TRANSCRIPTIONAL AND POSTTRANSLATIONAL REGULATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-5 IN RAT ARTICULAR CHONDROCYTES

The aim of this study was to assess the regulation of insulin-like gro wth factor-binding proteins (IGFBPs) by IGFs in primary cultures of ra t articular chondrocytes (RAC). Employing Western ligand blotting, imm unoprecipitation and Northern blot analysis, RAC were found to secrete IGFBP-5 (29 kDa) and IGFBP-4 (24 kDa) as the predominant IGFBPs, as w ell as IGFBP-2 (32-30 kDa) and IGFBP-3 (43-39 kDa) as the minor specie s. Treatment of cells with IGF-I and IGF-II resulted in a dose-depende nt increase of IGFBP-5 and a small increase in IGFBP-4 in conditioned media (CM). Des(1-3) IGF-I and [Gln(6), Ala(7), Tyr(18), Leu(19)] IGF- II ([QAYL] IGF-II), which bind to the type 1 IGF receptor but not to I GFBPs, also induced IGFBP-5 peptide, although the increase was less th an with IGF-I or IGF-II treatment of RAC. [Leu(27)] IGF-II, which does not bind to the type 1 IGF receptor but binds to IGFBPs, resulted in little induction of IGFBP-5, while [QAYL-Leu(27)] IGF-II, which has re duced affinity for both the type 1 IGF receptor and IGFBPs, did not in crease IGFBP-5. These data suggest that the increase in IGFBP-5 in CM is modulated by both the type 1 IGF receptor and the interaction betwe en IGFs and IGFBPs. Northern blotting analysis showed that IGF-I, IGF- II and des(1-3) IGF-I treatment of RAC increased steady state levels o f IGFBP-5 mRNA, suggesting that the IGF-mediated increase in IGFBP-5 i s transcriptionally modulated. Interestingly, the increase in IGFBP-5 peptide levels and mRNA were not parallel, suggesting the possibility of post-translational modifications of IGFBP-5, such as those seen wit h IGFBP-5 protease. IGFBP-5 protease activity was detectable in untrea ted CM, whereas treatment with IGF-I and IGF-II partially protected IG FBP-5 from proteolysis. In summary, treatment of RAC with IGF-I and IG F-II results in dose-dependent increases in both IGFBP-5 peptide in th e CM and mRNA levels. These changes are mediated by interactions via t he type 1 IGF receptor as well as IGFBPs, both transcriptionally and p ost-translationally.