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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.

Andela VB, Rosier RN - Mol. Cancer (2004)

Bottom Line: Nuclear factor kappa B (NFkappaB) is a pro-malignant transcription factor with reciprocal effects on pro-metastatic and anti-metastatic gene expression.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics & The James P, Wilmot Cancer Center, University of Rochester Medical Center, 601 Elmwood Avenue Box 665, Rochester, New York, 14642, USA. Valentine_Andela@urmc.rochester.edu

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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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.
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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.

Mentions: WT-Line1 cells, with high basal NFκB-DNA binding activity, demonstrate considerably lower RAR-DNA binding activity in contrast to mIκB-Line1 tumor cells, with lower basal NFκB-DNA binding activity [9]. Activation of NFκB with phorbol myristate acetate [9] results in a decrease in RAR-DNA binding activity in both WT and mIκB-Line1 cells (Fig 3A). These data suggest that NFκB activation (nuclear translocation) precludes RAR-DNA binding. Identical experiments performed in the presence of an RXR DR-1 type oligonucleotide sequence demonstrate no appreciable change in RXR-DNA binding (Fig 3A), although an increase in the expression of RXR subtypes was documented in the mIκB-Line1 cells (data not shown). Furthermore, there is no change in RXR-DNA binding, following induction of NFκB translocation by PMA. Given that RXR is a heterodimeric partner for multiple members of the nuclear hormone receptor superfamily [22], it is conceivable that this assay system is overwhelmed by mass effect.


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.

Andela VB, Rosier RN - Mol. Cancer (2004)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC398417&req=5

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.
Mentions: WT-Line1 cells, with high basal NFκB-DNA binding activity, demonstrate considerably lower RAR-DNA binding activity in contrast to mIκB-Line1 tumor cells, with lower basal NFκB-DNA binding activity [9]. Activation of NFκB with phorbol myristate acetate [9] results in a decrease in RAR-DNA binding activity in both WT and mIκB-Line1 cells (Fig 3A). These data suggest that NFκB activation (nuclear translocation) precludes RAR-DNA binding. Identical experiments performed in the presence of an RXR DR-1 type oligonucleotide sequence demonstrate no appreciable change in RXR-DNA binding (Fig 3A), although an increase in the expression of RXR subtypes was documented in the mIκB-Line1 cells (data not shown). Furthermore, there is no change in RXR-DNA binding, following induction of NFκB translocation by PMA. Given that RXR is a heterodimeric partner for multiple members of the nuclear hormone receptor superfamily [22], it is conceivable that this assay system is overwhelmed by mass effect.

Bottom Line: Nuclear factor kappa B (NFkappaB) is a pro-malignant transcription factor with reciprocal effects on pro-metastatic and anti-metastatic gene expression.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics & The James P, Wilmot Cancer Center, University of Rochester Medical Center, 601 Elmwood Avenue Box 665, Rochester, New York, 14642, USA. Valentine_Andela@urmc.rochester.edu

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.

Show MeSH
Related in: MedlinePlus