Krüppel-like factor 6 (KLF6) promotes cell proliferation in skeletal myoblasts in response to TGFß/Smad3 signaling.

ABSTRACT

BACKGROUND: Krüppel-like factor 6 (KLF6) has been recently identified as a MEF2D target gene involved in neuronal cell survival. In addition, KLF6 and TGFß have been shown to regulate each other's expression in non-myogenic cell types. Since MEF2D and TGFß 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, TGFß 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 TGFß, which is ordinarily repressed by TGFß treatment. Conversely, MEK/ERK (1/2) inhibition had no effect on TGFß-induced KLF6 expression whereas Smad3 inhibition negated this effect, together supporting the existence of two separable arms of TGFß signaling in myogenic cells. Loss of function analysis using siRNA-mediated KLF6 depletion resulted in enhanced myogenic differentiation whereas TGFß stimulation of myoblast proliferation was reduced in KLF6 depleted cells. CONCLUSIONS: Collectively these data implicate KLF6 in myoblast proliferation and survival in response to TGFß with consequences for our understanding of muscle development and a variety of muscle pathologies.