Regulation of thyroid hormone receptor beta-2 mRNA levels by retinoic acid.

The thyroid hormone receptor, TR beta-2, whose expression is limited to the pituitary and parts of the central nervous system, is strongly negatively regulated at the pre-translational level by thyroid hormone (T3). We have investigated whether retinoic acid (RA), whose receptors (RARs) share a high degree of homology with the thyroid hormone receptors (TRs), can regulate this gene in a manner similar to T3, as has been shown for the growth hormone (GH) gene. GH3 cells were incubated with 10 nM T3, 1 microM RA or both for 48 h and then TR beta-2 mRNA levels determined by RNA blot hybridization analysis. We observed a 73% decrease in TR beta-2 mRNA levels after incubation with T3 and a two-fold increase in TR beta-2 mRNA levels after incubation with RA alone. In the presence of RA, the T3 effect on TR beta-2 mRNA levels was blunted with mRNA levels decreasing by only 20%. We investigated the mechanism by which retinoic acid increases and opposes the effects of T3 on levels of TR beta-2 mRNA. In transient transfection experiments using a reporter plasmid containing the TR beta-2 promoter and in nuclear run on assays, we found no effect of RA on TR beta-2 gene transcription. We then investigated whether the effects of RA were mediated at the post-transcriptional level. Determination of the apparent half-life of TR beta-2 mRNA using the transcriptional inhibitor, actinomycin D, showed that RA had no effect on TR beta-2 mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal regulation of the growth hormone gene. Relationship of the rate of transcription to the level of nuclear thyroid hormone-receptor complexes.

Using cultured GH1 cells, we reported that stimulation (3- to 5-fold) of growth hormone synthesis and mRNA levels by thyroid hormone is mediated by a chromatin-associated receptor. Thyroid hormone also elicits a rapid reduction of homologous receptor in GH1 cells primarily by decreasing the synthetic rate of receptor ( Raaka , B. M., and Samuels , H. H. (1981) J. Biol. Chem. 256, 6883-6889). Without 3,5,3'-triiodo-L-thyronine (L-T3), glucocorticoid agonists induced a limited and delayed effect while L-T3 + glucocorticoid synergistically stimulated the response an additional 2- to 4-fold compared to L-T3. In this study, we utilized GC cells, a related cell line, to compare the abundance of L-T3-receptor complexes to the rate of growth hormone mRNA synthesis and gene transcription. Gene transcription was assessed by in vitro labeling of nuclei with [alpha-32P]UTP which were derived from cells incubated with hormone(s), while mRNA synthesis was determined in intact cells by [3H]uridine labeling. Labeled growth hormone mRNA and gene transcripts were quantitated by filter hybridization to plasmid containing growth hormone cDNA. L-T3 rapidly decreased receptor levels in GC cells with kinetics similar to that in GH1 cells. Both the L-T3 and the synergistic L-T3 + glucocorticoid stimulation of growth hormone mRNA synthesis changed in parallel with the level of L-T3-receptor complexes. Glucocorticoid hormones alone elicited a variable response which resulted in minimal stimulation or inhibition of growth hormone mRNA synthesis or gene transcription rates. No apparent lag was identified between the kinetics of L-T3 binding to receptor and stimulation of growth hormone gene transcription. L-T3 stimulated growth hormone gene transcription rates maximally in 1 h which then progressively decreased in parallel with L-T3-receptor levels. Using [3H]uridine pulse-chase, growth hormone mRNA was found to have a half-life of approximately 50 h in agreement with the decay curve of growth hormone production of deinduced cells. Our studies suggest that regulation of the growth hormone response is predominantly determined by positive control of growth hormone gene transcription which is proportional to the concentration of thyroid hormone-receptor complexes.

Thyroid and glucocorticoid hormones synergistically control growth hormone mRNA in cultured GH1 cells.

We have previously demonstrated that thyroid hormone controls growth hormone synthesis in GH1 cells and that the induction of the growth hormone response by glucocorticoid appears to be highly dependent on thyroid hormone action. Thyroid hormone induces growth hormone synthesis approximately 5- to 20-fold and cortisol increases this response 2- to 6-fold further. Long-term kinetics of the growth hormone response show that, without added thyroid hormone, cortisol can induce a small-growth hormone response after 48 hr of incubation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of proteins synthesized in intact cells demonstrates that the cortisol enhancement of growth hormone synthesis in cells incubated with thyroid hormone is a relatively selective process. Quantitation of growth hormone mRNA levels by cell-free protein synthesis demonstrates that the regulation of growth hormone synthesis by thyroid and glucocorticoid hormones is explained by a synergistic pretranslational control mechanism, presumably at the level of the growth hormone gene.