Proteasome inhibition induces nuclear translocation and transcriptional activation of the dioxin receptor in mouse embryo primary fibroblasts in the absence of xenobiotics

Abstract

The aryl hydrocarbon receptor (AHR) is a transcription factor that is highly conserved during evolution and shares important structural features with the Drosophila developmental regulators Sim and Per. Although much is known about the mechanism of AHR activation by xenobiotics, little information is available regarding its activation by endogenous stimuli in the absence of exogenous ligand. In this study, using embryonic primary fibroblasts, we have analyzed the role of proteasome inhibition on AHR transcriptional activation in the absence of xenobiotics. Proteasome inhibition markedly reduced cytosolic AHR without affecting its total cellular content. Cytosolic AHR depletion was the result of receptor translocation into the nuclear compartment, as shown by transient transfection of a green fluorescent protein-tagged AHR and by immunoblot analysis of nuclear extracts. Gel retardation experiments showed that proteasome inhibition induced transcriptionally active AHR-ARNT heterodimers able to bind to a consensus xenobiotic-responsive element. Furthermore, nuclear AHR was transcriptionally active in vivo, as shown by the induction of the endogenous target gene CYP1A2. Synchronized to AHR activation, proteasome inhibition also induced a transient increase in AHR nuclear translocator (ARNT) at the protein and mRNA levels. Since nuclear levels of AHR and ARNT are relevant for AHR transcriptional activation, our data suggest that proteasome inhibition, through a transient increase in ARNT expression, could promote AHR stabilization and accumulation into the nuclear compartment. An elevated content of nuclear AHR could favor AHR-ARNT heterodimers able to bind to xenobiotic-responsive elements and to induce gene transcription in the absence of xenobiotics. Thus, depending on the cellular context, physiologically regulated proteasome activity could participate in the control of endogenous AHR functions.

FIG. 1

AHR is depleted from the cytosol of proteasome inhibitor-treated MEF. Cells were plated and treated with the indicated chemicals for 12 h. AHR expression was analyzed by immunoblotting with anti-mouse AHR antibody using 10 μg of cytosolic or total-cell extracts. (Upper panel) Immunoblot analysis for AHR levels in cytosolic fractions from MEF treated with solvent (DMSO), 10 μM BP, 10 nM TCDD, 8 μM MG132, or 50 μM LLnL. (Lower panel) Immunoblot analysis for AHR levels in the corresponding total-cell extracts. Experiments were performed in duplicate in at least three different MEF preparations.

FIG. 2

Proteasome inhibitors induce AHR-EGFP nuclear translocation in the absence of xenobiotics. Swiss 3T3 fibroblasts were grown and transiently transfected with the EGFP control plasmid (A to D) or the AHR-EGFP fusion protein (panels E to H). Following a 24-h incubation to allow for protein expression, cells were treated for an additional 6 h with DMSO (A and E), 8 μM MG132 (C and G), or 50 μM LLnL (D and H) or for 90 min with 10 μM BP (B and F). Micrographs are representative of transfections performed in at least six different Swiss 3T3 cultures.

FIG. 3

The AHR-EGFP fusion protein is transcriptionally active. MEF cultures isolated from AHR-null mouse embryos were transfected with the AHR-EGFP fusion construct as indicated in Materials and Methods. Reporter gene expression was analyzed following CYP1A2 induction by immunoblotting using 10 μg of protein. AHR-null MEF cultures were transfected and treated for 6 h with DMSO (lane 3) or 10 nM TCDD (lane 2). Nontransfected AHR-null MEF cultures were used as a negative control (lane 1). Vacciniavirus-expressed mouse CYP1A2 was included as a positive control (lane 4). Expression of mouse β-actin was included as a control for protein loading. The experiment was done in duplicate with two different AHR-null MEF preparations.

FIG. 4

Proteasome inhibition induces AHR accumulation in the nucleus of MEF in absence of exogenous ligand. MEF were plated and treated with 8 μM MG132 for the indicated times. Nuclear and total-cell extracts were analyzed by immunoblotting using 10 μg of protein and an anti-mouse AHR antibody. (A) Total-cell extracts from cultures treated with MG132 for 0, 3, 6, or 12 h. (B) Nuclear extracts from MEF cultures treated as for panel A. AHR levels in nuclear extracts from wild-type and AHR-null MEF, both treated with 10 nM TCDD for 90 min, are included as positive and negative controls, respectively. Note the presence of basal levels of nuclear AHR in the absence of any treatment in wild-type MEF cultures (lane 0 h) but not in AHR-null MEF (lane Ahr−/−). The experiment was performed in duplicate with two different MEF preparations.

FIG. 5

Proteasome inhibition induces AHR-ARNT binding to a consensus XRE3 element in vitro. Nuclear extracts from MEF cultures treated with 8 μM MG132 for 0 h (lane 2), 3 h (lane 3), 6 h (lane 4), or 12 h (lane 5) were prepared and analyzed by EMSA using the ³²P-labeled XRE3. Nuclear extracts from wild-type (lane 6) and AHR-null (lane 7) MEF cultures, both treated with 10 nM TCDD for 90 min, were used as positive and negative controls, respectively. The specificity of the shifted band (arrow) was confirmed by preincubating nuclear extracts from MEF cultures treated for 6 h with 8 μM MG132 or 90 min with 10 nM TCDD with a 100-fold molar excess of unlabeled XRE3 (lanes 8 and 11, respectively) or with 2 μg of anti-AHR antibody (lanes 9 and 12, respectively). Nuclear extracts from MEF cultures treated for 6 h with 8 μM MG132 were also incubated with 2 μg of anti-ARNT antibody (lane 10). Lane 1 contains a binding-reaction mixture in the absence of nuclear extract; 10 and 7 μg of nuclear protein were used for MG132- and TCDD-treated MEF cultures, respectively. The position of the specific AHR-ARNT-XRE3 complex is indicated by the arrow at the top of the gel. The position of the free probe is shown at the bottom of the gel. Preincubation of nuclear extracts with preimmune IgG did not affect specific AHR-ARNT binding to XRE3. EMSA was performed in duplicate with nuclear extracts from three different MEF preparations.

FIG. 6

CYP1A2 protein induction by xenobiotics and proteasome inhibitors in MEF cultures. (A) MEF cultures from wild-type (lanes 1 to 3) or AHR-null (lanes 4 to 6) mice were treated with the indicated AHR ligands, and total-cell extracts were analyzed by immunoblotting using 10 μg of protein and an anti-CYP1A2 antibody. Total-cell extracts from cultures treated for 12 h with DMSO (lanes 1 and 4), 10 μM BP (lanes 2 and 5), or 10 nM TCDD (lanes 3 and 6) were used. (B) Wild-type MEF cultures were treated with 8 μM MG132 for the indicated times or with 10 nM TCDD for 6 h, and CYP1A2 expression was analyzed by immunoblotting. (C) To determine if CYP1A2 induction was an AHR-dependent process, AHR-null MEF cultures were treated with 8 μM MG132 for the indicated times or with 10 nM TCDD for 6 h and CYP1A2 expression was analyzed by immunoblotting. Expression of mouse β-actin was used as a control for protein loading. Vaccinia-expressed mouse CYP1A2 (r1A2) was used as positive control (lane 7). Experiments were done in duplicate with three different MEF preparations.

FIG. 7

CYP1A2 induction by proteasome inhibition is due to AHR-dependent transcriptional activation. Wild-type and AHR-null MEF cultures were treated with 8 μM MG132 for the indicated times or with 10 nM TCDD for 6 h, and CYP1A2 mRNA expression was measured by Northern blot analysis as indicated in Materials and Methods. mCYP1A2 mRNA expression steadily increased with time by proteasome inhibition or TCDD treatment in control (Ahr+/+) but not in AHR-null (Ahr−/−) MEF cultures. Note that although AHR-null cells were not responsive for CYP1A2 induction by any of the treatments, their basal CYP1A2 mRNA levels (0 h) appeared to be higher than for wild-type (Ahr+/+) MEF. Expression of mouse β-actin was used as a control for RNA integrity and loading. The experiment was repeated twice with two different MEF preparations.

FIG. 8

The proteasome inhibitor induces ARNT protein expression. Wild-type MEF cultures were treated with 8 μM MG132 for the indicated times. ARNT and AHR expression were analyzed by immunoblotting using the corresponding primary antibodies. (A) Representative immunoblots obtained for each of the proteins. (B) Quantitative analysis by volumetric integration of the raw data from the immunoblots shown in panel A. Data were normalized by the levels of mouse β-actin expression. The experiment was performed in duplicate with three different MEF preparations.

FIG. 9

The proteasome inhibitor induces ARNT transcription. Wild-type MEF cultures were treated with 8 μM MG132 for the indicated times. ARNT and AHR mRNA levels were analyzed by RT-PCR using 7 μg of total RNA and oligo (dT) priming. The specific oligonucleotides for PCR amplification of each gene are indicated in Materials and Methods. The control lane corresponds to an RT-PCR reaction performed in the absence of RNA template. Mouse β-actin mRNA expression was analyzed to verify total RNA integrity and concentration. Note that low but detectable levels of ARNT mRNA could be detected constitutively (0 h) and 12 h after proteasome inhibition. ARNT levels increased by about twofold at 3 h of MG132 treatment and by close to fourfold after 6 h of proteasome inhibition. The experiment was done in duplicate with three different MEF preparations.