Studies On Sema4C Gene Expression, Function And Mechanisms During The Process Of Myogenic Differentiation

Human Sema4C gene is a novel gene cloned and isolated from fetal brain that belongs to the class IV subgroup of the transmembrane semaphorin family. The gene is divided into 13 exons and mapped on chromosome 2q11.2. Sema4C is a single transmembrane protein encoding a protein of 833 amino acids, the molecular weight of which is about 92.6 kD. Here we show the systematic studies on Sema4C gene including the subcellular location, multi-tissue expression, biological function on myogenic differentiation and the possible mechanisms.By deletion analysis, we firstly showed that the integrity of extracellular domain of Sema4C including a sema domain and a PSI domain is necessary for the appropriate post-translational modification and subcellular localization. In situ hybridization and immunohistochemistry data showed that Sema4C gene was expressed in the forebrain, optic primordium, dorsal root ganglion and arch artery of E11.5 embryo, and also slightly expressed in the upper limb buds of E13.5 embryo. Sema4C gene was highly expressed in the cerebral cortex, cerebellar Purkinje cell layer, retinal ganglion cells and epithelial cells of renal tubule in adult mouse. However its expression in skeletal muscle, heart, lung, spleen, liver and other organs was very low or undetectable. The above data suggested that Sema4C might play certain roles during the development of other tissues except for nervous system such as skeletal muscle.To probe the function of Sema4C during skeletal muscle development and myogenic differentiation, we firstly examined the expression patterns during the differentiation of myoblasts cultivated in vitro and the process of rattus skeletal muscle injury and regeneration in vivo. RT-PCR and Western blot results showed that the expression of Sema4C was dramatically induced not only during the differentiation of myoblasts in vitro, but also during the injury-induced skeletal muscle regeneration process in vivo. To further investigate the effects of Sema4C gene on the proliferation and differentiation of C2C12 myoblasts, we constructed human Sema4C gene eukaryotic expression vector and recombinant Ad5-Sema4C adenovirus vector respectively. It was demonstrated that C2C12 cells either stably or transiently overexpressing Sema4C showed increased terminal myogenic differentiation with higher expression of muscle-specific protein myosin, muscle creatine kinase and increased the expression of myogenin. While depletion of Sema4C in C2C12 cells by specific small interference RNA resulted in marked inhibition of terminal myogenic differentiation and decreased expression of myosin and myogenin.Further investigation on the mechanism showed that overexpression of Sema4C in C2C12 cells directly elicited the phosphorylation of p38 MAPK but not ERK1/2 and JNK in MAPK signaling pathway. In addition, the positive effects of Sema4C gene on myogenic differentiation could be abolished by p38 specific inhibitor SB203580. Besides these, knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed with dramatically diminished myotube formation. By using dual-luciferase reporter assay system, we also found that Sema4C could activate and enhance the transcriptional activity of myogenin promoter during myogenic differentiation which could be abolished by SB203580. Taken together, these observations reveal for the first time that Sema4C promotes the terminal myogenic differentiation in a p38 MAPK-dependent manner.