Insulin-like growth factor binding protein-5 proteolytic activity in ovine articular chondrocyte culture.

We have previously demonstrated that ovine articular chondrocytes synthesise and release insulin-like growth factor binding protein-5 (IGFBP-5) which subsequently undergoes proteolysis in the tissue culture medium. The IGFBP-5 proteolytic activity has now been characterised and its substrate specificity analysed using recombinant IGFBP-5 and purified chondrocyte-derived IGFBPs. Iodinated human recombinant IGFBP-5 was incubated with chondrocyte culture or conditioned medium in the presence or absence of various inhibitors. Serine protease inhibitors aprotinin and heparin effectively inhibited the breakdown of IGFBP-5. Furthermore, insulin-like growth factor-I (IGF-I) but not its structural analogues with reduced affinity for IGFBP-5, was also able to partially protect IGFBP-5 from degradation indicating that the association of IGF with the binding protein was required for the inhibition of the proteolytic activity. The inflammatory cytokine interleukin-1 did not have any effect on IGFBP-5 proteolysis. The proteolytic activity appears to be IGFBP-5-specific since the incubation of chondrocyte-derived IGFBPs with chondrocyte conditioned medium resulted in the loss of IGFBP-5 while the levels of the other two IGFBPs (IGFBP-2 and a 24 kDa IGFBP) remained unchanged. In conclusion, we show that IGFBP-5 is specifically cleaved by a serine protease released by primary cultures of ovine articular chondrocytes and also demonstrate the ability of IGF-I to inhibit the proteolytic activity both in cell culture and in cell-free conditions.

Regulation of insulin-like growth factor-binding protein-5 by insulin-like growth factor I and interleukin-1alpha in ovine articular chondrocytes.

Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes' responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1-3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1-3)IGF-I or LR3IGF-I. Basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor had no effect on OAC IGFBPs. However, IL-1alpha increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1alpha, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1alpha resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1-3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1alpha reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1alpha were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1alpha synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1alpha in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.

Insulin-like growth factor binding proteins (IGF-BPs) in bovine articular and ovine growth-plate chondrocyte cultures: their regulation by IGFs and modulation of proteoglycan synthesis.

Cultured chondrocytes respond to insulin-like growth factors (IGFs) by increasing the production of proteoglycans and insulin-like growth factor binding proteins (IGF-BPs). To investigate the biological effects of IGFs and IGF-BPs, isolated bovine articular and ovine growth-plate chondrocytes were cultured at high density in the presence of IGF-1, and its truncated form, des (1-3) IGF-I. Both growth factors stimulated the production of IGF-BPs in articular and growth-plate chondrocyte monolayers. Western ligand blots showed that bovine articular chondrocytes released two forms of IGF-BPs into conditioned medium with molecular weights of 29 and 31 kDa. Ovine growth-plate chondrocytes released four different forms of IGF-BPs of approx. 22, 24; 29-30 and 34 kDa. IGF-I and des (1-3) IGF-I stimulated total proteoglycan synthesis by articular chondrocytes up to 1.5-fold. The truncated analogue was more potent at lower concentrations, particularly in stimulating incorporation of newly synthesized proteoglycans into the cell-layer. The maximal stimulation of proteoglycan synthesis in ovine growth-plate chondrocyte culture was 3-fold with des (1-3) IGF-I, while IGF-I enhanced proteoglycan production by only 2-fold over the concentrations used. Our results suggest that endogenous IGF-BPs in chondrocyte cultures act as a part of a feed-back mechanism which diminishes the bioactivity of IGF-I.