In contribution of our past research interests, we plan to carry out an integrated series of experiments designed to understand the regulation and function of Matrix Gla Protein (MGP). Matrix Gla protein is a 10 kDa protein which contains 5 residues of the vitamin K-dependent Ca2+-binding amino acid, gamma-carboxyglutamic acid (Gla). MGP was originally discovered in bone but is now known to be synthesized by all vertebrate tissues tested, with the highest levels of synthesis in ling, heart, kidney, and cartilage. We recently sequenced the human MGP gene and discovered a putative retinoic acid responsive element (RARE) in the MGP promoter which is virtually identical to the RARE recently identified in the retinoic acid receptor B (RARB) promoter. We subsequently found that retinoic acid (RA) treatment increased the level of MGP mRNA by over 10- fold in all human cells tested, which include normal and transformed bone cells, articular cartilage cells, and dermal fibroblasts. We plan to assess the MGP mRNA response to RA in a number of additional human cells. We also plan to investigate the molecular mechanisms of the MGP mRNA response to RA, including: the use of CAT constructs of defined portions of the MGP gene promoter to identify the sequence(s) that mediate retinoic acid responsiveness; the use of mobility shift DNA binding assays, methylation interference assays, and site directed mutagenesis to characterize the RARE in the hMGP gene; and the use of cotransfection of the MGP gene promoter and expression vectors for the different RAR's to characterize the receptor specificity of the response. To better understand the physiological significance of the MGP mRNA response to RA, we will determine the effect of RA administration and vitamin A deficiency on MGP synthesis in the rat. We will also investigate the possible regulation of MGP synthesis by transcription factors that may act through the putative AP1 and AP2 sites in the MGP gene promoter, and by growth factors such as TGFBeta and FGF. We will further investigate the proteolytic cleavage at the Arg 80-Arg81 dibasic site which may precede MGP secretion from the cell and the coagulation-related cleavages in blood at the Arg18-Arg19 site in order to determine the effect of these cleavages on biochemical properties of the protein which may be important to its function, including Ca2+- dependent and Gla-dependent binding to phospholipid vesicles, and the ability of the protein to strongly self associate. We plan to identify the macromolecule(s) which bind MGP in cartilage and in plasma and to use immunohistology to localize MGP antigen in the extracellular matrix of tissues and cells. We will test our hypothesis that MGP is a locally acting factor that mediates actions of RA on differentiation and development by investigating the binding of labeled MGP to target cells and the effects of binding on the cell, as well as the effect of vitamin K-deficiency on possible functions of MGP, particularly those associated with RA. Finally, we will measure the levels of plasma MGP in people with diseases which may involve altered synthesis of the protein and in children who are vitamin A deficient, and will further investigate the strong age dependence of plasma MGP in men and women.
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