergistically to inhibit Rb-hyperphosphorylation and BrdU incorporation, indicating that both pathways are required for growth inhibition. Consistent with this, constitutively active Epac increased Rap1 activity and synergised with 6-Benzoyl-cAMP to inhibit VSMC proliferation. PKA and Epac synergised to inhibit phosphorylation of ERK and JNK. Induction of stellate morphology, previously associated with cAMP-mediated growth arrest, was also dependent on activation of both PKA and Epac. Rap1 inhibition with Rap1GAP or siRNA silencing did not negate forskolin-induced inhibition of Rb-hyperphosphorylation, BrdU incorporation or stellate morphology. This data demonstrates for the first time that Epac synergises with PKA via a Rap1-independent mechanism to mediate cAMP-induced growth arrest in VSMC. This work highlights the role of Epac as a major player in cAMP-dependent growth arrest in VSMC.
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22
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Campbell JS, Argast GM, Yuen SY, Hayes B, Fausto N. Inactivation of p38 MAPK during liver regeneration. Int J Biochem Cell Biol 2010; 43:180-8. [PMID: 20708092 DOI: 10.1016/j.biocel.2010.08.002] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2009] [Revised: 07/27/2010] [Accepted: 08/03/2010] [Indexed: 12/22/2022]
Abstract
There is increasing evidence that p38 MAPK, which is classified as a stress-activated kinase, also participates in cell cycle regulation, functioning as a suppressor of cell proliferation and tumorigenesis. We conducted a study of p38 MAPK phosphorylation during liver regeneration in mice to determine whether p38 MAPK activation or inactivation may correlate with events that lead to DNA replication after partial hepatectomy (PH), and whether p38 MAPK activation may be required for hepatocyte DNA replication in vivo and in culture. We report that active p38 (Pi-p38 MAPK) is present in normal liver, is rapidly inactivated starting 30 min after PH, and is re-activated by 12h. Although levels of Pi-MKK 3/6, the upstream kinases that activate p38 MAPK increase after PH, the expression of the dual protein phosphatase 1 is also elevated, and may be responsible for Pi-p38 MAPK dephosphorylation after PH. Inactivation and re-activation of p38 MAPK inversely correlates with the stimulation of protein synthesis and translation pathways, as indicated by activation of p70S6 kinase, increases in the phosphorylation of initiation factor elF-4E and translational repressor, 4E-BP. The activity of a p38 MAPK downstream substrate, MAPKAPK2 (MK2), did not reflect the changing levels of Pi-p38 MAPK during liver regeneration. Pi-p38 MAPK may be involved in TNF-stimulated DNA replication of murine hepatocytes in culture, but is not necessary for hepatocyte DNA replication after PH. Our results suggest that p38 MAPK inactivation plays a permissible role in DNA replication during liver regeneration and is consistent with a role for p38 MAPK in the maintenance of hepatocyte cell cycle arrest in adult liver.
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Affiliation(s)
- Jean S Campbell
- Department of Pathology, University of Washington, School of Medicine, Seattle, WA 98195, USA.
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23
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Kumar D, Konkimalla S, Yadav A, Sataranatarajan K, Kasinath BS, Chander PN, Singhal PC. HIV-associated nephropathy: role of mammalian target of rapamycin pathway. THE AMERICAN JOURNAL OF PATHOLOGY 2010; 177:813-21. [PMID: 20581056 PMCID: PMC2913356 DOI: 10.2353/ajpath.2010.100131] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 04/16/2010] [Indexed: 12/22/2022]
Abstract
Both glomerular and tubular lesions are characterized by a proliferative phenotype in HIV-associated nephropathy. We hypothesized that mammalian target of rapamycin (mTOR) contributes to the development of the HIVAN phenotype. Both glomerular and tubular epithelial cells showed enhanced expression of phospho (p)-mTOR in HIV-1 transgenic mice (Tgs). In addition, renal tissues of transgenic mice (RT-Tg) showed enhanced phosphorylation of p70S6 kinase and an associated diminished phosphorylation of eEF2. Moreover, RT-Tgs showed enhanced phosphorylation of 4EBP1 and eIF4B; these findings indicated activation of the mTOR pathway in RT-Tgs. To test our hypothesis, age- and sex-matched control mice and Tgs were administered either saline or rapamycin (an inhibitor of the mTOR pathway) for 4 weeks. Tgs receiving rapamycin not only showed inhibition of the mTOR-associated downstream signaling but also displayed attenuated renal lesions. RT-Tgs showed enhanced expression of hypoxia-inducible factor-alpha and also displayed increased expression of vascular endothelial growth factor; on the other hand, rapamycin inhibited RT-Tg expression of both hypoxia-inducible factor-alpha and vascular endothelial growth factor. We conclude that the mTOR pathway contributes to the HIVAN phenotype and that inhibition of the mTOR pathway can be used as a therapeutic strategy to alter the course of HIVAN.
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Affiliation(s)
- Dileep Kumar
- Department of Immunology, Feinstein Institute for Medical Research, Manhasset, New York, USA
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24
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Blancquaert S, Wang L, Paternot S, Coulonval K, Dumont JE, Harris TE, Roger PP. cAMP-dependent activation of mammalian target of rapamycin (mTOR) in thyroid cells. Implication in mitogenesis and activation of CDK4. Mol Endocrinol 2010; 24:1453-68. [PMID: 20484410 DOI: 10.1210/me.2010-0087] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
How cAMP-dependent protein kinases [protein kinase A (PKA)] transduce the mitogenic stimulus elicited by TSH in thyroid cells to late activation of cyclin D3-cyclin-dependent kinase 4 (CDK4) remains enigmatic. Here we show in PC Cl3 rat thyroid cells that TSH/cAMP, like insulin, activates the mammalian target of rapamycin (mTOR)-raptor complex (mTORC1) leading to phosphorylation of S6K1 and 4E-BP1. mTORC1-dependent S6K1 phosphorylation in response to both insulin and cAMP required amino acids, whereas inhibition of AMP-activated protein kinase and glycogen synthase kinase 3 enhanced insulin but not cAMP effects. Unlike insulin, TSH/cAMP did not activate protein kinase B or induce tuberous sclerosis complex 2 phosphorylation at T1462 and Y1571. However, like insulin, TSH/cAMP produced a stable increase in mTORC1 kinase activity that was associated with augmented 4E-BP1 binding to raptor. This could be caused in part by T246 phosphorylation of PRAS40, which was found as an in vitro substrate of PKA. Both in PC Cl3 cells and primary dog thyrocytes, rapamycin inhibited DNA synthesis and retinoblastoma protein phosphorylation induced by TSH and insulin. Although rapamycin reduced cyclin D3 accumulation, the abundance of cyclin D3-CDK4 complexes was not affected. However, rapamycin inhibited the activity of these complexes by decreasing the TSH and insulin-mediated stimulation of activating T172 phosphorylation of CDK4. We propose that mTORC1 activation by TSH, at least in part through PKA-dependent phosphorylation of PRAS40, crucially contributes to mediate cAMP-dependent mitogenesis by regulating CDK4 T172-phosphorylation.
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Affiliation(s)
- Sara Blancquaert
- Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium
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25
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Richmond SR, Touchberry CD, Gallagher PM. Forskolin attenuates the action of insulin on the Akt–mTOR pathway in human skeletal muscle. Appl Physiol Nutr Metab 2009; 34:916-25. [DOI: 10.1139/h09-096] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Forskolin (FSK) is capable of both stimulating and inhibiting the intracellular signaling pathways of protein synthesis tissues other than skeletal muscle. The purpose of this investigation was to determine if FSK administration affects various elements of the protein kinase B (Akt)–mammalian target of rapamycin (mTOR) pathway in human skeletal muscle. Ten (n = 10) healthy, young (21.6 ± 1.3 years), nonobese (body mass index = 25.5 ± 3.5 kg·m–2), recreationally active males were selected for participation. Following an 8 h fast, 2 muscle biopsies of the vastus lateralis were performed. The samples were sectioned and exposed to 4 in vitro treatment conditions: basal, FSK, insulin (INS), and FSK+INS. The samples were then analyzed for total and phosphorylated levels of Akt, mTOR, S6 kinase (S6K1), and 4E binding protein (4EBP1). Akt phosphorylation was significantly greater in the INS-treated samples compared with the basal and FSK conditions (p = 0.007). Furthermore, the ratio of phosphorylated Akt to total Akt (P/T) was higher in the INS samples compared with the basal and FSK samples (p = 0.001). There were no differences in mTOR phosphorylation among the 4 groups; however, total mTOR was significantly greater in the FSK+INS group (p = 0.006). There were also no differences in phosphorylated or total levels of S6K1 among the 4 groups. However, 4EBP1 phosphorylation was significantly greater in the INS-treated samples compared with the basal (p = 0.003) and FSK (p = 0.004) treatments. There were no differences in the ratio of phosphorylated 4EBP1 to total 4EBP1 (P/T) among the 4 groups. These results indicate that FSK does not activate the Akt–mTOR pathway in human skeletal muscle; however, these results suggest that FSK may inhibit the actions of INS on this pathway.
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Affiliation(s)
- Scott R. Richmond
- Applied Physiology Laboratory, University of Kansas, Lawrence, KS 66045, USA
| | - Chad D. Touchberry
- Applied Physiology Laboratory, University of Kansas, Lawrence, KS 66045, USA
| | - Philip M. Gallagher
- Applied Physiology Laboratory, University of Kansas, Lawrence, KS 66045, USA
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26
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Chen S, Nakahara T, Uchi H, Takeuchi S, Takahara M, Kido M, Dugu L, Tu Y, Moroi Y, Furue M. Immunohistochemical analysis of the mammalian target of rapamycin signalling pathway in extramammary Paget’s disease. Br J Dermatol 2009; 161:357-63. [PMID: 19438435 DOI: 10.1111/j.1365-2133.2009.09179.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Affiliation(s)
- S Chen
- Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashiku, Fukuoka, Japan
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Lee MJ, Fried SK. Integration of hormonal and nutrient signals that regulate leptin synthesis and secretion. Am J Physiol Endocrinol Metab 2009; 296:E1230-8. [PMID: 19318513 PMCID: PMC2692400 DOI: 10.1152/ajpendo.90927.2008] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
This review summarizes recent advances in our understanding of the pre- and posttranscriptional mechanisms that regulate leptin production and secretion in adipocytes. Basal leptin production is proportional to the status of energy stores, i.e., fat cell size, and this is mainly regulated by alterations in leptin mRNA levels. Leptin mRNA levels are regulated by hormones, including glucocorticoids and catecholamines, but little is known about the transcriptional mechanisms involved. Leptin synthesis and secretion is also acutely modulated in response to hormones such as insulin and the availability of metabolic fuels. Acute variations in leptin production over a time course of minutes to hours are mediated at the levels of both translation and secretion. Increases in amino acids and insulin after a meal activate the mammalian target of rapamycin (mTOR) pathway, leading to an increase in specific rates of leptin biosynthesis. Cross-talk among mTOR, PKA, and AMP-activated protein kinase pathways appears to integrate hormonal and nutrient signals that regulate leptin mRNA translation, at least in part through mechanisms involving its 5'- and 3'-untranslated regions. In addition, the rate of leptin secretion from preformed stores in response to hormonal cues is also regulated. Insulin stimulates, and adrenergic agonists inhibit, leptin secretion, and this likely contributes to variations in the magnitude of nutrition-related leptin excursions and oscillations. Overall, the study of leptin production has contributed to a deepening understanding of leptin biology and, more broadly, to our understanding of the cellular and molecular mechanisms by which the adipocyte integrates hormonal and nutrient signals to regulate adipokine production.
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Affiliation(s)
- Mi-Jeong Lee
- Division of Endocrinology, Diabetes, and Nutrition, Department of Medicine, University of Maryland, School of Medicine, Baltimore, MD, USA
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Santa-Catalina MO, Garcia-Marin LJ, Bragado MJ. Lovastatin effect in rat neuroblasts of the CNS: inhibition of cap-dependent translation. J Neurochem 2008; 106:1078-91. [PMID: 18466319 DOI: 10.1111/j.1471-4159.2008.05458.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Mevalonate biosynthesis pathway is important in cell growth and survival and its blockade by 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, statins, arrest brain neuroblasts growth and induce apoptosis. Translation is among the main biochemical mechanisms that controls gene expression and therefore cell growth or apoptosis. In the CNS, translation regulates synaptic plasticity. Thus, our aim was to investigate the effect of lovastatin in protein translation in rat neuroblasts of the CNS and the biochemical pathways involved. Lovastatin treatment in rat brain neuroblasts causes a significant time- and concentration-inhibition of protein synthesis, which is partially mediated by phosphatydilinositol 3-kinase/mammalian target of rapamycin (mTOR) pathway inhibition. Lovastatin treatment decreases the phosphorylation state of mTOR substrates, p70S6K and eukaryotic translation initiation factor (eIF) 4E-binding protein 1 and simultaneously increases eIF4E-binding protein 1 in a time-dependent manner. Concomitantly, lovastatin causes a decrease in eIF4G cellular amount, which is partially mediated by caspase(s) activity excluding caspase 3. These biochemical pathways affected by lovastatin might explain the protein translation inhibition observed in neuroblasts. Cycloheximide treatment, which blocked protein synthesis, does not induce neuroblasts apoptosis. Therefore, we suggest that lovastatin-induced protein synthesis inhibition might not contribute to the concomitant neuroblasts apoptosis previously observed.
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Affiliation(s)
- Marta Olivera Santa-Catalina
- Research group of Intracellular Signalling and Technology of Reproduction, Department of Biochemistry, Molecular Biology and Genetics, Cáceres, Spain
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29
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Kimball SR, Do AND, Kutzler L, Cavener DR, Jefferson LS. Rapid turnover of the mTOR complex 1 (mTORC1) repressor REDD1 and activation of mTORC1 signaling following inhibition of protein synthesis. J Biol Chem 2008; 283:3465-3475. [PMID: 18070882 PMCID: PMC2654224 DOI: 10.1074/jbc.m706643200] [Citation(s) in RCA: 83] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity.
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Affiliation(s)
- Scot R Kimball
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the.
| | - A N Dang Do
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the
| | - Lydia Kutzler
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the
| | - Douglas R Cavener
- Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
| | - Leonard S Jefferson
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 and the
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Fernández N, González A, Valera I, Alonso S, Crespo MS. Mannan and peptidoglycan induce COX-2 protein in human PMN via the mammalian target of rapamycin. Eur J Immunol 2007; 37:2572-82. [PMID: 17683115 DOI: 10.1002/eji.200737262] [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] [Indexed: 01/21/2023]
Abstract
The induction of cyclooxygenase-2 (COX-2) protein expression was assessed in human polymorphonuclear leukocytes (PMN) stimulated via receptors of the innate immune system. Peptidoglycan (PGN) and mannan, and at a lower extent the bacterial lipoprotein mimic palmitoyl-3-cysteine-serine-lysine-4, induced COX-2 protein expression. In contrast, lipoteichoic acid and muramyldipeptide were irrelevant stimuli. The mRNA encoding COX-2 was present in resting PMN at an extent quite similar to that detected in stimulated PMN, whereas the expression of COX-2 protein was undetectable. Treatment with the phosphatidylinositol 3-kinase inhibitor (PI3K) wortmaninn, the mammalian target of rapamycin (mTOR) inhibitor rapamycin, and the translation inhibitor cycloheximide blocked the induction of COX-2 protein in response to mannan and PGN, whereas the transcriptional inhibitor actinomycin D did not show a significant effect. These results disclose a capability of pathogen-associated molecular patterns to induce the oxidative metabolism of arachidonic acid more robust than that of PMN archetypal chemoattractants, since mannan and PGN make it coincidental the release of arachidonic acid with a rapid induction of COX-2 protein regulated by a signaling cascade involving PI3K, mTOR, and the translation machinery. This mechanism of COX-2 protein induction expression in PMN is substantially different from that operative in mononuclear phagocytes, which is highly dependent on transcriptional regulation.
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Affiliation(s)
- Nieves Fernández
- Instituto de Biología y Genética Molecular, Consejo Superior de Investigaciones Científicas, Valladolid, Spain
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31
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Peffley DM, Sharma C, Hentosh P, Buechler RD. Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells. Arch Biochem Biophys 2007; 465:266-73. [PMID: 17601486 DOI: 10.1016/j.abb.2007.05.022] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2007] [Revised: 05/26/2007] [Accepted: 05/30/2007] [Indexed: 11/30/2022]
Abstract
Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific. Perillyl alcohol and genistein suppressed 4E-BP1(Ser65) phosphorylation in prostate tumor cell lines, DU145 and PC-3, and in Caco2 adenocarcinoma cells. Suppressive effects were similar to or greater than that observed with a PI3 kinase inhibitor or rapamycin, an mTOR inhibitor. 4E-BP1(Thr37) phosphorylation was reduced by perillyl alcohol and genistein in DU145, but not in PC-3. Conversely, perillyl alcohol but not genistein decreased 4E-BP1(Thr37) phosphorylation in Caco2. PKB/Akt activation via Ser473 phosphorylation was enhanced in DU145 by perillyl alcohol and in PC-3 by gamma-tocotrienol, but was suppressed by genistein. Importantly, perillyl alcohol disrupted interactions between eIF4E and eIF4G, key components of eIF4F (m(7)GpppX) cap binding complex. These results demonstrate that "pure" isoprenoids and genistein differentially impact cap-dependent translation in tumor cell lines.
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Affiliation(s)
- Dennis M Peffley
- Department of Biochemistry, Kansas City University of Medicine and Biosciences, 1750 Independence Avenue, Kansas City, MO 64106-1453, USA.
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32
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Elia A, Constantinou C, Clemens MJ. Effects of protein phosphorylation on ubiquitination and stability of the translational inhibitor protein 4E-BP1. Oncogene 2007; 27:811-22. [PMID: 17653084 DOI: 10.1038/sj.onc.1210678] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The availability of the eukaryotic polypeptide chain initiation factor 4E (eIF4E) for protein synthesis is regulated by the 4E-binding proteins (4E-BPs), which act as inhibitors of cap-dependent mRNA translation. The ability of the 4E-BPs to sequester eIF4E is regulated by reversible phosphorylation at multiple sites. We show here that, in addition, 4E-BP1 is a substrate for polyubiquitination and that some forms of 4E-BP1 are simultaneously polyubiquitinated and phosphorylated. In Jurkat cells inhibition of proteasomal activity by MG132 enhances the level of hypophosphorylated, unmodified 4E-BP1 but only modestly increases the accumulation of high-molecular-weight, phosphorylated forms of 4E-BP1. In contrast, inhibition of protein phosphatase activity with calyculin A reduces the level of unmodified 4E-BP1 but strongly enhances the amount of phosphorylated, high-molecular-weight 4E-BP1. Turnover measurements in the presence of cycloheximide show that, whereas 4E-BP1 is normally a very stable protein, calyculin A decreases the apparent half-life of the normal-sized protein. Affinity chromatography on m(7)GTP-Sepharose indicates that the larger forms of 4E-BP1 bind very poorly to eIF4E. We suggest that the phosphorylation of 4E-BP1 may play a dual role in the regulation of protein synthesis, both reducing the affinity of 4E-BP1 for eIF4E and promoting the conversion of 4E-BP1 to alternative, polyubiquitinated forms.
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Affiliation(s)
- A Elia
- Translational Control Group, Division of Basic Medical Sciences, Centre for Molecular and Metabolic Signalling, St George's, University of London, London, UK
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33
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Kim SK, Novak RF. The role of intracellular signaling in insulin-mediated regulation of drug metabolizing enzyme gene and protein expression. Pharmacol Ther 2006; 113:88-120. [PMID: 17097148 PMCID: PMC1828071 DOI: 10.1016/j.pharmthera.2006.07.004] [Citation(s) in RCA: 123] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2006] [Accepted: 07/18/2006] [Indexed: 12/28/2022]
Abstract
Endogenous factors, including hormones, growth factors and cytokines, play an important role in the regulation of hepatic drug metabolizing enzyme expression in both physiological and pathophysiological conditions. Diabetes, fasting, obesity, protein-calorie malnutrition and long-term alcohol consumption produce changes in hepatic drug metabolizing enzyme gene and protein expression. This difference in expression alters the metabolism of xenobiotics, including procarcinogens, carcinogens, toxicants and therapeutic agents, potentially impacting the efficacy and safety of therapeutic agents, and/or resulting in drug-drug interactions. Although the mechanisms by which xenobiotics regulate drug metabolizing enzymes have been studied intensively, less is known regarding the cellular signaling pathways and components which regulate drug metabolizing enzyme gene and protein expression in response to hormones and cytokines. Recent findings, however, have revealed that several cellular signaling pathways are involved in hormone- and growth factor-mediated regulation of drug metabolizing enzymes. Our laboratory has reported that insulin and growth factors regulate drug metabolizing enzyme gene and protein expression, including cytochromes P450 (CYP), glutathione S-transferases (GST) and microsomal epoxide hydrolase (mEH), through receptors which are members of the large receptor tyrosine kinase (RTK) family, and by downstream effectors such as phosphatidylinositol 3-kinase, mitogen activated protein kinase (MAPK), Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR), and the p70 ribosomal protein S6 kinase (p70S6 kinase). Here, we review current knowledge of the signaling pathways implicated in regulation of drug metabolizing enzyme gene and protein expression in response to insulin and growth factors, with the goal of increasing our understanding of how disease affects these signaling pathways, components, and ultimately gene expression and translational control.
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Affiliation(s)
- Sang K. Kim
- Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201, USA
- College of Pharmacy and Research Center for Transgenic Cloned Pigs, Chungnam National University, Daejeon 305-764, South Korea
| | - Raymond F. Novak
- Institute of Environmental Health Sciences, Wayne State University, 2727 Second Avenue, Room 4000, Detroit, MI 48201, USA
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34
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Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006; 5:671-88. [PMID: 16883305 DOI: 10.1038/nrd2062] [Citation(s) in RCA: 741] [Impact Index Per Article: 39.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Mammalian target of rapamycin (mTOR) is a kinase that functions as a master switch between catabolic and anabolic metabolism and as such is a target for the design of anticancer agents. The most established mTOR inhibitors--rapamycin and its derivatives--showed long-lasting objective tumour responses in clinical trials, with CCI-779 being a first-in-class mTOR inhibitor that improved the survival of patients with advanced renal cell carcinoma. This heralded the beginning of extensive clinical programmes to further evaluate mTOR inhibitors in several tumour types. Here we review the clinical development of this drug class and look at future prospects for incorporating these agents into multitarget or multimodality strategies against cancer.
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Affiliation(s)
- Sandrine Faivre
- Service Inter Hospitalier de Cancrologie, Beaujon University Hospital, 100 Boulevard du General Leclerc, 92118 Clichy Cedex, France
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35
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Abstract
Even apparently healthy patients on dialysis have significant loss of lean body mass. Patients with chronic renal failure without coexisting metabolic acidosis or inflammation have decreased protein turnover, with balanced reduction in protein synthesis and breakdown. However, regional and whole-body protein kinetic studies indicate that hemodialysis (HD) induces net increase in protein breakdown. Whole-body protein turnover studies show that HD is associated with decreased protein synthesis, but proteolysis is not increased. Muscle protein kinetics studies, however, identify enhanced muscle protein breakdown with inadequate compensatory increases in synthesis as the cause of the catabolism. Transmembrane amino acid-transport kinetics studies show that the outward transport is increased more than the inward transport of amino acids during HD. Altered intracellular amino acid transport kinetics and protein turnover during HD could be caused by the loss of amino acids in the dialysate or cytokine activation. Cytokines may be released from peripheral blood mononuclear cells and skeletal muscle during HD. Preliminary evidence indicates that intradialytic increase in cytokines activates the ubiquitin-proteasome pathway. An intradialytic increase in albumin and fibrinogen synthesis is facilitated by interleukin-6 and the constant supply of amino acids derived from skeletal muscle catabolism. Protein anabolism can be induced in end-stage renal disease patients by repletion of amino acids, and perhaps treatment with recombinant human insulin-like growth factor.
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Affiliation(s)
- Dominic S C Raj
- Division of Nephrology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
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36
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Wang L, Rhodes CJ, Lawrence JC. Activation of mammalian target of rapamycin (mTOR) by insulin is associated with stimulation of 4EBP1 binding to dimeric mTOR complex 1. J Biol Chem 2006; 281:24293-303. [PMID: 16798736 DOI: 10.1074/jbc.m603566200] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.
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Affiliation(s)
- Lifu Wang
- Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
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37
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Crozier SJ, Sans MD, Guo L, D'Alecy LG, Williams JA. Activation of the mTOR signalling pathway is required for pancreatic growth in protease-inhibitor-fed mice. J Physiol 2006; 573:775-86. [PMID: 16613881 PMCID: PMC1779746 DOI: 10.1113/jphysiol.2006.106914] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2006] [Accepted: 04/11/2006] [Indexed: 12/22/2022] Open
Abstract
Cholecystokinin (CCK)-induced pancreatic growth in mice involves parallel increases in DNA and protein. The mammalian target of rapamycin (mTOR) signalling pathway regulates mRNA translation and its activation is implicated in growth of various tissues. The aim of this study was to elucidate whether mTOR activation is required for pancreatic growth in a mouse model of increased endogenous CCK release. In mice fed chow containing the synthetic protease inhibitor camostat, protein synthetic rates and phosphorylation of two downstream targets of mTOR, eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and the ribosomal protein S6 (S6), increased in comparison with fasted controls. The camostat-induced increases in protein synthesis and 4E-BP1 and S6 phosphorylation were almost totally abolished by administration of the mTOR inhibitor rapamycin 1 h prior to camostat feeding. In contrast, the phosphorylation of ERK1/2 and JNK and the expression of the early response genes c-jun, c-fos, ATF3 and egr-1 induced by camostat feeding were not affected by rapamycin. In mice fed camostat for 7 days, the ratio of pancreatic to body weight increased by 143%, but when rapamycin was administered daily this was reduced to a 22% increase. Changes in pancreatic mass were paralleled by protein and DNA content following camostat feeding and rapamycin administration. Moreover, while BrdU incorporation, an indicator of DNA synthesis, was increased to 448% of control values after 2 days of camostat feeding, rapamycin administration completely inhibited this increase. We conclude that the mTOR signalling pathway is required for CCK-induced cell division and pancreatic growth.
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Affiliation(s)
- Stephen J Crozier
- Department of Molecular and Integrative Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA.
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Abstract
Aging is associated with remarkable changes in body composition. Loss of skeletal muscle, a process called sarcopenia, is a prominent feature of these changes. In addition, gains in total body fat and visceral fat content continue into late life. The cause of sarcopenia is likely a result of a number of changes that also occur with aging. These include reduced levels of physical activity, changing endocrine function (reduced testosterone, growth hormone, and estrogen levels), insulin resistance, and increased dietary protein needs. Healthy free-living elderly men and women have been shown to accommodate to the Recommended Dietary Allowance (RDA) for protein of 0.8 g . kg(-1) . d(-1) with a continued decrease in urinary nitrogen excretion and reduced muscle mass. While many elderly people consume adequate amounts of protein, many older people have a reduced appetite and consume less than the protein RDA, likely resulting in an accelerated rate of sarcopenia. One important strategy that counters sarcopenia is strength conditioning. Strength conditioning will result in an increase in muscle size and this increase in size is largely the result of increased contractile proteins. The mechanisms by which the mechanical events stimulate an increase in RNA synthesis and subsequent protein synthesis are not well understood. Lifting weight requires that a muscle shorten as it produces force (concentric contraction). Lowering the weight, on the other hand, forces the muscle to lengthen as it produces force (eccentric contraction). These lengthening muscle contractions have been shown to produce ultrastructural damage (microscopic tears in contractile proteins muscle cells) that may stimulate increased muscle protein turnover. This muscle damage produces a cascade of metabolic events which is similar to an acute phase response and includes complement activation, mobilization of neutrophils, increased circulating an skeletal muscle interleukin-1, macrophage accumulation in muscle, and an increase in muscle protein synthesis and degradation. While endurance exercise increases the oxidation of essential amino acids and increases the requirement for dietary protein, resistance exercise results in a decrease in nitrogen excretion, lowering dietary protein needs. This increased efficiency of protein use may be important for wasting diseases such as HIV infection and cancer and particularly in elderly people suffering from sarcopenia. Research has indicated that increased dietary protein intake (up to 1.6 g protein . kg(-1) . d(-1)) may enhance the hypertrophic response to resistance exercise. It has also been demonstrated that in very old men and women the use of a protein-calorie supplement was associated with greater strength and muscle mass gains than did the use of placebo.
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Affiliation(s)
- William J Evans
- Nutrition, Metabolism, and Exercise Laboratory, Donald W. Reynolds Center on Aging, Slot 806, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
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Cheng J, Thompson MA, Walker HJ, Gray CE, Warner GM, Zhou W, Grande JP. Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells. Exp Biol Med (Maywood) 2006; 231:288-95. [PMID: 16514175 DOI: 10.1177/153537020623100308] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.
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Affiliation(s)
- Jingfei Cheng
- Renal Pathophysiology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA
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40
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Law BK. Rapamycin: an anti-cancer immunosuppressant? Crit Rev Oncol Hematol 2005; 56:47-60. [PMID: 16039868 DOI: 10.1016/j.critrevonc.2004.09.009] [Citation(s) in RCA: 187] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2004] [Revised: 08/30/2004] [Accepted: 09/24/2004] [Indexed: 12/13/2022] Open
Abstract
Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressant and anti-tumor properties. These rapamycin actions are mediated through the specific inhibition of the mTOR protein kinase. mTOR serves as part of an evolutionarily conserved signaling pathway that controls the cell cycle in response to changing nutrient levels. The mTOR signaling network contains a number of tumor suppressor genes including PTEN, LKB1, TSC1, and TSC2, and a number of proto-oncogenes including PI3K, Akt, and eIF4E, and mTOR signaling is constitutively activated in many tumor types. These observations point to mTOR as an ideal target for anti-cancer agents and suggest that rapamycin is such an agent. In fact, early preclinical and clinical studies indicate that rapamycin derivatives have efficacy as anti-tumor agents both alone, and when combined with other modes of therapy. Rapamycin appears to inhibit tumor growth by halting tumor cell proliferation, inducing tumor cell apoptosis, and suppressing tumor angiogenesis. Rapamycin immunosuppressant actions result from the inhibition of T and B cell proliferation through the same mechanisms that rapamycin blocks cancer cell proliferation. Therefore, one might think that rapamycin-induced immunosuppression would be detrimental to the use of rapamycin as an anti-cancer agent. To the contrary, rapamycin decreases the frequency of tumor formation that occurs in organ transplant experiments when combined with the widely used immunosuppressant cyclosporine compared with the tumor incidence observed when cyclosporine is used alone. The available evidence indicates that with respect to tumor growth, rapamycin anti-cancer activities are dominant over rapamycin immunosuppressant effects.
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Affiliation(s)
- Brian K Law
- Department of Pharmacology and Therapeutics, University of Florida, P.O. Box 100267, R5-136, ARB, 1600 SW Archer Road, Gainesville, FL 32610, USA
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41
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Yeshao W, Gu J, Peng X, Nairn AC, Nadler JL. Elevated glucose activates protein synthesis in cultured cardiac myocytes. Metabolism 2005; 54:1453-60. [PMID: 16253633 DOI: 10.1016/j.metabol.2005.05.010] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2004] [Accepted: 05/08/2005] [Indexed: 11/25/2022]
Abstract
Diabetes mellitus results in chronic hyperglycemia, a serious metabolic disorder associated with a markedly increased risk of cardiovascular disease. However, the effects of high glucose (HG) on cardiac myocyte growth have not been fully clarified. In this study, the effect of glucose on cardiac myocyte growth was examined using leucine incorporation as an index of protein synthesis. High glucose (HG, 25 mmol/L) increased leucine incorporation (167% +/- 0.2% over normal glucose, n=4, P<.01) compared with a physiological glucose concentration (5.5 mmol/L, normal glucose). The HG-induced increase in leucine incorporation was time- and dose-dependent and was not due to osmotic changes because 25 mmol/L mannitol did not change leucine incorporation. High glucose also significantly reduced elongation factor 2 phosphorylation, an effect known to result in increased protein synthesis at the elongation step. Western blot analysis showed that HG-activated protein kinase B (PKB), also called Akt (PKB/Akt), at 18 hours. High glucose-induced leucine incorporation was attenuated with phosphatidylinositol 3-kinase (PI3K) inhibition using wortmannin and LY294002 and by rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, 72%, 64%, and 65% (P<.05), respectively. High glucose also activated extracellular signal-regulated kinase 1/2 activity with peak stimulation at 5 minutes. In addition, PD98059, an inhibitor of mitogen-activated protein kinase kinase, attenuated HG-induced leucine incorporation. These data show for the first time that elevated glucose increases protein synthesis in cardiac myocytes. The increase appears to be mediated by activation of PI3K-PKB/Akt and/or PI3K-mTOR as well as extracellular signal-regulated kinase 1/2. These results provide new evidence for a direct effect of glucose independent of insulin on cardiac myocyte growth.
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Affiliation(s)
- Wen Yeshao
- Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia Health Science Center, Charlottesville, VA 22908, USA.
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Shen W, Boyle DW, Liechty EA. Changes in 4E-BP1 and p70S6K phosphorylation in skeletal muscle of the ovine fetus after prolonged maternal fasting: effects of insulin and IGF-I. Pediatr Res 2005; 58:833-9. [PMID: 16183812 DOI: 10.1203/01.pdr.0000182588.20368.12] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
This study was conducted to investigate fasting-induced alterations in insulin signaling to the regulatory components of the translation machinery. Insulin (890 mIU/h) and IGF-I (40 nM/h) were infused into a chronically catheterized ovine fetus (0.85 gestation) for 7 h following a 5-d maternal fast. Amino acid and glucose concentrations were clamped to minimize the effects of alterations in circulating substrate concentrations. The IGF-I induced increase in 4E-BP1 phosphorylation (percentage in the gamma form) increased from 28% in control to 44% (NS). The insulin-induced increase in 4E-BP1 phosphorylation was more pronounced, and the gamma percentage was 56% on average in the insulin group. The insulin-induced increase in 4E-BP1 phosphorylation was lower than in fed animals and did not result in significant changes in eIF4E.4E-BP1 binding or eIF4E.eIF4G binding. Insulin increased PKB/Akt phosphorylation and p70S6K phosphorylation to a similar extent as in fed animals. We conclude that maternal fasting resulted in reduced insulin sensitivity of 4E-BP1 phosphorylation and eIF4F formation. This reduced insulin-induced 4E-BP1 phosphorylation was not due to a global defect in insulin signaling; the defects underlying the reduced basal phosphorylation and insulin-responsiveness of 4E-BP1 in fasted animals may be in signaling components other than, or downstream of, PKB/Akt. Selective inhibition of downstream components of insulin signaling allows fetuses to adapt to nutritional stress by decreasing the anabolic response to insulin and other growth factors, so that more amino acids can be used as oxidative substrate to compensate for shortage of energy due to reduced glucose supply.
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Affiliation(s)
- Weihua Shen
- Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
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Pelletier S, Julien C, Popoff MR, Lamarche-Vane N, Meloche S. Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway. J Cell Physiol 2005; 204:412-22. [PMID: 15706595 DOI: 10.1002/jcp.20308] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Cyclic AMP (cAMP) is a pleiotropic second messenger that regulates numerous cellular processes. In vascular smooth muscle cells (VSMCs), these include cell proliferation, migration, and contractility. Here we show that cAMP-elevating agents induce dramatic morphological changes in VSMCs, characterized by cell rounding and formation of long branching processes. The stellate morphology is associated with disassembly of actin stress fibers and lamellipodia, loss of focal adhesions, and the formation of small F-actin rings. Because of the importance of Rho family GTPases in regulating actin dynamics, we analyzed their individual roles in the cAMP phenotype. We found that pharmacological or genetic inhibition of Rac mimics cAMP effect in inducing a stellate morphology of VSMCs. Expression of activated Rac1 prevents forskolin-induced cAMP stellation, suggesting that cAMP affects cell morphology by inhibiting Rac function. Consistent with this, treatment with forskolin inhibits agonist-stimulated Rac activation in VSMCs. We further show that activated Rac1 containing the F37A effector loop substitution fails to rescue the cAMP phenotype. Our results suggest that cAMP modulates the morphology of VSMCs by inhibiting a Rac-dependent signaling pathway.
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Affiliation(s)
- Stéphane Pelletier
- Institut de Recherche en Immunovirologie et Cancérologie, Université de Montréal, Montreal, Quebec, Canada
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Crozier SJ, Vary TC, Kimball SR, Jefferson LS. Cellular energy status modulates translational control mechanisms in ischemic-reperfused rat hearts. Am J Physiol Heart Circ Physiol 2005; 289:H1242-50. [PMID: 15894572 DOI: 10.1152/ajpheart.00859.2004] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Mechanisms regulating ischemia and reperfusion (I/R)-induced changes in mRNA translation in the heart are poorly defined, as are the factors that initiate these changes. Because cellular energy status affects mRNA translation under physiological conditions, it is plausible that I/R-induced changes in translation may in part be a result of altered cellular energy status. Therefore, the purpose of the studies described herein was to compare the effects of I/R with those of altered energy substrate availability on biomarkers of mRNA translation in the heart. Isolated adult rat hearts were perfused with glucose or a combination of glucose plus palmitate, and effects of I/R on various biomarkers of translation were subsequently analyzed. When compared with hearts perfused with glucose plus palmitate, hearts perfused with glucose alone exhibited increased phosphorylation of eukaryotic elongation factor (eEF)2, the alpha-subunit of eukaryotic initiation factor (eIF)2, and AMP-activated protein kinase (AMPK), and these hearts also exhibited enhanced association of eIF4E with eIF4E binding protein (4E-BP)1. Regardless of the energy substrate composition of the buffer, phosphorylation of eEF2 and AMPK was greater than control values after ischemia. Phosphorylation of eIF2alpha and eIF4E and the association of eIF4E with 4E-BP1 were also greater than control values after ischemia but only in hearts perfused with glucose plus palmitate. Reperfusion reversed the ischemia-induced increase in eEF2 phosphorylation in hearts perfused with glucose and reversed ischemia-induced changes in eIF4E, eEF2, and AMPK phosphorylation in hearts perfused with glucose plus palmitate. Because many ischemia-induced changes in mRNA translation are mimicked by the removal of a metabolic substrate under normal perfusion conditions, the results suggest that cellular energy status represents an important modulator of I/R-induced changes in mRNA translation.
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Affiliation(s)
- Stephen J Crozier
- Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, USA
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Girard A, Madani S, El Boustani ES, Belleville J, Prost J. Changes in lipid metabolism and antioxidant defense status in spontaneously hypertensive rats and Wistar rats fed a diet enriched with fructose and saturated fatty acids. Nutrition 2005; 21:240-8. [PMID: 15723754 DOI: 10.1016/j.nut.2004.04.022] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2003] [Revised: 03/13/2003] [Accepted: 04/20/2004] [Indexed: 11/21/2022]
Abstract
OBJECTIVE Larger doses of fructose and saturated fat have been associated with oxidative stress and development of hypertension. The effects of modest amounts of fructose and saturated fatty acids on oxidative stress are unknown. METHODS To increase knowledge on this question, 10-wk-old spontaneously hypertensive rats and Wistar rats were fed for 8 wk with a control diet or an experimental diet enriched with fructose (18%) and saturated fatty acids (11%; FS diet). The total antioxidant status of organs and red blood cells was assayed by monitoring the rate of free radical-induced red blood cell hemolysis. Sensitivity of very low-density lipoprotein and low-density lipoprotein (VLDL-LDL) to copper-induced lipid peroxidation was determined as the production of thiobarbituric acid-reactive substances. Antioxidant enzymes and vitamins were also measured to establish the oxidative stress effect. RESULTS The FS diet did not affect blood pressure in either strain, but it increased plasma insulin concentrations only in Wistar rats without affecting those of glucose of either strain. The FS diet significantly enhanced plasma and VLDL-LDL triacylglycerol concentrations without affecting concentrations of VLDL-LDL thiobarbituric acid-reactive substances. The decreased content of arachidonic acid and total polyunsaturated fatty acids in VLDL-LDL by the FS diet may have prevented lipid peroxidation in this fraction. Moreover, FS consumption by both strains was accompanied by a significant increase in total antioxidant capacity of adipose tissue, muscle, heart, and liver. This may have resulted from increased tissue ascorbic acid levels and glutathione peroxidase and glutathione reductase activities in tissues. CONCLUSIONS These findings clearly indicate that the FS diet did not alter blood pressure of spontaneously hypertensive rats and Wistar rats. The FS diet resulted in hypertriglyceridemia but increased the total antioxidant status, which may prevent lipid peroxidation in these rats.
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Affiliation(s)
- Aurélie Girard
- Université de Bourgogne, UPRES Lipides Nutrition EA 2422, Faculté des Sciences Gabriel, Dijon, France
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Inoki K, Ouyang H, Li Y, Guan KL. Signaling by target of rapamycin proteins in cell growth control. Microbiol Mol Biol Rev 2005; 69:79-100. [PMID: 15755954 PMCID: PMC1082789 DOI: 10.1128/mmbr.69.1.79-100.2005] [Citation(s) in RCA: 251] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Target of rapamycin (TOR) proteins are members of the phosphatidylinositol kinase-related kinase (PIKK) family and are highly conserved from yeast to mammals. TOR proteins integrate signals from growth factors, nutrients, stress, and cellular energy levels to control cell growth. The ribosomal S6 kinase 1 (S6K) and eukaryotic initiation factor 4E binding protein 1(4EBP1) are two cellular targets of TOR kinase activity and are known to mediate TOR function in translational control in mammalian cells. However, the precise molecular mechanism of TOR regulation is not completely understood. One of the recent breakthrough studies in TOR signaling resulted in the identification of the tuberous sclerosis complex gene products, TSC1 and TSC2, as negative regulators for TOR signaling. Furthermore, the discovery that the small GTPase Rheb is a direct downstream target of TSC1-TSC2 and a positive regulator of the TOR function has significantly advanced our understanding of the molecular mechanism of TOR activation. Here we review the current understanding of the regulation of TOR signaling and discuss its function as a signaling nexus to control cell growth during normal development and tumorigenesis.
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Affiliation(s)
- Ken Inoki
- Life Science Institute, University of Michigan Medical School, 5450 Medical Science I Bldg., Ann Arbor, MI 48109-0606, USA
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Crozier SJ, Kimball SR, Emmert SW, Anthony JC, Jefferson LS. Oral leucine administration stimulates protein synthesis in rat skeletal muscle. J Nutr 2005; 135:376-82. [PMID: 15735066 DOI: 10.1093/jn/135.3.376] [Citation(s) in RCA: 248] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Oral administration of a single bolus of leucine in an amount equivalent to the daily intake (1.35 g/kg body wt) enhances skeletal muscle protein synthesis in food-deprived rats. To elucidate whether smaller amounts of leucine can also stimulate protein synthesis, rats were administered the amino acid at concentrations ranging from 0.068 to 1.35 g/kg body wt by oral gavage. Thirty minutes following the administration of doses of leucine as low as 0.135 g/kg body wt, skeletal muscle protein synthesis was significantly greater than control values. The increase in protein synthesis was associated with changes in the regulation of biomarkers of mRNA translation initiation as evidenced by upregulated phosphorylation of the translational repressor, eukaryotic initiation factor (eIF)4E-binding protein 1 (4E-BP1), the association of eIF4G with the mRNA cap binding protein eIF4E, and the phosphorylation of the 70-kDa ribosomal protein S6 kinase. Alterations in the phosphorylation of eIF4G, as well as the association of 4E-BP1 with eIF4E, were observed following leucine administration; however, these changes appeared to be biphasic with maximal changes occurring when circulating insulin concentrations were elevated. Thus it appears that leucine administration affects mRNA translation and skeletal muscle protein synthesis through modulation of multiple biomarkers of mRNA translation. The ability of small doses of leucine to stimulate skeletal muscle protein synthesis suggests that future research on the regulation of skeletal muscle protein synthesis by orally administered leucine will be feasible in humans.
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Affiliation(s)
- Stephen J Crozier
- Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
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Lindsley JE, Rutter J. Nutrient sensing and metabolic decisions. Comp Biochem Physiol B Biochem Mol Biol 2004; 139:543-59. [PMID: 15581787 DOI: 10.1016/j.cbpc.2004.06.014] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2004] [Revised: 06/18/2004] [Accepted: 06/19/2004] [Indexed: 12/20/2022]
Abstract
Cells have several sensory systems that detect energy and metabolic status and adjust flux through metabolic pathways accordingly. Many of these sensors and signaling pathways are conserved from yeast to mammals. In this review, we bring together information about five different nutrient-sensing pathways (AMP kinase, mTOR, PAS kinase, hexosamine biosynthesis and Sir2), highlighting their similarities, differences and roles in disease.
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Affiliation(s)
- Janet E Lindsley
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132-3201, USA.
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Lisik W, Kahan BD. Inhibitors of mammalian target of rapamycin: mechanism of action explains efficacy and toxicity. Curr Opin Organ Transplant 2004. [DOI: 10.1097/01.mot.0000146725.34815.ea] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Kimball SR, Jefferson LS. Amino acids as regulators of gene expression. Nutr Metab (Lond) 2004; 1:3. [PMID: 15507151 PMCID: PMC524028 DOI: 10.1186/1743-7075-1-3] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2004] [Accepted: 08/17/2004] [Indexed: 02/04/2023] Open
Abstract
The role of amino acids as substrates for protein synthesis is well documented. However, a function for amino acids in modulating the signal transduction pathways that regulate mRNA translation has only recently been described. Interesting, some of the signaling pathways regulated by amino acids overlap with those classically associated with the cellular response to hormones such as insulin and insulin-like growth factors. The focus of this review is on the signaling pathways regulated by amino acids, with a particular emphasis on the branched-chain amino acid leucine, and the steps in mRNA translation controlled by the signaling pathways.
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Affiliation(s)
- Scot R Kimball
- Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
| | - Leonard S Jefferson
- Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA
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