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1.
Cell Death Differ ; 27(8): 2344-2362, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32051546

RESUMO

Rab5 is a master regulator for endosome biogenesis and transport while its in vivo physiological function remains elusive. Here, we find that Rab5a is upregulated in several in vivo and in vitro myogenesis models. By generating myogenic Rab5a-deficient mice, we uncover the essential roles of Rab5a in regulating skeletal muscle regeneration. We further reveal that Rab5a promotes myoblast differentiation and directly interacts with insulin receptor substrate 1 (IRS1), an essential scaffold protein for propagating IGF signaling. Rab5a interacts with IRS1 in a GTP-dependent manner and this interaction is enhanced upon IGF-1 activation and myogenic differentiation. We subsequently identify that the arginine 207 and 222 of IRS1 and tyrosine 82, 89, and 90 of Rab5a are the critical amino acid residues for mediating the association. Mechanistically, Rab5a modulates IRS1 activation by coordinating the association between IRS1 and the IGF receptor (IGFR) and regulating the intracellular membrane targeting of IRS1. Both myogenesis-induced and IGF-evoked AKT-mTOR signaling are dependent on Rab5a. Myogenic deletion of Rab5a also reduces the activation of AKT-mTOR signaling during skeletal muscle regeneration. Taken together, our study uncovers the physiological function of Rab5a in regulating muscle regeneration and delineates the novel role of Rab5a as a critical switch controlling AKT-mTOR signaling by activating IRS1.


Assuntos
Diferenciação Celular , Proteínas Substratos do Receptor de Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/fisiologia , Mioblastos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/fisiologia , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células HEK293 , Membro Posterior/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Mioblastos/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Regulação para Cima/genética , Proteínas rab5 de Ligação ao GTP/genética
2.
Aging Cell ; 18(5): e13003, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31313490

RESUMO

Cellular senescence plays both beneficial and detrimental roles in embryonic development and tissue regeneration, while the underlying mechanism remains elusive. Recent studies disclosed the emerging roles of heat-shock proteins in regulating muscle regeneration and homeostasis. Here, we found that Hsp90ß, but not Hsp90α isoform, was significantly upregulated during muscle regeneration. RNA-seq analysis disclosed a transcriptional elevation of p21 in Hsp90ß-depleted myoblasts, which is due to the upregulation of p53. Moreover, knockdown of Hsp90ß in myoblasts resulted in p53-dependent cellular senescence. In contrast to the notion that Hsp90 interacts with and protects mutant p53 in cancer, Hsp90ß preferentially bound to wild-type p53 and modulated its degradation via a proteasome-dependent manner. Moreover, Hsp90ß interacted with MDM2, the chief E3 ligase of p53, to regulate the stability of p53. In line with these in vitro studies, the expression level of p53-p21 axis was negatively correlated with Hsp90ß in aged mice muscle. Consistently, administration of 17-AAG, a Hsp90 inhibitor under clinical trial, impaired muscle regeneration by enhancing injury-induced senescence in vivo. Taken together, our finding revealed a previously unappreciated role of Hsp90ß in regulating p53 stability to suppress senescence both in vitro and in vivo.


Assuntos
Senescência Celular , Proteínas de Choque Térmico HSP90/metabolismo , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSP90/química , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/química
3.
Mol Cell Biol ; 38(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30275345

RESUMO

The regenerative process of injured muscle is dependent on the fusion and differentiation of myoblasts derived from muscle stem cells. Hsp70 is important for maintaining skeletal muscle homeostasis and regeneration, but the precise cellular mechanism remains elusive. In this study, we found that Hsp70 was upregulated during myoblast differentiation. Depletion or inhibition of Hsp70/Hsc70 impaired myoblast differentiation. Importantly, overexpression of p38 mitogen-activated protein kinase α (p38MAPKα) but not AKT1 rescued the impairment of myogenic differentiation in Hsp70- or Hsc70-depleted myoblasts. Moreover, Hsp70 interacted with MK2, a substrate of p38MAPK, to regulate the stability of p38MAPK. Knockdown of Hsp70 also led to downregulation of both MK2 and p38MAPK in intact muscles and during cardiotoxin-induced muscle regeneration. Hsp70 bound MK2 to regulate MK2-p38MAPK interaction in myoblasts. We subsequently identified the essential regions required for Hsp70-MK2 interaction. Functional analyses showed that MK2 is essential for both myoblast differentiation and skeletal muscle regeneration. Taken together, our findings reveal a novel role of Hsp70 in regulating myoblast differentiation by interacting with MK2 to stabilize p38MAPK.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Regeneração/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Mioblastos/fisiologia , Regulação para Cima/fisiologia
4.
Biomed Res Int ; 2018: 5238760, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29687002

RESUMO

TMZ resistance remains one of the main reasons why treatment of glioblastoma (GBM) fails. In order to investigate the underlying proteins and pathways associated with TMZ resistance, we conducted a cytoplasmic proteome research of U87 cells treated with TMZ for 1 week, followed by differentially expressed proteins (DEPs) screening, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and validation of key candidate proteins in TCGA dataset. A total of 161 DEPs including 65 upregulated proteins and 96 downregulated proteins were identified. Upregulated DEPs were mainly related to regulation in actin cytoskeleton, focal adhesion, and phagosome and PI3K-AKT signaling pathways which were consistent with our previous studies. Further, the most significant module consisted of 28 downregulated proteins that were filtered from the PPI network, and 9 proteins (DHX9, HNRNPR, RPL3, HNRNPA3, SF1, DDX5, EIF5B, BTF3, and RPL8) among them were identified as the key candidate proteins, which were significantly associated with prognosis of GBM patients and mainly involved in ribosome and spliceosome pathway. Taking the above into consideration, we firstly identified candidate proteins and pathways associated with TMZ resistance in GBM using proteomics and bioinformatic analysis, and these proteins could be potential biomarkers for prevention or prediction of TMZ resistance in the future.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Biologia Computacional/métodos , Dacarbazina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteômica/métodos , Proteína Ribossômica L3 , Transdução de Sinais/efeitos dos fármacos , Temozolomida , Regulação para Cima/efeitos dos fármacos
5.
Stem Cells ; 36(4): 527-539, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29315990

RESUMO

Tendon repair is a clinical challenge because of the limited understanding on tenogenesis. The synthesis of type I collagen (Collagen I) and other extracellular matrix are essential for tendon differentiation and homeostasis. Current studies on tenogenesis focused mostly on the tenogenic transcriptional factors while the signaling controlling tenogenesis on translational level remains largely unknown. Here, we showed that mechanistic target of rapamycin (mTOR) signaling was activated by protenogenic growth factor, transforming growth factors beta1, and insulin-like growth factor-I. The expression of mTOR was upregulated during tenogenesis of mesenchymal stem cells (MSCs). Moreover, mTOR was downregulated in human tendinopathy tissues and was inactivated upon statin treatment. Both inhibition and depletion of AKT or mTOR significantly reduced type I collagen production and impaired tenogenesis of MSCs. Tendon specific-ablation of mTOR resulted in tendon defect and reduction of Collagen I. However, there is no evident downregulation of tendon associated collagens at the transcription level. Our study demonstrated that AKT-mTOR axis is a key mediator of tendon differentiation and provided a novel therapeutic target for tendinopathy and tendon injuries. Stem Cells 2018;36:527-539.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Tendões/metabolismo , Animais , Células-Tronco Mesenquimais/citologia , Camundongos , Tendões/citologia , Fator de Crescimento Transformador beta1/metabolismo
6.
Nan Fang Yi Ke Da Xue Xue Bao ; 37(9): 1183-1189, 2017 Sep 20.
Artigo em Chinês | MEDLINE | ID: mdl-28951359

RESUMO

OBJECTIVE: To investigate the role of microtubule-actin crosslinking factor 1 (MACF1) in the response of glioma cells to temozolomide (TMZ). METHODS: TMZ was applied to a human gliomablastoma cell line (U87) and changes in the protein expression and cellular localization were determined with Western blot, RT-PCR, and immunofluorescence. The responses of the cells with MACF1 expression knockdown by RNA interference to TMZ were assessed. TMZ-induced effects on MACF1 expression were also assessed by immunohistochemistry in a nude mouse model bearing human glioblastoma xenografts. RESULTS: TMZ resulted in significantly increased MACF1 expression (by about 2 folds) and changes in its localization in the gliomablastoma cells both in vitro and in vivo (P<0.01). Knockdown of MACF1 reduced the proliferation (by 45%) of human glioma cell lines treated with TMZ (P<0.01). TMZ-induced changes in MACF1 expression was accompanied by cytoskeletal rearrangement. CONCLUSION: MACF1 may be a potential therapeutic target for glioblastoma.

7.
Am J Cancer Res ; 5(5): 1741-50, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26175942

RESUMO

Metabolomic research has revealed that metabolites play an important role in prostate cancer development and progression. Previous studies have suggested that prostate cancer cell proliferation is induced by advanced glycation end products (AGEs) exposure, but the mechanism of this induction remains unknown. This study investigated the molecular mechanisms underlying the proliferative response of prostate cancer cell to the interaction of AGEs and the receptor for advanced glycation end products (RAGE). To investigate this mechanism, we used Western blotting to evaluate the responses of the retinoblastoma (Rb), p-Rb and PI3K/Akt pathway to AGEs stimulation. We also examined the effect of knocking down Rb and blocking the PI3K/Akt pathway on AGEs induced PC-3 cell proliferation. Our results indicated that AGE-RAGE interaction enhanced Rb phosphorylation and subsequently decreased total Rb levels. Bioinformatics analysis further indicated a negative correlation between RAGE and RB1 expression in prostate cancer tissue. Furthermore, we observed that AGEs stimulation activated the PI3K/Akt signaling pathway and that blocking PI3K/Akt signaling abrogated AGEs-induced cell proliferation. We report, for the first time, that AGE-RAGE interaction enhances prostate cancer cell proliferation by phosphorylation of Rb via the PI3K/Akt signaling pathway.

8.
Proteome Sci ; 12(1): 13, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24580839

RESUMO

OBJECTIVE: To identify the protein regulation profile of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced osteogenic differentiation in beagle bone marrow stem cells (BMSCs). METHODS: Beagle BMSCs were isolated and cultured with or without rhBMP-2. Two-dimensional gel electrophoresis was used to determine the differences in protein expression in rhBMP-2-induced and non-induced BMSCs. Real-time PCR and western blotting analyses were used to verify the expression patterns of selected proteins. RESULTS: After the induction, the osteogenic differentiation of beagle BMSCs was activated successfully. Nine and 11 proteins were found to be down- and up-regulated by rhBMP-2, respectively. The increase in Lim and SH3 domain protein 1(LASP1) and the decrease in ferritin were verified by real-time PCR and western blotting analyses. CONCLUSIONS: Among the 20 rhBMP-2-regulated factors, there is empirical evidence supporting the involvement of LASP1 and ferritin in osteogenic differentiation. LASP1 plays an important role in the regulation of the activity of the cytoskeleton, and ferritin is an important molecule in cellular iron homeostasis. Further studies focused on these 20 proteins will help elucidate the molecular mechanism(s) through which rhBMP-2 induces osteogenic differentiation of BMSCs.

9.
Huan Jing Ke Xue ; 29(2): 446-53, 2008 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-18613519

RESUMO

Evaluating the contribution of maize growth to soil organic carbon is important for the understanding of the relationship of farmland carbon balance and agriculture production. 4 times of 13C pulse-labelling were used to estimate the photosynthesized carbon distribution at different development stages (seedling, elongation, heading and grain-filling) in maize-soil system, and quantify the carbon inputs into each part of belowground in whole growth season. The result indicated that the 13C retained aboveground reached its maximum: 80.01% among net assimilated 13C at grain-filling stage labelling. For the 4 labelling stages, the 13C transferred into belowground is 43.24%, 46.46%, 30.30% and 19.99% respectively, and of the 13C input into belowground, 34.68%-77.56% was respired by rhizosphere, 16.63%-57.02% was remain in roots and 5.05%-8.30% was incorporated into soil organic carbon by rhizodeposition. During the whole growth season of maize, the photosynthesized carbon allocated to aboveground, roots, rhizosphere respiration and soil organic carbon was 62.39%, 17.88%, 17.07% and 2.67% of the net assimilated carbon. At elongation, heading and grain-filling stages, maize's rhizosphere respiration accounted for 67.07%, 63.31% and 28.82% of the total CO2 efflux from the soil respectively, during the same period rhizosphere priming effect led to 31.11%, 79.09% and 120.83% increase of decomposition of original soil organic carbon respectively. Based on the calculation of 18 t x hm(-2) dry matter of maize for farmland production and its C content is 42%, the total carbon transferred into belowground is 4.6 t x hm(-2), among which 2.1 t x hm(-2) was respired by rhizoshphere, 2.2 t x hm(-2) was retained in roots and 0.33 t x hm(-2) was incorporated into soil organic carbon.


Assuntos
Carbono/metabolismo , Fotossíntese/fisiologia , Raízes de Plantas/metabolismo , Solo/análise , Zea mays/metabolismo , Isótopos de Carbono , Compostos Orgânicos/metabolismo
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