Overexpression of Id2 Results in a Decrease in MyoD in Proliferating C2C12 Myoblasts

Abstract

Inhibitor of differentiation protein-2 (Id2) is a dominant negative helix-loop-helix (HLH) protein, and a positive regulator of proliferation, in various cells. Id2 may also have a role in apoptosis, because the N-terminal region induces apoptosis in myeloid 32d.3 cells. Furthermore, elevated levels of Id2 and apoptotic markers have been identified in aged rat skeletal muscles. The role of Id2 in skeletal muscle cells is unknown. PURPOSE Because Id2 is elevated in aged skeletal muscle, the objective of this study was to determine if overexpression of Id2 would alter MyoD levels in proliferating C2C12 myoblasts, a mouse satellite cell line. METHODS The full length Id2 gene was generated by PCR amplification from cDNA of rat hindlimb muscles. Primers with Nde1 and HindIII restrictions sites were used to subclone Id2 into a pDNR-1r Donor Vector that contained two loxP sites. Cre recombinase mediated the transfer of the Id2 fragment located between the two loxP sites of the Id2-pDNR-1r donor vector, to the loxP acceptor vector pLP-IRES2-eGFP to yield Id2-IRES-eGFP. The internal ribosomal entry sequence (IRES) allows both the Id2 gene and the enhanced green fluorescent protein (eGFP) gene to be translated simultaneously from a single bicistronic mRNA. For control experiments, pDNR-1r and Luciferase-pDNR-1r vectors were recombined with the pLP-IRES-eGFP vector to generate pDNR-IRES-eGFP and Luc-IRES-eGFP expression vectors. RESULTS Luciferase activity in Luc-IRES-eGFP transfected cells is significantly (p = 0.0001) higher (81,568-fold increase) compared to the average luciferase activity of C2C12 cells, pDNR-IRES-eGFP and Id2-IRES-eGFP transfected myoblasts. Florescence activated cell sorter (FACS) analysis revealed that Id2 expression is significantly (p > 0.05) elevated (383% & 275%) in Id2-IRES-eGFP transfected C2C12 myoblasts. Id2 expression was significantly (p > 0.05) elevated (383%) in Id2-IRES-eGFP transfected C2C12 myoblasts through western analysis. FACS analysis showed that MyoD expression was significantly (p > 0.05) lower (20%) in C2C12 myoblasts transfected with Id2. There were no significant differences in E47/E12 expression between groups. CONCLUSION These data suggest that the overexpression of Id2 in vitro maybe responsible for the decrease of MyoD, because Id2 has been shown to preferentially bind to Class A basic HLH proteins E47 and E12. This would leave MyoD without a dimerization partner and subject to proteolysis. Supported by NIH R01AG021530