COUNTER-REGULATORY MECHANISMS IN BONE MINERALIZATION

通过基因敲除模型，已确定成骨细胞中存在的三种分子影响骨矿物质的控制沉积，即碱性磷酸酶(TNAP)、PC-1(或NPPS，一种核苷三磷酸焦磷酸水解酶同工酶，NTPPPH)及其基因产物。我们的初步结果表明，TNAP-/-低磷小鼠的原代成骨细胞在其基质小泡(MVS)中增加了无机焦磷酸(PPI)的水平，PPI是一种已知的矿化抑制物。PPI是由ANK蛋白的作用产生的。在这项建议中要检验的中心假设是，TNAP在PPI的骨降解中的关键作用是去除矿化抑制物。我们进一步假设PC-1不仅是TNAP功能的直接拮抗剂，而且ANK可能拮抗TNAP依赖的基质钙化。此外，我们认为，两种不同的骨骼TNAP拮抗剂PC-1和ANK的功能丧失将在体内改善与TNAP缺乏相关的骨软化。相反，我们认为与PC-1基因缺失和ANK基因缺陷型(ANK/ANK)小鼠相关的骨质疏松症将通过体内TNAP功能的丧失而得到改善。因此，我们的具体目标是：1.检验PC-1和TNAP缺陷通过杂交相互挽救的假设；2.检验ank/ank和TNAP缺失小鼠也通过杂交相互拯救的假设；3.检验细胞内和细胞外PPI水平是调节PPI产生、降解和分泌的基因(PC-1、TNAP和ANK)表达的中枢调节因子的假设。该提案将确定TNAP和PC-1/ANK通过对PPI代谢的影响而成为控制骨矿化的逆调控因子的潜力，从而也阐明了这些对PPI代谢的影响对TNAP、PC-1和ANK调节骨矿化能力的相对贡献。这些研究的完成将可能为基质矿化减少和增加的疾病提供治疗方法。

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.