Endochondral bone formation is a multistep process involving chondrocyte proliferation, hypertrophy, calcification, degradation and replacement of cartilage by bone. The bone morphogenetic proteins (BMPs) have been shown to induce this process both in vivo and in vitro . To better define the molecular mechanisms of BMP induction of chondrocyte hypertrophy, we examined the effects of BMP-2, -4 and -7 on embryonic chick sternal chondrocytes cultured under serum-free conditions. Two populations of chondrocytes were analyzed: those from the cephalic or upper sternum (USCs), which hypertrophy in the embryo, and those from the caudal or lower sternum (LSCs), which remain immature during embryonic life. Northern analysis and biochemical assays were utilized to monitor BUT induction of common markers of chondrocyte maturation. Transient transfections of plasmid constructs containing the chicken collagen X promoter and various 5 ' flanking regions fused to a reporter gene were utilized to identify a BMP responsive transcriptional region. Electrophorectic mobility shift assays identified potential regions involved in transcriptional control of this promoter by BMPs.
All three BMPs effectively promoted maturation of USCs, but not LSCs. A 533bp region of the collagen X promoter located 2.4-2.9kb upstream from the transcriptional start site was both necessary and sufficient to direct strong (>20-fold) BMP responsiveness in USCs, but not LSCs. Deletion of a 180bp region from the 533bp region abolished all BMP responsiveness; however, this fragment alone was unable to confer BMP responsiveness, suggesting multiple elements may be responsible for BMP regulation of this gene.
The effects of BMPs on USCs could be mimicked by retrovirally expressing a constitutively active BMP receptor type IB (CA-IB) and blocked with a dominant negative receptor type IB. Despite the fact that BMPs do not induce hypertrophy in LSCs, the expression of CA-IB in LSCs induced small but significant changes associated with hypertrophy. Furthermore, LSCs contain transcripts for BMP-2, -4 and -7 and BMP receptors IA, IB and II. Thus, despite differences in response to BMPs by USCs and LSCs, both possess BMP signaling components.
