An HMGB1/RAGE/p38 MAPK/Myogenin Axis Modulates Pax7 Expression in Myoblasts by Both Transcriptional and Post-Transcriptional Mechanisms

RAGE (receptor for advanced glycation end products), activated by its ligand HMGB1, transduces a myogenic and anti-proliferative signal in differentiating myoblasts (Mol Cell Biol 24 (2004) 4880-4894; J Biol Chem 281 (2006) 8242-8253; Am J Pathol 171 (2007) 947-961). Satellite cells (SCs), the adult muscle stem cells, and quiescent primary myoblasts do not express RAGE; however, RAGE is rapidly expressed in activated myoblasts and, to a larger extent, in differentiating myoblasts, and its expression is directly related to the expression of myogenin, a muscle-specific transcription factor essential for myogenic differentiation, and inversely related to the expression of Pax7, a transcription factor which is required for quiescence and proliferation of myoblasts/SCs and has to be downregulated for myoblasts/SCs to differentiate. We found that either the blockade of RAGE activity or inactivation of p38 MAPK resulted in downregulation of myogenin and upregulation of Pax7 in myoblasts in differentiation medium (DM), suggesting that RAGE might participate in repression of Pax7 expression via p38 MAPK-dependent induction of myogenin expression. The murine Pax7 promoter contains six myogenin recognition sites, and by chromatin immunoprecipitation assay myogenin bound to three of them in growth medium (GM) and to all of them in DM. Transfection of myoblasts with myogenin caused inhibition of Pax7 (mRNA and protein) expression in GM and DM, while knockdown of myogenin by RNAi resulted in upregulation of Pax7 (mRNA and protein) in DM. Thus, the HMGB1/RAGE/p38 MAPK/myogenin axis might modulate Pax7 transcription. However, blocking the proteasome with MG132 at 6h by the switch of myoblasts to DM (when the myogenin and Pax7 abundances were relatively high and the two proteins were co-expressed in myoblasts) resulted in accumulation of Pax7. at this same time point Pax7 co-immunoprecipitated with myogenin pointing to myogenin-Pax7 interaction. These results suggest that the HMGB1/RAGE/p38 MAPK/myogenin axis modulates Pax7 abundance in differentiating myoblasts via both transcriptional and posttranscriptional mechanisms.