BIOLOGICAL EFFECTS OF BASIC CALCIUM PHOSPHATE CRYSTALS

美国人口普查局估计，到2000年，将有3500万美国人65岁或以上。骨关节炎和假性关节炎是最常见的衰老疾病。结晶二水合焦磷酸钙（CPPD）和碱性磷酸钙（BCP）是两种最常见的病理性关节矿物质。每一种都经常发生在OA关节中，并且每一种都可能是炎症性的，引起急性假性关节炎（CPPD）和急性钙化性周炎（BCP）。这两种钙晶体关节炎的发病率都随着年龄的增长而显著增加。本提案的总体目标是确定与关节组织中CPPD和BCP晶体沉积相关的退行性关节病的发病机制，最终目标是开发一种合理的方法来预防或逆转这种沉积的后果。我们的研究范式是晶体会导致关节组织退化。晶体诱导成纤维细胞样滑膜细胞增殖并产生金属蛋白酶[MMP]和前列腺素[PG]，最终导致关节组织变性。磷酸柠檬酸盐是钙晶体的一种特殊抑制剂，可以逆转晶体的退化作用。我们正在申请资金来阐明晶体和质膜相互作用的机制以及随后导致各种转录因子激活的信号通路，最终导致MMP合成和有丝分裂。总体假设是晶体与质膜中特定成分的相互作用主要通过Ras/ erk1和2丝裂原活化蛋白激酶(MAPK)/血清反应因子(SRF)/c-fos /AP-1依赖性信号通路导致MMP表达。关于晶体如何形成以及它们如何发挥其生物效应的新信息可能会带来新的治疗策略，这些策略可能会改善退化过程。我们将检验5个相互关联的子假设：假设1：晶体对细胞的激活是由于晶体表面与细胞膜的特定成分（如磷脂）的物理相互作用。假设2:SRF介导BCP晶体诱导的细胞活化。假设3:BCP晶体通过ras依赖的p42/44 MAPK信号转导途径诱导MMP-1。假设4：在MMP-1和-13的启动子中有特定的顺式元件调节晶体诱导的MAP合成。假设5：组织金属蛋白酶抑制剂（Tissue Inhibitor of Metalloproteinase， TIMP）-2启动子中有特定的顺式元件调节晶体抑制TIMP- 2的表达。

The US Census Bureau estimated that 35 million American will be 65 years or older by the Year 2000. Osteoarthritis [OA] and pseudogout are the most common afflictions of aging. Crystalline calcium pyrophosphate dihydrate [CPPD] and basic calcium phosphate [BCP] are the 2 most common forms of pathologic articular mineral. Each occurs frequently in OA joints, and each may be phlogistic, causing acute attacks of pseudogout (CPPD) and acute calcific periarthritis (BCP). The incidence of both calcium crystal arthritides significantly increases with age. The overall objective of this proposal is to determine the pathogenesis of the degenerative arthropathies associated with the deposition of CPPD and BCP crystals in articular tissues with the eventual goal of developing a rational means of preventing or reversing the consequences of such deposition. The paradigm of our work is that crystals can cause the degeneration of articular tissues. Crystals induce fibroblast-like synoviocytes to proliferate and produce metalloproteinases [MMP] and prostaglandins [PG], which eventually cause the degeneration of articular tissues. Phosphocitrate, a specific inhibitor of calcium crystals, reverses the degenerative effects of crystals. We are requesting funding to elucidate the mechanism of interaction of crystals and plasma membrane and the subsequent signal pathways leading the activation of various transcription factors and ultimately MMP synthesis and mitogenesis. The overall Hypothesis is that the interaction of crystals with specific components in plasma membrane resulted in MMP expression primarily through a Ras/ERK 1 and 2 Mitogen-activated Protein Kinase (MAPK)/Serum Response Factor (SRF)/c-fos /AP-1 dependent signaling pathway. New treatment strategies that might ameliorate the degenerative process may ensue from new information about how crystals form and how they exert their biologic effects. We shall test 5 inter-related sub-hypothesis: Hypothesis 1: Cell activation by crystals is due to the physical interaction of crystal surface with specific components of the cell membrane e.g. phospholipid. Hypothesis 2: SRF mediates BCP crystal- induced cell activation. Hypothesis 3: BCP crystals induce MMP-1 via a Ras-dependent p42/44 MAPK signal transduction pathway. Hypothesis 4: There are specific cis elements in the promoter of MMP-1 and -13 that regulates crystal-induced MAP synthesis. Hypothesis 5: There are specific cis elements in the promotor of Tissue Inhibitor of Metalloproteinase (TIMP)-2 that regulate crystal inhibition of TIMP- 2 expression.