Osteoarthritis is a severe debilitating disease manifested by degeneration of articular cartilage-lining joint cavities. The maintenance of healthy cartilage depends on balancing the synthetic and degradative processes involved in turnover of cartilage extracellular matrix. Cartilage extracellular matrices are composed predominately of collagen and proteoglycans tethered to single filaments of another matrix macromolecule, hyaluronan (HA). This HA, in turn, is bound or anchored directly to the chondrocyte membrane via interaction with an HA receptor protein, recently identified as CD44. Most studies of cartilage catabolism have centered on the extracellular processing of matrix components via proteases and protease inhibitors. However, enzymes(s) involved in the extracellular degradation of cartilage HA have never been documented, nor have other cellular mechanisms for HA catabolism. This knowledge is lacking despite the fact that HA plays a central role in the organization of cartilage extracellular matrix. Preliminary studies have shown that CD44 receptors mediate the endocytosis of HA for intracellular degradation. The binding and internalization of HA is blocked by HA hexasaccharides (competitive inhibitors of HA binding), as well as anti-CD44 antibodies. Small degradation fragments of HA are also detected intracellularly and the generation of these fragments is inhibited by the lysosomotropic agent, chloroquine. Catabolic cell mediators up-regulate CD44 expression, resulting in increased capacity of chondrocytes to accumulate intracellular HA. Thus for the first time, a mechanism for the catabolism of HA by the chondrocyte is evolving.
The Specific Aims of this proposal are to: (i) determine the exact rates of HA endocytosis, including HA of various sizes and HA """"""""decorated"""""""" with proteoglycan fragments and link protein; (ii) determine how the level of expression of CD44 affects the rate of HA endocytosis.
This second aim will involve the use of antisense inhibition of CD44 in both chondrocyte cultures, and cultures of intact cartilage tissue slices; and (iii) characterize changes in CD44 and isoform expression in human cartilage that occur during aging and disease.
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