Bone Abstracts

The G protein-coupled receptor, protease-activated receptor 2 (PAR₂), is expressed by osteoblasts and required for normal skeletal growth and repair. Prostate cancer (PCa) cells commonly secrete proteolytic activators of PAR₂ (including matriptase and kallikrein-related peptidase 4) and frequently form osteogenic metastases in bone. This study was undertaken to investigate the hypothesis that PAR₂ activators released by PCa cells modulate osteoblast behaviour in such a way as to support the formation of osteogenic metastases. Primary calvarial osteoblasts derived from wild-type (WT) and PAR₂-null mice were cultured in medium conditioned by the MDA-PCa-2b cell line (MDA-CM); proliferation was assessed by BrdU incorporation, and differentiation was assessed using assays for alkaline phosphatase activity and mineralization, and quantitative PCR analysis of osteoblast-associated genes. MDA-CM stimulated proliferation of osteoblasts independently of PAR₂, but promoted osteoblast differentiation in a PAR₂-dependent manner. Alkaline phosphatase activity and expression of Runx2 and Col1 mRNA in 1-day cultures, and mineralization in long-term cultures were all stimulated by MDA-CM in WT but not in PAR₂-null osteoblast cultures. A surprising observation was that long-term cultures of MDA-CM-treated PAR₂-null osteoblasts contained significantly more adipocytes than did matching WT cultures. In agreement with this observation, MDA-CM stimulated expression of the adipogenesis-associated genes encoding peroxisome proliferator-activated receptor-γ (Pparγ) and lipoprotein lipase (Lpl) in 4-day cultures of PAR₂-null but not WT osteoblasts. Moreover, expression of Pparγ mRNA was significantly greater in untreated PAR₂-null cultures than in WT cultures. These results indicate that expression of PAR₂ favours osteoblast differentiation over adipogenesis in mesenchymal cells capable of both osteoblast and adipocyte differentiation.