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Department of Environmental Toxicology (C.F.A.V., E.S., W.L., P.W., F.M.), University of California, Davis, Davis, California 95616; and Institut National de la Santé et de la Recherche Médicale Unité 844 (G.L.), Molecular and Cellular Endocrinology of Cancers, Montpellier F-34295, France
Address all correspondence and requests for reprints to: Christoph F. A. Vogel, Department of Environmental Toxicology, University of California, Davis, One Shields Avenue, Davis, California 95616. E-mail: cfvogel{at}ucdavis.edu.
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ABSTRACT |
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B (NF-B) transcription factor family has a crucial role in rapid responses to stress and pathogens. We show that the NF-B subunit RelB is functionally associated with the aryl hydrocarbon receptor (AhR) and mediates transcription of chemokines such as IL-8 via activation of AhR and protein kinase A. RelB physically interacts with AhR and binds to an unrecognized RelB/AhR responsive element of the IL-8 promoter linking two signaling pathways to activate gene transcription. We found a time-dependent recruitment of AhR to the RelB/AhR responsive element site of IL-8 mediated by the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and via activation of protein kinase A. Furthermore, NF-B-binding sites that are preferentially recognized by RelB/p52 are a target for RelB/AhR complexes without addition of any stimuli, implicating the endogenous function of the AhR. RelB/AhR complexes are also found to bind on xenobiotic responsive element, and RelB drastically increases the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced xenobiotic responsive element reporter activity. The interaction of RelB with AhR signaling, and AhR with NF-B RelB signaling pathways represent a new mechanism of cross talk between the two transcription factors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B)/Rel transcription factors play critical roles in diverse cellular processes including adaptive and innate immunity, cell differentiation, proliferation, and apoptosis. Transcriptionally active NF-B dimers are formed by combinatorial association of five subunits: p50, RelA (p65), p52, c-Rel, and RelB (1). The classic inducible NF-B heterodimer consists of the p50 and RelA subunits, each contacting one half of the DNA binding site. The slight variations in the 10-bp consensus sequence, 5'-GGGGYNNCCY-3', confers a preference for selected Rel combinations (2). Compared with other members of the NF-B family, the biological mode of action of the RelB subunit has remained elusive. RelB does not express the functional properties common to the Rel family, and no exclusive DNA binding activity had been discovered until recently. In vivo analysis revealed that the IB kinase (IKK)
activates an alternative NF-B pathway based on processing of NF-B2/p100 and release of RelB/p52 dimers in response to lymphotoxin β receptor (LTβR) trimers (3). Gene induction by IKK depends on selective activation of RelB/p52 dimers, which recognize a unique type of NF-B binding site (5'-NGGAGAYTTN-3') regulating organogenic chemokines such as B lymphocyte chemoattractant (BLC) or the B cell-activating factor of the TNF family (BAFF) (4). Unlike p50 or RelA, which are expressed in virtually all cell types, RelB is predominantly present in lymphoid tissue and can be constitutively expressed in the nucleus (5). The AhR is a member of basic helix-loop-helix-Pev-ARNT-Sim (PAS) transcription factors including Period, AhR nuclear translocator (ARNT), and single minded, regulating hypoxia, circadian rhythm, and cellular processes such as differentiation and apoptosis (6). The AhR is well described as a ligand-dependent activated transcription factor. About 15 yr ago Hankinson and co-workers (7) identified the encoded protein ARNT, which is required for ligand-dependent translocation of the AhR into the nucleus and its binding to xenobiotic responsive element (XRE), mediating induction of xenobiotic metabolizing enzymes (classical AhR/ARNT pathway). Numerous exogenous compounds (e.g. polycyclic aromatic hydrocarbons, benzimidazoles, and flavonoids) with various binding affinities have been shown to bind to and activate the AhR (8), but the physiological ligand or function of the AhR remained a key question. However, the conservation of the receptor in a wide range of animal species (including humans) suggests a fundamental role in cellular physiology. The nonactivated form of the AhR is complexed with heat shock protein 90 and X-associated protein 2 (XAP2) in the cytosol but, depending on cell type and physiological conditions, the AhR is also located in the nucleus in the absence of exogenous ligand (9). XAP2 may enhance the rate of nuclear translocation of the ligand-bound human AhR complex and modulates the subcellular localization of the mouse AhR (10, 11). The AhR has a critical role in development: AhR null mice show deficiencies in liver development, increased apoptosis in liver, and decreased accumulation of lymphocytes in the spleen and lymph nodes (12). This further indicates that the AhR is also located in the nucleus to regulate these physiological processes in the absence of exogenous ligands. Nuclear localization and activity of the AhR during embryonic development have also been reported (13). Recently, a protein kinase A (PKA)-dependent activation and nuclear translocation of the AhR by forskolin (FSK)/cAMP have been reported (14). However, the PKA-activated form of AhR was found to be different from the ligand-activated AhR and does not dimerize with ARNT, although the dimerization partner of the PKA-activated AhR and its regulatory function remained undiscovered.
In recent reports we have shown that the induction of IL-8 in vitro (15) as well as the induction of keratinocyte chemoattractant (KC) (homolog of human IL-8) in mice (16) by TCDD requires a functional AhR. By analyzing the mechanism of the AhR-mediated induction of IL-8, this study demonstrates the physical and functional association of the AhR and the NF-B subunit RelB, resulting in transcriptional activation of IL-8. IL-8 promoter studies with human macrophages U937 and human hepatoma cell line HepG2 revealed a novel RelB/AhR responsive element (RelBAhRE) required for transcriptional activation of IL-8 by FSK as well as by the prototype of AhR ligands, TCDD. Using EMSA and chromatin immunoprecipitation (ChIP) assays, we demonstrate the recruitment of AhR to the RelBAhRE region of the IL-8 promoter stimulated by FSK and TCDD. Furthermore, supershift analysis revealed clear binding activity of AhR in complex with RelB on a recently identified NF-B-binding site located on promoter regions of chemokines such as BLC and BAFF that are induced by FSK and TCDD.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). FSK was included in our study as an alternative activator of the AhR and inducer of IL-8 because FSK has been reported to activate AhR through a PKA-dependent mechanism (14) and has been shown to increase IL-8 (20). The FSK- and TCDD-induced mRNA expression in macrophages correlated with elevated protein level and secretion of IL-8 (Fig. 1, B and C). Results from transfection studies with short interfering RNA (siRNA) into U937 macrophages to target AhR suggest that TCDD as well as FSK activate IL-8 through an AhR-dependent mechanism (Fig. 1, D and E).
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, IL-1β, lipopolysaccharide (LPS), metals, hypoxia, reactive oxygen species, or cellular stress (21, 22). Several studies have shown that the sequence spanning –1 to –133 bp within the 5'-upstream regulatory region of the IL-8 gene is essential for transcriptional regulation of the gene. Previous studies identified three promoter binding sites for transcription factors of the AP-1, Oct-1, and NF-B family, which are involved in the transcriptional control of the IL-8 gene (23, 24). We were interested in the relevance of these binding sites and performed transfection experiments with deletion reporter constructs of the IL-8 promoter. Our data revealed that the region spanning –1 to –50 bp upstream of the start site of the IL-8 promoter is sufficient to induce promoter activity of the IL-8 gene mediated by TCDD or FSK (Fig. 2A). When this short promoter sequence was compared with consensus binding elements, we could identify an 8-bp sequence that contains an AhR/ARNT- and NF-B-like binding site (5'-GGGTGCAT-3'). Using EMSA we were interested in verifying changes in the binding activities of this XRE/NF-B-like sequence as well as identifying corresponding binding proteins that may bind on XRE or NF-B sites. We found that DNA binding activity to the XRE/NF-B-like sequence was enhanced in nuclear extracts from TCDD- as well as FSK-treated U937 macrophages, compared with control cells (Fig. 2, B and C). Supershift analysis in Fig. 2B revealed that AhR together with RelB are the dominant proteins binding to this unrecognized binding element of the IL-8 promoter, whereas p50 or RelA does not bind to the RelB/AhR responsive element, which is called RelBAhRE from here on. Furthermore, we could not observe any binding activity of ARNT (Fig. 2B) or p52 (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend/endojournals.org) on the RelBAhRE site. ARNT is well described as the dimerization partner of the ligand-activated form of the AhR binding to XRE, which is essential for TCDD-induced cytochrome P4501a1 (CYP1A1) activity (25). To determine the importance of the XRE-like component in the newly identified RelBAhRE sequence for the TCDD-mediated IL-8 activation, a T-to-C point mutation was introduced (5-GGGCGCAT-3', M1) as shown in Fig. 2D. The T residue is a total requirement for the activity of XRE consensus elements, and a T-to-C mutation is known to fully eliminate binding of the AhR/ARNT complex (26). In contrast to AhR/ARNT complexes, binding activity of the RelB/AhR complex is not reduced but even further increased (Fig. 2E) by this point mutation. This was confirmed by an elevated promoter activity of the mutation construct M1 (Fig. 2F). Supershift analysis confirmed that M1, like the wild-type (wt) RelBAhRE oligonucleotide binds RelB and AhR, but not p50, RelA, and ARNT (Fig. 2G). To investigate the importance of the first and second G, which are conserved in consensus NF-B sites as well as in consensus XRE, two point mutations (5'-CGCTGCAT-3', M2; Fig. 2D) were introduced similar to an earlier report (27). The two G-to-C point mutations drastically reduced the TCDD- and FSK-induced binding activity of RelBAhRE as well as activation of the IL-8 promoter (Fig. 2, E and F).
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) and induction of IL-8 mRNA expression (Fig. 1E
), thus underlining the requirement of AhR and RelB to mediate the activation of IL-8 by TCDD or FSK. These results are supported by overexpression of AhR and RelB, which enhanced the activation of the IL-8 promoter in a dose-dependent manner (Fig. 3, B and C). To verify the specificity of RelB and AhR, cells were transfected with small interfering ARNT (Fig. 3A) or ARNT expression plasmid (Fig. 3D), which did not significantly change the IL-8 promoter activity. Nuclear proteins from cells transfected with small interfering AhR or small interfering RelB showed significant decreased binding activity and no effect of TCDD treatment on RelBAhRE in EMSA, which supports the role of AhR and RelB (Fig. 3E). To determine whether activation of IL-8 by FSK and TCDD is PKA dependent, U937 macrophages were transfected with the IL-8 reporter in the presence or absence of a PKA wild type, a PKA mutant expression plasmid, and the PKA inhibitor H89. The requirement of PKA for TCDD and FSK to activate the IL-8 promoter was evident (data not shown), which is supported by EMSA showing decreased binding activity of RelBAhRE in cells pretreated with H89 (Fig. 3F).
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). To verify the effect of the vehicle Me2SO, we compared the vehicle controls with medium controls (untreated cells). No significant effect of Me2SO at a concentration of 0.1% was observed on the interaction or binding activity of RelB and AhR on a RelBAhRE oligonucleotide (data not shown). Although TCDD did not affect the apparent association between AhR and RelB, the functional activity of this complex has been clearly stimulated by TCDD or FSK as shown in EMSA and transient transfection studies (Fig. 2, A to F). Because ligand-activated AhR is known to dimerize with ARNT in the nucleus, we tested the possible interaction of ARNT with RelB. ARNT was found complexed with AhR in TCDD-treated cells, as expected, and no interaction of ARNT and RelB could be detected (Fig. 4A), which is supported by results obtained from gel shift studies (Figs. 2B and 5E). No association of AhR or RelB with NF-B proteins p50 or RelA could be detected by coimmunoprecipitation (data not shown) or EMSA (Fig. 5A) studies.
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). These results are confirmed by ChIP assays with U937 human macrophages to study the recruitment of AhR and RelB proteins to the RelBAhRE of the IL-8 promoter (Fig. 4C). For quantification, the ChIP samples were analyzed by real-time PCR; their relative enrichment levels are shown in Fig. 4D. Our data demonstrate the enhanced recruitment of AhR to the RelBAhRE of IL-8 stimulated by TCDD and FSK. Increased occupancy of the RelBAhRE promoter region by AhR was evident after 30 min, peaking at approximately 90 min and sustained thereafter during the course of the treatment. The ChIP analysis demonstrates a higher increase of AhR binding at RelBAhRE compared with results from EMSA, which might be due to the fact that ChIP includes the chromatin context and the dynamic of the cell, which is not the case in EMSA. No apparent significant kinetic differences were observed in the occupancy of the RelBAhRE region by RelB under these conditions. The critical role of PKA to recruit AhR was verified by significantly less occupancy of the RelBAhRE region by AhR in cells treated with TCDD or FSK in the presence of H89, which is in line with EMSA showing a lower binding activity of RelBAhRE by pretreatment with H89 (Fig. 3F). No recruitment of ARNT was observed to the RelBAhRE region (data not shown) within the IL-8 promoter, demonstrating the specific binding of RelB and recruitment of AhR to this promoter region of IL-8 (Fig. 4C).
FSK and TCDD Signaling Induces Binding of RelB/AhR Complexes to NF-B Binding Sites
Because RelB is a subunit of the NF-B family that binds to NF-B consensus sequences, we were interested in the possible coexistence of RelB and AhR complex binding to a NF-B consensus element. EMSA in Fig. 5A shows that TCDD and FSK stimulate binding activity of the lower NF-B complex. Supershift analyses with AhR-specific antibodies revealed that AhR indeed binds to a NF-B consensus element present in the lower complex of the classical TNF- or LPS-activated NF-B complex, which also contains the NF-B subunits RelB and p50. However, a physical interaction of AhR with RelA, which forms RelA homodimers or heterodimers with p50 after treatment with LPS, has not been observed (Fig. 5A). Binding of p52 or ARNT could not be detected on the NF-B consensus probe in supershift assays (supplemental Fig. S2). A 100-fold excess of cold NF-B oligonucleotide completely abolished formation of both NF-B complexes (Fig. 5A, lane 21), whereas excess of cold XRE consensus (lane 22) or RelBAhRE oligonucleotide (lane 23) abolished specifically the lower complex. The LPS-induced upper complex formed by RelA and p50 was not affected, indicating the specific binding of AhR and RelB on these DNA binding sequences (Fig. 5A, lanes 22 and 23). In contrast to the classical activation pathway of NF-B and inflammatory signaling by TNF or LPS through their respective receptors TNFR1/2 and TLR/IL-1R, TCDD obviously activates NF-B through an enhanced recruitment of AhR, which is complexed with RelB and binds to NF-B response elements. This suggestion is supported by the TCDD-induced binding activity of the lower complex containing AhR and RelB (Fig. 5B). As expected, RelA increased the constitutive NF-B-reporter activity drastically whereas p50 overexpression inhibited NF-B activity. Overexpression of ARNT had no significant effect on NF-B reporter activity. RelB and AhR increased the TCDD- but not FSK-stimulated NF-B-activity (Fig. 5C).
To address whether a recently identified B-binding site (5'-GGGAGATTTG-3') located on the promoter of chemokines such as BLC and BAFF that is preferentially recognized by RelB/p52 dimers and not RelA/p50 dimers (4) is also a target for AhR- and RelB-containing dimers, we performed EMSA with the specific B-binding site located on promoters of BLC at position –115 bp and BAFF at position –71 bp. Both probes exhibited strong binding activity to nuclear extracts of U937 macrophages, which was further increased using nuclear extracts of FSK- or TCDD-stimulated cells (Fig. 5, D and E). Supershift analysis revealed clear binding activity of AhR in complex with RelB. The presence of p50 or p52 subunits dimerized with RelB or AhR could not be detected in FSK-, TCDD-, or LPS-stimulated cells on the B-binding site of BLC (Fig. 5D) or BAFF (supplemental Fig. S3). The result agrees with previous studies showing that binding of p52/RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by LTβR signaling and IKK, but not by TNF or LPS and IKKβ or IKK
(28). Dimerization of ARNT with AhR or RelB uld not be detected in supershift assays with the B-binding site of BAFF (Fig. 5E, lane 5 and 6) or BLC (supplemental Fig. S4) as in the case of the RelBAhRE site of IL-8 (Fig. 2B, lanes 11 and 12). Migration and binding activity of the RelB/p52 probes was very similar to that of the RelBAhRE probe of the IL-8 promoter. In all cases, the detected protein-DNA complexes were specific, as indicated by competition experiments with RelB/p52 (Fig. 5D, lane 17) and RelBAhRE probes (lane 18). These results suggest that the RelB/AhR complex is also involved in the regulation of other chemokines such as BLC or BAFF containing the specific RelB/p52 B-binding site on their promoter. As suspected, treatment with FSK or TCDD led to induction of BLC and BAFF mRNA in U937 macrophages, and overexpression of AhR and RelB further increased the level of BLC and BAFF in control, FSK-, and TCDD-stimulated cells (Fig. 5F). These results and previous EMSA strongly suggest that BLC and BAFF gene induction is mediated not only via RelB/p52 but also via RelB/AhR complexes.
RelB Binds on a XRE Consensus Site of the CYP1A1 Promoter and Increases XRE Activity
Using EMSA we investigated the possible interaction of RelB with an XRE consensus element of a CYP1A1 promoter sequence. Supershift analyses with RelB-specific antibodies revealed clear binding activity of RelB in complex with AhR in nuclear extracts of U937 macrophages (Fig. 5G). Excess of cold XRE oligonucleotide completely abolished formation of both XRE complexes (lane 13), whereas excess of unlabeled RelBAhRE oligonucleotide (lane 14) abolished specifically the lower complex, which does not contain ARNT protein complexed with AhR (Fig. 5G). Binding of the RelB partner p50 or p52 on the XRE consensus probe could not be detected (supplemental Fig. S5). A very similar pattern of AhR/ARNT and AhR/RelB binding on consensus XRE was found in the human and mouse hepatoma cell lines HepG2 and Hepa1c1c7, respectively (our unpublished data). The data indicate the specific binding of AhR and RelB on DNA binding sequences that do not involve binding of AhR/ARNT complexes. Because the ligand-activated AhR preferably forms heterodimers with ARNT in the nucleus as shown in EMSA of TCDD-treated cells (Fig. 5G), RelB might bind to a different active form of the AhR located in the nucleus. The existence of a non-ligand-activated form of the AhR in the nucleus has been described previously (9, 14, 29). Overexpression of RelA had an inhibitory effect on TCDD-induced XRE activity, whereas overexpression of RelB, like AhR, drastically increased the TCDD-induced activity of the XRE reporter construct. ARNT overexpression had no significant effect on constitutive, TCDD-, or FSK-induced XRE activity (Fig. 5H). Compared with TCDD, FSK had only a small but statistically significant effect on XRE activity, which was further increased by overexpression of RelB. These results underline the cross talk between RelB and AhR and indicate the supportive action of RelB on AhR-mediated transcriptional activation.
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B binding site than the original RelBAhRE site. The enhanced recruitment of AhR and binding of RelB/AhR dimers to RelBAhRE induced by FSK or TCDD require PKA activity for the full induction of IL-8. In line with these findings, an increased FSK/cAMP-stimulated activation followed by nuclear localization of AhR, which does not dimerize with ARNT, has been reported previously (14), although the dimerization partner of the PKA-mediated nuclear AhR could not be revealed by these authors. A recent report demonstrated that the cochaperone XAP2 targets phosphodiesterase (PDE)2A to the AhR complex, which might be responsible for the cAMP-dependent regulation of AhR (30). PDE2A binding may ensure AhR retention in the cytoplasm, possibly by lowering the local cAMP concentrations under a level required for AhR complex translocation into the nucleus. Previously we could show that TCDD treatment is associated with an early increase of cAMP/PKA activity leading to the induction of C/EBPβ (18). Thus, it seems reasonable that TCDD induces nuclear translocation of cytosolic AhR through an elevation of cAMP/PKA activity, in addition to the classical well-described ligand-dependent activation and nuclear translocation of the AhR, which forms heterodimers with ARNT (31). A direct phosphorylation of the AhR protein, however, is obviously not responsible for the PKA-mediated AhR activation as reported by de Oliveira et al. (30). Although ligand binding is an important mechanism of nuclear receptor activation, other receptors, including estrogen receptor (ER) and ERβ, can be activated by specific kinases as well (32). Interestingly, a recent report found that the ligand-activated AhR forms also a complex with a novel cullin 4B ubiquitin ligase that targets sex steroid receptors such as ER for degradation (33), which provides mechanistic insight into the way AhR ligands such as TCDD disturb endocrine signaling.
Furthermore, we observed an enhanced binding activity of RelB/AhR complexes on the RelB/p52 consensus element of the BLC and BAFF promoter by TCDD or FSK. The enhanced binding activity was associated with an increased expression of BLC and BAFF mRNA, which was further elevated in AhR and RelB overexpressing cells. The chemokine BLC and the B cell activating factor BAFF are known to contain RelB/p52-responsive sites on their promoter and have been shown to be regulated through the alternative NF-B pathway (4). Thus, our study establishes an example of how an activated AhR pathway connects to the NF-B subunit RelB (alternative AhR/RelB pathway, Fig. 6) to cooperatively regulate inflammatory gene expression.
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B and AhR signaling and also reveals an important role for AhR in NF-B-dependent transcription, as well as for RelB in AhR-dependent transcription through XRE sites. There are conflicting reports on the effect of NF-B activation through TNF or LPS on TCDD-induced expression of AhR target genes. Some studies report an inhibition (34), whereas other groups (35, 36, 37) and our own results show an activation of NF-B activity. Other investigators observed that TCDD leads to induction of the proinflammatory gene IL-1β (38) and a sustained induction of NF-B binding activity (39). These differences may be due to different cell types, culture conditions, serum lots, and treatment regimes. Puga et al. (39) concluded that an increased formation of p50/p50 complexes might be responsible for the TCDD-mediated effect on NF-B reporter activity. Our results collectively demonstrate that the AhR interacts with RelB and that activation of the AhR leads to an increased NF-B activity. Current data also show that the increased binding activity of the lower complex of the NF-B element mediated by TCDD or FSK is obviously due to an increased nuclear accumulation of AhR complexed with RelB rather than increased binding of p50/p50 homodimers, which are believed to repress NF-B activity (40). This hypothesis is in line with other reports showing an increased NF-B binding in mouse hepatoma cells (Hepa1c1c7) transfected with an AhR expression plasmid (34). In the case of FSK, we observed a relatively weak effect on the reporter activity of NF-B or XRE compared with TCDD, and no additional effect of FSK in cells coexpressing AhR or RelB has been observed. One explanation might be the fact that the overexpression of AhR and RelB by itself leads to an increased NF-B- or XRE-reporter activity, which cannot be further enhanced by FSK. Another possibility is that binding of TCDD to AhR and subsequent release of PDEA2 from the cytosolic AhR complex (30) leads to more significant changes of the local cAMP levels than FSK stimulation.
In the case of the chemokines IL-8, BLC, and BAFF, the interaction of AhR and the NF-B member RelB enhances the gene activity by TCDD or FSK. The supportive action of RelB on AhR signaling is also clearly indicated by a distinct increase of TCDD-mediated XRE-Luc reporter activity through overexpression of RelB and the binding of RelB on a XRE consensus sequence. Similar results were received from parallel IL-8- and XRE-Luc reporter studies with the human hepatoma cell line HepG2 (our unpublished data) indicating that the observed mechanism of RelB and AhR interaction is not limited to macrophages and exists in other cell types as well. Recently we could show that TCDD induces KC (homolog of human IL-8) in various tissues of mice of C57Bl/6 mice (16). Two structurally distinct B sequence motifs have been identified for mouse KC (41) and the second B motif (3'-GGGTGT-5') of KC shows sequence homology to RelBAhRE. Results from AhRnls mice show that the induction of KC by TCDD depends on the nuclear translocation of the AhR. The induction of KC in liver of C57Bl/6 wt mice was associated with an increased expression of F4/80 (16), indicating the infiltration of macrophages, which suggest the physiological relevance and biological consequence of the AhR/RelB pathway.
Despite the present study our understanding of the molecular mechanisms of AhR and RelB cross talk is far from complete. For instance, the PKA-mediated signal(s) that stimulates nuclear translocation of AhR and complex formation with RelB are unknown and may include the activation of other signaling pathways and kinases. We believe that the PKA-stimulated association of AhR with RelB represents an important mechanism mediating cross talk between NF-B and AhR signaling pathways, but also a new mechanism of RelB and AhR action. Because AhR associates not only with ARNT (classical AhR/ARNT pathway) but also with RelB, we propose a model of an alternative AhR/RelB pathway in which AhR and RelB regulate inflammatory genes such as IL-8, BLC, or BAFF (Fig. 6).
Some of the major future questions are: how is the functional separation between the two AhR signaling pathways regulated? The alternative AhR/RelB pathway obviously overlaps with the alternative NF-B pathway and has a regulatory function on the expression of especially chemokines. Organogenic chemokines such as BLC and BAFF are regulated by the alternative NF-B pathway and are required for the recruitment of macrophages, T cells, and B cells to secondary lymphoid organs (3). Studies with AhR-deficient mice are show a distinct decrease of lymphocytes in spleen and lymph nodes (12), suggesting the critical role of AhR in the alternative NF-B pathway. By contrast, the classical AhR/ARNT pathway is mostly responsible for rapid responses to xenobiotics and activation of genes encoding xenobiotic metabolizing enzymes including CYP1A1, CYP1A2, and CYP1B1 through XRE sites. Another open question is whether the identified RelBAhRE site of IL-8 is a unique sequence that is selectively recognized by RelB/AhR dimers and not by RelB/p52 dimers, the ubiquitous target of the alternative NF-B pathway, and the possible existence on promoters of other target genes. Current data indicate that AhR influences NF-B signaling and RelB regulates AhR signaling and XRE activity. Thus, it will also be important to determine whether RelB/AhR complexes are recruited to other AhR target genes or restricted strictly to consensus XREs of CYP1A1 promoters. Even with the differences in the mechanisms and type of NF-B and AhR activation, our model suggests that the role of cAMP/PKA-dependent activation in assembling a RelB/AhR signaling complex is a conserved strategy that has evolved to regulate genes in response to environmental stressors and inflammatory signals.
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, and LPS were obtained from Sigma (St. Louis, MO). [y-32P]ATP (6000 Ci/mmol) was purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). FSK and N-{2-[(p-bromocinamyl)amino]ethyl}-5-isoquinolinesulfonamide·2 HCl (H89) was purchased from Calbiochem (San Diego, CA). TCDD (>99% purity) was originally obtained from Dow Chemical Co. (Midland, MI). Other molecular biological reagents were purchased from QIAGEN (Valencia, CA) and Roche Clinical Laboratories (Indianapolis, IN). Monoclonal ARNT, polyclonal RelA, p52 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), NF-B member p50, RelB, c-Rel (Active Motif, Carlsbad, CA), and polyclonal AhR (Novus Biologicals, Littleton, CO) antibodies were used for Western blot analyses, Supershift in EMSA, and ChIP assays.
Plasmids and Site-Directed Mutagenesis
Details concerning the cloning of a 1.5-kb IL-8 promoter fragment, deletion and mutation constructs into pGL3 Basic are described elsewhere (17). Mutation of RelBAhRE sequences of human IL-8 (GGGTGCAT to M1, GGGCGCAT or M2, GGCTCCAT) was carried out by site-directed mutagenesis (Stratagene, La Jolla, CA) using the following primers synthesized by Integrated DNA Technologies Inc. (Coralville, IA): M1–50-Mutant, 5'-GATGAGGGCGCATAAGTTCTCTAG-3'; and M2–50-Mutant, 5'-GATGAGGCTCCATAAGTTCTCTAG-3'. Insertion of mutations was confirmed by direct sequencing. The NF-B luciferase reporter was from CLONTECH Laboratories, Inc. (Mountain View, CA), and XRE luciferase reporter was kindly provided by J. Abel (University of Duesseldorf, Duesseldorf, Germany). The AhR and ARNT expression plasmid were a kind gifts of C. Bradfield (McArdle Laboratory for Cancer Research, Madison, WI). Expression vectors for p50 and RelA were kindly provided by W. Greene (J. David Gladstone Institute, San Francisco, CA). The RelB expression plasmid was kindly provided by U. Siebenlist (National Institutes of Health, Bethesda, MD).
Cell Culture, Transfection Experiments, and Luciferase Assay
Human U937 monocytic cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA) supplemented with 4.5 g/liter glucose, 1 mM sodium pyruvate, and 10 mM HEPES. Cell culture was maintained at a cell concentration between 2 x 105 and 2 x 106 cells/ml. HepG2 cells from ATTC were maintained in MEM medium 10% fetal bovine serum (FBS). Hepa1c1c7 cells were a kind gift from O. Hankinson (University of California, Los Angeles) and maintained in -MEM (Invitrogen). For transient transfection of U937 macrophages, cells were plated in RPMI with 10% FBS and 0.5 µg/ml TPA, which promotes differentiation into macrophages after 2 d. Transfection of plasmid DNA or siRNA into U937 macrophages was performed via Nucleofector technology. Briefly, 106 U937 macrophages were resuspended in 100 µl Nucleofector Solution V (Amaxa GmbH, Köln, Germany) and nucleofected with 1.0 µg plasmid DNA or 1.5 µg of the corresponding siRNA using program V-001, which is preprogrammed into the Nucleofector device (Amaxa GmbH). After nucleofection, the cells were immediately mixed with 500 µl of prewarmed RPMI 1640 and transferred into six-well plates containing 1.5 ml RPMI 1640 medium per well. Cells were treated 24 h after transfection with 10 nM TCDD, 10 µM FSK, or 0.1% Me2SO (control) for 24 h. In the case of siRNA transfection, the reduction of the target RNA and protein was detected by quantitative real-time RT-PCR and Western blot. siRNA to target human AhR (catalog no. M-004990) was designed and synthesized by Dharmacon (Lafayette, CO). siRNA to target human RelB (5'-GGAUUUGCCGAAUUAACAA-3') and a negative control siRNA (catalog no. 10272280) were synthesized by QIAGEN. For transient transfection experiments in HepG2, cells were plated in 24-well plates (1 x 105 cells per well) and transfected using jetPEI (PolyTransfection; Qbiogene, Irvine, CA), according to the manufacturer’s instructions. Briefly, 0.3 µg of the IL-8 construct was suspended in 25 µl of 150 mM sterile NaCl solution. Also 0.3 µl of jetPEI solution was suspended in 25 µl of 150 mM sterile NaCl solution. The jetPEI/NaCl solution was then added to the DNA/NaCl solution and incubated at room temperature for 30 min. The medium in the wells was changed to fresh medium, and 50 µl of the DNA/jetPEI was added to each well. The transfection was allowed to proceed for 6 h, and cells were treated with 10 nM TCDD, 10 µM FSK, or 0.1% Me2SO (control) for 24 h. To control the transfection efficiency, cells were cotransfected with 0.1 µg per well β-galactosidase reporter construct. Luciferase activities were measured with the Luciferase Reporter Assay System (Promega Corp., Madison, WI) using a luminometer (Berthold Lumat LB 9501/16; Pittsburgh, PA). Relative light units are normalized to β-galactosidase activity and to protein concentration, using Bradford dye assay (Bio-Rad Laboratories, Inc., Hercules, CA).
IL-8 ELISA
The IL-8 concentration in culture supernatants was determined by ELISA as recommended by the manufacturer. Briefly, samples were added to 96-well microtiter plates, which were coated with monoclonal anti-IL-8 antibody (MAB-208; R&D Systems, Minneapolis, MN). After 2 h, the wells were washed four times, and biotinylated anti-IL-8 antibody was added. After 1 h of incubation, the plates were washed three times, and streptavidin-horseradish peroxidase conjugate (RPN1231, Amersham, Buckinghamshire, UK) was supplied, and the plates were incubated for 20 min. Plates were washed again and chromogen substrate (Sigma Fast OPD; Sigma) was added. The plates were read at 450 nm.
Quantitative Real-Time RT-PCR Analysis
Total RNA was isolated from U937 macrophages using a high pure RNA isolation kit (QIAGEN), and cDNA synthesis was carried out as previously described (18). Quantitative detection of β-actin and IL-8 was performed with a LightCycler Instrument (Roche Diagnostics, Mannheim, Germany) using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the manufacturer’s instructions. DNA-free total RNA (1.0 µg) was reverse transcribed using 4 U Omniscript reverse transcriptase (QIAGEN) and 1 µg oligo(dT)15 in a final volume of 40 µl. The primers for each gene were designed on the basis of the respective cDNA or mRNA sequences using OLIGO primer analysis software, provided by Steve Rosen and Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research. The following primer sequences for human β-actin were used: (forward primer, 5'-GGACTTCGAGCAAGAGATGG-3'; reverse primer, 5'-AGCACTGTGTTGGCGTACAG-3'); human IL-8 (forward primer, 5'-CTGCGCCAACACAGAAATTA-3'; reverse primer, 5'-ATTGCATCTGGCAACCCTAC-3'); human BAFF (forward primer, 5'-CGTTCAGGGTCCAGAAGAAA-3'; reverse primer, 5'-GTCCCATGGCGTAGGTCTTA-3'); and human BLC (forward primer, 5'-GAGGCAGATGGAACTTGAGC-3'; reverse primer, 5'-CTGGGGATCTTCGAATGCTA-3'). All PCR assays were performed in triplicate. The intraassay variability was less than 7%. For quantification, data were analyzed with the LightCycler analysis software according to the manufacturer’s instructions. The variables were examined for one-sided Student’s t test. The results are given as the mean ± SEM.
ChIP
U937 macrophages were seeded in 150-mm dishes and cultured in RPMI containing 10% FBS. FSK, TCDD, and H89 were added for the indicated times, and protein-DNA complexes were cross-linked with 1% formaldehyde for 10 min. Cells were washed with PBS, harvested, and resuspended in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate] containing protease inhibitors (Roche Diagnostics) and sonicated with five sets of 10-sec pulses. The soluble chromatin was collected by centrifugation, and an aliquot of the chromatin was put aside and represented the input fraction. The supernatants were incubated with 30 µl of protein A/G Sepharose (50% slurry; Pharmacia Biotech, Piscataway, NJ) under gentle agitation for 2 h at 4 C. The supernatant was transferred to a new microcentrifuge tube, and 1 µg of antibody was added and incubated overnight at 4 C. Protein A/G-Sepharose (20 µl of a 50% slurry) was then added and incubated for 1.5 h. The pellets were successively washed for 10 min in 1 ml of buffer 1 [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS)], 1 ml of buffer 2 [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS], 1 ml of LiCl buffer [20 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 1% Nonidet P-40, 1% Na-deoxycholate], and 2 x 1 ml of TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. Protein-DNA complexes were eluted in 120 µl of elution buffer (TE, 1% SDS) for 30 min, and the cross-links were reversed by overnight incubation at 65 C. DNA was purified using a PCR purification kit (QIAGEN) and eluted in 50 µl distilled water. ChIP DNA (5 µl) was amplified by real-time PCR with primers 5'-AATGAAAAGATGAGGGTGCAT-3' and 5'-GCCAGCTTGGAAGTCATGTT-3' covering the specified region RelBAhRE of IL-8. For real-time PCR, SYBR green qPCR supermix (QIAGEN) was used to amplify a 182-bp fragment of the IL-8 promoter.
Nuclear Complex Coimmunoprecipitation Assay and Western Blot Analyses
Preparation of nuclear extracts and coimmunoprecipitation were performed according to the manufacturer’s protocol (Active Motif). To analyze the level of AhR and RelB protein in nuclei, nuclear protein extracts (15 µg) were separated on a 10% SDS-polyacrylamide gel and blotted onto a polyvinylidinedifluoride membrane (Immuno-Blot; Bio-Rad Laboratories). The antigen-antibody complexes were visualized using the chemoluminescence substrate SuperSignal, West Pico (Pierce Chemical Co., Rockford, IL) as recommended by the manufacturer.
EMSA
Nuclear extracts were isolated from U937 cells as described previously (18). In brief, 5 x106 cells were treated with 10 nM TCDD, 10 µM FSK, or 2 µg/ml LPS for 90 min unless noted otherwise in the figure legends, and harvested in Dulbecco’s PBS containing 1 mM phenylmethylsulfonylfluoride and 0.05 µg/µl of aprotinin. After centrifugation the cell pellets were gently resuspended in 1 ml of hypotonic buffer [20 mM HEPES, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulfonylfluoride, 0.13 µM okadaic acid, 1 mM dithiothreitol (pH 7.9), and 1 µg/ml each leupeptin, aprotinin, and pepstatin]. The cells were allowed to swell on ice for 15 min and then homogenized by 25 strokes of a Dounce homogenizer. After centrifugation for 1 min at 16,000 x g, nuclear pellets were resuspended in 300 µl ice-cold high-salt buffer (hypotonic buffer with 420 mM NaCl, and 20% glycerol). The samples were passed through a 21-gauge needle and stirred for 30 min at 4 C. The nuclear lysates were microcentrifuged at 16,000 x g for 20 min, aliquoted, and stored at –80 C. Protein concentrations were determined by the method of Bradford. Sequences for double-stranded oligonucleotides used in EMSA are shown in Table 1. DNA-protein binding reactions were carried out in a total volume of 15 µl containing 10 µg nuclear protein, 60,000 cpm of DNA oligonucleotide, 25 mM Tris buffer (pH 7.5), 50 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, and 1 µg polydeoxyinosinic deoxycytidylic acid. The samples were incubated at room temperature for 20 min. Supershift analysis was performed by adding 2 µg of monoclonal ARNT, polyclonal RelA (Santa Cruz Biotechnology, Inc.), NF-B member p50, RelB, c-Rel (Active Motif), or polyclonal AhR (Novus Biologicals) antibodies to the reaction mixtures. Competition experiments were performed in the presence of a 100-fold molar excess of unlabeled DNA fragments. Protein-DNA complexes were resolved on a 4% nondenaturating polyacrylamide gel and visualized by exposure of the dehydrated gels to x-ray films. For quantitative analysis, respective bands were quantified using a ChemiImager 4400 (Alpha Innotech Corp., San Leandro, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online September 6, 2007
Abbreviations: AhR, Aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; BAFF, B cell-activating factor of the TNF family; BLC, B lymphocyte chemoattractant; ChIP, chromatin immunoprecipitation; CYP1A1, cytochrome P4501a1; ER, estrogen receptor; FBS, fetal bovine serum; FSK, forskolin; IKK, IB kinase; KC, keratinocyte chemoattractant; LPS, lipopolysaccharide; NF-B, nuclear factor-B; PDE, phosphodiesterase; RelBAhRE, RelB/AhR responsive element; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; XAP2, X-associated protein 2; XRE, xenobiotic responsive element; wt, wild type.
Received for publication April 24, 2007. Accepted for publication August 28, 2007.
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