Three molecules present in osteoblasts have been identified, by means of gene knock-out models, as affecting the controlled deposition of bone mineral i.e., alkaline phosphatase (TNAP); PC-1 (or Npps, a nucleoside triphosphate pyrophosphate hydrolase isozyme, NTPPPH) and the and gene product. Our preliminary results indicate that primary osteoblasts from TNAP-/- hyphosphastasia mice have increased levels of inorganic pyrophosphate (PPi), a known inhibitor of mineralization, in their matrix vesicles (MVs). PPi is produced by the action of the ANK protein. The central hypotheses to be tested in this proposal are that TNAP's key function in bone degradation of PPi to remove the mineralization inhibitor. We further hypothesize that PC-1 is a direct antagonist of TNAP function but also that ANK may antagonize TNAP-dependent matrix calcification. Furthermore, we propose that loss of function of two distinct skeletal TNAP antagonists, PC-1 and ANK, will ameliorate TNAP deficiency-associated osteomalacia in vivo. Conversely, we propose that the hyperossification associated with both PC-1 null mice and ANK-deficient (ank/ank) mice will be ameliorated by loss of function of TNAP in vivo. Thus, our Specific Aims are: I. To test the hypothesis that the PC-1 and TNAP deficiencies are mutually rescued by cross-breeding; II. To test the hypothesis that ank/ank and TNAP null mice are also mutually rescued by cross breeding; III. To test the hypothesis that levels of intracellular and extracellular PPi are central regulators of the expression of the genes (PC-1, TNAP, and ANK) that regulate PPi production, degradation and secretion. The proposal will define the potential for TNAP and PC-1/ANK to serve as counter-regulatory factors controlling bone mineralization through effects on PPi metabolism, and thereby also address the relative contribution of the effects on PPi metabolism to the ability of TNAP, PC-1 and ANK to regulate bone mineralization. Completion of these studies will potentially provide therapeutic approaches to disorders of both decreased and increased matrix mineralization.
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