Endocrinology, Vol 127, 643-650, Copyright © 1990 by Endocrine Society

ARTICLES

Three distinct forms of insulin-like growth factor binding proteins are released by decidual cells in culture

DR Clemmons, KM Thrailkill, S Handwerger and WH Busby Jr
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599.

Recent studies have shown that human decidual explants synthesize a 25,272 dalton form of insulin-like growth factor binding protein (IGFBP- 1) that is identical to the most abundant form of IGFBP detected in human amniotic fluid. To determine whether decidual cells also secrete other structurally distinct forms of IGFBP, conditioned medium obtained from human decidual cells was analyzed by ligand blotting and immunoblotting using antisera that were specific for IGFBP-1 or 2. IGFBP-2 is a form of binding protein that is structurally distinct from IGFBP-1. Ligand blotting analysis (which detects all forms of IGFBPs) showed that 3 forms of IGFBP with Mr estimates of 34,000, 30,000 and 24,000 were present in basal, unstimulated, conditioned media. Immunoblotting showed that the 30,000 Mr form reacted with an antibody that is specific for IGFBP-1, whereas the 34,000 Mr form reacted with an antibody that is specific for IGFBP-2. The 24,000 Mr band did not react with antisera to either IGFBP-1 or 2. Basal IGFBP-1 release from human decidual cultures, as measured by RIA, showed a significant decrease during the 5 day period from 34 +/- 1.6 on day 1 to 12 +/- 1.9 ng/ml on day 5. Exposure of the cells to (Bu)2cAMP (1.0 mM) for 5 days had no significant effect on IGFBP-1 release during the first day of exposure, but caused a progressive stimulation of release during the subsequent 4 days. By day 5, exposure to (Bu)2cAMP induced a 5- to 6- fold increase in IGFBP-1 release that was completely inhibited by exposure to cycloheximide. Exposure of the cells to cholera toxin (0.1 micrograms/ml) for 4 days caused a 3.7-fold increase in IGFBP-1 release. The stimulation of IGFBP-1 release by (Bu)2cAMP was completely inhibited by simultaneous exposure to insulin (100 ng/ml) and/or IGF-I (100 ng/ml) (day 5 control = 35 ng/ml, (Bu)2cAMP alone = 248 ng/ml, insulin + (Bu)2cAMP = 36 ng/ml, or IGF-I + (Bu)2cAMP = 10.1 ng/ml). Hydrocortisone and the phorbol ester (phorbol myristate acetate) caused no significant changes in basal IGFBP-1 release but prevented the decrease in the basal rate of release that occurred between day 1 and 5. Ligand blotting studies showed that the 24,000 Mr form of binding protein was also stimulated by (Bu)2cAMP (3.3-fold increase); however, cholera toxin and phorbol myristate acetate were without effect. In contrast, the release of the 34,000 Mr form was unaffected by exposure to any of these agents, suggesting that regulation of this form of IGFBP is distinct from the other 2 forms.(ABSTRACT TRUNCATED AT 400 WORDS)

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