Proteolytic processing of myostatin is auto-regulated during myogenesis

Abstract

Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-beta superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide.

Increased Secretion and Expression of Myostatin in Skeletal Muscle From Extremely Obese Women

“…Although the mature 26-kDa myostatin peptide was clearly increased in extremely obese cells, it was only sporadically detected (three of eight samples). The processing of the 52-kDa myostatin precursor protein produces an NH 2 -terminal, 40-kDa latency-associated peptide (LAP) and the biologically active 26-kDa COOH-terminal dimer (19). These findings are consistent with those of previous studies: that myostatin is rapidly processed and secreted from cultured muscle cells compared with mature muscle (19,22).…”

“…The processing of the 52-kDa myostatin precursor protein produces an NH 2 -terminal, 40-kDa latency-associated peptide (LAP) and the biologically active 26-kDa COOH-terminal dimer (19). These findings are consistent with those of previous studies: that myostatin is rapidly processed and secreted from cultured muscle cells compared with mature muscle (19,22). Because both the expression and secretion of myostatin was increased in cells derived from extremely obese human donors, we also studied its expression in skeletal muscle and plasma in a separate patient cohort.…”

“…5, we show a 23% (P Ͻ 0.05) increase in myostatin 52-kDa precursor protein and a 35% increase in the 26-kDa dimer, respectively (P Ͻ 0.05), in extremely obese muscle and plasma relative to lean samples. Unlike myotubes, the 52-kDa precursor protein has been shown to be the most abundant immunoreactive species in mature skeletal muscle (19,21). Linear regression analysis of myostatin protein levels relative to HOMA (Fig.…”

“…Myostatin protein levels in conditioned media, muscle cells, whole skeletal muscle, and plasma were verified using Western immunoblot. To collect secreted proteins for quantifying myostatin levels, cells (9-day myotubes) were washed five times with PBS and incubated with serum-free Optimem (Invitrogen) for an additional 24 h as described previously (18,19). Optimem medium contains growth factors that promote cell survival with extended incubations and is specifically designed for the characterization of secreted proteins.…”

“…Protein concentration was determined using the Bio-Rad protein assay dye reagent, following the manufacturer's instructions (Bio-Rad, Hercules, CA). Twenty micrograms cellular protein (19,21) was then separated by SDS-PAGE and transferred to Immobilon-P PVDF Membrane (Millipore). Membranes were stained with Ponceau S to confirm equal loading and transfer efficiency.…”