Specific blockage of ligand-induced degradation of the Ah receptor by proteasome but not calpain inhibitors in cell culture lines from different species

Abstract

To firmly establish the pathway involved in ligand-induced degradation of the AHR, cell lines derived from mouse rat or human tissues were exposed to inhibitors specific to the proteasome or calpain proteases and exposed to TCDD. The level of endogenous AHR and CYP1A1 protein was then evaluated by quantitative Western blotting. Treatment of cells with the calpain inhibitors: calpeptin, calpain inhibitor III, or PD150606 either individually or in combinations up to 75 microM did not reduce TCDD-induced degradation of the AHR, the induction of endogenous CYP1A1 or the nuclear accumulation of the AHR. The activity of the inhibitors was verified with an in vivo calpain assay. In contrast, exposure of cells to the specific proteasome inhibitors: epoxomicin (1-5 microM), proteasome inhibitor I (5-10 microM) or lactacystin (5-15 microM) completely inhibited TCDD-induced degradation of the AHR. Inhibition of AHR degradation with these compounds did not reduce the induction of endogenous CYP1A1. In addition, exposure of the Hepa-1 line to the various proteasome inhibitors caused an accumulation of the AHR in the nucleus in the absence of TCDD exposure. Finally, Western blot analysis of the DNA bound AHR showed that its molecular mass was unchanged in comparison to the unliganded (cytoplasmic) AHR. Thus, these studies conclusively implicate the proteasome and not calpain proteases in the ligand-induced degradation of the mouse, rat and human AHR and suggest that the pharmacological use of proteasome inhibitors may impact the time course and magnitude of gene regulatory events mediated through the AHR.

Figure 1. Effect of individual calpain inhibitors on TCDD-induced degradation of the AHR and induction of CYP1A1

Duplicate plates of Hepa-1 cells were treated with 0.05% DMSO or the indicated calpain inhibitors for 1 hr at 37°C. Cells were then exposed to TCDD (2nM) for an additional 4 hrs and total cell lysates prepared. Equal amounts of total cell lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml), anti-ß-actin IgG (1:1000) or anti-CYP1A1 IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). A) Each calpain inhibitor was used at 15μM. B) Results with 25μM and 50μM calpeptin. C) Results with 25μM and 50μM calpain inhibitor III. D) Results with 25μM and 50μM PD150606. E) The level of AHR protein was normalized to the level of actin as detailed [29, 30]. Three independent experiments for each calpain inhibitor were then averaged and plotted as the mean +/- SE with the DMSO treated cells in each experiment set to 100%. * = statistically different from DMSO treated cells (p<0.001).

Figure 2. Effect of combinations calpain inhibitors on TCDD-induced degradation of the AHR and induction of CYP1A1

A) Duplicate plates of mouse Hepa-1 cells were treated with 0.05% DMSO or a combination of calpeptin (15μM or 25μM), calpain inhibitor III (15μM or 25μM) and PD15606 (15μM or 25μM) for 1 hr at 37°C. Cells were then exposed to TCDD (2nM) for an additional 4 hrs and total cell lysates prepared. Equal amounts of total cell lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml), anti-ß-actin IgG (1:1000) or anti-CYP1A1 IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). B) Hepa-1 cells were treated and analyzed as described in A, except the incubation with the calpain inhibitors was carried out for 1, 2 or 3 hrs prior to the exposure of TCDD. C) Human RPE cells were treated and analyzed as described in A. D) Rat RPE cells were treated and analyzed as described in A. E) The level of AHR protein was normalized to the level of actin as detailed [29, 30]. Three independent experiments for each cell line were then averaged and plotted as the mean +/- SE with the DMSO treated cells in each experiment set to 100%. * = statistically different from DMSO treated cells (p<0.001).

Figure. 3. In vivo calpain assay in the presence and absence of caplain inhibitors

Human RPE or Hepa-1 cells were grown on glass coverslips and treated with 0.05% DMSO (0.05%) or a combination of calpeptin (15μM), calpain inhibitor III (15μM) and PD15606 (15μM) for 1 hr at 37°C. Cells were then incubated with t-BOC-L-leucyl-L-methionine (10μM) for 20 minutes and wet mounted on slides in phosphate buffered saline. Fluorescence was observed at 405nm and individual fields photographed for identical times. Bar in the control panels = 5μm.

Figure. 4

Subcellular localization of the AHR in Hepa-1 cells exposed to calpain inhibitors. Hepa-1 cells were grown on glass coverslips and treated with DMSO (0.05%), or a combination of calpeptin (15μM), calpain inhibitor III (15μM) and PD15606 (15μM) for 1 hr at 37°C. Cells were then incubated with TCDD (2nM) for and additional hour at 37°C, fixed and then incubated with A-1 anti-AHR IgG (1.0μg/ml) and visualized with GAR-RHO IgG (1:400). All panels were photographed with identical exposures. Bar in the control panel = 5μm.

Figure 5. Effect of individual proteasome inhibitors on TCDD-induced degradation of the AHR and induction of CYP1A1

A) Duplicate plates of Hepa-1 cells were treated with 0.05% DMSO or the indicated proteasome inhibitors for 1 hr at 37°C. Cells were then exposed to TCDD (2nM) for an additional 4 hrs and total cell lysates prepared. Equal amounts of total cell lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml), anti-ß-actin IgG (1:1000) or anti-CYP1A1 IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). The level of AHR protein at each time point was divided by the corresponding level of actin and the average +/-SD plotted with DMSO treated cells set to 100%. PSI-1 = proteasome inhibitor 1; LAC = lactacystin. B) Hepa-1 cells were treated with MG-132 (1μM or 7.5μM) for 1 hr and then exposed to TCDD (2nM) for an additional 4 hrs. AHR and CYP1A1 protein was evaluated as detailed in A.

Figure 6. Effect of epoxomicin on TCDD-induced degradation of the AHR and induction of CYP1A1

A) Hepa-1 or human RPE cells were treated with 0.05% DMSO or epoxomicin (1μM or 5μM) for 1 hr at 37°C. Cells were then exposed to TCDD (2nM) for an additional 4 hrs and total cell lysates prepared. Equal amounts of total cell lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml), anti-ß-actin IgG (1:1000) or anti-CYP1A1 IgG (1:1000). Reactivity was visualized by ECL with GAR-HRP (1:10,000). B) The level of AHR protein was normalized to the level of actin as detailed [29, 30]. Three independent experiments for each cell line were then averaged and plotted as the mean +/- SE with the DMSO treated cells in each experiment set to 100%. * = statistically different from DMSO treated cells (p<0.001). ** = statistically different from TCDD treated control cells (p<0.001). C) Hepa-1 cells were grown on glass coverslips and treated with DMSO (0.05%), MG-132 (7.5μM), epoxomicin (5μM), or proteasome inhibitor 1 (20μM) for 1 hr at 37°C. Cells were then incubated with t-BOC-L-leucyl-L-methionine (10μM) for 20 minutes and wet mounted on slides in phosphate buffered saline. Fluorescence was observed at 405nm and individual fields photographed for identical times. Bar in the control panel = 5μm.

Figure 7. Subcellular localization of the AHR in Hepa-1 cells exposed to proteasome inhibitors

Hepa-1 cells were grown on glass coverslips and treated with DMSO (0.05%), MG-132 (7.5μM), lactacystin (15μM), proteasome inhibitor 1 (20μM) or epoxomicin (5μM), or for 2 hrs at 37°C. A sample was also treated with TCDD (2nM) for 1 hr at 37°C. Cells were then fixed and incubated with A-1 anti-AHR IgG (1.0μg/ml) and visualized with GAR-RHO IgG (1:400). All panels were photographed with identical exposures. Bar in the control panel = 5μm. Nuclear fluorescence intensities were determined for each of the treatments. 50-75 cells in 3-4 distinct fields of view were quantified using MicroSuite image analysis software and the average +/-SE plotted as relative pixel density. * = statistically different from DMSO treated cells (p<0.001). ** = statistically different from DMSO treated cells and TCDD treated cells (p<0.001).

Figure 8. Analysis of nuclear AHR following TCDD exposure

A) Hepa-1, rat RPE, and human RPE cells were treated with TCDD (5nM) for 1 hr and nuclear and cytoplasmic lysates prepared as detailed in Materials and Methods. Equal amounts of cytosol, or nuclear lysates were resolved by SDS-PAGE, blotted and stained with A-1A anti-AHR IgG (1.0μg/ml). Reactivity was visualized by ECL with GAR-HRP (1:10,000). Note the ability to resolve the different species of AHR and lack of lower molecular mass bands that show changes between the control and TCDD treated samples (asterisk). B) Hepa-1 or rat RPE cells were treated with TCDD as in A and nuclear extracts prepared as detailed in Materials and Methods. Equal amount of cytosol or nuclear extracts were resolved by SDS-PAGE and evaluated as detailed in A. As in (A), asterisks mark lower molecular mass bands that show no changes between control and TCDD treated samples.