Abstract

This proposal examines the role of proteolipids in biologic mineral formation and the mechanism of their action in situ. Matrix vesicle proteolipid (MVP) produced by rat chondrocytes in culture is used as a model system to test the hypothesis that MVP is a site of initial hydroxyapatite formation and that changes in the membrane environment, such as those produced by the action of vitamin D, will alter its functional properties. A model membrane system will be used to mimic the membrane environment in vitro. MVP will be purified by differential extraction in organic solvents of varying polarities, reverse phase and chromatofocusing HPLC. Heterogeneity will be assessed by ISF and SDS-PAGE. Polyclonal antibodies generated to MVP will be affinity purified and then used to affinity purify specific subsets of MVP, to characterize multimeric aggregates, to purify mRNA for individual proteolipids, and to chart MVP synthesis and incorporation into matrix vesicles. MVP will be incorporated into planar bilayers of known composition and ion transport compared to that of the intact matrix vesicle membrane. The ability of specific functional units to support hydroxyapatite formation will be tested in vitro. To examine the effect of regulation of the membrane microenvironment on MVP structure and function in culture and in vitro, reserve zone (RC) or growth region (GC) chondrocytes will be incubated with 1,25(OH)2D3 or 24R,25(OH)2D3. Changes in matrix vesicle and plasma membrane fluidity will be assessed using flourescence spectroscopy; membrane structure and function monitored by enzymatic activity (alkaline phosphatase, 5' nucleotidase, Na+/K+ ATPase) and phospholipid composition. Incorporation of vitamin D into the membrane will be measured using 3H-labeled metabolites. Phospholipid turnover will also be assessed as acylation and hydrolysis of radiolabeled fatty acids. Synthesis of MVP and its incorporation into matrix vesicles will be charted using specific antibodies. Membrane and MVP function will be measured using planar bilayer membranes or by in vitro hydroxyapatite formation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
2R01DE005937-07A1
Application #
3219662
Study Section
Oral Biology and Medicine Study Section (OBM)
Project Start
1981-03-01
Project End
1991-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
7
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
University of Texas Health Science Center San Antonio
Department
Type
Schools of Dentistry/Oral Hygn
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229

  Publications

Kinney, R C; Schwartz, Z; Week, K et al. (2005) Human articular chondrocytes exhibit sexual dimorphism in their responses to 17beta-estradiol. Osteoarthritis Cartilage 13:330-7
Schwartz, Z; Carney, D H; Crowther, R S et al. (2005) Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation. J Cell Physiol 202:336-43
Schwartz, Z; Graham, E J; Wang, L et al. (2005) Phospholipase A2 activating protein (PLAA) is required for 1alpha,25(OH)2D3 signaling in growth plate chondrocytes. J Cell Physiol 203:54-70
Gay, I; Schwartz, Z; Sylvia, V L et al. (2004) Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix. Biochim Biophys Acta 1684:18-28
Boyan, B D; Schwartz, Zvi (2004) Rapid vitamin D-dependent PKC signaling shares features with estrogen-dependent PKC signaling in cartilage and bone. Steroids 69:591-7
Boyan, B D; Jennings, E G; Wang, L et al. (2004) Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. J Steroid Biochem Mol Biol 89-90:309-15
Schwartz, Z; Shaked, D; Hardin, R R et al. (2003) 1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid. Steroids 68:423-37
Boyan, Barbara D; Sylvia, V L; McKinney, N et al. (2003) Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice. J Cell Biochem 90:1207-23
Lohmann, C H; Schwartz, Z; Liu, Y et al. (2003) Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells. J Orthop Res 21:326-34
Boyan, B D; Sylvia, V L; Frambach, T et al. (2003) Estrogen-dependent rapid activation of protein kinase C in estrogen receptor-positive MCF-7 breast cancer cells and estrogen receptor-negative HCC38 cells is membrane-mediated and inhibited by tamoxifen. Endocrinology 144:1812-24

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