Regulation of immunoreactive insulin-like growth factor binding protein-6 in normal and transformed human fibroblasts.

Insulin-like growth factor-binding proteins (IGFBPs) have been shown to both potentiate and inhibit IGF bioactivity in vitro; thus, changes to the type or amount of IGFBPs present in the cellular environment will ultimately affect insulin-like growth factor action. In this study, we have investigated the production of immunoreactive IGFBP-6 by normal human fibroblasts (NHF) and an SV-40-transformed human fibroblast line (AG2804). When analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, IGFBP-6 appeared as a doublet of 32-34-kDa in conditioned medium of both cell lines, with the lower molecular mass band predominating in the NHF cell line. Measured by a specific radioimmunoassay, serum-free NHF, and AG2804 cultures secreted IGFBP-6 at 1.44 +/- 0.09 and 1.23 +/- 0.08 ng/10(4) cells (mean +/- S.E.), respectively. Despite a relatively weak IGFBP-6 signal by ligand blot compared with IGFBP-3, the two proteins were secreted in similar molar concentrations by NHF. Retinoic acid increased IGFBP-6 by 3-fold in NHF and AG2804-conditioned media, maximal at approximately 100 nM retinoic acid. In contrast, IGFBP-6 production was inhibited by transforming growth factor-beta 1 and agents that increase intracellular cAMP concentrations, including dibutyryl cAMP, forskolin, isobutylmethylxanthine, and cholera toxin. This study indicates that IGFBP-6 has a pattern of regulation unique among the IGFBPs, supporting the concept of specific roles for each binding protein in regulating cell growth and metabolism.