Mechanisms of initation of skeletal mineralization

骨骼矿化的引发机制

• 批准号: 7902149
• 负责人: JOSE LUIS MILLAN
• 金额: $ 32.72万
• 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 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7902149
• 关键词: Affect; Alkaline Phosphatase; Area; Biochemical Process; Cartilage; Cells; Chemicals; Chondroblast; Collagen; Collagen Fibril; Collagen Type I; Data; Degenerative polyarthritis; Deposition; Development; Diphosphates; Disease; Extracellular Matrix; Future; Genes; Genetic; Hydroxyapatites; In Vitro; Mediating; Metabolic; Modeling; Mus; Osteoblasts; Osteomalacia; Osteoporosis; Phosphoric Monoester Hydrolases; Physiologic calcification; Play; Precipitation; Public Health; Reagent; Research Personnel; Role; Seeds; Series; Skeleton; Small Interfering RNA; Testing; Tissues; Vesicle; Work; bone; calcification; extracellular; in vivo; inhibitor/antagonist; insight; knockout gene; mineralization; mouse model; research study; skeletal; soft tissue; tool

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，这些过程共同促进羟基磷灰石在细胞外基质（ECM）的特定区域中的沉积。实验证据表明，沿着 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 (MV) 的管腔内存在羟基磷灰石 (HA) 晶体。我们的工作模型是骨矿化首先在 MV 管腔内启动。第二步，HA 晶体生长超出 MV 的范围，并暴露于细胞外环境，在那里它们继续沿着胶原纤维繁殖。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)（一种矿化抑制剂）的浓度以维持正常骨矿化所需的 Pi/PPj 比率方面发挥着至关重要的作用。通过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 增加而导致 MV 外 HA 晶体增殖停滞，从而表现出骨软化症。浓度。然而，在 Akp2-/- 小鼠的 MV 内部，HA 晶体仍然存在。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，这表明 HA 晶体在骨 ECM 中的增殖密切依赖于 I 型胶原蛋白的存在。但为什么 Akp2-/- 小鼠天生就有矿化骨骼，并且其 MV 中含有 HA 晶体？我们假设 MV 中存在一种新鉴定的可溶性磷酸酶 PHOSPHO1，它负责增加 MV 内 Pi 的局部浓度，从而改变 Pi/PPi 比率，有利于 HA 晶种的沉淀。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检验这一假设。通过实验，我们将表征与 Akp2-/- 小鼠相比，Phospho1 表达缺陷的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。我们还将研究消除或抑制 PHOSPHO1 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。我们的工作将为正常骨矿化机制提供基本见解。我们的项目还将生产有价值的工具和试剂，以促进未来的研究，旨在了解各种骨矿化和软组织骨化异常的发展，其中包括一些引起重大公共卫生关注的疾病，例如骨关节炎、骨质疏松症和动脉钙化。