Release of insulin-like growth factors and binding protein activity into serum-free medium of cultured human fibroblasts.
We have studied insulin-like growth factors (IGFs) and IGF-binding proteins released by human fibroblasts. Conditioned medium was obtained after incubation of 2 X 10(6) cells in 2 ml serum-free medium for 72 h. IGF binding protein was identified in aliquots of conditioned medium at 4 C for 16 h with [125]IGF II after charcoal separation. After gel filtration in neutral phosphate buffer through Sephadex G-150, the binding activity eluted with an apparent size greater than 100,000 daltons. After gel filtration through Bio-Rad P-100 in 1 M acetic acid, binding activity had a molecular size of about 50,000 daltons. When [125I]IGF-II bound to conditioned medium binding protein was cross-linked with disuccimidyl suberate and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex had an estimated molecular size of 67,000 daltons. Competitive binding studies with labeled and unlabeled IGF-I and IGF-II showed that IGF-II was preferentially bound by fibroblast binding protein. The above findings are characteristic of serum binding protein but not shed IGF surface receptors. To eliminate possible interference from binding proteins in the IGF-I RIA and the IGF-II radioreceptor assay, conditioned medium was subjected to acid gel filtration, and the peptide fractions were pooled. We found that conditioned medium of seven fibroblast lines contained 0.20 +/- 0.06 ng/ml IGF-I. After the addition of 20 ng/ml human GH (hGH), the conditioned medium contained 0.48 +/- 0.09 ng/ml. These results are lower than those previously reported. One of the two lines of fibroblasts from patients apparently resistant to GH had a minimal increase in IGF-I in conditioned medium after hGH addition. We were able to detect IGF-II in fibroblast conditioned medium in concentrations of 4.4 to 21 ng/ml but there was no consistent response to GH either in the normal fibroblast lines or in fibroblasts obtained from children with short stature.
D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D013002:Somatomedins / Q000378:metabolism*
D002352:Carrier Proteins; D003470:Culture Media; D007328:Insulin; D010455:Peptides; D013002:Somatomedins; D003513:Cycloheximide