Cell density regulates intracellular localization of aryl hydrocarbon receptor.
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 Ca(2+) 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.
Research Institute and Department of Pathology, Saitama Cancer Center, Saitama, 362-0806, Japan.
Togo
T
Ikuta
Yasuhito
Y
Kobayashi
Kaname
K
Kawajiri
D021581:Active Transport, Cell Nucleus; D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D002451:Cell Compartmentation; D002452:Cell Count; D002453:Cell Cycle; D002460:Cell Line; D004268:DNA-Binding Proteins; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D021382:Protein Sorting Signals; D021381:Protein Transport; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis / Q000378:metabolism*; D014157:Transcription Factors; D015533:Transcriptional Activation; D014945:Wound Healing; D048051:p38 Mitogen-Activated Protein Kinases
C497396:ARNT protein, human; C497397:Arnt protein, mouse; D004268:DNA-Binding Proteins; D021382:Protein Sorting Signals; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases