Objective. To determine whether aggrecan fragments bound to hyaluronan (HA) can be retained and internalized by articular chondrocytes and whether these events are dependent on HA and its receptor, CD44. An additional objective was to determine whether partial degradation of aggrecan is a prerequisite for internalization.Methods. Binding and internalization of a variety of fluorescein isothiocyanate (FITC)-or biotin-labeled HA/proteoglycan probes were investigated on normal bovine articular cartilage chondrocytes, bovine articular chondrocytes transfected with a dominant-negative construct of CD44, or COS-7 cells transfected with wildtype CD44. The probes were defined as being internalized by the presence of label associated with the cells following extensive trypsinization of the cell surface.Results. Biotinylated aggrecan fragments bound to FITC-HA were cointernalized in bovine articular chondrocytes or COS-7 cells transfected with CD44. Intracellular vesicles containing FITC-HA colocalized with a fluorescent probe for lysosomes. The internalization of the aggrecan fragments was dependent on the presence of HA as well as the presence of functional CD44. Intact aggrecan/FITC-HA complexes bound to the cell surface but were not internalized. However, following brief trypsin digestion of the aggrecan/HA complex, the remaining proteoglycan fragments were bound and internalized.Conclusion. Partially degraded aggrecan fragments (e.g., aggrecan G1 domains bound to HA) can be internalized by articular chondrocytes via a mechanism involving HA/CD44-mediated endocytosis. Further, the presence of an intact aggrecan monomer bound to HA inhibits the internalization of HA as well as HA-bound fragments.One of the features of osteoarthritis is the loss of the proteoglycan aggrecan from the cartilage extracellular matrix (1). It has been suggested that this loss is due to the extracellular proteolytic cleavage of aggrecan by enzymes such as matrix metalloproteinases (2) and aggrecanases (3). Following proteolytic cleavage, the C-terminal, chondroitin sulfate-rich portion of degraded aggrecan is readily lost from the cartilage, and newly generated neoepitopes can be detected within synovial fluid (4). The fate of the N-terminal domain of the aggrecan degradation product, presumably still bound to HA and stabilized by link protein, is less clear. In previous studies using organ culture of normal bovine articular cartilage slices, the turnover kinetics of newly synthesized, radiolabeled aggrecan and HA were found to be nearly identical, with half-lives in the range of 17-25 days (5,6). However, whereas released aggrecan monomers could be quantitatively recovered in the culture medium, little intact HA and no small HA fragments were recovered in the medium or within guanidine HCl extracts of the tissue. These data suggested that HA was not being degraded extracellularly, but rather undergoing turnover by another distinct mechanism such as receptor-mediated endocytosis. Recently, Fosang and colleagues demonstrated by confocal micros...

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