ABSTRACT
BACKGROUND: Severe peripheral nerve injury leads to skeletal muscle atrophy and impaired limb function that is not sufficiently improved by existing treatments. Fibroblast growth factor 6 (FGF6) is involved in tissue regeneration and is dysregulated in denervated rat muscles. However, the way that FGF6 affects skeletal muscle repair after peripheral nerve injury has not been fully elucidated. METHODS: In this study, we investigated the role of FGF6 in the regeneration of denervated muscles using myoblast cells and an in vivo model of peripheral nerve injury. RESULTS: FGF6 promoted the viability and migration of C2C12 and primary myoblasts in a dose-dependent manner through FGFR1-mediated upregulation of cyclin D1. Low concentrations of FGF6 promoted myoblast differentiation through FGFR4-mediated activation of ERK1/2, which upregulated expression of MyHC, MyoD, and myogenin. FGFR-1, FGFR4, MyoD, and myogenin were not upregulated when FGF6 expression was inhibited in myoblasts by shRNA-mediated knockdown. Injection of FGF6 into denervated rat muscles enhanced the MyHC-IIb muscle fiber phenotype and prevented muscular atrophy. CONCLUSION: These findings indicate that FGF6 reduces skeletal muscle atrophy by relying on the ERK1/2 mechanism and enhances the conversion of slow muscle to fast muscle fibers, thereby promoting functional recovery of regenerated skeletal muscle after innervation.
Subject(s)
Fibroblast Growth Factor 6/genetics , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Muscle, Skeletal/metabolism , Peripheral Nerve Injuries/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Regeneration/genetics , Animals , Cell Differentiation , Cell Line , Cell Movement , Cell Proliferation , Cyclin D1/genetics , Cyclin D1/metabolism , Fibroblast Growth Factor 6/antagonists & inhibitors , Fibroblast Growth Factor 6/metabolism , Gene Expression Regulation , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Muscle Denervation/methods , Muscle, Skeletal/innervation , Muscle, Skeletal/pathology , MyoD Protein/genetics , MyoD Protein/metabolism , Myoblasts/metabolism , Myoblasts/pathology , Myogenin/genetics , Myogenin/metabolism , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Peripheral Nerve Injuries/metabolism , Peripheral Nerve Injuries/pathology , Primary Cell Culture , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Receptor, Fibroblast Growth Factor, Type 4/genetics , Receptor, Fibroblast Growth Factor, Type 4/metabolism , Sciatic Nerve/injuriesABSTRACT
The sirtuin proteins are broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes including DNA recombination and repair, transcriptional silencing, longevity, apoptosis, axonal protection, insulin signaling, and fat mobilization. Because of these associations, the identification of small molecule sirtuin modulators has been of significant interest. Here we report on high throughput screening against the yeast sirtuin, Hst2, leading to the identification of four unique inhibitor scaffolds that also inhibit the human sirtuins, SIRT1-3, and are able to inhibit telomeric silencing of yeast Sir2 in vivo. The identified inhibitor scaffolds range in potency from IC(50) values of 6.5-130 microM against Hst2. Each of the inhibitor scaffolds binds reversibly to the enzyme, and kinetic analysis reveals that each of the inhibitors is non-competitive with respect to both acetyl-lysine and NAD(+) binding. Limited SAR analysis of the scaffolds also identifies which functional groups may be important for inhibition. These sirtuin inhibitors are low molecular weight and well-suited for lead molecule optimization, making them useful chemical probes to study the mechanism and biological roles of sirtuins and potential starting points for optimization into therapeutics.
