Mechanisms of initation of skeletal mineralization

描述(申请人提供)：软骨和骨骼的矿化通过一系列物理化学和生化过程共同促进羟基磷灰石在细胞外基质(ECM)的特定区域沉积。实验证据表明，羟基磷灰石(HA)晶体沿胶原纤维存在于ECM中，也存在于软骨母细胞和成骨细胞来源的基质小泡(MVS)的管腔内。我们的工作模型是，骨矿化首先在MVS的管腔内启动。在第二步中，HA晶体超出MVS的限制生长，并暴露在细胞外环境中，在那里它们继续沿着胶原纤维生长。我们最近的数据表明，组织非特异性碱性磷酸酶(TNAP)在限制矿化抑制剂细胞外无机焦磷酸(PPI)的浓度以维持正常骨矿化所允许的PI/PPJ比率方面起着至关重要的作用。利用各种单基因和双基因敲除实验，我们发现TNAP功能缺陷的小鼠，即Akp2-/-小鼠，由于细胞外ppj浓度增加导致HA晶体在MVS外的生长受阻而表现出骨软化。然而，在MVS内，Akp2-/-小鼠体内仍然存在HA晶体。我们还发现，TnAP和I型胶原的共同表达是导致任何ECM矿化所必需的，这表明HA晶体在骨ECM中的生长密切依赖于I型胶原的存在。但为什么Akp2-/-小鼠出生时就有矿化的骨骼，并且在它们的MVS中有HA晶体？我们推测，存在于MVS中的一种名为PHOSPHO1的新发现的可溶性磷酸酶负责增加MVS中PI的局部浓度，以改变PI/PPI的比率，从而有利于HA种子晶体的沉淀。我们将使用遗传学和药理学方法，通过影响MV介导的矿化的第一步和第二步来检验这一假说。在实验上，我们将与Akp2-/-小鼠相比，描述Phosph1表达缺陷小鼠的矿化异常和相关的代谢变化，并评估Phosphone1和Akp2基因同时失活对骨骼矿化的影响。我们还将研究消融或抑制PHOSPHO1和/或TNAP活性对成骨细胞来源的血管在体外启动和增殖钙化能力的影响。我们的工作将对正常骨矿化的机制提供基本的见解。我们的项目还将生产有价值的工具和试剂，以促进未来旨在了解各种骨矿化和软组织骨化异常发展的研究，这些异常包括一些严重影响公众健康的疾病，如骨关节炎、骨质疏松症和动脉钙化。

DESCRIPTION (provided by applicant): Mineralization of cartilage and bone occurs by a series of physicochemical and biochemical processes that together facilitate the deposition of hydroxyapatite in specific areas of the extracellular matrix (ECM). Experimental evidence has pointed to the presence of hydroxyapatite (HA) crystals along collagen fibrils in the ECM and also within the lumen of chondroblast- and osteoblast-derived matrix vesicles (MVs). Our working model is that bone mineralization is first initiated within the lumen of MVs. In a second step, HA crystals grow beyond the confines of the MVs and become exposed to the extracellular milieu where they continue to propagate along collagen fibrils. Our recent data have indicated that tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role in restricting the concentration of extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor, to maintain a Pi/PPj ratio permissive for normal bone mineralization. Using a variety of single and double gene knockout experiments we have found that mice deficient in TNAP function, i.e., Akp2-/- mice, display osteomalacia due to an arrest in the propagation of HA crystals outside the MVs caused by an increase in extracellular PPj concentrations. Inside the MVs, however, HA crystals are still present in Akp2-/- mice. We have also found that the co-expression of TNAP and type I collagen is necessary to cause mineralization of any ECM indicating that propagation of HA crystals in the bone ECM is intimately dependent on the presence of type I collagen. But why are Akp2-/- mice born with a mineralized skeleton and have HA crystals in their MVs? We hypothesize that a newly identified soluble phosphatase called PHOSPHO1, present in the MVs, is responsible for increasing the local concentration of Pi inside the MVs to change the Pi/PPi ratio to favor precipitation of HA seed crystals. We will test this hypothesis by affecting the first and second steps of MV-mediated mineralization using a genetic and pharmacological approach. Experimentally we will characterize the mineralization abnormalities and related metabolic changes in mice deficient in Phospho1 expression compared to Akp2-/- mice and assess the effect of the simultaneous inactivation of the Phospho1 and Akp2 genes on skeletal mineralization. We will also study the effects of ablating or inhibiting PHOSPHO1 and/or TNAP activity on the ability of osteoblast- derived MVs to initiate and propagate calcification in vitro. Our work will provide fundamental insights into the mechanisms of normal bone mineralization. Our project will also produce valuable tools and reagents that will facilitate future studies aimed at understanding the development of diverse bone mineralization and soft tissue ossification abnormalities that include some diseases of great public health concern, e.g., osteoarthritis, osteoporosis, and arterial calcification.