Mechanisms of initation of skeletal mineralization

骨骼矿化的引发机制

• 批准号: 7210171
• 负责人: JOSE LUIS MILLAN
• 金额: $ 36.6万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7210171
• 关键词: bone; calcification; collagen; extracellular; genes; model; normal ossification; osteoblasts; tissues

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.

描述（由申请人提供）：软骨和骨骼的矿化是通过一系列物理化学和生化过程发生的，这些过程共同促进了细胞外基质（ECM）特定区域中羟基磷灰石的沉积。实验证据表明，沿ECM中的胶原原纤维以及软骨细胞和成骨细胞衍生的基质囊泡（MVS）的液体存在羟基磷灰石（HA）晶体。我们的工作模型是，骨骼矿化首先是在MVS的腔内启动的。在第二步中，HA晶体生长在MV的范围之外，并暴露于细胞外环境中，在那里它们继续沿着胶原纤维传播。我们最近的数据表明，非特异性碱性磷酸酶（TNAP）在限制细胞外无机焦磷酸盐（PPI）（一种矿化抑制剂）中起着至关重要的作用，以维持正常骨矿化的PI/PPJ比率。使用多种单基因敲除实验，我们发现缺乏TNAP功能的小鼠，即Akp2 - / - 小鼠，由于在细胞外PPJ浓度的增加引起的MVS之外的HA晶体传播引起的HA晶体传播引起的骨质乳酸。但是，在MVS内部，HA晶体仍然存在于Akp2 - / - 小鼠中。我们还发现，TNAP和I型胶原蛋白的共表达对于引起任何ECM的矿化是必要的，表明骨ECM中HA晶体的传播密切取决于I型胶原蛋白的存在。但是，为什么AKP2 - / - 小鼠出生时带有矿物质骨骼并在其MV中有HA晶体？我们假设MVS中存在的一种新鉴定的称为Phospho1的可溶性磷酸酶负责增加MVS内PI的局部浓度，以改变PI/PPI之比，以有利于HA种子晶体的沉淀。我们将使用遗传和药理学方法来影响MV介导的矿化的第一和第二步骤来检验这一假设。在实验上，我们将表征与AKP2 - / - 小鼠相比，缺乏磷酸表达的小鼠的矿化异常和相关代谢变化，并评估磷酸1和AKP2基因对骨骼矿化的同时失活的影响。我们还将研究消融或抑制磷酸1和/或TNAP活性对成骨细胞衍生的MV在体外启动和传播钙化的能力的影响。我们的工作将提供对正常骨矿化机制的基本见解。我们的项目还将生产有价值的工具和试剂，这些工具和试剂将促进未来的研究，旨在了解各种骨矿化和软组织骨化异常，包括一些极大的公共健康问题疾病，例如骨关节炎，骨质疏松症和动脉钙化。