Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1)

The mammalian target of rapamycin (mTOR) is essential for skeletal myogenesis through controlling distinct cellular pathways. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In this study we have investigated the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. We find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 protein levels, and decreased Ser-307 (human Ser-312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis.

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Role of PRAS40 in Akt and mTOR signaling in health and disease

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00660.2011 ◽

2012 ◽

Vol 302 (12) ◽

pp. E1453-E1460 ◽

Cited By ~ 92

Author(s):

Claudia Wiza ◽

Emmani B. M. Nascimento ◽

D. Margriet Ouwens

Keyword(s):

Mammalian Target Of Rapamycin ◽

Mtor Signaling ◽

Cellular Growth ◽

S6 Kinase ◽

Binding Partners ◽

Health And Disease ◽

Mtor Complex 1 ◽

Mtor Activity

The proline-rich Akt substrate of 40 kDa (PRAS40) acts at the intersection of the Akt- and mammalian target of rapamycin (mTOR)-mediated signaling pathways. The protein kinase mTOR is the catalytic subunit of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTORC2, that link energy and nutrients to the regulation of cellular growth and energy metabolism. Activation of mTOR in response to nutrients and growth factors results in the phosphorylation of numerous substrates, including the phosphorylations of S6 kinase by mTORC1 and Akt by mTORC2. Alterations in Akt and mTOR activity have been linked to the progression of multiple diseases such as cancer and type 2 diabetes. Although PRAS40 was first reported as substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity. Adding to the complexity is that gene silencing studies indicate that PRAS40 is also necessary for the activity of the mTORC1 complex. This review summarizes the regulation and potential function(s) of PRAS40 in the complex Akt- and mTOR-signaling network in health and disease.

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mTOR-targeted cancer therapy: great target but disappointing clinical outcomes, why?

Frontiers of Medicine ◽

10.1007/s11684-020-0812-7 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shi-Yong Sun

Keyword(s):

Cancer Therapy ◽

Kinase Inhibitors ◽

Mammalian Target Of Rapamycin ◽

Mtor Inhibitors ◽

Targeted Cancer Therapy ◽

Biological Functions ◽

New Findings ◽

Survival Signaling ◽

Mtor Complex 1

Abstract The mammalian target of rapamycin (mTOR) critically regulates several essential biological functions, such as cell growth, metabolism, survival, and immune response by forming two important complexes, namely, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTOR signaling is often dysregulated in cancers and has been considered an attractive cancer therapeutic target. Great efforts have been made to develop efficacious mTOR inhibitors, particularly mTOR kinase inhibitors, which suppress mTORC1 and mTORC2; however, major success has not been achieved. With the strong scientific rationale, the intriguing question is why cancers are insensitive or not responsive to mTOR-targeted cancer therapy in clinics. Beyond early findings on induced activation of PI3K/Akt, MEK/ERK, and Mnk/eIF4E survival signaling pathways that compromise the efficacy of rapalog-based cancer therapy, recent findings on the essential role of GSK3 in mediating cancer cell response to mTOR inhibitors and mTORC1 inhibition-induced upregulation of PD-L1 in cancer cells may provide some explanations. These new findings may also offer us the opportunity to rationally utilize mTOR inhibitors in cancer therapy. Further elucidation of the biology of complicated mTOR networks may bring us the hope to develop effective therapeutic strategies with mTOR inhibitors against cancer.

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Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1420477112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3505-3510 ◽

Cited By ~ 1

Author(s):

Yongqiang Gou ◽

Hyewon Byun ◽

Adam E. Zook ◽

Gurvani B. Singh ◽

Andrea K. Nash ◽

...

Keyword(s):

Stress Responses ◽

Mammalian Cells ◽

Lentiviral Vector ◽

Mammalian Target Of Rapamycin ◽

Inducible Promoter ◽

Retroviral Vectors ◽

Signaling Mechanism ◽

Protein Levels ◽

Vector Sequences ◽

Standard Plasmid

Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.

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mTOR, p70S6K, AKT, and ERK1/2 levels predict sensitivity to mTOR and PI3K/mTOR inhibitors in human bronchial carcinoids

Endocrine Related Cancer ◽

10.1530/erc-13-0042 ◽

2013 ◽

Vol 20 (4) ◽

pp. 463-475 ◽

Cited By ~ 40

Author(s):

Teresa Gagliano ◽

Mariaenrica Bellio ◽

Erica Gentilin ◽

Daniela Molè ◽

Federico Tagliati ◽

...

Keyword(s):

Cell Viability ◽

Cell Lines ◽

Mtor Inhibitor ◽

Primary Cultures ◽

Mammalian Target Of Rapamycin ◽

Mtor Inhibitors ◽

Response To Treatment ◽

Akt Activation ◽

Protein Levels ◽

Bronchial Carcinoids

Bronchial carcinoids (BCs) are rare neuroendocrine tumors that are still orphans of medical treatment. Human BC primary cultures may display resistance to everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), in terms of cell viability reduction. Our aim was to assess whether the novel dual phosphatidylinositol 3-kinase (PI3K)/mTOR inhibitor NVP-BEZ235 is effective in everolimus-resistant human BC tissues and cell lines. In addition, we searched for possible markers of the efficacy of mTOR inhibitors that may help in identifying the patients who may benefit from treatment with mTOR inhibitors, sparing them from ineffective therapy. We found that NVP-BEZ235 is twice as potent as everolimus in reducing cell viability and activating apoptosis in human BC tissues that display sensitivity to mTOR inhibitors, but is not effective in everolimus-resistant BC tissues and cell lines that bypass cyclin D1 downregulation and escape G0/G1 blockade. Rebound AKT activation was not observed in response to treatment with either mTOR inhibitor in the ‘resistant’ BC cells. In addition to total mTOR levels, putative markers of the sensitivity of BCs to mTOR inhibitors are represented by AKT, p70S6K (RPS6KB2), and ERK1/2 (MAPK3/1) protein levels. Finally, we validated these markers in an independent BC group. These data indicate that the dual PI3K/mTOR inhibitor NVP-BEZ235 is more potent than everolimus in reducing the proliferation of human BC cells. ‘Resistant’ cells display lower levels of mTOR, p70S6K, AKT, and ERK1/2, indicating that these proteins may be useful as predictive markers of resistance to mTOR and PI3K/mTOR inhibitors in human BCs.

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A20 Promotes Ripoptosome Formation and TNF-Induced Apoptosis via cIAPs Regulation and NIK Stabilization in Keratinocytes

Cells ◽

10.3390/cells9020351 ◽

2020 ◽

Vol 9 (2) ◽

pp. 351 ◽

Cited By ~ 2

Author(s):

Maria Feoktistova ◽

Roman Makarov ◽

Sihem Brenji ◽

Anne T. Schneider ◽

Guido J. Hooiveld ◽

...

Keyword(s):

Cell Death ◽

Immune Responses ◽

Skin Diseases ◽

Signaling Mechanism ◽

Protein Levels ◽

Genome Wide ◽

Multiple Levels ◽

Subsequent Activation ◽

Induced Apoptosis

The ubiquitin-editing protein A20 (TNFAIP3) is a known key player in the regulation of immune responses in many organs. Genome-wide associated studies (GWASs) have linked A20 with a number of inflammatory and autoimmune disorders, including psoriasis. Here, we identified a previously unrecognized role of A20 as a pro-apoptotic factor in TNF-induced cell death in keratinocytes. This function of A20 is mediated via the NF-κB-dependent alteration of cIAP1/2 expression. The changes in cIAP1/2 protein levels promote NIK stabilization and subsequent activation of noncanonical NF-κB signaling. Upregulation of TRAF1 expression triggered by the noncanonical NF-κB signaling further enhances the NIK stabilization in an autocrine manner. Finally, stabilized NIK promotes the formation of the ripoptosome and the execution of cell death. Thus, our data demonstrate that A20 controls the execution of TNF-induced cell death on multiple levels in keratinocytes. This signaling mechanism might have important implications for the development of new therapeutic strategies for the treatment of A20-associated skin diseases.

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Requirement for PINCH in Skeletal Myoblast Differentiation

10.21203/rs.3.rs-560801/v1 ◽

2021 ◽

Author(s):

Siyi Xie ◽

Chushan Fang ◽

Yujie Gao ◽

Jie Yan ◽

Lina Luo ◽

...

Keyword(s):

Skeletal Muscle ◽

Skeletal Muscles ◽

Molecular Mechanisms ◽

Myogenic Differentiation ◽

Critical Role ◽

The Body ◽

Myoblast Differentiation ◽

Skeletal Myogenesis ◽

Congenital Myopathies

Abstract Background: Skeletal muscle is composed of bundles of myofibers ensheathed by extracellular matrix networks. Malformation of skeletal muscle during embryonic development results in congenital myopathies. Disease mechanisms of congenital myopathies remain unclear. PINCH, an adaptor of focal adhesion complex, plays essential roles in multiple cellular processes and organogenesis. Elucidation of the molecular mechanisms underlying skeletal myogenesis will offer new insights into pathogenesis of myopathies.Methods: We generated muscle-specific PINCH knock-out mice to study the functional role of PINCH in skeletal myogenesis. Histologic and Transmission Electron Microscopy analysis demonstrated that Impaired myogenic differentiation and maturation in mice with PINCH1 being ablated in skeletal muscle progenitors, and Ablation of PINCH1 and PINCH2 resulted in reduced size of muscle fibers and impaired multinucleation; Cell culture and immunostaining showed that defects in myoblast fusion and cytoskeleton assembly in PINCH double mutant mice; Western blotting showed that defects in expression of cytoskeleton proteins and proteins involved in myogenesis in DMUT skeletal muscles.Results: Double ablation of PINCH1 and PINCH2 resulted in early postnatal lethality with reduced size of skeletal muscles and detachment of diaphragm muscles from the body wall. Myofibers of PINCH mutant myofibers failed to undergo multinucleation and exhibited disrupted sarcomere structures. The mutant myoblasts in culture were able to adhere to newly formed myotubes, but impeded in cell fusion and subsequent sarcomere genesis and cytoskeleton organization. Consistent with this, expression of integrin β1 and some cytoskeleton proteins, and phosphorylation of ERK and AKT were significantly reduced in PINCH mutants. Expression of MRF4, the most highly expressed myogenic factor at late stages of myogenesis, was abolished in PINCH mutants, that could contribute to observed phenotypes. In addition, mice with PINCH1 being ablated in myogenic progenitors exhibited only mild centronuclear myopathic changes, suggesting a compensatory role of PINCH2 in myogenic differentiation, indicating a critical role of PINCH proteins in myogenic differentiation.Conclusion: Our results demonstrated an essential role of PINCH in skeletal myogenic differentiation.

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SIRT5 regulates autophagy and apoptosis in gastric cancer cells

Journal of International Medical Research ◽

10.1177/0300060520986355 ◽

2021 ◽

Vol 49 (2) ◽

pp. 030006052098635

Author(s):

Wen Gu ◽

Qinyi Qian ◽

Yinkai Xu ◽

Xiaolan Xu ◽

Liping Zhang ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Cells ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Mammalian Target Of Rapamycin ◽

Gastric Cancer Cells ◽

Protein Levels ◽

Amp Activated Protein Kinase ◽

Related Proteins

Objective Accumulating evidence illustrates that sirtuins (SIRTs) regulate autophagy and apoptosis in cancer cells; however, the role of SIRT5 in gastric cancer (GC) cells remains unknown. In this study, we examined the role of SIRT5 in GC cells. Methods We detected SIRT5 protein levels in freshly collected samples from patients with GC. Next, we studied the function of SIRT5 in autophagy. Furthermore, the signaling pathway through which SIRT5 enhanced autophagy in GC cells was detected. In addition, we established a GC cell apoptosis model to analyze the role of SIRT5 in apoptosis. Results SIRT5 expression was downregulated in GC tissues. We discovered that SIRT5 promoted autophagy in GC cells. We demonstrated that SIRT5 enhanced autophagy in GC cells via the AMP-activated protein kinase–mammalian target of rapamycin signaling pathway. In addition, SIRT5 was degraded during apoptosis in GC cells. Meanwhile, we observed that calpains and caspase-related proteins were associated with SIRT5-related GC cell apoptosis. Conclusions SIRT5 is a crucial regulator of autophagy and apoptosis in GC cell lines that can maintain the balance of autophagy and apoptosis.

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LKB1 negatively regulates AKT1 signaling via DBC1 and TRB3

10.1101/691402 ◽

2019 ◽

Author(s):

Zarka Sarwar ◽

Sameer Bhat ◽

Qaaifah Gillani ◽

Irfana Reshi ◽

Misbah Un Nisa ◽

...

Keyword(s):

Cell Proliferation ◽

Critical Role ◽

T Antigen ◽

Cellular Functions ◽

Signaling Mechanism ◽

Protein Levels ◽

Survival Pathways ◽

Small T Antigen ◽

Insight Into

AbstractDBC1 plays a critical role in various cellular functions notably cell proliferation, transcription, histone modification and adipogenesis. Current reports about the role of DBC1 in tumorigenesis are paradoxical and designate DBC1 both as a tumor suppressor or an oncogene. Here, using small T antigen of polyoma virus (PyST) as a tool, we have delineated a signaling mechanism that connects LKB1 to AKT1 via DBC1. We report that PyST associates with DBC1 and leads to its down-regulation. Our results also show that PyST expression promotes LKB1 activation which in turn leads to in the downregulation of DBC1 protein. Absence of DBC1 results in transcriptional upregulation and consequently enhanced protein levels of TRB3. TRB3 sequesters AKT1, and consequently the phosphorylation and activity of AKT1 is compromised. This ultimately results in inactivation of pro-survival pathways triggered via AKT1 signaling. Our studies thus provide an insight into a signaling pathway that connects LKB1, DBC1, TRB3 and AKT1.

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Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

International Journal of Molecular Sciences ◽

10.3390/ijms22168536 ◽

2021 ◽

Vol 22 (16) ◽

pp. 8536

Author(s):

Lakmini Kumari Senavirathna ◽

Yurong Liang ◽

Chaoqun Huang ◽

Xiaoyun Yang ◽

Gayan Bamunuarachchi ◽

...

Keyword(s):

Cell Proliferation ◽

Noncoding Rna ◽

Mammalian Target Of Rapamycin ◽

Lung Fibroblasts ◽

Mrna Levels ◽

Fibroblast Proliferation ◽

Protein Levels ◽

Key Characteristics ◽

Ribosomal S6 Kinase

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

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Regulatory effects of TLR2 on megakaryocytic cell function

Blood ◽

10.1182/blood-2010-09-304949 ◽

2011 ◽

Vol 117 (22) ◽

pp. 5963-5974 ◽

Cited By ~ 60

Author(s):

Lea M. Beaulieu ◽

Elaine Lin ◽

Kristine M. Morin ◽

Kahraman Tanriverdi ◽

Jane E. Freedman

Keyword(s):

Cell Function ◽

Mammalian Target Of Rapamycin ◽

Matrix Proteins ◽

Protein Levels ◽

Erk Mapk ◽

Normal Platelet ◽

Immune Mediated ◽

Regulatory Effects ◽

Specific Ligand

Abstract TLR2, a functional, inflammatory-related receptor, is known to be expressed on megakaryocytes and platelets and to lead to infection and immune-mediated activation of platelets; however, the role of this receptor in megakaryocytes is not understood. Using Meg-01 cells and mouse megakaryocytes, we found that NFκB, ERK-MAPK, and PI3K/Akt pathways, known downstream pathways of TLRs, are activated by Pam3CSK4, a TLR2-specific ligand. In addition, transcription factors associated with megakaryocyte maturation, GATA-1, NF-E2, and mammalian target of rapamycin (mTOR), are all increased in the presence of Pam3CSK4. The effect of Pam3CSK4 on megakaryocyte maturation was verified by the increase in DNA content and adhesion to extracellular matrix proteins by TLR2-dependent stimulation. In addition, TLR2 stimulation resulted in an increase in reactive oxygen species (ROS) production. Gene expression and protein levels of GP1b, CD41, MCP-1, COX2, NFκB1, and TLR2 were up-regulated in megakaryocytes after TLR2 stimulation through NFκB, PI3K/Akt, and ERK-MAPK pathways. Treatment of wild-type mice with Pam3CSK4 resulted in a return to normal platelet levels and an increase in megakaryocyte maturation, which did not occur in the TLR2−/− mice. Therefore, inflammation, through TLR2, can increase maturation and modulate the phenotype of megakaryocytes, contributing to the interrelationship between inflammation and hemostasis.

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