Scleraxis messenger ribonucleic acid is expressed in C2C12 myoblasts and its level is down-regulated by bone...

We examined the mRNA expression of scleraxis, a non-myogenic helix-loop-helix type transcription factor in C2C12 myogenic cells. Scleraxis mRNA has been shown to be expressed in sclerotome and perichondrium of the embryos. We found that C2C12 cells express 1.2 kb scleraxis mRNA constitutively. Since BMP was reported to induce ectopic bone formation when implanted in muscle, we examined the effects of BMP on scleraxis expression. Scleraxis mRNA expression in C2C12 cells was suppressed by the treatment with BMP2. This suppression was observed at 200 ng/ml but not at the lower concentrations. BMP2 treatment suppressed scleraxis mRNA level within 24 h and lasted at least up to 48 h. Electrophoresis mobility shift assay showed that the proteins in the crude nuclear extracts prepared from C2C12 cells bound to an Scx-E-box sequence, CATGTG, which is preferentially recognized by scleraxis. This binding was competed out by 100-fold molar excess of cold Scx-E-box sequence but not by the one with mutations in the E-box. This band was supershifted by the addition of antiserum raised against scleraxis. BMP2 treatment suppressed the Scx-E binding activity in C2C12 cells. This suppression of the Scx-E-box binding activity was in parallel to the BMP2 suppression of the transcriptional activity of the Scx-E-CAT reporter gene transfected into C2C12 cells. These data indicated that although the default pathway for C2C12 cells is to differentiate into muscle cells, these cells do express non-myogenic transcription factor, scleraxis, whose expression is suppressed by BMP2.