COUNTER-REGULATORY MECHANISMS IN BONE MINERALIZATION

描述(申请人提供)：我们的研究表明，钙化通常仅限于骨骼组织，这是由于组织非特异性碱性磷酸酶(TNAP)和纤维性胶原蛋白在骨和软骨中共同表达。TNAP的主要功能是降解胞外无机焦磷酸(EPPj)，ePPj是一种强有力的矿化抑制剂，由核苷酸焦磷酸酶/磷酸二酯酶-1(NPP1)的胞外活性产生，也通过强直蛋白(ANK)的作用运输到细胞外环境。缺乏TNAP(Akp2-/-)的小鼠会出现以ePPj水平显著升高为特征的低磷酸盐血症，从而导致骨软化。然而，同时消融NPP1或ANK功能，即在[Akp2-/-；Enpp1-/-]和[Akp2-/-；ank/ank]小鼠中，导致ePPj水平的纠正和骨软化的改善。我们还发现ePPj的水平与另一种有效的矿化抑制物骨桥蛋白(OPN)之间存在严格的相关性，我们发现ePPj上调成骨细胞中OPN基因的表达。然而，在没有OPN的情况下，小鼠不会发生骨软化症，事实上，尽管它们的Eppi比缺乏TNAP的小鼠有更多的Eppi，但它们实际上略有高矿化。在这一竞争性更新应用中，我们将重点检验这一中心假设，即PI/PPI介导的OPN调节和OPN介导的PI/PPI调节是控制这两个重要矿化抑制剂OPN和Eppi浓度的逆调节机制。我们的具体目标是检验假设：1)OPN控制产生、运输和降解Eppi的分子的表达，并在这样做的过程中作为反馈机制，使成骨细胞能够感知PI/PPI比率的变化并做出反应；2)在OPN-/-小鼠中，存在代偿性过量的Eppi，有助于避免骨骼病理。我们预测，上调Eppi水平将导致骨软化，而降低ePPj水平将导致OPN-/-小鼠的高矿化；3)正是Eppi和OPN水平的结合正常化导致[Akp2-/-；Enpp1-/-和[Akp2-/-；ank/ank]小鼠的钙化纠正；4)TNAP在整合的Eppi-OPN反调节机制中发挥多种相互关联的功能，包括a)促进PJ池，b)通过降解和产生PPj而促进Eppi池，从而与NPP1-/-和c)通过脱磷调节OPN的功能竞争。我们的工作将阐明ppj和OPN作为钙化抑制剂在健康和疾病中的协同和分级作用，以及骨形成细胞能够响应这些抑制剂浓度的变化以维持生理动态平衡的机制。

DESCRIPTION (provided by applicant): Our work has shown that calcification is normally restricted to skeletal tissue due to the co-expression in bone and cartilage of tissue-nonspecific alkaline phosphatase (TNAP) and fibrillar collagens. TNAP's primary function is to degrade extracellular inorganic pyrophosphate (ePPj), a potent mineralization inhibitor, which is produced ectoplasmically by the enzymatic activity of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and also transported to the extracellular milieu by the action of the ankylosis protein (ANK). Mice deficient in TNAP (Akp2-/-) develop hypophosphatasia characterized by greatly elevated levels of ePPj, which lead to osteomalacia. However, the simultaneous ablation of NPP1 or ANK function, i.e., in [Akp2-/-; Enpp1-/-] and [Akp2-/-; ank/ank] mice, lead to a correction in ePPj levels and an amelioration of osteomalacia. We have also discovered a strict correlation between the levels of ePPj and osteopontin (OPN), another potent inhibitor of mineralization and we uncovered that ePPj upregulates Opn gene expression in osteoblasts. However, in the absence of OPN, mice do not develop osteomalacia but in fact slightly hypermineralize despite them having even larger amounts of ePPi than TNAP-deficient mice. In this competitive renewal application we will focus on testing the central hypothesis that the Pi/PPi-mediated regulation of OPN and the OPN-mediated regulation of Pi/PPi are linked counter-regulatory mechanisms that control the concentrations of these two important mineralization inhibitors, OPN and ePPi. Our Specific Aims are to test the hypotheses: 1) that OPN controls the expression of the molecules that produce, transport and degrade ePPi and in so doing acts as a feedback mechanism to the ability of osteoblasts to sense and respond to changes in the Pi/PPi ratio; 2) that in Opn-/- mice there is a compensatory overproduction of ePPi that serves to avert skeletal pathology. We predict that manipulating ePPi levels upwards will lead to osteomalacia while reducing ePPj levels will cause hypermineralization in Opn-/- mice; 3) that it is the combined normalization of ePPi and OPN levels that lead to corrections in calcification in the [Akp2-/-; Enpp1-/- and [Akp2-/-; ank/ank] mice; 4) that TNAP plays multiple, interrelated functions in the integrated ePPi-OPN counter-regulatory mechanism including a) contributing to the Pj pool, b) contributing to the ePPi pool by both degrading and producing PPj, and thus competing with the function of NPP1 and c) modulating the biological function OPN through dephosphorylation. Our work will clarify the synergistic and hierarchical role of PPj and OPN as inhibitors of calcification in health and disease and the mechanisms by which bone-forming cells are able to respond to changes in the concentrations of these inhibitors to maintain physiological homeostasis.