HORMONAL-REGULATION AND BIOLOGICAL ACTIONS OF INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEINS IN ISOLATED OVINE THYROID-FOLLICLES

The ability of TSH to stimulate synthesis and release of thyroid hormones in ovine thyroid follicles in vitro depends partially on a synergy with insulin-like growth factors (IGFs). The cellular availability of IGFs may be influenced by the release of several IGF binding proteins (IGFBPs). The purposes of these studies was to 1) further characterize the species of IGFBPs synthesized by thyroid follicles, 2) examine the ability of TSH and cortisol to alter IGFBP gene expression and protein release, and 3) investigate the actions of exogenous IGFBPs on thyroid cell function. Adult sheep thyroid follicles were isolated after collagenase digestion, grown to confluence in Coon's modified Ham's F12M medium (OH) with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), cortisol and insulin, and maintained in serum-free test media with or without further supplements for up to 48 h. Conditioned media were analyzed for IGFBP presence by Western ligand blotting, and by immunoblotting using specific antisera against bovine IGFBP-2 and human IGFBP-5. IGFBP mRNAs from follicles were identified by Northern blot hybridization using [P-32]labeled complementary DNAs encoding ovine IGFBP-1 or -2, and rat IGFBP-4 -5, or -6. Uptake and organification of Na[I-125] were measured by incorporation into trichloroacetic acid-precipitable material. Isolated thyroid follicles synthesized four species of IGFBPs in either OH or 3H medium as detected by ligand blotting, of sizes 40-46, 34, 28, and 18 kilodaltons (kDa), respectively. The 32 kDa IGFBP was identified immunologically as IGFBP-2, whereas the 28 kDa and 18 kDa species were identified as IGFBP-5. Northern blot hybridization of total RNA from cells in 3H medium demonstrated an IGFBP-2 messenger RNA (mRNA) [1.4 kilobase (kb)], an IGFBP-4 mRNA (2.6 kb), and two IGFBP-5 mRNAs (6 kb and 1.8 kb). No IGFBP-1 or -6 mRNAs were detected. Incubation of cultured follicles with TSH (30-500 mu U/ml) caused a dose-dependent decrease in the abundance of all IGFBP mRNAs and released proteins, which were reduced further by TSH together with cortisol (10 nM). When the inhibitory effect of TSH and cortisol was removed, IGFBP-2 mRNA increased within 3 h and was 7-fold greater within 12 h. IGFBP-2 did not appear in the conditioned medium until 12 h after TSH removal, along with the other IGFBP species. Incubation of follicles with TSH, in either OH or 3H medium, with or without the further addition of cortisol alone, or in combination with either IGF-I (6.7 nM) or insulin (1.67 mu M), caused an increase in iodine uptake and incorporation which was blocked in the presence of exogenous bovine IGFBP-2, but not by human IGFBP-3. The results show that ovine thyroid follicles synthesize two major species of IGFBPs, IGFBPs -2 and -5, which are regulated acutely by TSH. The rapid changes in local release of IGFBP-2 may modulate the actions of exogenous or endogenous IGFs, and may be one important mechanism whereby TSH regulates iodide transport and its organification in thyroid follicles.