Correlation between ALK-6 (BMPR-IB) distribution and responsiveness to osteogenic protein-1 (BMP-7) in embryonic mouse bone rudiments.

Osteogenic protein-1 (OP-1) or bone morphogenetic protein-7 (BMP-7) stimulates cartilage formation in mouse bone rudiments in vitro but arrests terminal differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes. In this study we report that these effects of OP-1 depend on the developmental stage of the bone rudiment, early stages (E14 and E15 metatarsals) being most responsive. E17 metatarsals that already contained a hypertrophic area that had initiated mineralization were no longer affected by OP-1. We then investigated whether the sensitivity of the early long bone rudiments to OP-1 correlated with high expression of the OP-1 binding type I serine/threonine kinase receptors (activin receptor-like kinase: ALK-2/ActR-I, ALK-3/BMPR-IA or ALK-6/BMPR-IB) at this early stage. We did not find any significant difference in overall mRNA levels of these ALKs between stages E14 through E17 as assessed by RNase protection assays. However, by immunohistochemistry we found that ALK-6 staining was strong in E14 early cartilage primordium and its future perichondrium but dropped sharply to low levels in these cell types until onset of chondrocyte (pre)hypertrophy at E16. By contrast, ALK-2 and ALK-3 immunostainings in E14 were barely detectable. We also examined by immunohistochemistry the local synthesis of OP-1. OP-1 was present in E14 early chondrocytes and forming perichondrium but in low amounts; however, production of OP-1 increased in these cell types with age. All three receptor types as well as OP-1 were present in significant amounts in prehypertrophic chondrocytes and late hypertrophic chondrocytes including those undergoing mineralization. The temporary high immunostaining for ALK-6 in the early proliferating chondrocytes and future perichondrium of E14 bone rudiments, and its absence in older bones correlated with the sensitivity of chondrocytes and perichondrium to (exogenous) OP-1. We therefore propose that the effects of OP-1 on these cells in vitro are mediated by ALK-6/BMPR-IB. We furthermore conclude that locally produced OP-1 is a potential autocrine/paracrine growth factor. Increased local production of OP-1 may be partially responsible for the age-related decrease in responsiveness to exogenous OP-1 with respect to hypertrophy and mineralization of cartilage.