The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that dimerizes with the AHR nuclear translocator protein to mediate gene regulation. However, the AHR protein is rapidly depleted in vitro and in vivo following exposure to ligands. The purpose of the studies in this report was to characterize the mechanism of AHR degradation and determine the consequence of blocking the degradation process. Western blot and immunological analysis of rat smooth muscle (A7), murine Hepa-1, and human HepG2 cells show that ligand-induced degradation of AHR is blocked when the proteasome is inhibited by MG-132. AHR degradation is also blocked in Hepa-1 and HepG2 cells when nuclear export is inhibited with leptomycin B. Mutation of a putative nuclear export signal present in the AHR results in the accumulation of AHR in the nucleus and reduced levels of degradation following ligand exposure. In addition, inhibition of AHR degradation results in an increase in the concentration of AHR⅐AHR nuclear translocator complexes associated with DNA and extends the duration that the complex resides in the nucleus. These findings show that nuclear export and degradation of the AHR protein are two additional steps in the AHR-mediated signal transduction pathway and suggest novel areas for regulatory control. The aryl hydrocarbon receptor (AHR)1 is a ligand-activated transcription factor that belongs to the growing family of basic helix-loop-helix/PER-ARNT-SIM proteins (1, 2). The AHR is ubiquitously expressed and is usually localized in the cytoplasm of cells in an inactive multiprotein complex that contains hsp90 (3-5). Upon ligand binding, the AHR complex translocates to the nucleus where the AHR can dimerize with the ARNT protein to mediate gene regulation through direct binding to xenobiotic response element (XRE) enhancer sequences (reviewed in Refs. 6 and 7). In addition, recent studies also show that ligand binding results in rapid depletion of the AHR protein in vivo and in vitro. AHR protein is reduced by 80 -95% in numerous cell culture models within 4 h of TCDD treatment and does not recover to basal levels as long as ligand is present in the medium (8 -10). AHR protein is also dramatically reduced in the male reproductive tissues, spleen, thymus, liver, and lung of rats given a single oral dose of TCDD (11, 12) and in male reproductive tissues of rats exposed to TCDD in utero and lactationally (13).At the molecular level, Western blot analysis shows that the concentration of the AHR and ARNT protein detected in nuclear lysates of culture cells is highest following 1 h of TCDD exposure but then rapidly declines (3,8). These findings are supported by gel mobility shift analysis showing that the association of the AHR⅐ARNT complex with the XRE is transient and becomes greatly reduced in cell culture models within 2-6 h of the initial agonist stimulation (14, 15). In addition, studies in rat LCS7 and mouse Hepa-1 cells indicate that protein association at the endogenous CYP1A1 promoter and CYP1A1 transcription a...

The aryl hydrocarbon nuclear translocator (ARNT) protein functions as a transcription factor after dimerization with other basic helix-loop-helix proteins. Thus, dimerization of ARNT within one pathway may limit the availability of this protein to others. To investigate this issue, aryl hydrocarbon receptor (AHR)-mediated signaling was investigated in mouse (Hepa-1), rat (H4IIE), and human (HepG2) hepatoma cell lines undergoing physiologically induced hypoxia (<1% O(2)). Basal levels of ARNT protein were not affected by hypoxia in any cell line, and ARNT remained exclusively nuclear. Furthermore, quantitative Western blotting revealed that the concentration of ARNT sequestered during hypoxia represented a small fraction of the total ARNT protein pool (12 and 15% in Hepa-1 and H4 cells, respectively). When the AHR-mediated signaling pathway was activated during hypoxia by 2,3,7,8-tetrachlorodibenzo-p-dioxin, the induction of P4501A1 protein was reduced by 55% without changes in the level of mRNA in Hepa-1 cells, whereas the levels of induction of both P4501A1 protein and CYP1A1 mRNA were reduced by >80% in the H4 cell line. Importantly, gel mobility shift analysis and Western blotting showed that the same level of AHR/ARNT complexes could be detected in cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin during hypoxia and normoxia. These data suggest that the effects of hypoxia on AHR-mediated gene regulation occur distal to the formation of AHR/ARNT complexes and imply that functional interference between hypoxia and AHR-mediated signaling does not occur through competition for ARNT protein.