Kruppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFβ/Smad3 signaling

Background: Kruppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGF beta have been shown to regulate each other's expression in non-myogenic cell types. Since MEF2D and TGF beta also fulfill crucial roles in skeletal myogenesis, we wanted to identify whether KLF6 functions in a myogenic context. Methods: KLF6 protein expression levels and promoter activity were analyzed using standard cellular and molecular techniques in cell culture. Results: We found that KLF6 and MEF2D are co-localized in the nuclei of mononucleated but not multinucleated myogenic cells and, that the MEF2 cis element is a key component of the KLF6 promoter region. In addition, TGF beta potently enhanced KLF6 protein levels and this effect was repressed by pharmacological inhibition of Smad3. Interestingly, pharmacological inhibition of MEK/ERK (1/2) signaling resulted in re-activation of the differentiation program in myoblasts treated with TGF beta, which is ordinarily repressed by TGF beta treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGF beta-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGF beta signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGF beta stimulation of myoblast proliferation was reduced in KLF6 depleted cells. Conclusions: Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGF beta with consequences for our understanding of muscle development and a variety of muscle pathologies.