Mechanisms of initation of skeletal mineralization

描述（由申请人提供）：软骨和骨的矿化是通过一系列物理化学和生物化学过程发生的，这些过程共同促进了羟基磷灰石在细胞外基质（ECM）特定区域的沉积。实验证据表明，羟基磷灰石（HA）晶体沿着ECM中的胶原原纤维存在，并且在成软骨细胞和成骨细胞来源的基质囊泡（MVs）的管腔内也存在。我们的工作模型是骨矿化首先在骨髓腔内开始。在第二步中，透明质酸晶体生长超出mv的范围，并暴露于细胞外环境中，在那里它们继续沿着胶原原纤维繁殖。我们最近的数据表明，组织非特异性碱性磷酸酶（TNAP）在限制细胞外无机焦磷酸盐（PPi）的浓度方面起着至关重要的作用，PPi是一种矿化抑制剂，可以维持正常骨矿化所需的Pi/PPj比率。通过多种单基因和双基因敲除实验，我们发现，缺乏TNAP功能的小鼠，即Akp2-/-小鼠，由于细胞外PPj浓度增加导致MVs外HA晶体的繁殖受阻而表现出骨软化症。然而，在mv中，Akp2-/-小鼠体内仍然存在HA晶体。我们还发现，TNAP和I型胶原蛋白的共同表达对于任何ECM的矿化都是必要的，这表明骨ECM中HA晶体的传播密切依赖于I型胶原蛋白的存在。但是，为什么Akp2-/-小鼠出生时骨骼矿化，并且在它们的mv中有透明质酸晶体？我们假设，一种新发现的可溶性磷酸酶PHOSPHO1存在于mv中，负责增加mv内Pi的局部浓度，从而改变Pi/PPi比率，有利于HA种子晶体的沉淀。我们将通过使用遗传和药理学方法影响mv介导的矿化的第一步和第二步来测试这一假设。通过实验，我们将描述与Akp2-/-小鼠相比，Phospho1表达不足小鼠的矿化异常和相关代谢变化，并评估Phospho1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MVs在体外启动和增殖钙化能力的影响。我们的工作将为正常骨矿化的机制提供基本的见解。我们的项目还将产生有价值的工具和试剂，以促进未来的研究，旨在了解各种骨矿化和软组织骨化异常的发展，包括一些具有重大公共卫生关注的疾病，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.