Abstract
Muscle formation is controlled by a number of key myogenic transcriptional regulators that govern stage-specific gene expression programs and act as terminal effectors of intracellular signaling pathways. To date, the role of phosphatases in the signaling cascades instructing muscle development remains poorly understood. Here, we show that a specific PP2A-B55δ holoenzyme is necessary for skeletal myogenesis. The primary role of PP2A-B55δ is to dephosphorylate histone deacetylase 4 (HDAC4) following myocyte differentiation and ensure repression of Myocyte enhancer factor 2D (MEF2D)-dependent gene expression programs during myogenic fusion. As a crucial HDAC4/MEF2D target gene that governs myocyte fusion, we identify ArgBP2, an upstream inhibitor of Abl, which itself is a repressor of CrkII signaling. Consequently, cells lacking PP2A-B55δ show upregulation of ArgBP2 and hyperactivation of CrkII downstream effectors, including Rac1 and FAK, precluding cytoskeletal and membrane rearrangements associated with myoblast fusion. Both in vitro and in zebrafish, loss-of-function of PP2A-B55δ severely impairs fusion of myocytes and formation of multinucleated muscle fibers, without affecting myoblast differentiation. Taken together, our results establish PP2A-B55δ as the first protein phosphatase to be involved in myoblast fusion and suggest that reversible phosphorylation of HDAC4 may coordinate differentiation and fusion events during myogenesis.
Highlights
Skeletal myogenesis, the process by which contractile muscle fibers are formed begins with specification of precursor cells towards the myoblastic lineage, followed by differentiation into postmitotic myocytes
Developmental progression in the skeletal muscle lineage is orchestrated by a group of basic helix-loop-helix transcription factors collectively referred to as myogenic regulatory factors (MRFs)
phosphatase 2A (PP2A) activity is necessary for myogenesis To explore a potential role for PP2A in myogenesis, C2C12 myoblasts were cultured in differentiation medium (DM) with and without low concentration (2 nM) of the PP2A inhibitor Okadaic Acid (OA) (IC50 = 0.1 nM)[23]
Summary
The process by which contractile muscle fibers are formed begins with specification of precursor cells towards the myoblastic lineage, followed by differentiation into postmitotic myocytes. The ability of MRFs to drive the myogenic program relies on their collaboration with non muscle-specific factors, such as MEF2 family members and chromatin-modifying enzymes[2]. Whereas MEF2 has acquired a central role in multiple aspects of the skeletal muscle differentiation program[3], its potential function during myoblast fusion remains essentially unaddressed. MEF2 transcriptional activity is negatively regulated by its interaction with class IIa histone deacetylases (HDACs), including HDAC4, −5, −7, and −94. Association with class IIa HDACs decreases MEF2 DNA-binding and leads to Official journal of the Cell Death Differentiation Association
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