Hyaluronan and the hyaluronan-binding region of aggrecan are co-internalized by articular chondrocytes

The HA/aggrecan complex is anchored to the chondrocyte cell surface via interaction of HA with the membrane receptor CD44. Aggrecan catabolism in cartilage is thought to be mediated by the extracellular action of specific neutral proteinases, such as aggrecanase. Aggrecan cleavage releases the chondroitin sulfate-rich domain from the HA-binding domain, allowing its diffusion out of the cartilage matrix. The fate of the N-terminal domain of aggrecan, presumably still bound to HA and stabilized by link protein, is less clear. CD44, besides functioning as an anchor, also mediates the internalization and intracellular degradation of HA by chondrocytes. In this study, fluorescein-conjugated HA decorated with biotinylated HA-binding-protein region of aggrecan (b-HABP), generated by partial trypsin digestion of aggrecan, was used to demonstrate that aggrecan fragments can be co-internalized with HA via an HA-dependent, CD44-mediated mechanism. However, when intact aggrecan is pre-bound to HA, the aggregate complex was retained at the cell surface, and not internalized by chondrocytes. Internalization could be re-established following degradation of aggrecan by a brief trypsinization. Although these results demonstrate the potential of chondrocytes to internalize HA with PG “stubs,” questions remain as to whether this process occurs naturally in cartilage. Specific antibodies to the aggrecan neoepitopes ITEGE and DIPEN-generated following the cleavage of aggrecan by matrix metalloproteinases were localized on the cell surface and intracellularly. Internalization of aggrecan fragments bearing endogenous ITEGE or DIPEN neoepitopes was enhanced upon treatment of the chondrocytes with the catabolic cytokine IL-1α. Purified G1-ITEGE and G1-DIPEN aggrecan fragments pre-bound to HA were bound and internalized by articular chondrocytes similar to the HA/b-HABP complexes. Two approaches were used to validate the requirement of CD44 in the retention and internalization of PG “stubs.” First, HA/b-HABP, HA/ITEGE-G1 and HA/DIPEN-G1 internalization was inhibited in chondrocytes transfected with a dominant negative mutant of CD44. Continuous treatment with Streptomyces hyaluronidase also prevented internalization. Second, CD44-negative COS-7 cells gained the capacity to internalize HA/b-HABP, HA/ITEGE-G1 and HA/DIPEN-G1 upon transient transfection with full-length CD44. Thus, N-terminal domain fragments of aggrecan that remain bound to HA can be bound and internalized via a mechanism that is HA-dependent and CD44-mediated.