Parathyroid hormone-related protein is required for normal intramembranousbone development

It is well established that parathyroid hormone-related protein (PTHrP) reg ulates chondrocytic differentiation and endochondral bone formation. Beside s its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP r eceptor) have been found in osteoblasts, suggesting an important role of PT HrP during the process of intramembranous; bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockou t mice histologically. We also analyzed phenotypic markers of osteoblasts a nd osteoclasts in vitro and in vivo. A well-organized branching and anastom osing pattern was seen in the wild-type mice. In contrast, marked disorgani zation of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the f emur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and s ome of the irregularly aligned osteoblastic cells in the homozygous mice ex pressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heter ozygous mutant and wild-type mice (+/? mice) and those from homozygous muta nt mice. However, both mRNA and protein levels of osteocalcin in the osteob lastic cells of homozygous mutant mice were lower than those of +/? mice, a nd exogenous PTHrP treatment corrected this suppression. Immunohistochemica l localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteobl astic cells and spleen cells of homozygous mutant mice generated an equival ent number of tartrate-resistant acid phosphatase-positive (TRAP(+)) mononu clear and multinucleated cells and of pit formation to that of +/? mice, su ggesting that osteoclast differentiation is not impaired in the homozygous; mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.