Constitutive Transactivation Research Articles

Estrogen receptor α isoforms generated by alternative use of cryptic exons.

Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

Cell differentiation modifies the p53 transcriptional program through a combination of gene silencing and constitutive transactivation

The p53 transcription factor is a master regulator of cellular responses to stress that is commonly inactivated in diverse cancer types. Despite decades of research, the mechanisms by which p53 impedes tumorigenesis across vastly different cellular contexts requires further investigation. The bulk of research has been completed using in vitro studies of cancer cell lines or in vivo studies in mouse models, but much less is known about p53 action in diverse non-transformed human tissues. Here, we investigated how different cellular states modify the p53 transcriptional program in human cells through a combination of computational analyses of publicly available large-scale datasets and in vitro studies using an isogenic system consisting of induced pluripotent stem cells (iPSCs) and two derived lineages. Analysis of publicly available mRNA expression and genetic dependency data demonstrated wide variation in terms of expression and function of a core p53 transcriptional program across various tissues and lineages. To monitor the impact of cell differentiation on the p53 transcriptome within an isogenic cell culture system, we activated p53 by pharmacological inhibition of its negative regulator MDM2. Using cell phenotyping assays and genome wide transcriptome analyses, we demonstrated that cell differentiation confines and modifies the p53 transcriptional network in a lineage-specific fashion. Although hundreds of p53 target genes are transactivated in iPSCs, only a small fraction is transactivated in each of the differentiated lineages. Mechanistic studies using small molecule inhibitors and genetic knockdowns revealed the presence of two major regulatory mechanisms contributing to this massive heterogeneity across cellular states: gene silencing by epigenetic regulatory complexes and constitutive transactivation by lineage-specific transcription factors. Altogether, these results illuminate the impact of cell differentiation on the p53 program, thus advancing our understanding of how this tumor suppressor functions in different contexts.

Identification of a novel C-terminally truncated estrogen receptor α variant (ERαi34) with constitutive transactivation and estrogen receptor antagonist resistance

Constitutively active estrogen receptor α (ERα) variants with C-terminal truncation are candidate molecules for gain of both endocrine- and chemotherapy-resistance in estrogen-sensitive tumors. Our previous reports using artificially truncated ERα constructs demonstrated that ERα variants encoded in 1-2-3-cryptic exon- and 1-2-3-4-cryptic exon-types of transcripts have potentials to display constitutive transactivation of an estrogen response element reporter. However, naturally occurring 1-2-3-cryptic exon-type ERα variants have not been cloned yet. Therefore, the present study was designed to identify naturally occurring ERα variants encoded in 1-2-3-cryptic exon-type ERα transcripts. We cloned a novel C-terminally truncated ERα variant (ERαi34) encoded in a 1-2-3-i34 transcript from MCF-7 cells and characterized its constitutive and ER antagonist-resistant transactivation in transfected cells. Stable transfection of the variant into MCF-7 cells augmented basal cell proliferation insensitive to fulvestrant. Collectively, we validated the structure-based mechanisms underlying constitutive and ER antagonist-resistant transactivation by C-terminally truncated ERα variants.

Palmitoylome profiling indicates that androgens regulate the palmitoylation of α‑tubulin in prostate cancer‑derived LNCaP cells and supernatants.

Prostate cancer is closely associated with constitutive transactivation of the androgen receptor (AR) signaling pathway. After treatment with androgen‑deprivation therapy (ADT), the majority of patients develop castration‑resistant prostate cancer within months or years. In order to investigate potential novel therapeutic targets in addition to ADT, the present study examined the regulatory mechanisms of the AR signaling pathway. In the present study, LNCaP cells were metabolically‑labeled with Alk‑C16, a palmitate probe. In addition, cells were treated with R1881, an androgen, or DMSO. Subsequently, click‑chemistry‑based palmitoylome profiling was performed in LNCaP cells and palmitoylated proteins were compared between cells treated with androgen and untreated cells. Androgen treatment was revealed to significantly increase the palmitoylation level of α‑tubulin. In addition, the palmitoylation level of Ras‑related protein Rab‑7a (Rab7a) was enhanced by androgen treatment. Palmitoylation of α‑tubulin and Rab7a were essential for cell proliferation. Notably, in the supernatant of LNCaP cells, the palmitoylation level of α‑tubulin was also increased following androgen treatment. Palmitoylation of α‑tubulin may provide a new potential target for the treatment of prostate cancer. In addition, the high level of α‑tubulin palmitoylation in the supernatant may represent a biomarker for early‑stage prostate cancer.

A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKβ inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

Prevalence of ESR1 E380Q mutation in tumor tissue and plasma from Japanese breast cancer patients

BackgroundESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations.MethodsWe screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations (E380Q, Y537S, Y537N, Y537C, and D538G) using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated.ResultsWe detected ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment.ConclusionsWe have shown that there are distinct populations of ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.

Characterization of rodent constitutively active estrogen receptor α variants and their constitutive transactivation mechanisms

Estrogen receptor α (ERα) mRNAs exhibit remarkable heterogeneity owing to complicated alternative splicing. Some encode C-terminally-truncated ERα proteins, which display ligand-independent transactivation or dominant-negative activity. We previously characterized C-terminally-truncated ERα mRNA variants with cryptic sequences in humans and mice, and demonstrated that helices in the ligand-binding domains (LBDs) of ERα variants contribute to ligand-independent transcriptional activity. However, existence of non-conventional coding exons and generation of constitutively active ERα variants have remained to be examined in rats. To comparatively analyze modular organization and splicing profiles of the ERα genes, the range of C-terminally-truncated ERα variants was explored in rats and mice using rapid amplification of cDNA ends and RT-PCR cloning. Furthermore, their functions were determined in transiently transfected cells using expression constructs and luciferase reporter assays. Multiple cryptic exons and C-terminally-truncated ERα variant mRNAs were identified in rats and mice. Naturally occurring and artificially truncated variants/constructs lacking helix 5 potentially exhibited gain-of-function in transfected cells. Although cryptic exons and splicing profiles were poorly conserved among humans, mice, and rats, constitutively active variants were generated from the ERα genes. Moreover, the primary mechanism of ligand-independent activation by C-terminally-truncated ERα variants is C-terminus to helix 5 deletion in the LBD. This comparative study documented the complexity of ERα genes, mRNAs, and proteins, and further determined the underlying structural basis of ligand-independent activation by C-terminally-truncated ERα variants.

Human C-terminally truncated ERα variants resulting from the use of alternative exons in the ligand-binding domain

The nuclear receptor genes contain alternative internal and terminal exons, with alternative exon incorporation yielding mRNA variants that encode various receptor types, including some with C-terminal truncation that exhibit constitutive activation or dominant-negative transcriptional transactivation. However, C-terminally truncated estrogen receptor α (ERα) variants with alternative sequences have rarely been reported in humans. Therefore, we assessed human ERα genomic organization and alternative splicing profiles, and identified both alternative exons and C-terminally truncated ERα variants. These naturally occurring C-terminally truncated ERα proteins were localized in the nuclei of transfected cells. In addition, ERαi45c and ERαΔ5 variants exhibited constitutive transactivation of an estrogen responsive element-driven promoter in transfected cells. We manufactured expression vectors encoding artificially truncated ERα constructs and evaluated their transactivation abilities to establish mechanisms determining the constitutive activity and dominant-negative properties of truncated variants. Lack of the region encoded in exon 8 eliminated basal and ligand-induced transcriptional transactivation. The C-terminally truncated ERα variants/constructs containing the helices 5 in their ligand-binding domains did not exhibit constitutive transactivation. Furthermore, we demonstrated that truncation from C-termini to helices 5 in the variant ligand-binding domains was required for constitutive activation and found that the remnant regions of the ligand-binding domains and variant-specific sequences influenced transcriptional transactivation efficiency. In conclusion, we elucidated the structural and functional features of novel C-terminally truncated ERα variants and revealed the mechanisms underlying constitutive transactivation by C-terminally truncated nuclear receptor variants.

Generation and characterization of a tomato DCL3-silencing mutant

DICER-like 3 (DCL3) is a major player in heterochromatic 24-nucleotide (nt) small RNA (sRNA) and long microRNA (lmiRNA) biogenesis, and higher plant DCL3 mutants have been characterized from Arabidopsis thaliana and rice. Here, a tomato DCL3 (SlDCL3) mutant was generated through the use of trans-activated artificial miRNA and characterized. Constitutive trans-activation knocked down SlDCL3 levels by ∼64%, resulting in dramatically decreased 24-nt sRNA levels and a significant increase in 21- and 22-nt sRNAs. The latter was correlated with specific upregulation of SlDCL4 and SlDCL2b, which function in the biogenesis of 21- and 22-nt sRNAs, respectively. Moreover, at the majority of sRNA-generating genomic loci, an almost complete overlap between small RNA signatures of control and silenced seedlings was observed, suggesting that the reductions in 24-nt sRNAs at these loci were compensated for by biogenesis of 21- and 22-nt sRNAs from the same double-stranded RNA substrates. In addition, bioinformatic analysis and reduced expression in SlDCL3-silenced seedlings identified four novel tomato lmiRNAs, two of which were found to be developmentally regulated. Taken together, these results establish the requirement of SlDCL3 for the biogenesis of 24-nt sRNAs and lmiRNAs in tomato and suggest SlDCL4 and SlDCL2b as surrogates for SlDCL3.

Global and local perturbation of the tomato microRNA pathway by atrans-activatedDICER-LIKE 1mutant

DICER-like 1 (DCL1) is a major player in microRNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22-nucleotide small RNAs. The increase in the 22-nucleotide small RNAs was correlated with specific up-regulation of SlDCL2b and SlDCL2d, which are probably involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, these results establish OP:SlDCL1IR as an efficient tool for analysing processes regulated by SlDCL1-mediated gene regulation in tomato.

Activation of two distinct MAPK pathways governs constitutive expression of matrix metalloproteinase-1 in human pancreatic cancer cell lines

Elevated matrix metalloproteinase-1 (MMP-1) expression is known to correlate with poor prognosis of pancreatic cancer. We investigated the molecular mechanisms of constitutive expression of MMP-1 in pancreatic cancer cell lines. Expression of MMP-1 mRNA and protein as well as its enzymatic activity were observed in three pancreatic cancer cell lines. Transient transfection assays of two MMP-1 promoter/luciferase constructs (full-length 4.4-kb or proximal 0.6-kb region) showed high levels of transcription in pancreatic cancer cells compared with non-MMP-1 producing cells. The 0.6-kb promoter region of MMP-1 gene contained three activator protein-1 (AP-1) sites and the strong AP-1 activity was detected by electrophoretic mobility shift assays (EMSAs). In these cells, production and phosphorylation of c-Jun were commonly observed. Phosphorylated c-Jun NH2-terminal kinase (p-JNK) and activator transcription factor-2 (p-ATF-2) were also detected in two of the three cell lines. Phosphorylated extracellular signal-regulated kinase (p-ERK) was observed in one. The promoter activity, AP-1-binding activity and MMP-1 production were suppressed by a specific inhibitor of JNK or MEK. K-ras mutation, reported to be present in three cell lines used, is known to activate JNK and ERK pathways. Considering the facts together, our results revealed that activation of JNK/AP-1 or ERK/AP-1 pathway plays crucial roles in constitutive transactivation of MMP-1 in these cancer cells. This study contributes to provide new insights into strategies for inhibiting tumor cell invasion in pancreatic cancer.

Characterization of the region of the aryl hydrocarbon receptor required for ligand dependency of transactivation using chimeric receptor between Drosophila and Mus musculus

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor. Although 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is high affinity and toxic to many vertebrate animals, invertebrate AhRs including Drosophila melanogaster AhR (spineless) have no ability to bind exogenous chemicals as ligands. To analyze the ligand-binding domain (LBD) of AhR, we used chimeras between mouse and Drosophila AhR. The chimeric AhR revealed that the LBD determines constitutive transactivation in Drosophila AhR or ligand-dependent activation in mouse AhR. The LBD was further divided into three blocks that corresponded to amino acids 230–300, 301–361, and 361–420 of the mouse sequence. Six chimeric proteins clarified that amino acids 291–350 of the Drosophila LBD, i.e. the middle region, were required to keep the protein in the active form in the absence of ligand binding, whereas in the mouse AhR, this region was required to maintain the protein in the inactive form in the absence of ligand. Furthermore, Arg346 in the middle region of the mouse LBD, was identified as amino acids that were critical for AhR activation by site-directed mutagenesis.

Role of hepatitis B virus X repression of C/EBPβ activity in the down-regulation of glutathione S-transferase A2 gene: implications in other phase II detoxifying enzyme expression

A genome-wide in silico screening rendered the genes of phase II enzymes in the rat genome whose promoters contain the putative DNA elements interacting with CCAAT/enhancer binding protein (C/EBP) and NF-E2-related factor (Nrf2). The hepatitis B virus X (HBx) protein strongly modulates the transactivation and/or the repression of genes regulated by some bZIP transcription factors.This study investigated the effects of HBx on the induction of phase II enzymes with the aim of elucidating the role of HBx interaction with C/EBPβ or Nrf2 bZIP transcription factors in hepatocyte-derived cells.Immunoblot and reporter gene analyses revealed that transfection of HBx interfered with the constitutive and inducible GSTA2 transactivation promoted by oltipraz (C/EBPβ activator), but not that by tert-butylhydroquinone (t-BHQ, Nrf2 activator). Moreover, HBx transfection completely inhibited GSTA2 reporter gene activity induced by C/EBPβ, but failed to inhibit that by Nrf2.Gel shift assays identified that HBx inhibited the increase in C/EBPβ−DNA complex formation by oltipraz, but not the increase in Nrf2−DNA complex by t-BHQ. Immunoprecipitation and immunoblot assays verified the direct interaction between HBx and C/EBPβ. Moreover, chromatin immunoprecipitation assays confirmed HBx inhibition of C/EBPβ binding to its binding site in the GSTA2 gene promoter. HBx repressed the induction of other phase II enzymes including GSTP, UDP-glucuronyltransferase 1A, microsomal epoxide hydrolase, GSTM1, GSTM2, and γ-glutamylcysteine synthase.These results demonstrate that HBx inhibits the induction of phase II detoxifying enzymes, which is mediated by its interaction with C/EBPβ, but not Nrf2, substantiating the specific role of HBx in phase II detoxifying capacity.

Loss of MLL PHD Finger 3 Is Necessary for MLL-ENL–Induced Hematopoietic Stem Cell Immortalization

Reciprocal chromosomal translocations at the MLL gene locus result in expression of novel fusion proteins, such as MLL-ENL, associated with leukemia. The three PHD finger cassette, one of the highly conserved domains in MLL, is absent in all fusion proteins. This domain has been shown to interact with Cyp33, a cyclophilin which enhances the recruitment of histone deacetylases (HDAC) to the MLL repression domain and mediates HOX gene repression. Insertion of the third PHD finger of MLL into MLL-ENL allows the recruitment of Cyp33 and, subsequently, HDAC1 to the fusion protein. Furthermore, expression of the fusion protein with the PHD finger insertion mediates the down-regulation of the HOXC8 gene expression in a Cyp33-dependent manner. Finally, the addition of the PHD finger domain or the third PHD finger alone into MLL-ENL blocks the hematopoietic stem cell immortalization potential of the fusion protein in serial plating colony assays. Insertion of only the first and second PHD fingers has no such effect. These data support the hypothesis that the binding of Cyp33 to the MLL third PHD finger switches the MLL function from transactivation to repression. In the immortalizing MLL fusion protein, the loss of the PHD fingers, in combination with the gain of the activation domain of ENL or of other partner proteins, makes the fusion protein a constitutive transactivator. This leads to constitutive overexpression of MLL target genes that block stem cell commitment and promote stem cell renewal, probably the first step in MLL-related leukemogenesis.

Induction of Nuclear Translocation of Constitutive Androstane Receptor by Peroxisome Proliferator-activated Receptor α Synthetic Ligands in Mouse Liver

Peroxisome proliferators activate nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) and enhance the transcription of several genes in liver. We report here that synthetic PPARalpha ligands Wy-14,643, ciprofibrate, clofibrate, and others induce the nuclear translocation of constitutive androstane receptor (CAR) in mouse liver cells in vivo. Adenoviral-enhanced green fluorescent protein-CAR expression demonstrated that PPARalpha synthetic ligands drive CAR into the hepatocyte nucleus in a PPARalpha- and PPARbeta-independent manner. This translocation is dependent on the transcription coactivator PPAR-binding protein but independent of coactivators PRIP and SRC-1. PPARalpha ligand-induced nuclear translocation of CAR is not associated with induction of Cyp2b10 mRNA in mouse liver. PPARalpha ligands interfered with coactivator recruitment to the CAR ligand binding domain and reduced the constitutive transactivation of CAR. Both Wy-14,643 and ciprofibrate occupied the ligand binding pocket of CAR and adapted a binding mode similar to that of the CAR inverse agonist androstenol. These observations, therefore, provide information for the first time to indicate that PPARalpha ligands not only serve as PPARalpha agonists but possibly act as CAR antagonists.

Regulation of porcine classical and nonclassical MHC class I expression

Major histocompatibility complex (MHC) class I molecules comprise a family of polymorphic cell surface receptors consisting of classical 1 a molecules that present antigenic peptides and nonclassical 1 b molecules. Gene expression for human classical and nonclassical MHC class I molecules has been shown to be differentially regulated by interferon, with variation in the nucleotide sequence of promoter regions, resulting in differences in interferon inducibility and basal levels of gene transcription. In this study on porcine classical and nonclassical swine leukocyte Ag (SLA) class I molecules, we show alignments of putative regulatory elements in the promoters of the three functional classical class I genes, SLA-1, SLA-2, and SLA-3; two nonclassical 1 b genes, SLA-6 and SLA-7; and a MIC-2 gene. Promoter elements were cloned upstream from a luciferase reporter gene, and the basal and inducible activities of each were characterized by expression in Max cells, an immortalized pig cell line that responds to interferon and tumor necrosis factor alpha (TNF-alpha). All three classical class I but not nonclassical promoters responded to interferon. This was confirmed by the transactivation of SLA-1, but not SLA-7, after the co expression with interferon regulatory factors (IRFs), IRF-1, IRF-2, IRF-3, IRF-7, and IRF-9. Classical class I genes were activated by cotransfection with nuclear factor kappa B (NF-kappaB) p65 and by treatment of cells with TNF-alpha, although, unlike human promoter there was no synergistic effect with interferon. The greatest effect on classical class I promoters was coexpression with the class II transactivator (CIITA), important for constitutive transactivation. These results determine the differential regulation of porcine classical and nonclassical MHC class I and reflects their importance in antigen presentation during infection.

Ganoderma lucidum inhibits proliferation of human breast cancer cells by down-regulation of estrogen receptor and NF-κB signaling

Ganoderma lucidum, an oriental medical mushroom, has been used in Asia for the prevention and treatment of a variety of diseases, including cancer. We have previously demonstrated that G. lucidum inhibits growth and induces cell cycle arrest at G0/G1 phase through the inhibition of Akt/NF-kappaB signaling in estrogen-independent human breast cancer cells. However, the molecular mechanism(s) responsible for the inhibitory effects of G. lucidum on the proliferation of estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cells remain to be elucidated. Here, we show that G. lucidum inhibited the proliferation of breast cancer MCF-7 and MDA-MB-231 cells by the modulation of the estrogen receptor (ER) and NF-kappaB signaling. Thus, G. lucidum down-regulated the expression of ERalpha in MCF-7 cells but did not effect the expression of ERbeta in MCF-7 and MDA-MB-231 cells. In addition, G. lucidum inhibited estrogen-dependent as well as constitutive transactivation activity of ER through estrogen response element (ERE) in a reporter gene assay. G. lucidum decreased TNF-alpha-induced (MCF-7) as well as constitutive (MDA-MB-231) activity of NF-kappaB. The inhibition of ER and NF-kappaB pathways resulted in the down-regulation of expression of c-myc, finally suppressing proliferation of estrogen-dependent as well as estrogen-independent cancer cells. Collectively, these results suggest that G. lucidum inhibits proliferation of human breast cancer cells and contain biologically active compounds with specificity against estrogen receptor and NF-kappaB signaling, and implicate G. lucidum as a suitable herb for chemoprevention and chemotherapy of breast cancer.

The constitutive androstane receptor mediates phenytoin induction of cyp2c37

Cytochrome P450 monoxygenases (P450) are responsible for the metabolism and detoxification of many therapeutics. Induction of P450 enzymes by drugs can lead to potentially harmful drug-drug interactions and alterations in drug therapy efficacy. Drug induction of P450 enzymes has been shown to involve the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR). Recently, we reported that the nuclear receptor CAR mediated phenytoin (DPH) induction of the murine Cyp2b10 and Cyp2c29 genes. Herein, we report the discovery of a second DPH inducible murine CYP2C, Cyp2c37. Quantitative RT-PCR and immunoblot data demonstrate that hepatic CYP2C37 mRNA and protein amounts are increased by DPH. We have identified a putative CAR response element (CAR-RE) at -2.8kb from the start of translation of the Cyp2c37 gene. Mutation of this CAR-RE in Cyp2c37 luciferase promoter constructs demonstrated that it is necessary for mCAR constitutive transactivation and that induction is governed by a transcriptional regulation mechanism. Furthermore in CAR-null mice, quantitative RT-PCR results indicate that the induction of CYP2C37 mRNA by DPH is abolished. These results suggest that DPH induction of the Cyp2c37 gene, like the Cyp2b10 and Cyp2c29 genes, is regulated by mCAR. However, in CAR-null mice induction of CYP3A11 mRNA is not abolished suggesting that both CAR and PXR can mediate DPH induction of P450 genes, depending on the gene promoter. This research was supported by the intramural division of the NIEHS.

CAR, The Continuously Advancing Receptor, in Drug Metabolism and Disease

The detoxification and elimination of potentially toxic foreign and endogenous compounds depends on the concerted action of xenobiotic metabolizing enzymes. Nuclear hormone receptors (NHRs) have emerged as key regulators of the expression of these enzymes and his review focuses on the xenosenor CAR (Constitutive Androstane Receptor, NR1I3). CAR is highly expressed in the liver and the small intestine, two key tissues expressing xenobiotic metabolizing enzymes, and mediates the induction of their expression by the widely used antiepileptic drug, phenobarbital (PB) and the potent synthetic inducer 1, 4-bis-(2-(3, 5, -dichloropyridyloxy)) benzene (TCPOBOP). TCPOBOP is an agonist ligand for CAR. PB induces its nuclear translocation, which results in increased expression of CAR target genes since, unlike the classical, ligand-dependent nuclear receptors, CAR is an apparently constitutive transactivator. This constitutive activity is inhibited by the inverse agonist ligands androstanol and androstenol. The CAR mediated induction of the expression of xenobiotic metabolizing enzymes is generally protective, but can be deleterious if toxic metabolites are produced. CAR also has a protective role in the stress response elicited by hyperbilirubinemia, as well as lithocholic acid induced cholestasis. In addition, recent studies show that CAR activation disrupts thyroid hormone homeostasis. Finally, CAR activation promotes hepatocyte proliferation and blocks apoptosis, and is essential for the tumorigenesis induced by its activators PB and TCPOBOP. The role of CAR in endobiotic and xenobiotics metabolism has clinical implications in disease prevention, drug-drug interactions, and the development of better drug treatments.

NuRD complex component Mi-2β binds to and represses RORγ-mediated transcriptional activation

RORγ is a nuclear receptor that binds to DNA motifs as a monomer to constitutively activate target genes. RORγ plays an important role in thymocyte development and lymph node organogenesis, while the regulation of RORγ-mediated transcriptional activation is currently unclear. The purpose of this study was to identify other nuclear proteins that interact with RORγ. A yeast two-hybrid screen with Y190 yeast cells under stringent conditions resulted in the identification of CHD4, also known as Mi-2β, as a RORγ-interacting protein. This interaction was confirmed by GST pull-down assays. This interaction occurred within the middle regulatory region (amino acids 719–1164) of Mi-2β. Transfection of Gal4-RORγ into HeLa cells resulted in constitutive transactivation of the MH100-tk-luc reporter. The addition of Mi-2β resulted in a dramatic 50% decrease in Gal4-RORγ-mediated transactivation. These data demonstrate that RORγ forms a protein–protein interaction with the regulatory region of Mi-2β, resulting in inhibition of RORγ transcriptional activity. These results may provide evidence as to how RORγ-mediated transactivation is regulated by other nuclear proteins.