Estrogen receptors (ERs alpha and beta) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ERalpha AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ERalpha utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ERalpha AF-1 and weakly inhibited by AF-2. Indeed, ERalpha truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ERbeta, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors.

Clinical evidence indicates that higher levels of estrogen receptor beta (ERbeta) predicts improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy. To better understand the mechanisms in which ERbeta can modulate breast cancer therapies, we introduced ERbeta under an inducible promoter into MCF-7 breast cancer cells. In these cells, induction of ERbeta expression led to a shift in the potency and an increase in the efficacy of tamoxifen to inhibit proliferation. A similar effect on breast cancer cells was observed for two other antiestrogens, raloxifene, and fulvestrant. Induced expression of ERbeta did not enhance the antiproliferative effects of small molecule inhibitors that target the epidermal growth factor receptor, insulin growth factor receptor-1 and histone deacetylase, indicating ERbeta specifically cooperates with antiestrogens. The combination of ERbeta expression, which arrests cells in G2, and tamoxifen, which arrests cells in G1, led to a potent blockade of the cell cycle. ERbeta also increased tamoxifen-induced cell death and cooperated with tamoxifen to induce expression of the pro-apoptotic gene bik. In summary, our data indicates that ERbeta increases the efficacy of antiestrogens by effects on apoptosis and on cell cycling and, together with clinical observations, suggests ERbeta could be a valuable prognostic marker and potential therapeutic target.

Here we report a novel potential therapeutic strategy using histone deacetylase (HDAC) inhibitors to enhance the action of hormonal therapy agents in estrogen receptor alpha (ER alpha)-positive breast cancer. HDAC inhibitors [trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA)], inhibited proliferation of MCF-7 breast cancer cells and, in combination with tamoxifen inhibited proliferation better than with either agent alone. VPA, an anti-convulsant drug with HDAC inhibitory activity, enhanced tamoxifen action at doses within the concentration range used for anti-convulsive therapy. VPA cooperated with tamoxifen in a variety of ER alpha-positive cell lines and was also effective when combined with other antiestrogens, and with aromatase inhibition. VPA enhanced antiestrogen action by promoting cell death via apoptosis without affecting cell cycling. Some of this action may be due to VPA's ability to induce the pro-apoptotic gene Bik, which is also induced by antiestrogens. Remarkably, VPA blocked the undesirable pro-proliferative action of tamoxifen on uterine endometrial cells. Our in vitro results suggest that VPA and other HDAC inhibitors have the potential to enhance hormonal therapy for ER alpha-positive breast cancer and simultaneously reverse the adverse effects of antiestrogens in the uterus.

The presence of stable multiprotein complexes containing adrenergic receptors is verified in live H9c2 cardiomyocyte-like cells by single-particle tracking. The immobilization of β-adrenergic receptors presumably contributes to the specificity of cardiac adrenergic responses.

Unliganded thyroid hormone receptors (TRs) repress transcription through recruitment of corepressors, including nuclear receptor corepressor (N-CoR). We find that N-CoR contains three interaction domains (IDs) that bind to TR, rather than the previously reported two. The hitherto unrecognized ID (ID3) serves as a fully functional TR binding site, both in vivo and in vitro, and may be the most important for TR binding. Each ID motif contains a conserved hydrophobic core (I/LXXII) that resembles the hydrophobic core of nuclear receptor boxes (LXXLL), which mediates p160 coactivator binding to liganded nuclear receptors. Although the integrity of the I/LXXII motif is required for ID function, substitution of ID isoleucines with leucines did not allow ID peptides to bind to liganded TR, and substitution of NR box leucines with isoleucines did not allow NR box peptides to bind unliganded TR. This indicates that the binding preferences of N-CoR for unliganded TR and p160s for liganded TR are not dictated solely by the identity of conserved hydrophobic residues within their TR binding motifs. Examination of sequence conservation between IDs, and mutational analysis of individual IDs, suggests that they are comprised of the central hydrophobic core and distinct adjacent sequences that may make unique contacts with the TR surface. Accordingly, a hybrid peptide that contains distinct adjacent sequences from ID3 and ID1 shows enhanced binding to TR.

Pseudomonas aeruginosa infections are associated with high mortality rates and occur in diverse conditions including pneumonias, cystic fibrosis and neutropenia. Quorum sensing, mediated by small molecules including N-(3-oxo-dodecanoyl) homoserine lactone (C12), regulates P. aeruginosa growth and virulence. In addition, host cell recognition of C12 initiates multiple signalling responses including cell death. To gain insight into mechanisms of C12-mediated cytotoxicity, we studied the role of endoplasmic reticulum stress in host cell responses to C12. Dramatic protection against C12-mediated cell death was observed in cells that do not produce the X-box binding protein 1 transcription factor (XBP1s). The leucine zipper and transcriptional activation motifs of XBP1s were sufficient to restore C12-induced caspase activation in XBP1s-deficient cells, although this polypeptide was not transcriptionally active. The XBP1s polypeptide also regulated caspase activation in cells stimulated with N-(3-oxo-tetradecanoyl) homoserine lactone (C14), produced by Yersinia enterolitica and Burkholderia pseudomallei, and enhanced homoserine lactone-mediated caspase activation in the presence of endogenous XBP1s. In C12-tolerant cells, responses to C12 including phosphorylation of p38 MAPK and eukaryotic initiation factor 2α were conserved, suggesting that C12 cytotoxicity is not heavily dependent on these pathways. In summary, this study reveals a novel and unconventional role for XBP1s in regulating host cell cytotoxic responses to bacterial acyl homoserine lactones.

Antiestrogens, including tamoxifen and raloxifene, block estrogen receptor (ER) action by blocking the interactions of an estrogen-dependent activation function (AF-2) with p160 coactivators. Although tamoxifen does show some agonist activity in the presence of ER␣, this stems from a distinct constitutive activation function (AF-1) that lies within the ER␣ N terminus. Previous studies identified a naturally occurring mutation (D351Y) that allows ER␣ to perceive tamoxifen and raloxifene as estrogens. Here, we examine the contributions of ER␣ activation functions to the D351Y phenotype. We find that the AF-2 function of ER␣ D351Y lacks detectable tamoxifen-dependent activity when tested in isolation but does synergize with AF-1 to allow enhanced tamoxifen response. Weak tamoxifen-dependent interactions between the ER␣ D351Y AF-2 function and GRIP1, a representative p160, can be detected in glutathione S-transferase binding assays and mammalian two-hybrid assays. Furthermore, tamoxifen-dependent AF-2 activity can be detected in the presence of ER␣ D351Y and high levels of overexpressed GRIP1. We therefore propose that the D351Y mutation allows weak tamoxifen-dependent AF-2 activity but that this activity is only detectable when AF-1 is strong, and AF-1 and AF-2 synergize, or when p160s are overexpressed. We discuss the possible structural basis of this effect.Estrogens act by binding two specific intracellular receptor proteins (ERs 1 ␣ and ␤, hereafter ER␣ and ER␤; reviewed in Refs.

SUMMARY P. aeruginosa infections are commonly associated with cystic fibrosis, pneumonias, neutropenia and burns. The P. aeruginosa quorum sensing molecule N-(3-oxo-dodecanoyl) homoserine lactone (C12) cause multiple deleterious host responses, including repression of NF-κB transcriptional activity and apoptosis. Inhibition of C12-mediated host responses is predicted to reduce P. aeruginosa virulence. We report here a novel, host-targeted approach for potential adjunctive anti-Pseudomonal therapy based on inhibition of C12-mediated host responses. A high-throughput screen was developed to identify C12 inhibitors that restore NF-κB activity in C12-treated, lipopolysaccharide (LPS)-stimulated cells. Triazolo[4,3-a]quinolines with nanomolar potency were identified as C12-inhibitors that restored NF-κB-dependent luciferase expression in LPS- and TNF-stimulated cell lines. In primary macrophages and fibroblasts, triazolo[4,3-a]quinolines inhibited C12 action to restore cytokine secretion in LPS-stimulated cells. Serendipitously, in the absence of an inflammatory stimulus, triazolo[4,3-a]quinolines prevented C12-mediated responses, including cytotoxicity, elevation of cytoplasmic calcium, and p38 MAPK phosphorylation. In vivo efficacy was demonstrated in a murine model of dermal inflammation involving intradermal C12 administration. The discovery of triazolo[4,3-a]quinolines provides a pharmacological tool to investigate C12-mediated host responses, and a potential host-targeted anti-Pseudomonal therapy.