REGULATION OF BONE FORMATION BY OSTEOCLASTS

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Bone resorption and bone formation appear to be tightly coupled. To explore osteoclast influences on osteoblast recruitment and differentiation, we examined osteoclast conditioned media (CM) for influences on osteoblasts. Osteoclast CM stimulated human mesenchymal stem (hMS) cell mineralized nodule formation. We identified candidate osteoclast-derived coupling factors using Affymetrix microarray. We observed induction of sphingosine kinase 1 (SPHK1), WntlOb, and BMP6 in mature multinucleated osteoclasts as compared to pre-osteoclasts. Stimulation of hMS cell nodule formation by the osteoclast CM was attenuated by the Wnt antagonist, Dkk1, a BMP-6 neutralizing antibody, and by a S1P antagonist. Sclerostin expression was elevated in osteoclast precursors and rapidly down-regulated during differentiation. CM from osteoclasts generated in vitro from 18-month old mice was unable to support hMS cell mineralization. TGF-beta treatment elevated SPHK1 and WntlOb expression. Our central hypothesis is that osteoclast production of SPHK1, WntlOb, and BMP6, combined with decreased Sclerostin expression, promotes osteoblast precursor recruitment, proliferation, differentiation, and survival and that each factor plays an integral role in maintaining coupling between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. In Aim 1, we will use functional cell assays and gene expression to ascertain the mechanisms by which osteoclast-derived coupling factors promote osteoblast gene expression, recruitment, proliferation, differentiation, and survival in vitro. In Aim 2 we will use molecular approaches to determine the mechanisms by which Sclerostin, SPHK1, WntlOb, and BMP6 are modulated during osteoclast differentiation in vitro. In Aim 3 we will use an in vivo model to examine the role of TGF-beta regulation of coupling factor production on osteoclast-mediated coupling of bone resorption to bone formation. In Aim 4 we will examine gene expression and cellular assays to resolve the contribution of age in osteoclast support of osteoblast maturation and mineralization. Together, these studies will provide important novel information on how osteoclasts control osteoblast-mediated bone formation.