Dalei Wu

Cross-talk between Aryl Hydrocarbon Receptor and the Inflammatory Response A ROLE FOR NUCLEAR FACTOR-κB

Background: Aryl hydrocarbon receptor (AhR) is a protein regulating differentiation and function of immune cells. Results: NF-κB activates transcription of AhR and enhances activity of AhR-regulated genes. Conclusion: Activation of NF-κB involves RelA-mediated expression of AhR. Significance: Inflammatory stimuli and cytokines that regulate NF-κB induce AhR expression during activation and differentiation of immune cells. The aryl hydrocarbon receptor (AhR) is involved in the regulation of immune responses, T-cell differentiation, and immunity. Here, we show that inflammatory stimuli such as LPS induce the expression of AhR in human dendritic cells (DC) associated with an AhR-dependent increase of CYP1A1 (cytochrome P4501A1). In vivo data confirmed the elevated expression of AhR by LPS and the LPS-enhanced 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated induction of CYP1A1 in thymus of B6 mice. Inhibition of nuclear factor-κB (NF-κB) repressed both normal and LPS-enhanced, TCDD-inducible, AhR-dependent gene expression and canonical pathway control of RelA-regulated AhR-responsive gene expression. LPS-mediated induction of AhR was NF-κB-dependent, as shown in mouse embryonic fibroblasts (MEFs) derived from Rel null mice. AhR expression and TCDD-mediated induction of CYP1A1 was significantly reduced in RelA-deficient MEF compared with wild type MEF cells and ectopic expression of RelA restored the expression of AhR and induction of CYP1A1 in MEF RelA null cells. Promoter analysis of the human AhR gene identified three putative NF-κB-binding elements upstream of the transcription start site. Mutation analysis of the AhR promoter identified one NF-κB site as responsible for mediating the induction of AhR expression by LPS and electrophoretic shift assays demonstrated that this NF-κB motif is recognized by the RelA/p50 heterodimer. Our results show for the first time that NF-κB RelA is a critical component regulating the expression of AhR and the induction of AhR-dependent gene expression in immune cells illustrating the interaction of AhR and NF-κB signaling.

Activation of Aryl Hydrocarbon Receptor Induces Vascular Inflammation and Promotes Atherosclerosis in Apolipoprotein E−/− Mice

Objective—Exposure to dioxins has been shown to contribute to the development of inflammatory diseases, such as atherosclerosis. Macrophage-mediated inflammation is a critical event in the initiation of atherosclerosis. Previously, we showed that treatment of macrophages with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to aryl hydrocarbon receptor (AhR)–dependent activation of inflammatory mediators and the formation of cholesterol-laden foam cells. However, the mechanisms responsible for the formation of atherosclerotic lesions mediated through AhR have not been identified. Methods and Results—An in vitro macrophage and an apolipoprotein E (ApoE)−/− mouse model were used to determine whether chemokines and their receptors are responsible for the AhR-mediated atherogenesis. Exposure of ApoE−/− mice to TCDD caused a time-dependent progression of atherosclerosis, which was associated with induction of inflammatory genes, including interleukin-8, as well as F4/80 and matrix metalloproteinase-12. A high-fat diet enhanced the TCDD-mediated inflammatory response and aggravated the formation of complex atheromas. Treatment with a CXCR2 inhibitor and an AhR antagonist reduced the TCDD-induced progression of early atherosclerotic lesions in ApoE−/− mice. Conclusion—The results suggest that CXCR2 mediates the atherogenic activity of environmental pollutants, such as dioxins, and contributes to the development of atherosclerosis through the induction of a vascular inflammatory response by activating the AhR-signaling pathway.

Multidomain integration in the structure of the HNF-4α nuclear receptor complex

The hepatocyte nuclear factor 4α (HNF-4α; also known as NR2A1) is a member of the nuclear receptor (NR) family of transcription factors, which have conserved DNA-binding domains and ligand-binding domains. HNF-4α is the most abundant DNA-binding protein in the liver, where some 40% of the actively transcribed genes have a HNF-4α response element. These regulated genes are largely involved in the hepatic gluconeogenic program and lipid metabolism. In the pancreas HNF-4α is also a master regulator, controlling an estimated 11% of islet genes. HNF-4α protein mutations are linked to maturity-onset diabetes of the young, type 1 (MODY1) and hyperinsulinaemic hypoglycaemia. Previous structural analyses of NRs, although productive in elucidating the structure of individual domains, have lagged behind in revealing the connectivity patterns of NR domains. Here we describe the 2.9 Å crystal structure of the multidomain human HNF-4α homodimer bound to its DNA response element and coactivator-derived peptides. A convergence zone connects multiple receptor domains in an asymmetric fashion, joining distinct elements from each monomer. An arginine target of PRMT1 methylation protrudes directly into this convergence zone and sustains its integrity. A serine target of protein kinase C is also responsible for maintaining domain–domain interactions. These post-translational modifications lead to changes in DNA binding by communicating through the tightly connected surfaces of the quaternary fold. We find that some MODY1 mutations, positioned on the ligand-binding domain and hinge regions of the receptor, compromise DNA binding at a distance by communicating through the interjunctional surfaces of the complex. The overall domain representation of the HNF-4α homodimer is different from that of the PPAR-γ–RXR-α heterodimer, even when both NR complexes are assembled on the same DNA element. Our findings suggest that unique quaternary folds and interdomain connections in NRs could be exploited by small-molecule allosteric modulators that affect distal functions in these polypeptides.

Structural integration in hypoxia-inducible factors.

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1β) subunits. Here we describe crystal structures for each of mouse HIF-2α–ARNT and HIF-1α–ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α–ARNT and HIF-1α–ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

Aryl hydrocarbon receptor signaling regulates NF-κB RelB activation during dendritic-cell differentiation.

How the aryl hydrocarbon receptor (AhR) regulates dendritic‐cell (DC) differentiation is unknown. We show that activation of AhR by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) caused enhanced differentiation from immature DCs (IDCs) to mature DCs (MDCs) in the bone‐marrow‐derived DCs (BMDC) from B6 wild‐type mice but not in the BMDCs from AhR‐null mice as indicated by the expression of CD11c and class II major histocompatibility complex (MHC). Enhanced maturation of BMDCs was associated with elevated levels of CD86 and an increased AhR‐dependent nuclear accumulation of nuclear factor‐kappa‐light‐chain enhancer of activated B cell (NF‐κB) member RelB in BMDCs. The expression of interleukin (IL) 10 and chemokine DC‐CK1 was suppressed, whereas that of CXCL2, CXCL3 and IL‐22 was significantly increased in AhR‐activated BMDCs. Furthermore, TCDD induced expression of the regulatory enzymes indoleamine 2,3‐dioxygenase (IDO1) and indoleamine 2,3‐dioxygenase‐like 1 (IDO2). Increased expression of IDO2 was associated with coexpression of the cell‐surface marker CCR6. Interestingly, mRNA expression of the chemokine receptor CCR6 was drastically decreased in AhR‐null IDCs and MDCs. Overall, these data demonstrate that AhR modifies the maturation of BMDCs associated with the induction of the regulatory enzyme IDO and altered expression of cytokine, chemokines and DC‐specific surface markers and receptors.

AhR deficiency impairs expression of LPS-induced inflammatory genes in mice

Recent reports suggest the participation of the aryl hydrocarbon receptor (AhR) in the induction mechanism of the NF-κB signaling pathway. In the current study we challenged C57BL/6 wild-type (WT) and AhR deficient (AhR(-/-)) mice with bacterial lipopolysaccharide (LPS) to investigate the role of the AhR in expression profiles of LPS and NF-κB target genes. Further, we analyzed the effect of LPS on the DNA binding activity of NF-κB, C/EBP and AP-1 transcription factors in liver and lung from WT and AhR(-/-) mice. The results show that the LPS-induced expression of several target genes was impaired in AhR(-/-) mice compared to WT mice. Depending on the target gene, the target tissue as well as the time of treatment, the deficiency of AhR may cause an inhibition or increase of the LPS-induced gene expression. The binding activity of NF-κB, C/EBP and AP-1 transcription factors was also affected in a time- and tissue-dependent manner. The current study shows that the AhR is implemented in LPS-induced inflammatory gene expression in vivo even in the absence of exogenous ligands of the AhR. The main implication of this finding is that the AhR functions in Toll-like receptor (TLR) and NF-κB signaling after activation by a classical stimulus, such as LPS.

Structure and Dimerization Properties of the Aryl Hydrocarbon Receptor PAS-A Domain

ABSTRACT The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds to xenobiotics and responds by regulating the expression of gene programs required for detoxification and metabolism. AHR and its heterodimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT) belong to the basic helix-loop-helix (bHLH)–PER-ARNT-SIM (PAS) family of transcription factors. Here we report the 2.55-Å-resolution crystal structure of the mouse AHR PAS-A domain, which represents the first AHR-derived protein structure. The AHR PAS-A domain forms a helix-swapped homodimer in the crystal and also in solution. Through a detailed mutational analysis of all interface residues, we identified several hydrophobic residues that are important for AHR dimerization and function. Our crystallographic visualization of AHR PAS-A dimerization leads us to propose a mode of heterodimerization with ARNT that is supported by both biochemical and cell-based data. Our studies also highlight the residues of other mammalian bHLH-PAS proteins that are likely involved in their homo- or heterodimerization.

Interaction of aryl hydrocarbon receptor and NF-κB subunit RelB in breast cancer is associated with interleukin-8 overexpression.

The aryl hydrocarbon receptor (AhR) has been best known for its role in mediating the toxicity of dioxin. Here we show that AhR overexpression is found among estrogen receptor (ER)α-negative human breast tumors and that its overexpression is positively correlated to that of the NF-κB subunit RelB and Interleukin (IL)-8. Increased DNA binding activity of the AhR and RelB is coupled to IL-8 overexpression in primary breast cancer tissue, which was also supported by in situ hybridization. Activation of AhR in vitro by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced IL-8 expression in MDA-MB 436 and MCF-7 cells in an AhR and RelB dependent manner. Consistently, downregulation of RelB or AhR by small interfering RNAs (siRNA) decreased the level of IL-8 but increased expression of ERα in vitro in MCF-7 cells. Our results strongly suggest that RelB and AhR have a critical role in the regulation of IL-8 and reveal a supportive role of RelB and AhR in the anti-apoptotic response in human breast cancer cells. AhR and RelB may present a novel therapeutic target for inflammatory driven breast carcinogenesis and tumor progression. Overexpression of pro-survival factors AhR and RelB may explain the process of the development of environmentally-induced type of breast cancers.

Non-genomic action of TCDD to induce inflammatory responses in HepG2 human hepatoma cells and in liver of C57BL/6J mice

Abstract To assess the significance of the non-genomic signaling of TCDD (=dioxin) on liver of C57BL/6 mice and HepG2 human hepatoma cells, we first determined the group of markers that are susceptible to inhibition by parthenolide, a compound known to specifically suppress NF-κB-mediated inflammation. Of those, the most consistent marker turned out to be SOCS3 (a suppressor of cytokine signaling) known to respond to inflammation. An early diagnostic test on the action of TCDD on HepG2 cells in vitro within 3–6 h indicated that Cox-2 and SOCS3 are mainly induced via a non-genomic route, whereas PAI-2 appears to be induced through the classical action route. More detailed diagnostic tests at later stages of action of TCDD in HepG2 cells revealed that induction of IL-1β, BAFF, and iNOS are largely mediated by the protein kinase-dependent non-genomic route. An in vivo study on the 7 day action of TCDD on liver of AhRNLS mice showed that several early markers (e.g., Cox-2, MCP-1 and SOCS3) are induced, but not late markers such as IL-1β. Together, these results show that the non-genomic pathway contributes significantly to the early stress response reactions to TCDD that includes inflammation in hepatoma cells as well as in the liver.

Effects of selected food phytochemicals in reducing the toxic actions of TCDD and p,p′-DDT in U937 macrophages

To assess the effectiveness of selected food phytochemicals in reducing the toxic effects of the environmental toxicants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and p,p′-DDT (DDT), we tested the potencies of auraptene, nobiletin, zerumbone, and (±)-13-hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) in reversing the inflammatory action of these toxicants in U937 human macrophages. Using quantitative RT–PCR as the initial screening assay, we identified antagonistic actions of zerumbone and auraptene against the action of TCDD and DDT in up-regulating the mRNA expressions of COX-2 and VEGF. The functional significance of the inhibitory action of zerumbone on COX-2 expression was confirmed by demonstrating its suppression of TCDD-induced activation of COX-2 gene expression in mouse MMDD1 cells. We tested auraptene on DDT-induced reactive oxygen species (ROS) formation in U937 macrophages and found that auraptene is a powerful agent antagonizing this action of DDT. To confirm the significance of these actions of zerumbone and auraptene at the cellular level, we assessed their influence on TCDD-induced apoptosis resistance in intact U937 macrophages and found that they are capable of reversing this action of TCDD. In conclusion, zerumbone and auraptene were identified to be the most effective agents in protecting U937 macrophages from developing these cell toxic effects of TCDD and DDT.