Bone anabolic effects of Sonic/Indian hedgehog are mediated by BMP-2/4-dependent pathways in the neonatal rat metatarsal model

A neonatal rat metatarsal organ culture model has been employed to study th e anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture syst em, a significant increase in endochondral ossification is measured by an i ncrease in length of mineralized bone, after daily treatment for 7 days wit h Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH rela ted protein (PTHrP) is a critical target of hedgehog function in endochondr al ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-relat ed increase in endochondral ossification is observed when metatarsals are t reated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4) . However, this activity is not evident at higher doses of BMP-4 (200 ng/ml ). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabol ic action of Shh-N. This observation suggests that BMP-I signaling can infl uence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossificati on is reversed by treatment with antisense oligonucleotides targeted agains t Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral o ssification that mediates its effect via BMP-4 and Cbfa1-dependent pathways .