Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C

Abstract

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage-specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T 80 by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation-deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain-of-function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In lossof-function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C-dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

Defined three-dimensional culture conditions mediate efficient induction of definitive endoderm lineage from human umbilical cord Wharton’s jelly mesenchymal stem cells

“…Both exendin-4 and EGF direct the DE differentiation toward primitive gut tube endoderm, and enhance proinsulin biosynthesis and expansion of pancreatic progenitor at later stages of hESC and hiPSC differentiation [18]. Recently, Nekoei et al [73] reported the generation of insulin-producing cells from WJ-MSC cultured in a serum-dependent differentiation containing exendin-4 [24], yet the efficiency of insulin induction was relatively low most likely due to absence of the stepwise differentiation process. Similarly, Kadam et al [74] reported a low yield of pancreatic β-cell-like clusters generated from monolayer cultures of placenta-derived MSCs.…”

“…Remarkably, WJ-MSCs generated stable spheroidal bodies upon culturing in AggreWell plates, which showed an improved differentiation capacity into DE lineage. In both ESCs and iPSCs this technique has been proven to efficiently mediate the differentiation toward different developmental lineages [8, 24, 25], but it has not been tested in WJ-MSCs. Notably, differentiating WJ-MSCs in monolayer cultures toward insulin-producing cells resulted in the generation of cell aggregates; while remaining attached to the plate surface, cells within the cluster-like morphology showed a significant differentiation property [73, 76].…”

“…First-strand cDNA was synthesized from 50–100 ng RNA by reverse transcription using the QuantiTect Reverse Transcription Kit (Qiagen Inc., USA). Quantitative real-time PCR (qRT-PCR) reactions were performed as described previously [24, 25]. Primer pairs with equivalent efficiencies (Additional file 1: Table S2) were selected from Primer Bank [26] or were designed using primer3web (http://primer3.ut.ee/) [27] and primer-BLAST tools (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) [28].…”

Collagen scaffolds with or without the addition of RGD peptides support cardiomyogenesis after aggregation of mouse embryonic stem cells

“…Cardiomyocyte formation (EB formation): Cardiomyocyte formation was induced by growing the mES cells in suspension culture conditions, in the absence of LIF, to form embryoid bodies (EBs), as described previously (Kennedy et al 2009;Al Madhoun et al 2011). Cells growing in monolayer (termed day 0) were brought into single cell suspension by treating for 5 min with 1× trypsin-EDTA (Invitrogen), and re-suspending in complete media, without LIF.…”

The mammalian target of rapamycin signaling pathway regulates myocyte enhancer factor‐2C phosphorylation levels through integrin‐linked kinase in goat skeletal muscle satellite cells

“…A member of the MADS box transcription factor regulator family, MEF2 is a protein found in developing skeletal muscle myotubes and of which there are four members: MEF2A, MEF2B, MEF2C, and MEF2D (Potthoff et al, 2007;Angelelli et al, 2008;Al Madhoun et al, 2011). The DNA-binding site of MEF2 is a segment of the conservative DNA sequence CTA(A/T) 4 TAG, which exists widely in the regulatory region of muscle tissue-specific expression genes, such as that for muscle creatine kinase (MCK) (Black and Olson, 1998).…”