Analysis of the Complex Relationship between Nuclear Export and Aryl Hydrocarbon Receptor-Mediated Gene Regulation

Abstract

The aryl hydrocarbon receptor (AHR) contains signals for both nuclear import and nuclear export (NES). The purpose of the studies in this report was to determine the relationship between the nuclear export of the AHR and AHR-mediated gene regulation. Blockage of nuclear export in HepG2 cells with leptomycin B (LMB) resulted in increased levels of AHR-AHR nuclear translocator (ARNT) complex in the nucleus and correlative reductions in agonist-stimulated AHR degradation. However, LMB exposure inhibited agonist-mediated induction of numerous AHR-responsive reporter genes by 75 to 89% and also inhibited induction of endogenous CYP1A1. LMB did not transform the AHR to a ligand binding species or affect activation by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin). Mutagenesis of leucines 66 and 71 of the putative AHR NES resulted in a protein with reduced function in dimerization to ARNT and binding to DNA, while alanine substitution at leucine 69 (AHR_A69) resulted in an AHR that bound with ARNT and associated with DNA. AHR_A69protein injected directly into the nuclei of E36 cells remained nuclear following 6 h of agonist stimulation. In transient-transfection assays, AHR_A69 accumulated within the nucleus was not degraded efficiently following agonist exposure. Finally, AHR_A69 supported induction of AHR-responsive reporter genes in an agonist-dependent manner. These findings show that it is possible to generate an AHR protein defective in nuclear export that is functional in agonist-mediated gene induction. This implies that the negative effect of LMB on agonist-mediated gene induction is independent of the nuclear export of the AHR.

ACKNOWLEDGMENTS

Several individuals deserve special thanks for their input in the project. We acknowledge the contribution of Steve Frawley and Bill Faught, who carried out the microinjections; William Greenlee, who provided the CYP1B1 reporter construct and shared unpublished data with the laboratory; Michael Carvan, who provided the rtCYP1A3 reporter construct; Michael Denison, who provided the minimal XRE construct; Chris Bradfield, for the original pSportM′AHR vector; Minoru Yoshida, who generously donated LMB; Brian Necela, who carried out the immunoprecipitation studies; and Alan Schwartz, for providing the E36 cell line and also offering his insights into protein degradation. Finally, we thank Nikos Davarinos for his participation in aspects of this project and Michael Kern for his support and helpful discussions of the work. The reviewers of this work are also acknowledged for their critical insights with regard to the work.

This work was supported in part by National Institutes of Health grants ES-08980 and ES-10401 to R.S.P., and by Institutional Research Funds to the Medical University of South Carolina for 1999-2000.