Fibronectin fragments have both catabolic and anabolic activities toward articular cartilage explants in vitro. Whereas a 1 nM concentration of an N-terminal 29 kDa fibronectin fragment (Fn-f) increases the proteoglycan (PG) content of cartilage without induction of matrix metalloproteinases (MMPs), 0.1Ő1 ƁM Fn-f temporarily suppresses PG synthesis and enhances MMP release. The higher concentrations cause an initially rapid PG depletion during the first week of culture, followed by much slower PG loss and gradually increasing rates of PG synthesis. To test for the involvement of mediators, human articular cartilage was cultured with Fn-f, and conditioned media were assayed for selected cytokines and factors. With 1 nM Fn-f, the release of the anabolic factors, insulin growth factor-I and transforming growth factor β1, from cultured cartilage was enhanced by 50Ő100% during the entire 28-day culture period and this was associated with both supernormal rates of PG synthesis and PG content. However, the higher concentrations of Fn-f additionally enhanced release, by at least 10-fold, of the cytokines, tumour necrosis factor α, interleukin-1α, interleukin-1β and interleukin-6 while causing depletion of cartilage PG. Release of tumour necrosis factor α, interleukin 1β and interleukin 1α peaked at days 2, 3 and 9 during or slightly after the period of maximal PG depletion and decreased to control levels by days 7, 7 and 21 respectively, whereas release of interleukin 6 was enhanced throughout the culture period. Neutralizing antibodies to the catabolic cytokines reduced Fn-f-mediated MMP-3 release and suppression of PG synthesis. The temporal aspects of this interplay between catabolic and anabolic factors are consistent with the kinetics of Fn-f-mediated cartilage damage and attempted repair and may be relevant to cartilage damage and repair in vivo.

These data demonstrate that although Fn-fs could be generated in vivo within synovial fluids and Fn-fs found in OA synovial fluid may contribute to cartilage damage in vivo, Fn-fs could also be generated within cartilage and amplify cartilage damage. Thus, Fn-fs may be both autocrine and paracrine regulators of cartilage metabolism.

Bovine articular chondrocytes cultured in alginate beads were used to study the effect of catabolic cellular mediators on CD44 expression. Treatment with either the 29-kDa fragment of fibronectin or interleukin-1 alpha results in a time- and dose-dependent inhibition of proteoglycan synthesis as well as a stimulation in the expression of CD44 mRNA level as determined by semi-quantitative polymerase chain reaction following reverse transcription. No noticeable effect at 6 h was observed. By 24 h, the major CD44 product (CD44H) from fibronectin fragment-treated cultures showed an 8-fold increase; CD44H from interleukin-1 alpha-treated cultures showed a 6-fold increase as compared to control cultures. In addition, a minor band, determined to be an isoform of CD44, was also shown to be up-regulated by both mediators. Stimulation of CD44 mRNA via interleukin-1 was also evident by in situ hybridization studies of bovine as well as human articular cartilage in organ culture. The increased in CD44 mRNA is matched by an increase at the protein level as determined by Western blot analysis. The Western blot reveals a doublet protein band at 80-90 kDa that corresponds to the molecular mass of CD44H. Cultures incubated with fibronectin fragments for 24 h had an 8.0-fold increase in CD44, while a 6.6-fold was observed for interleukin-1 alpha. Fluorescein-conjugated hyaluronan binding and internalization studies indicate that the increase in CD44 protein, induced by interleukin-1 alpha, closely correlates with an increase in functional hyaluronan receptors present at the chondrocyte cell surface. Taken together these results indicate that conditions that up-regulate chondrocyte catabolism also up-regulate the expression of CD44, a cell surface hyaluronan receptor involved in hyaluronan endocytosis.

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