REGULATION OF INSULIN-LIKE GROWTH-FACTOR (IGF)-BINDING PROTEIN-6 AND MANNOSE-6-PHOSPHATE IGF-II RECEPTOR EXPRESSION IN IGF-II-OVEREXPRESSING NIH 3T3 CELLS/

Insulin-like growth factor II (IGF-II)-overexpressing NIH 3T3 cells we re used to examine regulation of insulin-like growth factor binding pr otein (IGFBP) and mannose 6-phosphate (M6P)/IGF-II receptor expression . Ligand blot analysis of conditioned media indicated a predominant IG FBP of 26-28 kilodaltons the abundance of which is 3- to 10-fold highe r 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/I GF-II receptors. Further proof that IGF-II regulated the IGFBP was obt ained by incubation of IGF-II overexpressing cells in the presence of antisense IGF-II oligomers or anti-IGF-II antibodies, which resulted i n significant reduction of the IGFBP in conditioned medium. Mouse IGFB P-6 mRNA expression was increased in IGF-II-overexpressing or IGF-ll-t reated control cells. The IGFBP contained O-linked carbohydrate residu es and was recognized by an antiserum to rat IGFBP-6. To determine whe ther IGFs were influencing proteolytic degradation of IGFBPs, cell-fre e conditioned media were incubated at 37 C with recombinant human IGFB Ps. 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 w as prevented in IGF-II-overexpressing cell-conditioned medium. Treatme nt of control cells with IGF-II for 24-48 h inhibited the acid-activat ed 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 p rotease inhibitors prevented degradation. The expression of MGP/IGF-II receptor protein and mRNA was increased in IGF-II-overexpressing cell s. By contrast, no change in the mRNA level of the 46- kilodalton M6P receptor lacking an IGF-II-binding site, was observed. These results s uggest that IGF-II may regulate IGFBP-6 level in NIH 3T3 cells both by transcriptional and posttranslational mechanisms and induce the MGP/I GF-II receptor expression.