Rab5a activates IRS1 to coordinate IGF-AKT-mTOR signaling and myoblast differentiation during muscle regeneration.

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.

Hsp90ß interacts with MDM2 to suppress p53-dependent senescence during skeletal muscle regeneration.

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.

Identification of Key Candidate Proteins and Pathways Associated with Temozolomide Resistance in Glioblastoma Based on Subcellular Proteomics and Bioinformatical Analysis.

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.

Activation of AKT-mTOR Signaling Directs Tenogenesis of Mesenchymal Stem Cells.

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.

[MACF1 knockdown in glioblastoma multiforme cells increases temozolomide-induced cytotoxicity].

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.

AGE/RAGE/Akt pathway contributes to prostate cancer cell proliferation by promoting Rb phosphorylation and degradation.

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.

Proteomic analysis on effectors involved in BMP-2-induced osteogenic differentiation of beagle bone marrow mesenchymal stem cells.

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.