Mef2C Regulate The Differentiation Of Myoblast Through AdipoR1 Pathway

Mef2C (Myocyte Enhacer Factor C) has been reported as a muscle development related genes in 1993, which is very conserved throughout evolution. Mef2C is the terminal factor of many signal pathways that regulate myoblast differentiation, there are plenty of researches in myocyte and nerve cell aiming at extracellular signaling and co-factors that modulate MEF2 activity. Studies in muscle cells have revealed the overlapped fuction of Mef2C and other muscle specific factors.Adiponectin is an newly discovered adipocytokine. Adiponectin was ubiquitously expressed in many issues, function by autocrine, paracrine and endocrine. Adiponectin was identified as an "Insulin-sensitizing" Hormone independently by four groups, and has been reported to have direct antiathero-sclerotic effects. There are two adiponectin receptors, AdipoRl and AdipoR2, and AdipoRl was ubiquitously expressed and most abundantly expressed in skeletal muscle.Adiponectin has been reported to promote the differentiation of myoblast, the relationship of adiponectin/AdipoRl and the classical myocyte factor Mef2C deserved a further exploration.In this study, by using Real-Time PCR, Dual luciferase experiment, Western Blot, etc, we studied the expression level variation in murine myoblasts differentiation process and the effect of Mef2C on AdipoRl expression and the downstream pathways of adiponectin. Here are the results:1. Using C2C12 murine myoblasts as a model, cultivated in differentiation medium for 5 days. Harvest cells for RNA and protein collection, Real-Time PCR was performed to detect the mRNA expression level of adiponectin and AdipoRl; Western Blot was performed to detect the protein level of adiponectin and AdipoRl. We found the expression of adiponectin and AdipoRl was up-regulated in defferentiated C2C12.2. An adiponectin steady-transfected C2C12 cell line was constructed to investigate the mRNA expression level of MHC by Real-Time PCR. Immunohistochemistry was performed to detect the MHC protein in C2C12. We found mRNA expression of MHC increased in tranfected cells, and lots of myotube could been seen under a Inverted Flurescence microscope. For further evidence, primary skeletal myoblasts were isolated from C57/B16 mice. These myoblasts were treated by recombinant adiponectin(2ug/ml) then observed under a microscope after immunohistochemical staining, remarkble myotube forming was observed.3. To demonstrate the function of Mef2C in adiponectin/AdipoRl pathway, we constructed a Mef2C steady-transfected C2C12 cell line. Real-Time PCR and Western Blot were performed to detect the expression level of adiponectin and AdipoRl. We found that the mRNA and protein expression level were up-regulated.4. MEF2 binding sites were found in the mucleotide sequences of the 5’-flanking region of the murine Mef2C gene. To clarify whether MEF2C bind on the promoter of adiponectin/AdipoRl directly, adiponectin-PGL3 vector and AdipoRl-PGL3 vector were constructed for a Luciferase Reporter Assay; Mef2C expression vector was constructed meanwhile. Co-transfect Mef2C vector and adiponectin/AdipoRl vector, we found Mef2C function on AdipoRl promoter only. ChIP assay was performed ulteriorly, these data illustrate MEF2C can bind on the MEF2 sites of AdipoRl directly.5. After transient transfection, C2C12 myoblasts were cultivated for Real-Time PCR. The results showed that the mRNA level of APPL1 and GLUT4 was up-regulated. Blocking AdipoRl on the membrane of C2C12 with AdipoRl antibody or knocking down AdipoRl with siRNA resulted in down-regulation of MHC. These data demonstrate Mef2C regulate AdipoRl then affect downstream sianals of adiponectin, finally influence myogenesis process.