Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus.

Thyroxine (T4) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T3 and T4in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH α and ß subunits from 4 to 66h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species.

Cyclic mRNA expression of thyrotropin subunits and deiodinases in red drum, Sciaenops ocellatus.

The role of thyrotropin (thyroid-stimulating hormone, TSH) in driving peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T4), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the teleost fish the red drum. Previous research suggests that the red drum T4 cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T4 feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with feedback inhibition by this T4 cycle, we used quantitative real time PCR (qPCR) to quantify the daily cycle of expression of the pituitary TSH subunits GSUα, and TSHß. We found that TSH expression cycled inversely to, and 6-12 h out of phase with, the T4 cycle, consistent with the hypothesis that TSH secretion drives the T4 cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, we also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner-ring deiodinase (Dio3). Dio2 was not expressed with an obvious daily cycle, whereas Dio3 expression mirrored the expression of TSH. These results are consistent with circulating T4 providing the negative feedback signal controlling both TSH production and Dio3 expression in the pituitary, and suggest that TH inactivation by inner ring deiodination is an important component of TSH negative feedback control.

Regulation of ornithine decarboxylase mRNA by phorbol esters and insulin in normal and C-kinase-deficient rat hepatoma cells.

Tumor-promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4-6-fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down-regulation or inhibition of protein kinase C (PKC), consistent with a PKC-mediated mechanism. In contrast, PKC down-regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC-depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.