Abstract
Isolated sheep thyroid follicles release insulin-like growth factors (IGF)-I and -II together with IGF-binding proteins (IGFBPs). We previously showed that TSH suppresses the biosynthesis and release of IGFBPs in vitro which may increase the tissue availability of IGFs, allowing a synergy with TSH which potentiates both thyroid growth and function. Many of the actions of TSH on thyroid cell function are dependent upon activation of adenylate cyclase, although increased synthesis of inositol trisphosphate and activation of protein kinase C (PKC) have also been implicated. We have now examined whether probable changes in intracellular cyclic adenosine monophosphate (cAMP) or PKC are involved in TSH-mediated suppression of IGFBP release. Confluent primary cultures of ovine thyroid cells were maintained in serum-free Ham's modified F-12M medium containing transferrin, somatostatin and glycyl-histidyl-lysine (designated 3H), and further supplemented with sodium iodide (10−8–10−3 mol/l), dibutyryl cAMP (0·25–1 mmol/l), forskolin (5–20 μmol/l) or 12-0-tetradecanoylphorbol-13-acetate (TPA; 10−11–10−6 mol/l), with or without exposure to TSH (200 μU/ml). The uptake and organification of Na [125I] by cells was examined after test incubations of up to 48 h, and IGFBPs in conditioned media were analysed by ligand blot using 125I-labelled IGF-II. The PKC activity in the cytosol and plasma membrane fractions of cells was measured by phosphorylation of histone using [γ-32P]ATP, and PKC immunoreactivity was visualized by Western immunoblot analysis. While dibutyryl cAMP or forskolin largely reproduced the stimulatory effect of TSH on iodine organification, they did not mimic the inhibitory effect of TSH on the secretion of IGFBPs of 43, 34, 28 and 19 kDa. Incubation with physiological or pharmacological concentrations of iodide (10−6–10−3 mol/l) for up to 48 h significantly decreased TSH action on iodide uptake and organification but did not alter the inhibitory action of TSH on IGFBP release. Incubation of cells with 10−11–10−6 mol TPA/l for 24 h inhibited the subsequent ability of TSH both to potentiate iodine organification and to suppress IGFBP release. In 3H medium, PKC activity was predominantly recovered from the membrane fraction but, following incubation for 48 h with TSH, the enzyme was no longer translocated to the membrane and was recovered predominantly from the cytosol. An 80 kDa species of immunoreactive PKC was recovered from the membranes of cells cultured in 3H medium, but its presence in membrane was decreased following incubation with TSH. The actions of TSH on intracellular PKC distribution were reversed by prior incubation with TPA, which itself stimulated the appearance of membrane PKC immunoreactivity. These results suggest that the ability of TSH to suppress IGFBP release does not depend primarily on cAMP stimulation, but may involve changes in the activation of PKC, possibly inhibition or down-regulation.
Journal of Endocrinology (1994) 141, 231–242
Control of interleukin-1 beta expression by protein kinase C and cyclic adenosine monophosphate in myeloid leukemia cells