Insulin-like growth factor-binding proteins (IGF-BPs) produced by human skin fibroblasts: immunological relationship to other human IGF-BPs.
We have characterized the insulin-like growth factor-binding protein (IGF-BP) produced by neonatal human skin fibroblasts in monolayer culture using antibodies specific for the acid-stable subunit of the 150K GH-dependent IGF-BP complex, BP-53, and the amniotic fluid IGF-BP, BP-28. Fibroblasts produced 65.3 +/- 10.4 ng/ml.72 h (SE; n = 6) immunoreactive BP-53 in serum-free medium; this was stimulated by increasing fetal bovine serum in the medium up to 385.3 +/- 49.0 ng/ml.72 h at 10% serum. Epidermal growth factor (EGF) also caused dose-dependent stimulation of BP-53 production, with a maximal effect (3-fold increase) at 30 ng/ml EGF. No immunoreactive BP-28 production was detectable in the presence or absence of serum or EGF. Neutral gel chromatography of serum-free medium revealed a peak of immunoreactive BP-53 at about 50K, with a smaller species at 20-30 K. Serum- and EGF-stimulated cells produced higher levels of about 50K BP-53, and an additional peak of immunoreactivity at 150K was present in serum-stimulated, but not EGF-stimulated, samples. Comparison of IGF-I and IGF-II binding by fibroblast BP-53 revealed slightly higher IGF-II than IGF-I binding, and association constants of 3-4 x 10(10) liter/mol for both IGFs, similar to BP-53 from human plasma. Affinity labeling of acid-stripped medium followed by nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specifically cross-linked IGF-binding species of 60K (identical to labeled plasma BP-53), 42K, and 37K. Only the 60K and 42K complexes were precipitable by antiserum to plasma BP-53, and none was precipitable by anti-BP-28 serum, suggesting that the 37K band might represent a third class of IGF-BP. We conclude that neonatal skin fibroblasts produce no BP-28, but do produce two IGF-BPs immunologically homologous to human plasma BP-53, one of which shows size and IGF-binding characteristics identical to the plasma protein.
Martin
J L
JL
Department of Medicine, University of Sydney, New South Wales, Australia.
Baxter
R C
RC
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D002478:Cells, Cultured; D003429:Cross Reactions; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007231:Infant, Newborn; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D008297:Male; D011863:Radioimmunoassay; D011972:Receptor, Insulin / Q000276:immunology / Q000378:metabolism*; D017451:Receptors, Somatomedin; D012867:Skin / Q000378:metabolism*; D013002:Somatomedins / Q000378:metabolism*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D011972:Receptor, Insulin