Roles Of IGF1Ec, A Splicing Isoform Of IGF1, And Relative Mechanism In C2C12 Cells

IGF1(Insulin-like growth factor1), as a growth factor, is critical for the growth, development and maintain of many types of tissue. IGF1 Ec is an alternative splicing variant of IGF1, and its expression level is significantly increased in muscle following stretch, mechanical damage resulting from electrical stimulation and exercise, bupivacaine injection, and so on. It has been reported that MGF E, a C-terminal E-domain with 24 amino acids of IGF1 Ec, was involved in the repair and treatment for diseases of muscle, nerves and heart. However, the reports about the biological functions of IGF1 Ec were very few, and the researches about the physiological functions of MGF E in muscle were not very consistent. Therefore, this study investigated physiological responses of C2C12 muscle cells to IGF1 Ec, IGF1 and MGF E, which is helpful for understanding of the structure-function relationships of IGF1 Ec. We also used site-directed mutagenesis PCR to optimize the gene sequence of IGF1 Ec, and got a better production of IGF1 Ec protein in BL21(DE3). Our research results will useful for the application of IGF1 Ec, IGF1 and MGF E in muscle tissue repair and regeneration engineering, and it also provided an economical method for industrial production of IGF1 Ec. The main works and conclusions are included as follows:(1) The rare codon in IGF1 Ec gene sequence was the main factor to result in non-expression of IGF1 Ec protein in BL21(DE3). Therefore, we optimized the rare codons into BL21(DE3) preferred ones through site-directed mutagenesis PCR, and constructed two recombinant plasmids p GEX-4T-1/IGF1Ec(Mut54-56) and pGEX-4T-1/IGF1Ec(Mut-total). To pioneer the commercial production of IGF1 Ec protein in Escherichia coli, we compared the production of IGF1 Ec between Rosetta(DE3) and BL21(DE3). Our data showed that the IGF1 Ec protein pGEX-4T-1/IGF1Ec(Mut54-56) and pGEX-4T-1/IGF1Ec(Mut-total) levles in BL21(DE3) were significantly increased as compared with that pGEX-4T-1/IGF1 Ec level in Rosetta(DE3). After that, we checked the effect of IGF1 Ec on proliferation, migration and differentiation in osteoblast. Our data showd that IGF1 Ec with low concentration could enhance osteoblast proliferation and migration, but repressed differentiaton, which certificated that we purified IGF1 Ec with high biological activity.(2) To understand of the structure-function relationships of IGF1 Ec, we first compared the biological reactions of C2C12 muscle cells to IGF1 Ec, IGF1 and MGF E. Our data showed that the proliferation of C2C12 cells was increased with treatment of IGF1 Ec and IGF1, but was not affected by MGF E. All three proteins were able to induce the expression of Myog in C2C12 cells, elongation of the cell body as well as formation of multi-nucleated myoblast fusion. However, IGF1 Ec and MGF E appeared to be more active than IGF1 in stimulating myogenic differentiation. And for migration, our results demonstrated that IGF1 Ec and MGF E were also more effective than IGF-1 in promoting migration of C2C12 cells. Then we examined the role of IGF1 R in mediating the functions of IGF1 Ec, IGF1 and MGF E. Our data indicated that IGF1 R was completely involved in proliferation effect of IGF1 Ec and IGF1 in C2C12 cells, but partially involved migration effect of IGF1 Ec and MGF E, which indicated that IGF1 Ec and MGF E could activate other receptors to mediate cell migration. Based on our results, we concluded that the N-terminal sequence of IGF1 Ec, which was homologue in part with IGF-1, promoted proliferation and the C-terminal sequence of IGF1 Ec, which was identical to MGF E, promoted differentiation and migration of C2C12 cells.(3) We constructed a fusion protein named IGF1-24, which linked the mature IGF1 and MGF E together with three Gly. Our data showed that IGF1-24 had a similar effect on proliferation, differentiation and migration in C2C12 cell as IGF1 Ec had. And the role of IGF1 R in IGF1-24 mediated cell biologic action was also as the same as IGF1 Ec. Therefore, our study confirmed that the function of IGF1 Ec was assuredly obtained from IGF1 and MGF E.(4) We investigated the existence form of IGF1 Ec. And our data showed that only intact IGF1 Ec was detected by antibody in cell lysis, which indicated that the intracellular IGF1 Ec existed mostly in full-length. And in cell medium, only IGF1 Ec in full- length was detected, suggesting that the form of IGF1 Ec was quite stable. Therefore, we inferred that the existence of IGF1 Ec was not in order to provide mature IGF1 and E peptide, but to function as a whole.(5) We performed GST-Pulldown and LC-MS/MS analysis to search proteins that interact with IGF1 Ec and MGF E, respectively, and identified a total of 46 proteins that interact with both IGF1 Ec and MGF E, but not with IGF1. For a better understanding of the function of IGF1 Ec and MGF E, we did bioinformation analysis via GOEAST and DAVID tool. Our results indicated that these identified interaction proteins regulated some biological processes, such as protein biosynthesis, protein transport, cell migration, cell junction and cell differentiation.