Phloretin inhibits cellular uptake and nuclear receptor binding of triiodothyronine in human hep G2 hepatocarcinoma cells.

Phloretin is an inhibitor of the mammalian glucose transporter and the iodothyronine-5'-deiodinase. We examined the effects of phloretin on cellular and nuclear uptake of [125I]T3 in cultured human Hep G2 hepatocarcinoma cells. The initial rate of T3 uptake was energy dependent and saturable with both a high affinity (Km = 3.6 nM) and a low affinity (Km = 503 nM) process. Phloretin produced a dose-dependent decrease in [125I]T3 uptake by Hep G2 cells (IC50 = 88 microM) and also inhibited nuclear uptake in intact cells and isolated nuclei. The solubilized nuclear receptor in the Hep G2 cells had a Kd of 0.14 nM for T3. Phloretin inhibited [125I]T3 binding to the solubilized nuclear receptor by competitive inhibition. Phlorizin, the beta-D-glucoside of phloretin, had no effect on T3 binding to the solubilized nuclear receptor. The inhibition of T3 cellular uptake and nuclear receptor binding is probably due to structural similarities between T3 and phloretin.

Active repression by thyroid hormone receptor splicing variant alpha2 requires specific regulatory elements in the context of native triiodothyronine-regulated gene promoters.

Structural requirements for the inhibitory action of thyroid hormone receptor splicing variant alpha2 (TR alpha2) on T3/TRbeta1-mediated transactivation were investigated in native promoters of two T3-regulated genes: the brain-specific myelin basic protein (MBP) and the housekeeping malic enzyme (ME). T3/TRbeta1 transactivation of MBP256-chloramphenicol acetyl transferase (CAT) and ME315-CAT constructs was inhibited and unaffected by TR alpha2, respectively. In electrophoretic mobility shift assays, TR alpha2 bound MBP-thyroid response element (TRE) as a monomer but failed to interact with ME-TRE. Mutations of ME-TRE allowed TR alpha2 binding but not inhibition of T3/TRbeta1-mediated transactivation. In the context of the MBP promoter, replacement of MBP-TRE with ME-TRE or exchange of MBP TATA-like box with the ME GC-rich region spanning the transcription start site abolished TR alpha2 dominant negative action. Simultaneous introduction of both MBP-TRE and MBP TATA-like box in the context of ME promoter, however, triggered TR alpha2 inhibition of T3/TRbeta1 transactivation, indicating that these regulatory elements are necessary, but not individually sufficient, to mediate TR alpha2 dominant negative activity. Functional studies at low TR alpha2/TRbeta1 ratios revealed that binding to TRE facilitates TR alpha2 dominant negative action while prevention of DNA interaction by altering TR alpha2 P-box structure preserved TR alpha2 inhibitory effect, although with lower potency. In conclusion, the results suggest that, in native promoters of T3-regulated genes, a dual molecular mechanism, with DNA-binding dependent and DNA-binding independent components, underlies TR alpha2 dominant negative activity.

Selective degradation of mRNA: the role of short-lived proteins in differential destabilization of insulin-induced creatine phosphokinase and myosin heavy chain mRNAs during rat skeletal muscle L6 cell differentiation.

This investigation concerns the combined effects of removal and readdition of insulin and inhibition of protein and RNA synthesis on the stability of insulin-induced mRNAs during and after differentiation of rat L6A1 myoblast cells in culture. Addition of insulin accompanying the withdrawal of the mitogenic stimulus of serum to myoblasts caused an 80-fold increase in creatine phosphokinase (CK) activity which was largely accounted for by a similar increase in the amount of CK mRNA. The latter was co-ordinately induced with myosin heavy chain (MHC) mRNA but not malic enzyme (ME) mRNA. Measurements of steady-state levels of mRNA showed that removal of insulin caused CK mRNA, but not MHC mRNA, to be rapidly degraded, the effect being reversed upon readdition of the hormone. Direct measurement of 3H-labeled CK, MHC and beta-actin mRNAs confirmed the selective stabilization and destabilization of CK mRNA by the hormone. Conditions were established for a time-window during which cycloheximide (Cx) produced a virtually total arrest of protein synthesis in myotubes that was reversible upon removal of the inhibitor. Under these conditions, Cx selectively prevented the degradation of CK mRNA in a reversible manner. Actinomycin D (Act D) also arrested the loss of this mRNA. Under the same conditions of mRNA stabilization during de-induction, a superinduction of CK mRNA, but not MHC mRNA, was observed if the two inhibitors were added during induction in the continuous presence of insulin. We conclude that a short-lived protein(s), encoded by a short-lived mRNA(s), selectively regulates the stability of reversibly inducible mRNA.

Divergent effects of 9-cis-retinoic acid receptor on positive and negative thyroid hormone receptor-dependent gene expression.

The thyroid hormone (TH)-inducible expression of some genes has recently been shown to be enhanced by 9-cis-retinoic acid (9-cis-RA) receptor (RXR). This effect appears to be at least partially elicited by the ability of RXR to heterodimerize with TH receptor (THR) and enhance its binding to the cis-acting thyroid hormone responsive elements (TREs) found within those genes. However, whether RXR beta enhances TH/THR-mediated transactivation of all Tre-containing genes, and if RXR has any effect on TH-dependent negative regulation are not known. In the present study, we show that the TH/THR-inducible expression of the myelin basic protein (MBP) gene is not enhanced by RXR beta, despite high affinity binding of the RXR beta.THR alpha heterodimer to the MBP-TRE. We also demonstrate that RXR beta reverses the TH/THR-dependent down-regulation mediated by the negative TRE found within the promoter of the mouse thyroid stimulating hormone gene (TSH). The ligand for RXR beta (9-cis-RA), either alone or in combination with TH, did not enhance the transcription mediated by either the MBP-TRE, TSH-TRE, or the malic enzyme (ME)-TRE. However, the ME-TRE is known to confer RXR beta-dependent enhancement of TH-induced gene expression. Thus, the capacity of RXR beta to modulate TH-dependent transcriptional regulation depends upon the nature of the TRE.

The N-terminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormone-dependent transactivation function.

In this study we have investigated the role of the N-terminal region of thyroid hormone receptors (TRs) in thyroid hormone (TH)-dependent transactivation of a thymidine kinase promoter containing TH response elements composed either of a direct repeat or an inverted palindrome. Comparison of rat TR beta 1 with TR beta 2 provides an excellent model since they share identical sequences except for their N termini. Our results show that TR beta 2 is an inefficient TH-dependent transcriptional activator. The degree of transactivation corresponds to that observed for the mutant TR delta N beta 1/2, which contains only those sequences common to TR beta 1 and TR beta 2. Thus, TH-dependent activation appears to be associated with two separate domains. The more important region, however, is embedded in the N-terminal domain. Furthermore, the transactivating property of TR alpha 1 was also localized to the N-terminal domain between amino acids 19 and 30. Using a coimmunoprecipitation assay, we show that the differential interaction of the N terminus of TR beta 1 and TR beta 2 with transcription factor IIB correlates with the TR beta 1 activation function. Hence, our results underscore the importance of the N-terminal region of TRs in TH-dependent transactivation and suggest that a transactivating signal is transmitted to the general transcriptional machinery via a direct interaction of the receptor N-terminal region with transcription factor IIB.

Differential 9-cis-retinoic acid-dependent transcriptional activation by murine retinoid X receptor alpha (RXR alpha) and RXR beta. Role of cell type and RXR domains.

The 9-cis-retinoic acid (9cRA)-inducible enhancer of the rat cellular retinol-binding protein type II gene (CRBP II) was shown to be differentially regulated by the murine retinoid X receptor alpha (RXR alpha) as compared with RXR beta. Transient transfection assays performed in NIH 3T3 fibroblast cells demonstrated that RXR alpha yielded a high level of 9cRA-dependent transcription of a reporter gene linked to the CRBP II enhancer, when compared with RXR beta. This effect was cell type-dependent, since both receptors elicited comparable transcriptional activation of the same reporter in P19 embryonal carcinoma cells. To further explore the structural determinants responsible for the differences between these two receptors, a series of chimeric receptor constructs were made. Co-transfection assays utilizing these chimeras demonstrated that both the N terminus and the hinge region connecting the DNA binding domain with the ligand binding domain of RXR alpha were responsible for the high level of 9cRA-dependent transcription observed in NIH 3T3 cells, Furthermore, the hinge region of RXR alpha was shown to be necessary to repress, in the absence of hormone, the transcriptional activation function located in the N-terminal domain of RXR alpha. These results stress the importance of functional links between different RXR domains and suggest an RXR subtype and cell type-dependent specificity in the control of the 9cRA response.