REGULATION OF MINERALIZATION IN SKELETAL TISSUES

生物矿化在骨骼和牙齿发育过程中起着至关重要的作用，使骨骼组织在成年期发挥其应有的功能。尽管它的重要性显而易见，但人们对其监管知之甚少。组织中矿物质形成的调节对于它们的正常功能至关重要。例如，过多的矿物质沉积伴随着动脉粥样硬化和骨关节炎。在后者中，关节表面区的晶体形成可能在炎症的发生和关节破坏的进展中起主要作用。另一方面，骨质疏松症中的矿物质丢失会导致脆弱的骨骼和骨折。基质囊泡是一种被膜包裹的小颗粒，在许多组织中具有启动矿化的关键作用，包括头面部骨骼、长骨、软骨和牙本质。虽然已经确定这些颗粒是从质膜中释放出来的，但尚不清楚基质小泡的释放是如何调节的，以及它们释放到细胞外基质后，小泡如何启动矿物形成。我们的初步研究首次表明，只有经历矿化的细胞才会释放富含膜联蛋白II和V的含钙/磷复合体(核核心)的基质小泡，这些小泡能够启动矿化，而非矿化细胞释放不能矿化的小泡。此外，我们提供的证据表明，膜联蛋白II和V在基质小泡中形成钙通道，允许钙离子流入这些颗粒，并形成和生长第一个腔内晶体。这些富含膜联蛋白II和V的基质小泡的释放伴随着膜联蛋白II和V表达的增加以及细胞内钙离子浓度的增加。我们的假设是：(I)细胞内钙浓度以及膜联蛋白II和V的表达都是释放具有矿化能力的基质小泡所必需的，(Ii)膜联蛋白II和V与核合作，使钙离子进入囊泡，形成第一矿物相，并启动矿化。该项目将通过使用不同的实验策略直接验证这些假设，包括细胞培养、钙通道研究、定点突变和突变的膜联蛋白分子的表达。目前的提议将为体内矿化调控的一些最有趣和最重要的特征提供新的见解。这些信息对于在病理条件下防止几种组织中不受控制的矿化至关重要。

Biomineralization plays a crucial role during skeletal and tooth development and allows skeletal tissues to exert their proper functions during adulthood. Despite its obvious importance little is known about its regulation. Regulation of mineral formation in tissues is critical for their proper function. For example, excessive mineral deposition accompanies atheriosclerosis and osteoarthritis. In the latter, crystal formation in the articular surface area may play a major role in the onset of inflammation and the progression of joint destruction. On the other hand, loss of mineralization as seen in osteoporosis leads to fragile bones and bone fractures. Matrix vesicles, small membrane-enclosed particles, have the critical role of initiating mineralization in many tissues, including craniofacial bones, long bones, cartilage and dentin. While it is well established that these particles are released from the plasma membrane, it is unclear how the release of matrix vesicles is regulated and how, following their release into the extracellular matrix, vesicles initiate mineral formation. In our Preliminary Studies it is shown for the first time that only cells undergoing mineralization release annexins II and V-rich, Ca2+/Pi complexes (nucleational core)-containing matrix vesicles which are able to initiate mineralization, while nonmineralizing cells release vesicles which do not mineralize. In addition, we provide evidence that annexins II and V form Ca2+ channels in matrix vesicles, allowing Ca2+ influx into these particles and the formation and growth of the first intralumenal crystals. Release of these annexins II and V-rich matrix vesicles is accompanied by increases in annexins II and V expression and cytosolic Ca2+ concentration, [Ca2+]i. Our hypotheses are that (i) increases in both cytosolic Ca2+ concentration and annexins II and V expression are required for the release of mineralization-competent matrix vesicles, and that (ii) annexins II and V cooperate with the nucleational core to enable Ca2+ influx into the vesicles, formation of the first mineral phase and initiation of mineralization. This project will test these hypotheses directly by the use of diverse experimental strategies, including cell culture, Ca2+ channel studies, site-directed mutagenesis and expression of mutated annexin molecules. The present proposal will provide new insights into some of the most intriguing and important features which regulate mineralization in vivo. This information will be of critical importance for the prevention of uncontrolled mineralization in several tissues during pathological conditions.