Transcriptional and post-translational 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 growth factor-binding proteins (IGFBPs) by IGFs in primary cultures of rat articular chondrocytes (RAC). Employing Western ligand blotting, immunoprecipitation and Northern blot analysis, RAC were found to secrete IGFBP-5 (29 kDa) and IGFBP-4 (24 kDa) as the predominant IGFBPs, as well as IGFBP-2 (32-30 kDa) and IGFBP-3 (43-39 kDa) as the minor species. Treatment of cells with IGF-I and IGF-II resulted in a dose-dependent increase of IGFBP-5 and a small increase in IGFBP-4 in conditioned media (CM). Des(1-3) IGF-I and [Gln6, Ala7, Tyr18, Leu19] IGF-II ([QAYL] IGF-II), which bind to the type 1 IGF receptor but not to IGFBPs, also induced IGFBP-5 peptide, although the increase was less than with IGF-I or IGF-II treatment of RAC. [Leu27] 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-Leu27] IGF-II, which has reduced affinity for both the type 1 IGF receptor and IGFBPs, did not increase 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 between 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 of IGFBP-5 mRNA, suggesting that the IGF-mediated increase in IGFBP-5 is 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 with IGFBP-5 protease. IGFBP-5 protease activity was detectable in untreated CM, whereas treatment with IGF-I and IGF-II partially protected IGFBP-5 from proteolysis. In summary, treatment of RAC with IGF-I and IGF-II results in dose-dependent increases in both IGFBP-5 peptide in the CM and mRNA levels. These changes are mediated by interactions via the type 1 IGF receptor as well as IGFBPs, both transcriptionally and post-translationally.
Matsumoto
T
T
Department of Pediatrics, Oregon Health Sciences University, Portland, USA.
Gargosky
S E
SE
Oh
Y
Y
Rosenfeld
R G
RG
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics / Q000378:metabolism*; D008297:Male; D011233:Precipitin Tests; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013002:Somatomedins / Q000378:metabolism*; D014158:Transcription, Genetic
D018975:Insulin-Like Growth Factor Binding Protein 5; D012333:RNA, Messenger; D013002:Somatomedins