Objectives: CD44 receptor mediated uptake of hyaluronan (HA) leading to its degradation intercellularly is the principal pathway for HA turnover by chondrocytes. When proteoglycan (PG) aggrecan is processed within the extracellular matrix by MMP or aggrecanases, residual PG and link protein domains remain attached to HA. We addressed the question as to whether these fragments were co-internalized with HA via CD44 mediated endocytosis by detection of the aggrecanase-derived ITEGE neoepitope. Methods: Primary cultures of bovine articular chondrocytes were incubated with IL-1beta for 0-5 days. To define the cellular distribution of ITEGE neoepitope, the membrane-bound pericellular matrix fraction was first separated from the intracellular fraction prior to cell lysis. ITEGE neoepitope in each fraction was then quantified by western blot analysis. In other experiments, CD44 expression was inhibited using a specific siRNA prior to incubation with IL-1beta or chondrocytes derived form CD44 knockout mice were stimulated with IL-1beta.
Results: The distribution of ITEGE neoepitope was validated by western blots of separated cellular fractions. IL-1beta increased the amounts of ITEGE in both fractions as compared with the control. Interestingly, the accumulation of intracellular ITEGE was coordinately diminished in chondrocytes transfected with CD44 siRNA, in proportion to the degree of CD44 inhibition and accumulation of ITEGE was diminished in CD44 knockout mouse chondrocytes.
Conclusions: The major goal of this study was to determine the regulation of CD44-mediated internalization of ITEGE. Results from CD44 knockout mouse study strongly support the siCD44 data. These results document that one aspect of aggrecan turnover in cartilage includes the local endocytosis of HA-bound aggrecan G1 domains - an internalization event mediated by CD44.