SYNTHESIS AND REGULATION OF INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-5 IN FRTL-5 CELLS

FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells, exhibit a marked mitogenic response to insulin-like growth factors (IGFs) when they are exposed to TSH. Because IGF binding proteins (IGFBPs) are important modulators of IGF actions, we investigated the capacity of FRTL-5 cells to synthesize IGFBPs. We found that FRTL-5 cell conditioned media contained a single band of approximately 31 kilodaltons on ligand blot analysis. This band represents IGFBP-5 because: 1) it can be immunostained with a specific antibody raised against human IGFBP-5; 2) by Northern analysis, total RNA from FRTL-5 cells contains a major 6-kilobase transcript when hybridized with a cDNA for rat IGFBP-5; and 3) no transcripts were observed when Northern blots of FRTL-5 cells were hybridized with complementary DNAs for rat IGFBP-1, -2, -3, -4 or -6. Conditioned media IGFBP-5 increased in response to IGF-I, IGF-II, and insulin in a dose-dependent fashion, compared with unstimulated FRTL-5 cells. At maximally effective concentrations IGF-I was 3.5- and 6-fold more potent than IGF-II and insulin, respectively. The addition of a monoclonal antibody (Sm 1.2) to IGF-I completely inhibited IGF-I stimulation of the IGFBP-5 6-kilobase transcript and the appearance of IGFBP-5 in FRTL-5 conditioned media. Stimulation of IGFBP-5 synthesis by the IGFs and insulin appeared to be regulated at the messenger RNA (mRNA) level, because each stimulated similar increases in both IGFBP-5 mRNA and media protein at maximally effective concentrations. TSH, on the other hand, inhibited basal levels of IGFBP-5 mRNA and attenuated the increase in IGFBP-5 mRNA stimulated by IGF-I. FRTL-5 cells provide a relatively uncomplicated model to study the regulation and action of IGFBP-5 and the mechanisms by which IGFs interact with this binding protein.