The Ah receptor (AhR) mediates many of the toxic responses induced by polyhalogenated and polycyclic hydrocarbons (PAHs) which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. NF-B is a pleiotropic transcription factor controlling many physiological functions adversely affected by PAHs, including immune suppression, thymus involution, hyperkeratosis, and carcinogenesis. Here, we show physical interaction and mutual functional repression between AhR and NF-B. This mutual repression may provide an underlying mechanism for many hitherto poorly understood PAH-induced toxic responses, and may also provide a mechanistic explanation for alteration of xenobiotic metabolism by cytokines and compounds that regulate NF-B.
Proinflammatory cytokines, such as tumor necrosis factor (TNF)-␣, interleukin-1, and lipopolysaccharides (LPS), suppress the gene expression of cytochrome P-450 1A1 (cyp1a1). The mechanism of the suppression is not well understood. In present study, we show that activation of nuclear factor-B (NF-B) is a critical event leading to the suppression of cyp1a1 gene expression, thus providing an underlying mechanism for the TNF-␣-and LPS-induced cyp1a1 suppression. We demonstrated that: (i) inducible RelA expression down-regulated aryl hydrocarbon receptor (AhR) activated reporter gene; (ii) the suppressive effects of LPS and TNF-␣ on the AhR-activated reporter gene could be blocked by pyrrolidine dithiocarbamate, which is known to inhibit NF-B action; and (iii) TNF-␣ and LPSimposed repression could be reversed by the NF-B super repressor (SRIB␣), thus demonstrating the specific involvement of NF-B. Furthermore, nuclear receptor coactivators p300/CBP and steroid receptor coactivator-1 act individually as well as cooperatively to reverse the suppressive effects by NF-B on the AhR-activated reporter gene, suggesting that these transcriptional coactivators serve as the common integrators for the two pathways, thereby mediating the cross-interactions between AhR and NF-B. Finally, using the chromatin immunoprecipitation assay, we demonstrated that AhR ligand induces histone H4 acetylation at the cyp1a1 promoter region containing the TATA box, whereas TNF-␣ inhibits this acetylation, suggesting that AhR/NF-B interaction converges at level of transcription involving chromatin remodeling.
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-B activation by lipopolysaccharide and tumor necrosis factor-␣ plays a pivotal role in the suppression of cyp3a4 through interactions of NF-B with the PXR⅐retinoid X receptor (RXR) complex. Inhibition of NF-B by NF-B-specific suppressor SRIB␣ reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-␣. Furthermore, we showed that NF-B p65 disrupted the association of the PXR⅐RXR␣ complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-B p65 directly interacted with the DNA-binding domain of RXR␣ and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR⅐RXR␣ complex. This mechanism of suppression by NF-B activation may be extended to other nuclear receptor-regulated systems where RXR␣ is a dimerization partner.Inflammatory responses and infections suppress the biotransformation of drugs and decrease the hepatointestinal capacity of drug clearance. This results in alterations of therapeutic indices and increases the toxicity of certain administered drugs. Inflammatory responses also play important roles in liver pathological conditions such as drug-induced hepatitis and cholestatic diseases (1, 2). The mechanisms of these clinically important effects have not been well understood.In human liver, the first pass of biotransformation is mainly carried out by cytochrome P-450 (CYP) 2 3A4, which is the predominant isoform of monooxygenases that are expressed in the adult hepatointestinal system. It is estimated that CYP3A4 is responsible for the metabolism of over 50% of drugs in use today, many of which are either metabolically activated and/or metabolically broken down (detoxified) through this enzyme. Therefore, transcriptional and post-transcriptional alterations of CYP3A4 activity have direct effects on the efficacy of drugs and detoxification of xenobiotics (reviewed in Refs. 3 and 4). Recent molecular and pharmacological studies have demonstrated that transcriptional activation of cyp3a4 is mediated by the nuclear receptor PXR (pregnane X receptor). The rodent PXR (5) and its human homolog hPXR (6), also known as steroid and xenobiotic receptor (7) or hPAR (8), were identified as xenobiotic receptors that can be activated by certain xenobiotics and endobiotics. PXR regulates the expression of cyp3a4 by associating with its obligate partner RXR, and the heterodimer binds to the nuclear receptor response elements found in the regulatory regions of these genes. Genes that are regulated by PXR include multiple drug-resistant genes such as MDR1 (9) and MRP2 (10) as wel...
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-a-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, Po0.01). 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 G 0 /G 1 cell-cycle arrest. p21 WAF1/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 G 0 /G 1 cell phase by regulating p21 WAF1/CIP1 and E2F/Rb pathways.
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 (PXR)2 is an orphan nuclear receptor that regulates metabolism and disposition of various xenobiotics and endobiotics (1). These physiological functions of PXR are achieved through coordinating transcriptional regulation of Phase I and Phase II drug-metabolizing enzymes as well as the "Phase III" transporters (2). The structural flexibility in the ligand binding pocket enables PXR to function as a xenobiotic receptor through interacting with a wide range of structurally diverse compounds (3). Xeno-and endobiotics that activate PXR include a variety of prescription and nonprescription drugs, herbal medicines, environmental toxicants, and bile acids (4). The transcriptional activity of PXR is not only regulated by ligands, but also by various signal transduction pathways including NF-B-regulated inflammatory pathway (5). We have shown that the transcriptional activity of PXR is negatively regulated by NF-B (5), which may play a role in the pathogenesis of inflammatory bowl diseases and colon cancer (6).Post-translational modifications on the N termini of histones have been shown to play critical roles in gene regulation including the regulation of transcriptional activity by nuclear receptors. These modifications include phosphorylation, acetylation, methylation, and ubiquitination (7). It is believed that the combination of modifications of the chromatin-associated histone and non-histone proteins, and the interplays between these modificat...
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