Mechanisms for differential endocytosis of CD44 involved in turnover and signaling in chondrocytes

In cartilage, hyaluronan (HA) is anchored to chondrocytes via binding to CD44. We have shown that CD44‐mediated endocytosis and lysosomal degradation of HA requires CD44 palmitoylation and subsequent transit into lipid raft microdomains. Interestingly, raft‐CD44 is also a substrate for MMP cleavage generating a 20kD C‐terminal fragment. In chondrocytes, CD44‐HA complexes are thought to be present in different membrane pools, some destined for endocytosis and others involved in signaling, migration or anchorage of matrix. Differentiating these pools is challenging due in part to the constant replenishment of CD44. Trypsin treatment of chondrocytes generates a unique 25kD fragment representative of CD44 present in all membrane pools prior to typsinization; the 20kD fragment only the lipid raft pool. Decay of both 20 and 25kD CD44 fragments is completely blocked by chloroquine suggesting lysosomal degradation as a common endpoint. No 20kD domains were derived from 25kD fragments suggesting the 25kD cannot subsequently move into lipid rafts and serve as a substrate for MMP and γ‐secretase. Chlorpromazine, 350 mM sucrose and Dynasore, inhibitors of clathrin coated vesicle formation, partially block the decay of the 25kD fragment. We hypothesize that raft‐CD44 is responsible for HA endocytosis; whereas CD44 involved in signaling may be downregulated by clathrin‐associated endocytosis. Supported by NIH R01‐AR39507 and R01‐AR43384. Grant Funding Source : NIH R01‐AR39507 and R01‐AR43384