Cell Density Regulates Intracellular Localization of Aryl Hydrocarbon Receptor

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR contains signals for both nuclear localization and nuclear export (NES). The objective of this study was to demonstrate how AhR intracellular distribution was regulated physiologically in cells. We found that cell density, but not the cell cycle, influenced the subcellular distribution of AhR in a keratinocyte cell line, HaCaT: AhR was predominantly nuclear at sparse cell densities, both nuclear and cytoplasmic at subconfluence, and predominantly cytoplasmic at confluence. Stable transfectants of HaCaT carrying a reporter gene fused with xenobiotic responsive element showed an association between xenobiotic responsive element-mediated transcription and AhR relocalization. Leptomycin B promoted nuclear accumulation of AhR irrespective of cell density, suggesting that this alteration may be because of a change of the regulation of the nuclear export of AhR. We found that Ser-68 in the NES of AhR was phosphorylated after nuclear accumulation of activated AhR and the nuclear export of a chimeric GST-AhR-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid, which mimics the negative charge of phosphorylation. This novel cell density-dependent AhR relocalization was affected by exposure to SB203580, okadaic acid, and low Ca2+ concentrations. These findings strongly suggest that cell density regulates the intracellular localization and function of AhR, because of modulation of nuclear export activity. The p38 MAPK-mediated phosphorylation of the NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR contains signals for both nuclear localization and nuclear export (NES). The objective of this study was to demonstrate how AhR intracellular distribution was regulated physiologically in cells. We found that cell density, but not the cell cycle, influenced the subcellular distribution of AhR in a keratinocyte cell line, HaCaT: AhR was predominantly nuclear at sparse cell densities, both nuclear and cytoplasmic at subconfluence, and predominantly cytoplasmic at confluence. Stable transfectants of HaCaT carrying a reporter gene fused with xenobiotic responsive element showed an association between xenobiotic responsive element-mediated transcription and AhR relocalization. Leptomycin B promoted nuclear accumulation of AhR irrespective of cell density, suggesting that this alteration may be because of a change of the regulation of the nuclear export of AhR. We found that Ser-68 in the NES of AhR was phosphorylated after nuclear accumulation of activated AhR and the nuclear export of a chimeric GST-AhR-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid, which mimics the negative charge of phosphorylation. This novel cell density-dependent AhR relocalization was affected by exposure to SB203580, okadaic acid, and low Ca2+ concentrations. These findings strongly suggest that cell density regulates the intracellular localization and function of AhR, because of modulation of nuclear export activity. The p38 MAPK-mediated phosphorylation of the NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization. The aryl hydrocarbon receptor (AhR) 1The abbreviations used are: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; NLS, nuclear localization signal; NES, nuclear export signal; bHLH, basic helix-loop-helix; PAS, PER-ARNT-SIM homology region; XRE, xenobiotic responsive element; MC, 3-methylcholanthrene; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; PP, protein serine/threonine phosphatase; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase; BrdUrd, bromodeoxyuridine; ERK, extracellular signal-regulated kinase. is a ligand-activated transcription factor belonging to the basic-helix-loop-helix (bHLH)/PER-ARNT-SIM homology region (PAS) family. AhR is an intracellular mediator of the xenobiotic signaling pathway and remains in the cytoplasm as a complex with Hsp90 (1Denis M. Cuthill S. Wikstrom A.C. Gustafsson J.A. Biochem. Biophys. Res. Commun. 1988; 155: 801-807Google Scholar, 2Perdew G.H. J. Biol. Chem. 1988; 263: 13802-13805Google Scholar), p23 (3Nair S.C. Toran E.J. Rimerman R.A. Hjermstad S. Smithgall T.E. Smith D.F. Cell Stress Chaperones. 1996; 1: 237-250Google Scholar, 4Kazlauskas A. Poellinger L. Pongratz I. J. Biol. Chem. 1999; 274: 13519-13524Google Scholar), and XAP2, also known as ARA9 or AIP (5Carver L.A. Bradfield C.A. J. Biol. Chem. 1997; 272: 11452-11456Google Scholar, 6Ma Q. Whitlock J.P. J. Biol. Chem. 1997; 272: 8878-8884Google Scholar, 7Meyer B.K. Pray-Grant M.G. Vanden Heuvel J.P. Perdew G.H. Mol. Cell. Biol. 1998; 18: 978-988Google Scholar). When environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene (MC) bind to AhR, the receptor complex subsequently translocates into the nucleus, where AhR dissociates from Hsp90 to bind to the heterodimer partner AhR nuclear translocator (ARNT) (8McGuire J. Whitelaw M.L. Pongratz I. Gustafsson J.A. Poellinger L. Mol. Cell. Biol. 1994; 14: 2438-2446Google Scholar, 9Eguchi H. Ikuta T. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1997; 272: 17640-17647Google Scholar, 10Heid S.E. Pollenz R.S. Swanson H.I. Mol. Pharmacol. 1999; 57: 82-92Google Scholar). In the nucleus, AhR/ARNT binds to xenobiotic responsive elements (XRE) (11Fujisawa-Sehara A. Sogawa K. Nishi C. Fujii-Kuriyama Y. Nucleic Acids Res. 1986; 14: 1465-1477Google Scholar), which are enhancer DNA elements located in the 5′-flanking region of target genes. In addition to the genes for xenobiotic metabolizing enzymes such as CYP1A1 or glutathione S-transferase Ya, several genes such as p27Kip1 (12Kolluri S.K. Weiss C. Koff A. Gottlicher M. Genes Dev. 1999; 13: 1742-1753Google Scholar), AhR repressor (13Mimura J. Ema M. Sogawa K. Fujii-Kuriyama Y. Genes Dev. 1999; 13: 20-25Google Scholar), Bax (14Matikainen T. Perez G.I. Jurisicova A. Pru J. Schlezinger J.J. Ryu H-Y. Laine J. Sakai T. Korsmeyer S. Casper R.F. Sherr D.H. Tilly J.L. Nat. Genet. 2001; 28: 355-359Google Scholar), and DNA polymerase κ (15Ogi T. Mimura J. Hikida M. Fujimoto H. Fujii-Kuriyama Y. Ohmori H. Genes Cells. 2001; 6: 943-953Google Scholar) have also been identified as target genes for the AhR/ARNT system. Analyses of AhR-deficient mice have shown that AhR is involved directly in chemical carcinogenesis caused by benzo-(a)pyrene (16Shimizu Y. Nakatsuru Y. Ichinose M. Takahashi Y. Kume H. Mimura J. Fujii-Kuriyama Y. Ishikawa T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 779-782Google Scholar), in teratogenesis such as cleft palate (17Mimura J. Yamashita K. Nakamura K. Morita M. Takagi N.T. Nakao K. Ema M. Sogawa K. Yasuda M. Katsuki M. Fujii-Kuriyama Y. Genes Cells. 1997; 2: 645-654Google Scholar), hepatotoxicity, and immunosuppression because of thymic involution caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (18Fernandez-Salguero P.M. Hilbert D.M. Rudikoff S. Ward J.M. Gonzalez F.J. Toxicol. Appl. Pharmacol. 1996; 140: 173-179Google Scholar). Moreover, AhR may participate in the resolution of fetal vascular structures during development (19Lahvis G. Lindell S.L. Thomas R.S. McCuskey R.S. Murphy C. Glover E. Bentz M. Southard J. Bradfield C.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10442-10447Google Scholar), and in the normal ovarian germ-cell dynamics of mice (20Robles R. Morita Y. Mann K.K. Perez G.I. Yang S. Matikainen T. Sherr D.H. Tilly J. Endocrinology. 2000; 141: 450-453Google Scholar). It is also likely that AhR is involved indirectly in adverse biological responses because of endocrine disrupters in the environment by a molecular interaction of activated AhR and nuclear steroid hormone receptors such as the estrogen receptor (21Klinge C.M. Kaur K. Swanson H.I. Arch. Biochem. Biophys. 2000; 373: 163-174Google Scholar, 22Ohtake F. Takeyama K. Matsumoto T. Kitagawa H. Yamamoto Y. Nohara K. Tohyama C. Krust A. Mimura J. Chambon P. Yanagisawa J. Fujii-Kuriyama Y. Kato S. Nature. 2003; 423: 545-550Google Scholar). However, despite an extensive study to elucidate the various roles of AhR, little is known of the physiological function of AhR because of a paucity of information of its endogenous ligands (23Adachi J. Mori Y. Matsui S. Takigami H. Fujino J. Kitagawa H. Miller III, C.A. Kato T. Saeki K. Matsuda T. J. Biol. Chem. 2001; 276: 31475-31478Google Scholar, 24Song J. Clagett-Dame M. Peterson R.E. Hahn M.E. Westler W.M. Sicinski R.R. DeLuca H.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14694-14699Google Scholar). We previously identified and characterized a nuclear localization signal (NLS) of AhR as amino acid residues 13-39 (25Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Google Scholar), the bipartite core that overlapped with domains of DNA or Hsp90 binding (26Lees M.J. Whitelaw M.L. Mol. Cell. Biol. 1999; 19: 5811-5822Google Scholar, 27Kazlauskas A. Sundstrom S. Poellinger L. Pongratz I. Mol. Cell. Biol. 2001; 21: 2594-2607Google Scholar). Moreover, we also showed that AhR has a leucine-rich nuclear export signal (NES) (28Fischer U. Huber J. Boelens W. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Google Scholar, 29Wen W. Meinkkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Google Scholar) that is dependent on chromosome region maintenance 1 composed of amino acid residues 55-75 in helix 2 (25Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Google Scholar), the hydrophobic core involved in heterodimer formation with ARNT. Using these two signals, AhR shuttles between the cytoplasm and the nucleus of a cell (30Ikuta T. Tachibana T. Watanabe J. Yoshida M. Yoneda Y. Kawajiri K. J. Biochem. (Tokyo). 2000; 127: 503-509Google Scholar), and the intracellular localization of AhR may be regulated by the masking and unmasking of NLS and NES with some interacting proteins. In general, the intracellular distribution of nucleo-cytoplasmic shuttling proteins such as transcription factors is also determined by the balance of nuclear import and export activity (31Nigg Nature. 1997; Scholar, I.W. L. Biochem. 1998; Scholar, E. 1998; Scholar, Cell Biol. 1999; Scholar), which is regulated by various physiological or environmental of phosphorylation or S. 1996; Scholar), to the NLS H. R. J. Scholar, F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: Scholar) or NES Y. Y. 2001; Scholar, H. W. Mol. Cell. Biol. 2002; Scholar), by the of signal are known to the intracellular distribution of such in and gene regulation by transcription elucidate how the nuclear import and export of AhR is regulated physiologically in we used a keratinocyte cell line, which both AhR and R. S. T. Biochem. Biophys. Res. Commun. 1995; Scholar). are a of the which is the of and the from various of 2,3,7,8-tetrachlorodibenzo-p-dioxin K. Scholar). The of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in is the development of a characterized by and of the caused by a of the normal of M. J. Acad. Scholar). or of is regulated in by cell density, we the subcellular localization of AhR in HaCaT various cell density and found that the cell density, but not the cell cycle, influenced not the intracellular localization of AhR but also AhR in HaCaT cells. It is likely that cell density may the AhR relocalization by the p38 mitogen-activated protein phosphorylation and of the Ser-68 in the Cell used for this study the keratinocyte HaCaT by P. J. A. J. Cell Biol. 1988; Scholar) and cells. in Dulbecco's modified Eagle's with fetal at with HaCaT also in in the of and okadaic acid the Ca2+ HaCaT also in with fetal that was of the fused with the and was by J. Mimura (13Mimura J. Ema M. Sogawa K. Fujii-Kuriyama Y. Genes Dev. 1999; 13: 20-25Google Scholar). The was into the of to The of the GST-AhR-GFP fusion gene was as previously H. Ikuta T. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1997; 272: 17640-17647Google Scholar). of amino acid residues was a to AhR and was by of AhR in HaCaT with sparse or on Leptomycin B or to the for 2 an and an with used for the and of was as previously H. K. J. 1994; 13: Scholar) an and a to with of and on with and the by to the cells. The cell in and on for The and at for the as the and the in the as the nuclear The protein of and nuclear used for for by in and the proteins on of by to a The used as and which was from The was with with and by an of Stable with and or by the to the basic or used for the and with a HaCaT in a and to the cell The activity was determined a to the and was to the protein of The protein of the determined by the protein In HaCaT to and the was to in L. S. P. J. J.M. G. Gonzalez F.J. J. Chambon P. W. J. Cell Biol. 2001; Scholar). was by fluorescent was into and the fusion proteins as previously H. Ikuta T. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1997; 272: 17640-17647Google Scholar). The of was into cell with which was to a nuclear (25Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Google Scholar). the at for with The protein was by of mice an of of in for of from or mice with for and with was by the of the and as and Cell AhR in HaCaT elucidate how nuclear import and export of AhR is regulated physiologically in we the subcellular localization of AhR in HaCaT the of sparse or HaCaT at on a with on and for the of and the AhR was predominantly in the the in subconfluence, the fluorescent signal of AhR was both in the cytoplasm and the However, the to on the AhR was predominantly in the Using was also shown that the of AhR in the nucleus was in to the cell density we the of LMB, a of nuclear on the intracellular localization of AhR of cell density with for 2 in a promoted nuclear accumulation of AhR with cell density, suggesting that both nuclear import activity and some endogenous ligands for the AhR the HaCaT are These to that AhR in HaCaT its intracellular distribution to cell density, and this alteration may be because of a change of the regulation of the nuclear export of AhR. of the HaCaT to be in the novel AhR relocalization by cell density, we the of Ca2+ on the AhR When to on in by in the or of the Ca2+ or with a nuclear localization of AhR was in the in which contact was by a low of Ca2+ The cell-cell interaction was by of subcellular localization of not study the AhR nuclear localization in sparse cell density was because of a distribution to the cell cycle, identified HaCaT cell on 2 by with shown in a nuclear localization of AhR was not with or negative that AhR relocalization was dependent on the cell density but not on a in the cell by Cell or not intracellular localization of AhR because of cell density we transfectants of HaCaT by which contains of the fused with the that the not a activity in the of The activity of was by with the activity of cells. This may be because of the of on the nuclear of AhR in HaCaT the activity in to the of cells. When was at on a on the to on and a activity was as shown in When the to on AhR was predominantly in the cytoplasm the activity was to not The transcription cell density was by an in which of on When a of in a and for the activity was to with a of We also the of the on the of When in on a with by of on a on the activity a of of the AhR/ARNT in with a target gene CYP1A1 cell shown in this the of AhR or not change of cell CYP1A1 was sparse or the of CYP1A1 was in the to the of activity the the of a of L. S. P. J. J.M. G. Gonzalez F.J. J. Chambon P. W. J. Cell Biol. 2001; Scholar), with cell is likely that the activity because of cell density from a novel of AhR, but not from a of AhR. in a the activity of the AhR/ARNT in transfectants of HaCaT with a fused with a of the of a various of cell The was on 1 or the at low In the green after the to or or the between cell density-dependent AhR and we an an in which of such as and P. 1997; 276: Scholar). or not the cell in a of at the the of was in and the for from the to both after but was not not of the of was the the These suggest that transcription was activated at the where the to be These the that transcription is influenced by cell density and suggest that the AhR/ARNT in this of Ser-68 NES of cell density-dependent AhR relocalization may be because of a change of the regulation of the nuclear export of AhR, we which amino acid of are for the modulation of nuclear export activity. In addition to the NES located in the of AhR (25Ikuta T. Eguchi H. Tachibana T. Yoneda Y. Kawajiri K. J. Biol. Chem. 1998; 273: 2895-2904Google Scholar), a novel of in the was P. Pongratz I. J. Biol. Chem. 2001; 276: Scholar), but nuclear export activity of the has not been a of the NES core of with the region of proteins nuclear export activity. we in Ser-68 or because some transcription factors a or in NES, which is phosphorylated in the activated or substitution mimics the of the NES, an aspartic acid substitution mimics the negative charge of the phosphorylated F. Mol. Cell. Biol. 2001; 21: Scholar). When into the nucleus of the fused with was found to be in the cytoplasm and was from the nucleus In was in the When into the nucleus, nuclear export was to the nuclear and in the nucleus suggesting that phosphorylation of Ser-68 nuclear export activity. We to or not Ser-68 of the AhR was phosphorylated in the Using in binding of the to phosphorylated or we that the with the phosphorylated of but not with the When HaCaT to in DMEM, by with a nuclear localization of AhR was as shown in was as a a with molecular of AhR was in cell from an with was from In with was in from DMEM, and a with was in from for the molecular of AhR in from is molecular modulation of phosphorylation. In of AhR was on from or mice In of AhR by was in the was in the of from These to that Ser-68 in the NES was phosphorylated in the role for the p38 signaling pathway in the nuclear export of the or estrogen receptor has been Y. Y. 2001; Scholar, H. W. Mol. Cell. Biol. 2002; Scholar). In this is likely that Ser-68 the NES of the AhR is the amino acid that is modified by the p38 the molecular modulation of the by the activated p38 is for the cell density-dependent of AhR, exposure to an of p38 in an subcellular localization of the endogenous AhR. HaCaT in the in which the AhR in the cytoplasm and the nucleus, with a p38 SB203580, and we a cytoplasmic distribution of AhR. with a of the signal the subcellular localization of AhR was not affected with that of of AhR by activated p38 MAPK, but not was also by a reporter as shown in In we that the p38 was phosphorylated by the change from to after with the HaCaT by CYP1A1 In HaCaT in which AhR was predominantly in the with a protein serine/threonine okadaic acid, a a nuclear distribution of AhR was we to the novel relocalization of AhR a cytoplasmic or nuclear suggesting that p38 phosphorylation of the and its may be involved in the novel relocalization of AhR caused by cell AhR contains both signals for nuclear import and to a intracellular localization physiological such as and for the physiological function of AhR. In the we showed that AhR intracellular localization is regulated by cell density because of nuclear export activity by modulation of the showed an association between the AhR relocalization regulated by cell density and activity. Using the keratinocyte cell we found that the cell density, but not the cell cycle, the intracellular distribution of AhR, with a nuclear localization of AhR in localization in both the nucleus and the cytoplasm in and cytoplasmic localization in in have that nuclear localization of protein is regulated not by nuclear import but also by export activity J.M. U. J. 1999; 18: Scholar, C. J. 1999; 18: Scholar). with nuclear accumulation of AhR irrespective of cell density, cell density may the nuclear export of AhR but not the nuclear import of AhR. localization of activated AhR may suggest the of an endogenous or a in HaCaT cells. It has been known that the nuclear export of protein is and regulated by of the NES Y. Y. 2001; Scholar, H. W. Mol. Cell. Biol. 2002; Scholar). the nuclear export of a chimeric fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid which mimics the negative charge of phosphorylation. the phosphorylation of Ser-68 in the NES of AhR, we and used a We found that the Ser-68 was phosphorylated in HaCaT with by with which a that of AhR because of molecular modulation by negative charge of was by in cell from and this was also with a to AhR In an phosphorylation of Ser-68 was also after nuclear accumulation of activated AhR in the of of mice The of used was by binding with the phosphorylated of but not with as shown in is likely that the NES of AhR a of nuclear by for known was be that the p38 signaling pathway J. L. 1994; Scholar, S. M.J. J.L. Nature. 1994; Scholar, J. P. S. M. A. T. Cell. 1994; Scholar), which is an mediator of the to environmental such as and is involved in nuclear accumulation of environmental gene such as Y. Y. 2001; Scholar) and estrogen receptor H. W. Mol. Cell. Biol. 2002; Scholar). In this is likely that Ser-68 the NES of AhR is the amino acid that is modified by the p38 to a of nuclear export activity. we the of a or an of in MAPK, on cell density-dependent novel relocalization of AhR. We found an association between the but not the signal and AhR relocalization It is that was a in of p38 between of such as and that of cell-cell contact The phosphorylation of p38 was in HaCaT with by with activated p38 after a exposure to the S. M. J. H. J. Biol. Chem. 2002; Scholar). the physiological of AhR is not nuclear import of AhR and of CYP1A1 at 2 of suggesting between the activated p38 and AhR. is likely that a p38 phosphorylation of may in the nucleus after the nuclear of activated p38 J. S. M. J. J. Biol. Chem. 1995; Scholar, M. U. Mol. Cell. Biol. 2002; Scholar). we have little information of which of participate in the of to export from the nucleus, a okadaic acid, but not not an of the of AhR, suggesting the of regulated by a in AhR relocalization may the regulation of cell of these is an activity in the nucleus with cell density, and the is a activity of showed that of 1 is in the nucleus of that are at a cell density from the nucleus C. M. J. Cell Sci. 2002; Scholar). of for may for cell novel relocalization of AhR, a between activity and cell density be The cytoplasmic localization of endogenous AhR with cell density was a nuclear distribution by the Ca2+ suggesting that signals from cell-cell contact may have pivotal roles in the nuclear export of AhR. In this is that the in of CYP1A1 and activity in are of xenobiotic AhR ligands C.M. J. Biol. Chem. 1994; Scholar). This may Hsp90 to be from AhR to nuclear import of AhR I. Poellinger L. J. Biol. Chem. Scholar). the cell-cell contact in of the may the AhR from the nucleus to the cell density-dependent AhR relocalization. for AhR relocalization is shown in a of transcription have been known to between the nucleus and the are a of cell regulated proteins. The S. W.M. Proc. Natl. Acad. Sci. U. S. A. 1996; Scholar) and F. Mol. Cell. Biol. 2001; 21: Scholar), both of which may cell and are known as shuttling and subcellular localization is regulated by cell These proteins also are predominantly in the nucleus with low cell density but in the cytoplasm with cell density, AhR. However, is to that was a in regulation between cell density-dependent and AhR relocalization. novel of was by phosphorylation of 2 and protein the NLS for the F. Mol. Cell. Biol. 2001; 21: Scholar), modulation of the NES was for a novel relocalization of AhR. The of AhR was to the cell and the and intracellular localization of the protein was in a in mice Perdew G.H. Dev. 1995; Scholar, Dev. 1995; Scholar), suggesting that the ligand-activated transcription factor may be in normal the regulated intracellular localization of transcription factors as a biological in to various signals, a novel relocalization of AhR caused by cell density to be an for the biological function of the AhR. AhR in and of the nucleus a and to responses to environmental or physiological and extracellular signals in cells.