A BMP RESPONSIVE TRANSCRIPTIONAL REGION IN THE CHICKEN TYPE-X COLLAGEN GENE

Bone morphogenetic proteins (BMPs) were originally identified by their ability to induce ectopic bone formation and have been shown to promo te both chondrogenesis and chondrocyte hypertrophy, BMPs have recently been found to activate a membrane serine/threonine kinase signaling m echanism in a variety of cell types, but the downstream effecters of B MP signaling in chondrocyte differentiation remain unidentified. We ha ve previously reported that BMP-2 markedly stimulates type X collagen expression in prehypertrophic chick sternal chondrocytes, and that typ e X collagen mRNA levels in chondrocytes cultured under serum-free (SF ) conditions are elevated 3- to 5-fold within 24 h, To better define t he molecular mechanisms of induction of chondrocyte hypertrophy by BMP s, we examined the effect of BMPs on type X collagen production by 15- day chick embryo sternal chondrocytes cultured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7, Two popula tions of chondrocytes were used: one representing resting cartilage is olated from the caudal third of the sterna and the second representing prehypertrophic cartilage from the cephalic third of the sterna, BMP- 2, BMP-4 and BMP-7 all effectively promoted chondrocyte maturation of cephalic sternal chondrocytes as measured by high levels of alkaline p hosphatase, diminished levels of type II collagen, and induction of th e hypertrophic chondrocyte-specific marker, type X collagen. To test w hether BMP control of type X collagen expression occurs at the transcr iptional level, we utilized plasmid constructs containing the chicken collagen X promoter and 5' flanking regions fused to a reporter gene. Constructs were transiently transfected into sternal chondrocytes cult ured under SF conditions in the presence or absence of 30 ng/ml BMP-2, BMP-4, or BMP-7, A 533 bp region located 2.4-2.9 kb upstream from the type X collagen transcriptional start site was both necessary and suf ficient for strong BMP responsiveness in cells destined for hypertroph y, but not in chondrocytes derived from the lower sterna.