Regulation of insulin-like growth factor (IGF)-binding protein-6 and mannose 6-phosphate/IGF-II receptor expression in IGF-IL-overexpressing NIH 3T3 cells.

Abstract

Insulin-like growth factor II (IGF-II)-overexpressing NIH 3T3 cells were used to examine regulation of insulin-like growth factor binding protein (IGFBP) and mannose 6-phosphate (M6P)/IGF-II receptor expression. Ligand blot analysis of conditioned media indicated a predominant IGFBP of 26-28 kilodaltons the abundance of which is 3-to 10-fold higher in media of IGF-II-overexpressing cells. The IGFBP level in control cell medium was increased by incubation in the presence of IGF-II, IGF-I, and mutant IGF-II forms with reduced affinities for IGF-I or M6P/IGF-II receptors. Further proof that IGF-II regulated the IGFBP was obtained by incubation of IGF-II overexpressing cells in the presence of antisense IGF-II oligomers or anti-IGF-II antibodies, which resulted in significant reduction of the IGFBP in conditioned medium. Mouse IGFBP-6 mRNA expression was increased in IGF-II-overexpressing or IGF-II-treated control cells. The IGFBP contained O-linked carbohydrate residues and was recognized by an antiserum to rat IGFBP-6. To determine whether IGFs were influencing proteolytic degradation of IGFBPs, cell-free conditioned media were incubated at 37 C with recombinant human IGFBPs. At neutral pH proteolysis of IGFBP-5 occurred during incubation in conditioned media from control and IGF-II-overexpressing cells. Upon acidification of the medium samples, only the degradation of IGFBP-6 was prevented in IGF-II-overexpressing cell-conditioned medium. Treatment of control cells with IGF-II for 2446 h inhibited the acid-activated IGFBP-6 protease activity in conditioned media while the incubation of media with IGF-II under cell-free conditions failed to affect the IGFBP-6 proteolysis. Chelating agents had no effect on acid-activated degradation of IGFBP-6, while several serine, cysteine, and aspartic protease inhibitors prevented degradation. The expression of MGPAGF-II receptor protein and mRNA was increased in IGF-II-overexpressing cells. By contrast, no change in the mRNA level of the 46-kilodafton M6P receptor lacking an IGF-IIbinding site, was observed. These results suggest that IGF-II may regulate IGFBP-6 level in NIH 3T3 cells both by transcriptional and posttranslational mechanisms and induce the MGPAGF-II receptor expression. © 1995 by The Endocrine Society.