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. 2004 Mar 22:3:8.
doi: 10.1186/1476-4598-3-8.

The proteosome inhibitor MG132 attenuates retinoic acid receptor trans-activation and enhances trans-repression of nuclear factor kappaB. Potential relevance to chemo-preventive interventions with retinoids

Valentine B Andela  1 , Randy N Rosier
Affiliations

The proteosome inhibitor MG132 attenuates retinoic acid receptor trans-activation and enhances trans-repression of nuclear factor kappaB. Potential relevance to chemo-preventive interventions with retinoids

Valentine B Andela et al. Mol Cancer. .

Abstract

Background: Nuclear factor kappa B (NFkappaB) is a pro-malignant transcription factor with reciprocal effects on pro-metastatic and anti-metastatic gene expression. Interestingly, NFkappaB blockade results in the reciprocal induction of retinoic acid receptors (RARs). Given the established property of RARs as negative regulators of malignant progression, we postulated that reciprocal interactions between NFkappaB and RARs constitute a signaling module in metastatic gene expression and malignant progression. Using Line 1 tumor cells as a model for signal regulation of metastatic gene expression, we investigated the reciprocal interactions between NFkappaB and RARs in response to the pan-RAR agonist, all-trans retinoic acid (at-RA) and the pan-RAR antagonist, AGN193109.

Results: At-RA [0.1-1 microM] dose-dependently activated RAR and coordinately trans-repressed NFkappaB, while AGN193109 [1-10 microM] dose-dependently antagonized the effects of at-RA. At-RA and AGN193109 reciprocally regulate pro-metastatic matrix metalloprotease 9 (MMP 9) and its endogenous inhibitor, the tissue inhibitor of metalloprotease 1 (TIMP 1), in a manner consistent with the putative roles of NFkappaB and RAR in malignant progression. Activation of RAR concurs with its ubiquitination and proteosomal degradation. Accordingly, the proteosome inhibitor, MG132 [5 microM], blocked RAR degradation, quelled RAR trans-activation and enhanced RAR trans-repression of NFkappaB.

Conclusion: We conclude that reciprocal interactions between NFkappaB and RARs constitute a signaling module in metastatic gene expression and malignant progression and propose that the dissociative effect of proteosome inhibitors could be harnessed towards enhancing the anticancer activity of retinoids.

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Figures

Figure 1
Figure 1
Suppression of NFκB signaling activity allows for the induction of retinoic acid receptor (RAR) message and protein levels and an increase in RAR signaling activity. WT-Line 1 tumor cells and their non-malignant counterparts (mIκB-Line 1) transduced with a dominant negative inhibitor of NFκB were assessed for the expression of RAR transcripts by RT-PCR, using primers specific for RAR subtypes. Exponential amplification of β-actin was utilized as a loading control (A). Western blot analysis for RAR expression in WT and mIκB-Line 1 tumor cells, using a pan-RAR antibody, demonstrates increased RAR protein levels in mIκB-Line 1 tumor cells (B) and correspondingly increased RAR reporter activity (C).
Figure 2
Figure 2
Ligand modulation of RAR activity with the pan-RAR agonist, at-RA and the pan-RAR antagonist AGN193109, induces reciprocal effects on NFκB signaling activity. WT-Line 1 tumor cells, transiently transfected with RAR or NFκB reporter constructs were exposed to at-RA +/- AGN193109 for 24 h at the indicated concentrations. The dose dependent induction of RAR reporter activity (A) and reciprocal repression of NFκB reporter activity (B) by at-RA is reversed by AGN193109. The results represent the average (+/- SEM) of 3 independent experiments. A consistent observation is the agonistic tendency of 10 μM AGN193109, in both RAR and NFκB reporter assays.
Figure 3
Figure 3
Activation (nuclear translocation) of NFκB precludes RAR-DNA interactions while RAR reversibly interacts with NFκB-DNA complexes in a ligand dependent manner. (A) RAR and RXR gelshift oligonucleotides were employed to contrast RAR and RXR-DNA binding activity in WT and mIκB-Line 1 tumor cells under basal and PMA (NFκB) induced conditions. Under basal conditions, we observe increased RAR-DNA binding activity in mIκB cells contrasted to their WT counterparts (1, 3), and a decrease in RAR-DNA binding activity upon activation of NFκB with PMA in both cell types (2, 4). No appreciable differences in RXR-DNA binding activity were observed under all experimental conditions. (B) NFκB gelshift oligonucleotides conjugated to agarose beads were used to pull down NFκB and NFκB associated proteins, from the total protein lysate of cells exposed to at-RA +/- AGN193109 for 24-h. We consistently pulled down comparable amounts of RelA-NFκB and observed a dose dependent increase in the association of RAR with NFκB-DNA complexes with increasing concentrations of at-RA, and its reversal by increasing concentration of AGN193109.
Figure 4
Figure 4
RAR reciprocally regulates anti-metastatic TIMP 1 and pro-metastatic MMP 9 in a ligand dependent and reversible manner. MMP 9 and TIMP 1 gene expression in WT-Line 1 tumor cells exposed to at-RA +/- AGN193109 for 24 hours at the indicated concentrations was assessed by real time PCR. The results represent the average of 3 independent experiments, normalized to β-actin loading controls.
Figure 5
Figure 5
The proteosome inhibitor, MG132, quells RAR trans-activation a potentiates RAR trans-repression of NFκB. (A) at-RA induces a dose dependent decrease in RAR protein levels while increasing concentrations of AGN193109 have a restorative effect. The expression of RelA and β-actin is however not affected by at-RA +/- AGN193109. (B) at-RA (1 μM) induced degradation of RAR is blocked by the proteosome inhibitor MG132 (5 μM). Correspondingly, induction of RAR reporter activity by at-RA (1 μM) is quelled by MG132 (5 μM). (C) Basal NFκB reporter activity in mIκB-Line 1 cells is further repressed by MG132 (5 μM) by mechanism independent of the IκB-NFκB signaling axis (* P value < 0.05).
Figure 6
Figure 6
A reductionist model for optimizing the anticancer property of retinoids. The classical IκB-NFκB signaling cascade proceeds through the sequential phosphorylation, ubiquitination (ub) and proteosomal degradation of IκBα and the coordinate release and nuclear translocation of NFκB. Hyper-activation of NFκB in cancer can be overridden by hyper-activating RAR with retinoids. As such retinoid therapy induces malignant reversion but is associated with retinoid toxicity. Proteosome inhibitors quell RAR trans-activation and enhance RAR trans-repression of NFκB.
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