Aryl Hydrocarbon Receptor Imported into the Nucleus following Ligand Binding Is Rapidly Degraded via the Cytosplasmic Proteasome following Nuclear Export

Abstract

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 Receptor Modulates Acute and Late Mast Cell Responses

“…1B). This finding is in accordance with previous works showing that the exposure to AhR agonists causes AhR-expressing cells to downregulate the receptor through the ubiquitin/ proteasome degradation pathway (18,19). To confirm that this pathway mediates AhR downregulation also in MCs, cells were treated with the proteasome inhibitor MG-132, together with FICZ for 4 h. This treatment blocked ligand-dependent degradation of AhR (Fig.…”

Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1

“…However, it remains possible that they become accessible under some specific situations. Such a situation has been described in the case of AhR, which is exported back to the cytoplasm after inactivation (65,71).…”

ERK Kinase Inhibition Stabilizes the Aryl Hydrocarbon Receptor

“…This downregulation of Ah receptor is associated with ubiquitination via the 26 S proteasome pathway. Proteasome inhibitors MG-132 or lactacystin, or the overexpression of a dominant-negative ubiquitin mutant, UbK48R, blocks Ah receptor degradation by TCDD leading to an increase in gene expression as demonstrated by enhanced binding to DNA and CYP1A1 induction (13,(42)(43)(44). This result implies that changes in the steadystate levels of the Ah receptor initiated by blocking 26 S proteasome-dependent proteolysis results in enhancement of functional activity.…”