This project seeks to define the role of the recently cloned factor, osteogenic protein-1 (OP-1), in bone cell biology. OP-1 and other bone morphogenetic proteins (BMP's) are homologous members of the TGF-beta superfamily of growth and differentiation factors which were originally isolated from demineralized bone powder. These proteins are strongly implicated as the key regulatory elements in the pathway of osteoblastic differentiation leading to osteoinductive bone formation. Osteoinduction is a developmental process in which bone tissue forms de novo by recruitment and commitment of relatively undifferentiated mesenchymal cells. Osteoinduction occurs naturally in skeletal development of the embryo or fetus, and it may be partially recapitulated in fracture healing, surgical repair of bone, and osteointegration of prosthetic devices and implants. Abnormal operation of the osteoinductive differentiation cascade may explain the pathophysiology of numerous skeletal disorders such as osteoporosis. We hypothesize that OP-1 induces new bone tissue by recruiting mesenchymal precursor cells to the osteoblastic pathway, and by stimulating osteoblastic phenotypic expression in committed cells. the major aim of this project is to characterize the regulatory effects of recombinant OP-1 on the osteoblast in vitro. Human and rat primary osteoblasts (and other osteoblastic and undifferentiated cell lines) will be used to examine OP-1 regulation of proliferation and expression of phenotypic markers (osteocalcin, alkaline phosphatase, mineralization, etc.). Methods include Northern analysis of mRNA levels, radioimmunoassay of osteocalcin, immunohistochemistry, in situ hybridization, and regulation of osteocalcin promoter-CAT constructs in ROS 17/2.8 cells.
The second aim i s to evaluate the role of OP-1 in the recruitment and commitment of mesenchymal cells to the osteoblastic phenotype. Mesenchymal cells from developing osteoinductive implants and limb buds will be exposed to OP-1 under various conditions. Shell-less chick embryos in vitro will be implanted with OP-1 in methylcellulose discs at different times and locations. Target cells for OP-1 will be labeled with 125I-OP-1 and visualized autoradiographically.
The third aim i s to characterize the mechanism of OP-1 osteoinduction, including matrix and cell surface receptor binding of OP-1, latent OP-1 activation and clearance, and synergism/antagonism of OP-1 biological activity by other bone-related polypeptide growth factors. These studies will proceed in vivo with rat implants and in vitro with osteoblasts, determining whether OP-1 enhances osteoblastic expression directly, or acts through secondary paracrine factors. OP-1 is produced at low levels in bone and at high levels in kidney. Thus the mechanism by which the mineralized extracellular matrix sequesters and presents OP-1 to bone cells is critical to the understanding of bone cell biology as it relates to skeletal development, maintenance, and healing.
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