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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.(ABSTRACT TRUNCATED AT 250 WORDS)

molecular endocrinology

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.