BTB-Kelch protein Krp1 regulates proliferation and differentiation of myoblasts

Abstract

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.

Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

“…In proliferating C2C12 myoblasts SARCOSIN was already expressed at considerable levels, which is in contrast to results from Paxton et al, (2011) who observed absence of SARCOSIN (Krp1) expression in proliferating C2 cells. This could indicate that in our experiments some cells were already differentiating in the growth medium.…”

“…A similar function for SARCOSIN in cultured cardiomyocytes has been reported (Greenberg et al, 2008). In differentiating C2C12 cells we observed SARCOSIN distributed rather evenly throughout the cytoplasm which indeed has been described very recently (Paxton et al, 2011), suggesting different functions for SARCOSIN in developing and adult muscle cells.…”

“…Very recently, it was described that SARCOSIN expression is upregulated in C2 cells differentiating to myoblastst. Intriguingly, both knockdown and overexpression of SARCOSIN in these cells inhibited myoblast differentiation (Paxton et al, 2011).…”

p38α MAPK Regulates Adult Muscle Stem Cell Fate by Restricting Progenitor Proliferation During Postnatal Growth and Repair

“…Immunofluorescence on cultured satellite cells and ex vivo muscle was performed as previously described [39,40].…”

“…Western blotting was performed as previously described [40], with the following antibodies (in addition to those above): p38 (Cell Signaling Technology), phospho-p38 (Cell Signaling Technology), p38a (R&D Systems, Abingdon, UK, http://www.rndsy stems.com), and GAPDH (Abcam, Cambridge, UK, http:// www.abcam.com).…”