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.
Sunic
D
D
Department of Medicine, Modbury Public Hospital, Smart Road, Modbury, SA 5092, Australia. dsunic@health.adelaide.edu.au
McNeil
J D
JD
Andress
D L
DL
Belford
D A
DA
D000818:Animals; D002358:Cartilage, Articular / Q000201:enzymology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000201:enzymology*; D017077:Culture Media, Conditioned; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000378:metabolism*; D018969:Insulin-Like Growth Factor Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012756:Sheep
D017077:Culture Media, Conditioned; D018975:Insulin-Like Growth Factor Binding Protein 5; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D012697:Serine Endopeptidases