Glycosaminoglycans inhibit degradation of insulin-like growth factor-binding protein-5.
Human dermal fibroblasts secrete insulin-like growth factor-binding protein-3 (IGFBP-3), -4, and -5. Fibroblast-conditioned medium contains minimal intact IGFBP-5, and this form of IGFBP is predominantely a 23-kilodalton fragment, suggesting that the IGFBP-5 fragment is derived from intact IGFBP-5 by proteolysis. In this study we investigated the effects of glycosaminoglycans on IGFBP-5 degradation in fibroblast-conditioned medium. The addition of heparin, heparan sulfate, and dermatan sulfate (100 micrograms/ml) to the medium of fibroblast monolayer cultures inhibited IGFBP-5 degradation, as determined by the conversion of intact IGFBP-5 to a 23-kilodalton fragment. In contrast, hyaluronic acid, keratan sulfate, and chondroitin sulfate-A and -C had no effect. Heparin and heparan sulfate inhibited IGFBP-5 degradation at concentrations of 1 or 2.5 micrograms/ml, but 100 micrograms/ml dermatan sulfate were required. Heparin was also inhibitory in vitro, that is when conditioned medium and heparin were incubated without cells. Experiments with modified forms of heparin showed that O-sulfate groups in the 2 or 3 carbon position were required for heparin to be inhibitory. Completely desulfated heparin had no activity, and N-resulfation of desulfated heparin had only a minimal effect. Dextran sulfate, pentosan polysulfate, and fucoidan, which are composed of different saccharide units but contain O-sulfate groups in the 2 or 3 carbon positions, also inhibited IGFBP-5 degradation. These results demonstrate that heparin-like molecules are important regulators of IGFBP-5 degradation. O-Sulfation of the 2 or 3 position of the saccharide ring is required for inhibitory activity. As glycosaminoglycan side-chains are present in proteoglycans that are present in extracellular matrix and on cell surfaces, these side-chains represent a potential mechanism for regulating IGFBP-5 proteolysis in vivo.
Arai
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Department of Medicine, University of North Carolina, Chapel Hill 27599.
Arai
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Busby
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Clemmons
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D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006025:Glycosaminoglycans / Q000494:pharmacology*; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D018975:Insulin-Like Growth Factor Binding Protein 5; D008024:Ligands; D010447:Peptide Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D012867:Skin / Q000166:cytology / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism
D002352:Carrier Proteins; D006025:Glycosaminoglycans; D018975:Insulin-Like Growth Factor Binding Protein 5; D008024:Ligands; D013002:Somatomedins; D013431:Sulfates; D006493:Heparin; D010447:Peptide Hydrolases