POSTER NO: 236

Chemokine-like factor 2 could promote the proliferation and differentiation of C2C12 skeletal muscle cells

¹Donglan Xia, ¹Xianting Li, ¹Yaxin Lou, ²Wenling Han, ²Quansheng Song, ²Yingmei Zhang, ¹Dalong Ma
¹Lab of Medical Immunology, School of Basic Medical Science, Peking University, Xueyuan Road 38, Beijing 100083, China, ²Peking University Center for Human Disease Genomics and Lab of Medical Immunology, School of Basic Medical Science, Peking University, Xueyuan Road 38, Beijing 100083, China

Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells (Han et al: Biochem J. 2001, 357, 127-135). It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of chemokine-like factor 2 (CKLF2), which is the largest isoform of CKLFs, we transfected C2C12, a mouse satellite cell line, with CKLF2 cDNA to analyze for the effects and possible mechanisms of CKLF activity on the proliferation and differentiation of muscle cells. Compared with control vector transfected C2C12 myoblasts, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [3H]-TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. The in vitro transfection assays provide evidence that the muscle-stimulating effect of CKLF2 is independent of other cytokines. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. These data provide strong evidence that CKLF2 promotes the proliferation and differentiation of C2C12 skeletal muscle cells. Since C2C12 is a murine immortal satellite cell line, we cloned the murine homologue of CKLF2, mCKLF2, and we tried to uncover the physiological action of mCKLF2 on C2C12 proliferation and myogenesis. Preliminary studies showed that the rat and mouse homologue of CKLF2, may participate in the growth of muscle cells and that murine and rattus CKLF2 were up-regulated while accompanied with muscle-specific molecular markers upon serum-deprivation in mouse or rat primary satellite cells. By transient transfection of mCKLF2, we have analyzed that mCKLF2 could promote the proliferation, as indicated by cell MTT assay and 3H-TdR incorporation. Our research also indicated overexpression of mCKLF2 in C2C12 could also promote differentiation of C2C12 cells, as is indicated by western blot of myogenin and myosin. This suggests that CKLF2's function on myogenesis is evolutionary conserved. In accordance of our former research, a phosphorothioate antisense oligonucleotide approach was utilized to determine if inhibition of mCKLF2 expression would block or perturb myocyte proliferation. Three phosphorothioate oligonucleotides including sense, antisense and random control were designated and generated. The antisense oligo is compelmentary to the first 17bp from ATG transcription start site. These oligos were added to proliferating cells by electroporation at a concentration of 10µM. Treatment of cells with rambling oligo had little effect on 3H-TdR incorporation of C2C12 and on cell cycle. In contrast, treatment with 10µM sense and antisense oligos inhibited the 3H-TdR incorporation of C2C12 and led to a G2/M arrest as detected by FACS scans. Semiquantitive RT-PCR and co-transfection assay have indicated that both of sense and antisense oligos could diminish the expression of mCKLF2. Combined, these results provide strong evidence supporting a functional role for mCKLF2 in C2C12 cell proliferation. In conclusion, our findings strongly suggest a role for CKLF2 in regulation of skeletal muscle proliferation and differentiation.