Regulation of insulin‐like growth factor (IGF) binding protein‐2 synthesis and degradation by platelet‐derived growth factor and the IGFs is enhanced by serum deprivation in vascular smooth muscle cells

Abstract

Growth factors such as platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) stimulated proliferation and migration of vascular smooth muscle cells (SMC). IGF-I bioactivity is modulated by high-affinity binding proteins (IGFBP) which are important regulators of these processes. Porcine vascular SMC synthesize IGFBP-2 and IGFBP-4 in vitro. In the present study, levels of IGFBP-2 in conditioned media (CM) were increased approximately 1.6 to 2.2-fold when cells were exposed to PDGF (20 ng/ml) or insulin (5 micrograms/ml) for 24 hr following a 24 hr incubation in serum-free media, or following a 72 hr exposure to either growth factor. Similar increases in IGFBP-2 mRNA levels were observed. Exposure of cells to PDGF for 24 hr without prior serum deprivation resulted in smaller (47 +/- 11%) increases in IGFBP-2 protein levels but failed to alter mRNA levels. IGF-I, FGF, TGF-beta and EGF failed to increase IGFBP-2 using either experimental paradigm. In contrast, IGFBP-2 protein levels were consistently decreased (75 +/- 14%) after 72 hr of exposure to IGF-II without corresponding decreases in IGFBP-2 mRNA levels. Immunoprecipitation of [35S]methionine-labeled IGFBP-2 indicated that this decrease was not due to a decrease in synthesis of IGFBP-2. Immunoblot analysis of CM from cells treated with IGF-II indicated that the decrease in intact protein corresponded with an increase in two non-IGF binding IGFBP-2 fragments of 22 and 14 kD. Increased abundance of these fragments was also observed following IGF-I exposure, although corresponding decreases in intact IGFBP-2 were not usually observed. The relative abundance of these fragments did not appear to be affected by treatment with PDGF or insulin. In contrast to IGFBP-2, regulation of the levels of IGFBP-4 in CM did not appear to be altered by serum deprivation. Insulin consistently increased IGFBP-4 mRNA and protein levels under all situations. PDGF tended to increase IGFBP-4 protein levels, although this effect was less consistent and not as great as the increase observed with insulin. Treatment with IGF-I or -II consistently decreased IGFBP-4 levels in CM but tended to increase their mRNA levels under all situations. These data indicate that insulin, PDGF, and the IGFs regulate both IGFBP-2 and IGFBP-4. While PDGF and insulin stimulate IGFBP-2 and 4 synthesis, the IGFs appear to activate protease(s) which regulate IGFBP-2 and -4 levels post-translationally.(ABSTRACT TRUNCATED AT 400 WORDS)