Cytochrome P450 1A1 gene regulation by UVB involves crosstalk between the aryl hydrocarbon receptor and nuclear factor κB

doi:10.1016/j.cbi.2010.01.038

Chemico-Biological Interactions

Volume 184, Issue 3, 30 March 2010, Pages 466-473

https://doi.org/10.1016/j.cbi.2010.01.038 Get rights and content

Abstract

UVB induces the expression of genes controlled by the aryl hydrocarbon receptor (AhR), a transcription factor that has been implicated in the UV stress response. In this study, we used the human hepatoma cell line HepG2 to investigate in more detail the effects of UVB irradiation on AhR activation and induction of cytochrome P450 1A1 (CYP1A1), a highly AhR-responsive gene. The CYP1A1 enzyme efficiently degrades 6-formylindolo[3,2-b]carbazole (FICZ), a high affinity ligand and suggested endogenous activator of the AhR. We show that physiologically relevant doses of UVB suppress CYP1A1 gene expression immediately after irradiation, but induce its expression later in an AhR-dependent manner. The initial repression phase of CYP1A1 transcription was mediated by another UVB-inducible transcription factor, the nuclear factor κB (NFκB). Crosstalk between AhR and NFκB signaling has earlier been implicated to control CYP1A1 expression following stimulation by xenobiotics and cytokines. Now, our findings clearly indicate a role of NFκB also in UVB-dependent AhR signaling. We also observed that UVB reduced the catalytic activity of the CYP1A1 enzyme. Thereby, UVB attenuated the clearance of FICZ, which led to prolonged AhR activation. We further noted that repeated irradiation with UVB or H₂O₂ treatment shifted the cells into a refractory state in which AhR signaling could not be efficiently activated by UVB or H₂O₂, but by ligands. Together, our results suggest that the NFκB-mediated initial suppression of CYP1A1 as well as the unresponsiveness of AhR signaling to repeated irradiation may be part of a protective cellular UV stress response.

Introduction

Ultraviolet radiation (UVR) elicits a broad range of effects on cells. UVR is absorbed by photon-sensing molecules and stimulates numerous signal transduction pathways. Many of the cellular responses following UVR are mediated by growth factor receptors e.g. epidermal growth factor receptor (EGFR) and protein kinases and involve the activation of transcription factors and changes in gene expression [1].

In the skin as well as in cells originated from other tissues, UVB (280–320 nm) induces the expression of genes controlled by the aryl hydrocarbon receptor (AhR) [2], [3], [4]. In particular, the expression of cytochrome P450s CYP1A1 and CYP1B1 is upregulated in response to UVB. The AhR, a basic helix-loop-helix (bHLH) transcription factor belonging to the Per-ARNT-Sim (PAS) family of proteins (for a review see [5]) functions both as a ligand-activated E3 ubiquitin ligase [6], [7] and a transcriptional regulator. In its inactive form, the AhR resides in the cytoplasm bound to a complex of molecular chaperones. Upon activation, the AhR is released from this complex and translocates to the nucleus, where it heterodimerizes with ARNT (AhR nuclear translocator) and binds to the xenobiotic response elements (XREs) in the promoter regions of AhR target genes (reviewed in [8]). Activation of AhR and subsequent induction of CYP1A1 gene expression by UVB has been shown to be mediated through the intracellular formation of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) [3], [9], a high affinity ligand and suggested endogenous activator of the AhR. FICZ is effectively degraded via AhR-regulated CYP1 enzymes [10], [11]. The rapid metabolic clearance of FICZ constitutes an autoregulatory feedback loop between the AhR and the CYP1 enzymes.

Even though CYP1A1 gene expression is primarily controlled by activated AhR, other pathways have been implicated in the transcriptional regulation of this gene. Down-regulation of CYP1A1 gene expression has for example been reported after oxidative stress [12], [13], [14]. Suppression of TCDD-induced CYP1A1 expression was also observed after treatment with proinflammatory cytokines or bacterial endotoxins and this suppression was shown to be mediated by nuclear factor κB (NFκB) [15], [16]. NFκB is a pleiotrophic transcription factor belonging to the family of Rel proteins. In the inactive state, NFκB resides in the cytoplasm associated with IκB, a member of the inhibitory family of proteins, masking its nuclear localization sequence. Upon stimulation, IκB is targeted for proteasomal degradation through the phosphorylation of its N-terminal serines 32 and 36 by IκB kinases. Subsequently, NFκB is released and translocates to the nucleus where it can affect transcription of target genes (for a review see [17]). Crosstalk between the AhR and the NFκB subunit RelA (p65) has been earlier described in the context of CYP1A1 gene regulation. The AhR and the NFκB p65 subunit have been shown to physically interact with each other [18] and interferences of those two pathways may occur via the use of common co-factors e.g. p300/CBP as well as involve chromatin remodeling [16].

In this study, we investigated in more detail the effects of UVB on AhR signaling and CYP1A1 gene induction using a human hepatoma-derived cell line stably transfected with a sensitive AhR-driven reporter. We observed a delayed activation of AhR signaling by UVB compared to activation by FICZ, the tryptophan photoproduct that is produced endogenously by UVB. Furthermore, we showed crosstalk between the AhR and NFκB which could explain the delayed activation of AhR signaling by UVB and which may be part of the protection against oxidative stress.

Section snippets

Materials

FICZ was synthesized by Drs. Jan Bergman and Niklas Wahlström (Novum, Karolinska Institutet, Sweden) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Chemsyn (Lenexa, KS). H₂O₂ was purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO). For inhibition studies, the NFκB inhibitors BAY 11-7085 (BAY) (Sigma-Aldrich) and SN50 (Calbiochem/Merck, Darmstadt, Germany) were used. All compounds, except H₂O₂, were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The final

UVB treatment activates AhR signaling and upregulates CYP1A1 gene expression after an initial transcriptional repression period

UVB has been shown previously to stimulate AhR translocation into the nucleus and induce transcriptional upregulation of the CYP1A1 gene in HaCaT cells [3], [9]. It is proposed that UVB acts through the formation of the tryptophan photoproduct FICZ. By using the hepatoma-derived cell line HepG2-XRE-Luc carrying a luciferase reporter under the control of two XRE sequences, we investigated the activation of AhR signaling by low, physiologically relevant doses of UVB and compared it to the

Discussion

CYP1A1 gene induction is a hallmark of the AhR signaling pathway (reviewed in [34]). Induction of CYP1A1 and CYP1B1 has been observed in the skin of rodents irradiated with UVB [35] and in UVB-exposed human skin [2]. Detailed studies revealed that the high affinity AhR ligand FICZ is formed as a photoproduct of the amino acid tryptophan when irradiated with UVB or visible light [11], [33], [36], [37]. Subsequently, the intracellular formation of FICZ was shown in UVB-irradiated HaCaT cells and

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We would like to thank Dr. Anastasia Simi and Dr. Niklas Schultz for help with the immunocytochemistry for p65, Dr. Katarina Gradin for providing the HepG2-XRE-Luc cells and Dr. Fedor Moncek for performing the EROD experiments. This work was supported by grants from the Swedish Research Council for Environment, Agricultural Science and Spatial Planning (FORMAS) and the Swedish Radiation Safety Authority.

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The aim of this study was to gain more information about the mechanisms that regulate expression of the aryl hydrocarbon receptor (AHR) target gene CYP1A1. Human hepatoma cells (HepG2 and Huh7) and human immortalized keratinocytes (HaCaT) were treated with different concentrations of the dietary polyphenolic compound curcumin (CUR) alone or in combination with the natural AHR agonist 6-formylindolo[3,2-b]carbazole (FICZ). In an earlier study, we described that CUR can activate the AHR indirectly by inhibiting metabolic clearance of FICZ. Here, we measured cell viability, activation of AHR signaling, oxidative stress and histone modifying activities in response to CUR at concentrations ranging from 0.1 to 50 μM. We observed apparent non-linear responses on cell viability and activation of AHR signaling. The CYP1A1 expression and the CYP1A1 enzyme activity in the presence of CUR reflected the histone acetylation efficiency observed in nuclear extracts. At the lowest concentration, CUR significantly decreased histone deacetylase activity and increased the FICZ-induced CYP1A1 activity. In contrast, at the highest concentration, CUR increased the formation of reactive oxygen species, significantly inhibited histone acetylation, and temporally decreased FICZ-induced CYP1A1 activity. The results suggest that CUR can both increase and decrease the accessibility of DNA and thereby influence transcriptional responses to the ligand-activated AHR. This suggestion was supported by the fact that chromatin remodeling treatments with trichostatin A, p300, or 5-aza-dC increased CYP1A1 transcription. We conclude that the AHR-dependent transcriptional efficiency is modified by factors that influence the cellular redox status and the chromatin structure.
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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.
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Again, bi-phasic effects of 50 μM Rv were observed: significant reduction of AhR nuclear levels and CYP1A1 synthesis at 1 h and an increase of these parameters at later time-points (Figs. 8D and E). Similar time-dependent bi-phasic expression of CYP1A1 has been previously observed in hepatoma cells exposed to UVB irradiation (Luecke et al., 2010). The authors concluded that the initial inhibitory phase was AhR independent and driven by early transient redox-dependent p65 phosphorylation and its nuclear translocation, while late up-regulation strictly depended on AhR activation.
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Cytochrome P450 1A1 gene regulation by UVB involves crosstalk between the aryl hydrocarbon receptor and nuclear factor κB

Abstract

Introduction

Section snippets

Materials

UVB treatment activates AhR signaling and upregulates CYP1A1 gene expression after an initial transcriptional repression period

Discussion

Conflict of Interest

Acknowledgments

J. Photochem. Photobiol. B.

J. Invest. Dermatol.

Chem. Biol. Interact.

Toxicol. Appl. Pharmacol.

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Gene

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Mutat. Res.