Thyroid hormone (triiodothyronine, T 3 ) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T 3 response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TR␤ activates transcription more efficiently than TR␣ at an inverted palindrome (F2), and this correlates with preferential TR␤ homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TR␤ homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TR␤ mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TR␤ homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TR␤ requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR⅐TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo.

There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/− T3 in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T3 or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T3 response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/− T3, TR regulation patterns and T3 dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).

Synthetic selective thyroid hormone (TH) receptor (TR) modulators (STRM) exhibit beneficial effects on dyslipidemias in animals and humans and reduce obesity, fatty liver, and insulin resistance in preclinical animal models. STRM differ from native TH in preferential binding to the TRβ subtype vs. TRα, increased uptake into liver, and reduced uptake into other tissues. However, selective modulators of other nuclear receptors exhibit important gene-selective actions, which are attributed to differential effects on receptor conformation and dynamics and can have profound influences in animals and humans. Although there are suggestions that STRM may exhibit such gene-specific actions, the extent to which they are actually observed in vivo has not been explored. Here, we show that saturating concentrations of the main active form of TH, T(3), and the prototype STRM GC-1 induce identical gene sets in livers of euthyroid and hypothyroid mice and a human cultured hepatoma cell line that only expresses TRβ, HepG2. We find one case in which GC-1 exhibits a modest gene-specific reduction in potency vs. T(3), at angiopoietin-like factor 4 in HepG2. Investigation of the latter effect confirms that GC-1 acts through TRβ to directly induce this gene but this gene-selective activity is not related to unusual T(3)-response element sequence, unlike previously documented promoter-selective STRM actions. Our data suggest that T(3) and GC-1 exhibit almost identical gene regulation properties and that gene-selective actions of GC-1 and similar STRM will be subtle and rare.

Background:Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family.Objectives:We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG).Methods:The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells.Results:Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene.Conclusion:Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation.Citation:Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM, Yaswen P, Vulpe CD, Leitman DC. 2016. Parabens and human epidermal growth factor receptor ligand cross-talk in breast cancer cells. Environ Health Perspect 124:563–569; http://dx.doi.org/10.1289/ehp.1409200

Glucocorticoids exert potent anti-inflammatory effects by repressing proinflammatory genes. We previously demonstrated that estrogens repress numerous proinflammatory genes in U2OS cells. The objective of this study was to determine if cross talk occurs between the glucocorticoid receptor (GR) and estrogen receptor (ER)α. The effects of dexamethasone (Dex) and estradiol on 23 proinflammatory genes were examined in human U2OS cells stably transfected with ERα or GR. Three classes of genes were regulated by ERα and/or GR. Thirteen genes were repressed by both estradiol and Dex (ER/GR-repressed genes). Five genes were repressed by ER (ER-only repressed genes), and another five genes were repressed by GR (GR-only repressed genes). To examine if cross talk occurs between ER and GR at ER/GR-repressed genes, U2OS-GR cells were infected with an adenovirus that expresses ERα. The ER antagonist, ICI 182780 (ICI), blocked Dex repression of ER/GR-repressed genes. ICI did not have any effect on the GR-only repressed genes or genes activated by Dex. These results demonstrate that ICI acts on subset of proinflammatory genes in the presence of ERα but not on GR-activated genes. ICI recruited ERα to the IL-8 promoter but did not prevent Dex recruitment of GR. ICI antagonized Dex repression of the TNF response element by blocking the recruitment of nuclear coactivator 2. These findings indicate that the ICI–ERα complex blocks Dex-mediated repression by interfering with nuclear coactivator 2 recruitment to GR. Our results suggest that it might be possible to exploit ER and GR cross talk for glucocorticoid therapies using drugs that interact with ERs.

Estrogens are frequently used in reproductive medicine. The Women's Health Initiative trial found that the risks of menopausal hormone therapy (MHT) exceed the benefits. The estrogens in MHT, however, were introduced prior to our understanding of the mechanism of action of estrogens. Estrogen signaling is highly complex, involving various DNA regulatory elements to which estrogen receptors bind. Numerous transcription factors and co-regulatory proteins modify chromatin structure to further regulate gene transcription. With a greater understanding of estrogen action, the major problem with the current estrogens in MHT appears to be that they are nonselective. This produces beneficial effects in bone, brain, and adipose tissue but increases the risk of breast and endometrial cancer and thromboembolism. Resurrecting MHT for long-term therapy will require the development of more selective estrogens, such as estrogen receptor (ER)β-selective estrogens and tissue-selective ERα agonists. These compounds will offer the best prospects to expand the indications of MHT and thus prevent the chronic conditions associated with menopause.

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Hep G2 cells were used to study the synthesis and secretion of phospholipid transfer protein (PLTP). Upon incubation of the cells at confluence with serum-free Dulbecco's modified Eagle's medium (DMEM), phosphatidylcholine (PC) transfer activity was found to accumulate in the culture media. The PC transfer activity in the media was effectively inhibited by rabbit anti-human PLTP immunoglobulin (Ig)G, thus indicating that the PC transfer activity was due to secreted PLTP. The molecular weight of Hep G2 PLTP was approximately 78 kDa by Western blot analysis, in agreement with the molecular weight obtained for purified human plasma PLTP. The PLTP secreted by Hep G2 also possessed an HDL conversion activity similar to that of human plasma PLTP. The addition of butyrate to the cell culture media resulted in a marked increase in the secretion of PLTP. After 24 h incubation with 4 mmol/L sodium butyrate, a more than twofold increase (P < 0.01) of PC transfer activity in the cell-conditioned media was obtained. The dose-dependent increase in the PC transfer activity in the media upon butyrate treatment was well correlated (r = 0.80, P < 0.01) with that of PLTP mass as determined by immuno-slot blot analysis of cell-conditioned media. The increased secretion of PLTP by Hep G2 treated with sodium butyrate was accompanied by a greater increase in the level of PLTP mRNA in the cells as determined by ribonuclease protection assay. In the presence of 4 mmol/L sodium butyrate, a fourfold increase (P < 0. 01) in mRNA level was obtained at 24 h. No stabilizing effect of butyrate on PLTP mRNA was apparent upon treatment of the cultured cells with the RNA synthesis inhibitor, actinomycin D. Thus, the up-regulatory effect of butyrate on PLTP gene expression seemed to have occurred at the transcriptional level.