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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR038349-07
Application #
2079276
Study Section
Orthopedics and Musculoskeletal Study Section (ORTH)
Project Start
1987-05-01
Project End
1997-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Hauschka, P V; Damoulis, P D (1998) Functions of nitric oxide in bone. Biochem Soc Trans 26:39-44
Damoulis, P D; Hauschka, P V (1997) Nitric oxide acts in conjunction with proinflammatory cytokines to promote cell death in osteoblasts. J Bone Miner Res 12:412-22
Asahina, I; Sampath, T K; Hauschka, P V (1996) Human osteogenic protein-1 induces chondroblastic, osteoblastic, and/or adipocytic differentiation of clonal murine target cells. Exp Cell Res 222:38-47
Hunter, G K; Hauschka, P V; Poole, A R et al. (1996) Nucleation and inhibition of hydroxyapatite formation by mineralized tissue proteins. Biochem J 317 ( Pt 1):59-64
Neugebauer, B M; Moore, M A; Broess, M et al. (1995) Characterization of structural sequences in the chicken osteocalcin gene: expression of osteocalcin by maturing osteoblasts and by hypertrophic chondrocytes in vitro. J Bone Miner Res 10:157-63
Damoulis, P D; Hauschka, P V (1994) Cytokines induce nitric oxide production in mouse osteoblasts. Biochem Biophys Res Commun 201:924-31
Maliakal, J C; Asahina, I; Hauschka, P V et al. (1994) Osteogenic protein-1 (BMP-7) inhibits cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat osteosarcoma (17/2.8) cells. Growth Factors 11:227-34
Asahina, I; Sampath, T K; Nishimura, I et al. (1993) Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria. J Cell Biol 123:921-33
Taichman, R S; Hauschka, P V (1992) Effects of interleukin-1 beta and tumor necrosis factor-alpha on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro. Inflammation 16:587-601
Spampata, R; Werther, J R; Hauschka, P V (1992) Accelerated endochondral osteoinduction in the absence of bone matrix particles in a rat model system. J Oral Maxillofac Surg 50:140-51;discussion 151-2

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