CD44 MEDIATED CATABOLISM OF HYALURONAN BY CHONDROCYTES

描述（由申请人提供）：本提案的潜在新假设是CD44受体介导的透明质酸（HA）内吞作用为软骨细胞调节聚集蛋白多糖（PG）周转中的局部蛋白水解事件提供了一种机制。在目前的资助期内，我们已经确定软骨中HA的局部周转是通过cd44介导的内吞作用发生的，因为膜相关透明质酸酶对细胞外HA降解没有贡献。此外，我们已经确定含有ITEGE和dipen1的聚集蛋白G1结构域（在PG蛋白水解裂解后仍与HA结合的结构域）通过相同的cd44介导机制与HA共同内化。此外，我们还表明，当代谢发生改变时，由于IL-1治疗，软骨细胞变得更加“分解代谢”，CD44在蛋白质水平上的表达增加了6-8倍，这一变化使HA的内化增加了3倍。我们所做的另一个重要观察是，用完整的蛋白聚糖单体修饰的透明质酸不能内化。因此，要进行HA转换，PG在G1-G2结构域内或附近的切割是一个先决条件。我们提出完整的、高度硫酸化的PG与HA结合的存在建立了一种抑制cd44介导的HA内吞作用的抗性。然而，驱动内吞作用的活跃细胞介导过程也会产生PG聚集体的焦点聚集，为靶向PG降解奠定了基础。我们新的令人兴奋的数据表明，软骨细胞CD44聚集成脂质筏微域是内吞作用的先决条件。我们现在可以选择性地操纵cd44介导的HA内吞作用，并确定对PG降解率的最终影响。我们还将确定CD44是否作为MMP和ADAMTS蛋白酶的筏式锚定平台，这些蛋白酶最近被证明是活跃的膜结合酶。因此，本提案的重点是确定HA内吞作用和PG降解之间的机制联系。

DESCRIPTION (provided by applicant): The underlying new hypothesis of this proposal is that CD44 receptor-mediated endocytosis of hyaluronan (HA) provides a mechanism for chondrocytes to regulate local proteolytic events in the turnover of aggrecan proteoglycan (PG). During the current funding period we have determined that the local turnover of HA in cartilage occurs by CD44-mediated endocytosis, as there is no contribution of membrane-associated hyaluronidases to extracellular HA degradation. In addition we have determined that the ITEGE and DIPEN-containing aggrecan G1 domains-domains that remain bound to HA following proteolytic cleavage of PG, are co-internalized with HA by the same CD44-mediated mechanism. Furthermore, we have also shown that when metabolism is altered, and chondrocytes become more "catabolic" due to IL-1 treatment, there is an increase in CD44 expression by 6-8 fold at the protein level -- a change that increases the internalization of HA by 3 fold above control. Another critical observation that we made was that HA decorated with intact proteoglycan monomers cannot be internalized. Thus, for HA turnover to proceed, cleavage of PG within or near the G1-G2 domains is a prerequisite event. We propose that the presence of intact, highly-sulfated PG bound to HA establishes a resistance that inhibits CD44-mediated HA endocytosis. However the active cell-mediated processes that drive endocytosis also generate a focal clustering of PG aggregates, setting the stage for targeted PG degradation. Our new exciting data demonstrates that chondrocyte CD44 clusters into lipid raft microdomains as a prerequisite for endocytosis. We can now selectively manipulate CD44-mediated endocytosis of HA and determine the resultant effect on the rate of PG degradation. We will also determine whether CD44 serves as a raft-anchoring platform for MMP and ADAMTS proteinases- proteinases that recently have been shown to be present as active membrane-bound enzymes. Thus, the focus of this proposal is to determine the mechanistic link between HA endocytosis and PG degradation.