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AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

ABSTRACT: BACKGROUND: Follistatin (FST) has been shown to bind to some TGF-? family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector. RESULTS: Western blot results showed that AAV induced the expression of FST protein in transduced OPM cells. Real-time quantitative PCR results indicated that over-expression of FST resulted in a dramatic increase in Akt I and CDK2 expression and a decrease in p21 expression. Moreover, cell cycle analysis confirmed that FST down-regulated p21, a CDK inhibitor, and increased the level of CDK2 expression in OPM cells. Hence, follistatin positively regulated the G1 to S progression. Our results showed that FST induced proliferation through a down-regulation of p21, as only the p21 expression level was down-regulated as a result of FST over-expression in myoblasts, whereas no change was observed in the level of p57 expression. CONCLUSIONS: These results expanded our understanding of the regulation mechanism of FST in ovine primary myoblasts. Our results provide the first evidence that the AAV viral system can be used for gene transfer in ovine myoblast cells. Moreover, the results showed that an AAV vector can successfully induce the expression of FST in OPM cells in vitro. These findings demonstrated that FST over-expression induces proliferation through a down-regulation of the p21 gene under proliferating conditions.

SUBMITTER: Nazari M

PROVIDER: S-EPMC4210504 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

Nazari Mahmood M Salabi Fatemeh F Zhang Li L Zhao Fuping F Wei Caihong C Du Lixin L

BMC biotechnology 20141021

<h4>Background</h4>Follistatin (FST) has been shown to bind to some TGF-β family members and can function as a potent myostatin (MSTN) antagonist. Recent studies have revealed that over-expression of FST by adeno-associated viruses increases muscle growth in mice, humans and nonhuman primates. In the present study, to determine the effect of FST on ovine primary myoblast (OPM) proliferation, FST was over-expressed using an adeno-associated virus serotype 2 (AAV 2) vector.<h4>Results</h4>Western ...[more]

PMID: 25330993

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Project description:Background: Follistatin (FS) is an activin-binding glycoprotein, known for its antagonistic action on the TGF-β super-family of cytokines. It has ability to prevent muscle atrophy and to stimulate muscle growth through binding of myostatin (GDF8). FS does not cause these changes only by inhibition of GDF8 but this mechanism is still not well elucidated. In order to identify GDF8-dependent and GDF8-independent action of FS, we performed comprehensive transcriptomic analysis of differentiating C2C12 mouse myoblasts. Results: To evaluate influence of FS-288 (200ng/ml) on differentiating C2C12 myoblasts was used immunofluorescence analysis of MyHC protein expression in the culture and counting of the number and type of myotubes. Differentiating myoblasts treated with FS showed increase in number and size of myotubes. Microarray studies performed by us identified 30 genes oppositely regulated by FS and GDF8. Analyses of the transcriptomic profiles showed that FS has highly significant influence, associated with GDF8 binding, on the regulation of cell cycle, transport processes, cell communication, cell adhesion, cell motion, developmental processes and binding functions. The signalling pathways that may play a primary role in the myogenesis stimulation by FS are associated with TGF-β, Delta-Notch and Wnt signalling pathways, integrin and actin cytoskeleton signalling and adipogenesis regulation. Changes in expression of Hgf, Sort1, Trdn, Stmn2, Hdac1 genes, significantly involved in the process of myoblasts differentiation were validated with Real-Time PCR method. Conclusions: Our results implicate which aspects of transcriptional regulation of myogenesis process by FS could have significant and protective influence. We have shown that FS activity is associated not only with GDF8 binding but also with inhibited expression of genes involved in TGF-β signalling and by regulating of genes associated with vesicle transport apoptosis signalling pathway, inflammatory pathways and adipogenesis expression. Our analysis showed that follistatin GDF8-independent on differentiating myotubes involves regulation of localization processes, cell-matrix adhesion, mRNA processing, apoptosis, fatty acid metabolic processes, SNAP receptor activity functions and partial regulation of transcription processes. These results give a new and complete image of the follistatin role in the muscle cell differentiation and indicate target genes for FS which in the future may be a good therapeutic approach.

Dataset Information

AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

Publications

AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation.

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