COUNTER-REGULATORY MECHANISMS IN BONE MINERALIZATION

描述（由申请人提供）：我们的工作表明，由于组织非特异性碱性磷酸酶（TNAP）和纤维状胶原在骨和软骨中的共同表达，钙化通常仅限于骨组织。TNAP的主要功能是降解细胞外无机焦磷酸盐（ePPj），这是一种有效的矿化抑制剂，它通过核苷酸焦磷酸酶/磷酸二酯酶-1 （NPP1）的酶活性在胞外产生，并通过强直蛋白（ANK）的作用运输到细胞外环境。缺乏TNAP （Akp2-/-）的小鼠会出现低磷酸症，其特征是ePPj水平大幅升高，从而导致骨软化。然而，同时消融NPP1或ANK功能，即[Akp2-/-；Enpp1-/-]和[Akp2-/-；ank/ank]小鼠，导致ePPj水平的纠正和骨软化症的改善。我们还发现了ePPj和骨桥蛋白（OPN）水平之间的严格相关性，骨桥蛋白是另一种有效的矿化抑制剂，我们发现ePPj上调成骨细胞中OPN基因的表达。然而，在缺乏OPN的情况下，小鼠不会发生骨软化症，但事实上，尽管它们比缺乏tnap的小鼠具有更多的epi，但它们的骨软化症实际上是轻微的高矿化。在这个竞争性更新申请中，我们将重点测试一个中心假设，即Pi/PPi介导的OPN调控和OPN介导的Pi/PPi调控是相互关联的反调控机制，控制这两种重要的矿化抑制剂OPN和ePPi的浓度。我们的具体目标是验证以下假设：1)OPN控制产生、运输和降解epi的分子的表达，并以此作为成骨细胞感知和响应Pi/PPi比率变化的反馈机制；2)在Opn-/-小鼠中存在代偿性epi的过量产生，这有助于避免骨骼病理。我们预测，在Opn-/-小鼠中，上调ePPj水平将导致骨软化，而降低ePPj水平将导致高矿化；3) epi和OPN水平的联合正常化导致了[Akp2-/-]钙化的纠正；Enpp1-/-和[Akp2-/-；⊙用途制造ank /]老鼠;4)在ePPi-OPN综合反调控机制中，TNAP发挥着多种相互关联的功能，包括：a)参与Pj库；b)通过降解和产生PPj参与ePPi库，从而与NPP1的功能竞争；c)通过去磷酸化调节OPN的生物学功能。我们的工作将阐明PPj和OPN在健康和疾病中作为钙化抑制剂的协同作用和分层作用，以及骨形成细胞能够响应这些抑制剂浓度变化以维持生理稳态的机制。

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.