Growth factor modulation of insulin-like growth factor-binding proteins in rat osteoblast-like cells.

Abstract

Insulin-like growth factors (IGFs) stimulate growth and differentiation of osteoblasts in culture, and these biological functions can be modulated by their binding proteins (IGFBPs). Previous studies have shown that IGFBP-2 is the major IGFBP synthesized by fetal rat osteoblast-like (ROB) cells, which also secret a minor 24-kilodalton IGFBP, presumably IGFBP-4. In this study we examined the modulation of the abundance of IGFBPs by various growth factors. By immunoprecipitation and ligand blotting, we have proved that the 24 kilodalton protein, which appeared at 25 kilodalton in the present study, is IGFBP-4, whose level was strongly enhanced by basic fibroblast growth factor (bFGF) and platelet-derived growth factor BB homodimer. Induction of IGFBP-4 by these factors was detected at 1 nM (10- to 30-fold) and increased to 50- to 70-fold of control at 10 nM. The abundance of IGFBP-4 was also increased but to a lesser degree by transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF). As opposed to the actions of other growth factors, TGF-beta 1 substantially lowered the levels of IGFBP-2 and IGFBP-4. PTH and PTH-related peptide did not induce IGFBPs in ROB cells. This is in contrast to the findings from a malignant rat osteoblast-like cell line, UMR 106-01. Since the level of IGFBP-4 after bFGF treatment remained elevated in the presence of hydroxyurea, the induction was likely to be independent of the stimulatory effects of these factors on mitogenesis. To further examine the mechanisms by which IGFBPs were regulated, cultures were treated with actinomycin D and cycloheximide with and without bFGF. Although the synthesis of IGFBP-2 and IGFBP-4 were both inhibited by cycloheximide, actinomycin D blocked the synthesis of basal and bFGF-induced IGFBP-4 but not IGFBP-2. Total RNA extracted from ROB cells and hybridized with specific rat complementary DNAs for IGFBP-2 and IGFBP-4 showed single transcripts of 1.3 and 2.2 kilobases, respectively. Regardless of the changes at the protein level, the abundance of IGFBP-4 transcripts was not different from the vehicle-treated controls at 2, 8, and 24 h after bFGF treatment. Similarly, the levels of messenger RNA for IGFBP-2 and IGFBP-4 did not change during the same time course in the TGF-beta 1 treatment. These data demonstrate that in ROB cells, the abundance of IGFBPs is differentially regulated by various growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)