Background: MEF2C is essential for vascular smooth muscle development, yet the signaling pathways that regulate its function in this cell type remain largely unknown. Results: We identify a novel regulator of MEF2C in vascular smooth muscle, called CPI-17. Conclusion: Our data identify a genetic pathway involving CPI-17, MEF2C, and myocardin. Significance: These findings have important ramifications during vascular development and for stem cell programming.
e Canonical Wnt/-catenin signaling plays a major role in various biological contexts, such as embryonic development, cell proliferation, and cancer progression. Previously, a connection between p38 mitogen-activated protein kinase (MAPK) signaling and Wnt-mediated activation of -catenin was implied but poorly understood. In the present study, we investigated potential cross talk between p38 MAPK and Wnt/-catenin signaling. Here we show that a loss of p38 MAPK ␣/ function reduces -catenin nuclear accumulation in Wnt3a-stimulated primary vascular smooth muscle cells (VSMCs). Conversely, active p38 MAPK signaling increases -catenin nuclear localization and target gene activity in multiple cell types. Furthermore, the effect of p38 MAPK ␣/ on -catenin activity is mediated through phosphorylation of a key p38 MAPK target, myocyte enhancer factor 2 (MEF2). Here we report a p38 MAPK-mediated, phosphorylation-dependent interaction between MEF2 and -catenin in multiple cell types and primary VSMCs that results in (i) increased -catenin nuclear retention, which is reversed by small interfering RNA (siRNA)-mediated MEF2 gene silencing; (ii) increased activation of MEF2 and Wnt/-catenin target genes; and (iii) increased Wnt-stimulated cell proliferation. These observations provide mechanistic insight into a fundamental level of cross talk between p38 MAPK/MEF2 signaling and canonical Wnt signaling. Characterization of the canonical Wnt signaling pathway over the last 2 decades has revealed a fundamental role in many physiological and pathophysiological processes. Molecular defects in Wnt genes or their associated downstream effectors, most notably -catenin, often have profound consequences linked with a myriad of developmental disorders and human diseases, including those involving hippocampal development, epithelial tube formation, and cancer (1-5).The canonical Wnt pathway involves a family of 19 Wnt ligands, which are cysteine-rich glycoproteins that bind to the Frizzled receptor proteins, of which there are 10 family members. The ligand-receptor interaction comprises part of a larger signaling complex containing other receptor-related proteins, such as the low-density lipoprotein receptor-related protein 5 (LRP5) and LRP6 single-pass transmembrane proteins. -Catenin, a bifunctional protein that serves as a component of the cell adhesion machinery in combination with E-cadherin and ␣-catenin, also performs an essential nodal function in the canonical Wnt pathway downstream of the receptor complex. In brief, without active Wnt signaling, -catenin is phosphorylated by glycogen synthase kinase 3 (GSK3) and casein kinase I (CKI) in an adenomatous polyposis coli (APC)/axin "destruction complex," which facilitates interaction with -transducin repeat-containing E3 ubiquitin protein ligase (-TrCP) and subsequent ubiquitin-mediated proteasomal degradation (6-8). Conversely, pathway activation by the Wnt-Frizzled interaction dismantles the destruction complex, leading to enhanced levels of cellular -catenin and su...
Canonical Wnt/β‐catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/β‐catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context‐dependent mechanisms regulating this pathway. Since protein–protein interactions underpin β‐catenin function and localization, we sought to identify novel β‐catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where β‐catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC β‐catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for β‐catenin, particularly in mRNA translation, and we confirm a direct interaction between β‐catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of β‐catenin to the messenger ribonucleoprotein and translational pre‐initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering β‐catenin away from the pre‐initiation complex. In conclusion, we present evidence that β‐catenin fulfills a previously unrecognized function in translational repression.
MYOGENIN is a member of the muscle regulatory factor family that orchestrates an obligatory step in myogenesis, the terminal differentiation of skeletal muscle cells. A paradoxical feature of alveolar rhabdomyosarcoma (ARMS), a prevalent soft tissue sarcoma in children arising from cells with a myogenic phenotype, is the inability of these cells to undergo terminal differentiation despite the expression of MYOGENIN. The chimeric PAX3-FOXO1 fusion protein which results from a chromosomal translocation in ARMS has been implicated in blocking cell cycle arrest, preventing myogenesis from occurring. We report here that PAX3-FOXO1 enhances glycogen synthase kinase 3β (GSK3β) activity which in turn represses MYOGENIN activity. MYOGENIN is a GSK3β substrate in vitro on the basis of in vitro kinase assays and MYOGENIN is phosphorylated in ARMS-derived RH30 cells. Constitutively active GSK3β(S9A) increased the level of a phosphorylated form of MYOGENIN on the basis of western blot analysis and this effect was reversed by neutralization of the single consensus GSK3β phosphoacceptor site by mutation (S160/164A). Congruently, GSK3β inhibited the trans-activation of an E-box reporter gene by wild-type MYOGENIN, but not MYOGENIN with the S160/164A mutations. Functionally, GSK3β repressed muscle creatine kinase (MCK) promoter activity, an effect which was reversed by the S160/164A mutated MYOGENIN. Importantly, GSK3β inhibition or exogenous expression of the S160/164A mutated MYOGENIN in ARMS reduced the anchorage independent growth of RH30 cells in colony-formation assays. Thus, sustained GSK3β activity represses a critical regulatory step in the myogenic cascade, contributing to the undifferentiated, proliferative phenotype in alveolar rhabdomyosarcoma (ARMS).
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