SCLERAXIS MESSENGER-RIBONUCLEIC-ACID IS EXPRESSED IN C2C12 MYOBLASTS AND ITS LEVEL IS DOWN-REGULATED BY BONE MORPHOGENETIC PROTEIN-2 (BMP2)

We examined the mRNA expression of scleraxis, a non-myogenic helix-loo p-helix type transcription factor in C2C12 myogenic cells. Scleraxis m RNA has been shown to be expressed in sclerotome and perichondrium of the embryos. We found that C2C12 cells express 1.2 kb scleraxis mRNA c onstitutively. 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 b y the treatment with BMP2. This suppression was observed at 200 ng/ml but not at the lower concentrations. BMP2 treatment suppressed sclerax is mRNA level within 24 h and lasted at least up to 48 h. Electrophore sis mobility shift assay showed that the proteins in the crude nuclear extracts prepared from C2C12 cells bound to an Scx-E-box sequence, CA TGTG, which is preferentially recognized by scleraxis. This binding wa s 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 supershifte d by the addition of antiserum raised against scleraxis. BMP2 treatmen t suppressed the Scx-E binding activity in C2C12 cells. This suppressi on of the Sex E-box binding activity was in parallel to the BMP2 suppr ession of the transcriptional activity of the Scx-E-CAT reporter gene transfected into C2C12 cells. These data indicated that although the d efault pathway for C2C12 cells is to differentiate into muscle cells, these cells do express non-myogenic transcription factor, scleraxis, w hose expression is suppressed by BMP2. (C) 1997 Wiley-Liss, Inc.