Yanan Tian

Role of NF-κB in Regulation of PXR-mediated Gene Expression A MECHANISM FOR THE SUPPRESSION OF CYTOCHROME P-450 3A4 BY PROINFLAMMATORY AGENTS

It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-κB activation by lipopolysaccharide and tumor necrosis factor-α plays a pivotal role in the suppression of cyp3a4 through interactions of NF-κB with the PXR·retinoid X receptor (RXR) complex. Inhibition of NF-κB by NF-κB-specific suppressor SRIκBα reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-α. Furthermore, we showed that NF-κB p65 disrupted the association of the PXR·RXRα complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with the DNA-binding domain of RXRα and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR·RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner.

Ah receptor and NF-κB interplay on the stage of epigenome

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family. Its ligands include many natural and synthetic compounds, some of which, such as polyhalogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons, are important environmental contaminants. NF-kappaB is a pleiotropic factor that regulates many physiological and pathophysiological processes including the immune and inflammatory responses. In the past decade, accumulating evidence suggests close interactions between AhR and NF-kappaB pathways, and these interactions are potentially important mechanisms for many pathological processes such as the chemical-induced immune dysfunctions, carcinogenesis and alteration of xenobiotic metabolism and disposition. AhR-NF-kappaB interaction has become a mechanistic linchpin linking certain pathological responses induced by environmental insults. Furthermore, the AhR-NF-kappaB interaction provides basis for therapeutic applications of certain AhR ligands to treat human diseases. The effects of AhR-NF-kappaB on the epigenome are an important area that is not well understood. In this review, I highlight current research regarding the AhR-NF-kappaB(RelA) interactions with emphasis on the epigenetic impacts of these interactions on chromatin modifications and transcription elongation control.

Interactions between the aryl hydrocarbon receptor and P-TEFb. Sequential recruitment of transcription factors and differential phosphorylation of C-terminal domain of RNA polymerase II at cyp1a1 promoter.

The expression of the cytochrome P450 1A1 gene (cyp1a1) is regulated by the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor that mediates most toxic responses induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). In the nucleus, ligand-activated AhR binds to the xenobiotic response elements, initiating chromatin remodeling and recruitment of coregulators, leading to the formation of preinitiation complex followed by elongation. Here, we report that ligand-activated AhR recruits the positive transcription elongation factor (P-TEFb) and RNA polymerase II (RNA PII) to the cyp1a1 promoter with concomitant phosphorylation of the RNA PII carboxyl domain (CTD). Interestingly, the serine 2 and serine 5 of the heptapeptide repeats (YSPTSPS) were sequentially phosphorylated upon TCDD treatment. Inhibition of P-TEFb kinase activity by 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) suppressed CTD phosphorylation (especially serine 2 phosphorylation) and abolished processive elongation without disrupting the assembly of the preinitiation complex at the cyp1a1 promoter. Remarkably, we found that activation of NF-κB by TNF-α selectively inhibited TCDD-induced serine 2 phosphorylation in mouse liver cells, suggesting that residue-specific phosphorylation of RNA PII CTD at the cyp1a1 promoter is an important regulatory point upon which signal “cross-talk” converges. Finally, we show that ligand-activated AhR associated with P-TEFb through the C terminus of cyclin T1, suggesting that AhR recruit the P-TEFb to the cyp1a1 promoter whereupon its kinase subunit phosphorylates the RNA PII CTD.

Epigenetic regulation of transcriptional activity of pregnane X receptor by protein arginine methyltransferase 1

Pregnane X receptor (PXR) is a ligand-dependent transcription factor, regulating gene expression of enzymes and transporters involved in xenobiotic/drug metabolism. Here, we report that protein arginine methyltransferase 1 (PRMT1) is required for the transcriptional activity of PXR. PRMT1 regulates expression of numerous genes, including nuclear receptor-regulated transcription, through methylating histone and non-histone proteins. Co-immunoprecipitation and histone methyltransferase assays revealed that PRMT1 is a major histone methyltransferase associated with PXR. The PXR ligand-binding domain is responsible for PXR-PRMT1 interaction as determined by mammalian two-hybrid and glutathione S-transferase (GST) pull-down assays. The chromatin immunoprecipitation (ChIP) assay showed that PRMT1 was recruited to the regulatory region of the PXR target gene cytochrome P450 3A4 (CYP3A4), with a concomitant methylation of arginine 3 of histone H4, in response to the PXR agonist rifampicin. In mammalian cells, small interfering RNA (siRNA) knockdown and gene deletion of PRMT1 greatly diminished the transcriptional activity of PXR, suggesting an indispensable role of PRMT1 in PXR-regulated gene expression. Interestingly, PXR appears to have a reciprocal effect on the PRMT1 functions by regulating its cellular compartmentalization as well as its substrate specificity. Taken together, these results demonstrated mutual interactions and functional interplays between PXR and PRMT1, and this interaction may be important for the epigenetics of PXR-regulated gene expression.

Pregnane X receptor protects HepG2 cells from BaP-induced DNA damage.

Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

Pregnane X receptor suppresses proliferation and tumourigenicity of colon cancer cells

Background:Pregnane X receptor (PXR) is a nuclear receptor that regulates the metabolism and disposition of various xenobiotics and endobioitics. We investigated a novel PXR function in regulating colon tumourigenesis in this study.Methods:Histochemistry, transfection, cell proliferation assay, anchorage-α-dependent assay, xenograft, immunohistochemistry, immunofluorescence flow cytometry.Results:Using histochemistry analysis, we found that PXR expressions were lost or greatly diminished in many colon tumours. Ectopic expression of human PXR through stable transfection of PXR into colon cancer cell line HT29 significantly inhibited cell proliferation as determined by cell proliferation assay and anchorage-independent assay. Pregnane X receptor suppressed significantly HT29 xenograft tumour growth in nude mice compared with control (310±6.2 vs 120±6 mg, P<0.01). Immunohistochemistry and immunofluorescence analysis of Ki-67 on excised xenograft tumour tissues showed that PXR inhibited cancer cell proliferation. Furthermore, expressions of PXR and Ki-67 were mutually exclusive. The flow cytometry analysis indicated that PXR caused G0/G1 cell-cycle arrest. p21WAF1/CIP1 expression was markedly elevated whereas E2F1 expression was inhibited by PXR.Conclusion:PXR inhibits the proliferation and tumourigenicity of colon cancer cells by controlling cell cycle at G0/G1 cell phase by regulating p21WAF1/CIP1 and E2F/Rb pathways.

Histopathology and the detection of avian bornavirus in the nervous system of birds diagnosed with proventricular dilatation disease.

Avian bornavirus (ABV) is currently considered a probable etiologic agent of proventricular dilatation disease (PDD) of psittacines. We tested 24 stored avian brain samples, processed for histopathology and retained following their submission for necropsy or histopathology to the Schubot Exotic Bird Center diagnostic laboratory in 1992. Thirteen of these samples were from birds diagnosed at that time as suffering from PDD. The remaining 11 samples were diagnosed as suffering from diseases other than PDD. Immunohistochemistry was performed using an antiserum directed against the ABV nucleoprotein (N-protein). Stained slides were read by an investigator unaware of their prior histopathology results. Cells containing ABV N-protein were present in the nervous tissues of all 13 PDD cases. One bird not previously diagnosed with PDD also had ABV N-protein in its brain. A review of this birds necropsy report indicated that it was, most probably, also suffering from PDD. The remaining 10 non-PDD birds had no detectable N-protein in their brains. The N-protein was present in the cerebrum, cerebellum and spinal cord. These findings support other studies that indicate that ABV is an etiological agent of PDD.

The Aldo-Keto Reductase Akr1b7 Gene Is a Common Transcriptional Target of Xenobiotic Receptors Pregnane X Receptor and Constitutive Androstane Receptor

Aldo-keto reductase (AKR) family 1, member 7 (AKR1B7), a member of the AKR superfamily, has been suggested to play an important role in the detoxification of lipid peroxidation by-products. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are xenosensors postulated to alleviate xeno- and endobiotic chemical insults. In this study, we show that the mouse Akr1b7 is a shared transcriptional target of PXR and CAR in the liver and intestine. Treatment of wild-type mice with the PXR agonist pregnenolone-16α-carbonitrile (PCN) activated Akr1b7 gene expression, whereas the effect was abrogated in PXR(-/-) mice. Similarly, the activation of Akr1b7 gene expression by the CAR agonist 1,4-bis[2-(3,5-dichlorpyridyloxyl)]-benzene, seen in wild-type mice, was abolished in CAR(-/-) mice. The promoter of Akr1b7 gene was activated by PXR and CAR, and this activation was achieved through the binding of PXR-retinoid X receptor (RXR) or CAR-RXR heterodimers to direct repeat-4 type nuclear receptor-binding sites found in the Akr1b7 gene promoter. At the functional level, treatment with PCN in wild-type mice, but not PXR(-/-) mice, led to a decreased intestinal accumulation of malondialdehyde, a biomarker of lipid peroxidation. The regulation of Akr1b7 by PXR was independent of the liver X receptor (LXR), another nuclear receptor known to regulate this AKR isoform. Because a major function of Akr1b7 is to detoxify lipid peroxidation, the PXR-, CAR-, and LXR-controlled regulatory network of Akr1b7 may have contributed to alleviate toxicity associated with lipid peroxidation.

Pregnane X Receptor as the “Sensor and Effector” in Regulating Epigenome

The pregnane X receptor (PXR, NR1I2) is a ligand‐activated nuclear receptor which plays an essential role in organisms metabolic detoxification system by sensing the presence of xenobiotics and triggering detoxification responses. In addition to its role in xenobiotic metabolism, PXR has pleiotropic functions in regulating immune/inflammatory responses, cell proliferation, bile acid/cholesterol metabolism, glucose and lipid metabolism, steroid/endocrine homeostasis, and bone metabolism. Recent research suggests that the PXR is required for maintaining healthy commensalism between microbiota and gut. Interestingly, the metabolites such as indole derivatives from commensal microbes serve as the ligands for the PXR in intestinal epithelium forming an intricate mutualistic interaction between host and microbiota. PXR‐regulated gene responses are controlled at epigenetic level by chromatin modifications, DNA methylation and noncoding RNA. Developmental alterations of the epigenome by exposure to the xenobiotics or diseases may produce persistent changes in PXR‐regulated physiological responses. These new areas of research promise to vastly increase our understanding of PXR‐regulated responses. In this review we highlight recent results on the epigenetic mechanisms for the PXR‐regulated gene expression and discuss the physiological significance of these findings. J. Cell. Physiol. 230: 752–757, 2015.

T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway

T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10 ng/ml) and androstenedione (3 ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100 nM, 24 h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1 ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.