REGULATION OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4 IN HUMAN ENDOMETRIAL STROMAL CELL-CULTURES - EVIDENCE FOR LIGAND-INDUCED PROTEOLYSIS

The present study investigates the regulation of IGFBP-4 levels by ins ulin-like growth factor (IGF) peptides in human endometrial stromal ce ll cultures. A 24-kilodalton (kDa) IGF-binding protein (IGFBP) secrete d by stromal cells was identified as IGFBP-4 by immunoprecipitation an d Western Ligand blotting. Western ligand blot analysis of conditioned medium showed that treatment of stromal cells with IGF-I or IGF-II in duced a dose-dependent reduction of detectable IGFBP-4 levels. Two IGF analogs that bind type I IGF receptors, but have reduced affinity for IGFBPs, increased detectable levels of IGFBP-4, and their ability to reduce IGFBP-4 at high concentrations was positively correlated with t heir affinity for this binding protein. LR(3)-IGF-I, which has 1000-fo ld lower binding affinity than IGF-I, increased detectable IGFBP-4 at all concentrations tested. Des-(13)-IGF-I, whose affinity for IGFBP-4 is 30-fold lower than that of IGF-I, increased detectable IGFBP-4 at l ow concentrations (0.1-10 ng/mL), but reduced its levels at 100 ng/mL, consistent with the significant binding to IGFBP-4 at this concentrat ion. In contrast, IGFBP-4 was undetectable in cultures receiving Leu(2 7)-IGF-II, which has reduced affinity for the type I IGF receptor but unaltered affinity for IGFBPs and the type II receptor. High concentra tions of insulin (100 ng/mL), which interacts with type I IGF receptor s without binding IGFBPs, also increased detectable levels of IGFBP-4 in stromal cultures. The addition of IGF-I or IGF-II to cell-free cond itioned medium from stromal cells cultured in the absence of IGFs resu lted in the reduction of detectable endogenous IGFBP-4 levels. The eff ects of IGFs on IGFBP-4 levels in this cell-free system were time, tem perature, and pH dependent and were prevented by the serine proteinase inhibitor, aprotinin, by the divalent cation chelator, EDTA, and by t he metal ion chelator, 1,10-phenanthroline. Western immunoblotting sho wed that the IGF-induced reduction of intact 24-kDa IGFBP-4 was accomp anied by the generation of an immunoreactive fragment of approximately 16 kDa, which was not detectable by Western ligand blotting. Cell-fre e conditioned medium from endometrial stromal cultures proteolyzed cov alently cross-linked [I-125]IGF-II-IGFBP-4 complexes in the absence of added IGFs, generating an 18-kDa radiolabeled fragment, and addition of free IGF peptide did not enhance the degradation of IGF-II-IGFBP-4 complexes. These results are consistent with the hypothesis that bindi ng of IGFs to IGFBP-4 induces increased susceptibility of this BP to p roteolysis, and that direct proteinase activation and/or repression of a proteinase inhibitor are less likely to contribute to IGF-induced I GFBP-4 proteolysis in medium conditioned by human endometrial stromal cells.