Retinoic acid receptors and retinoid X receptor-alpha down-regulate the transforming growth factor-beta 1 promoter by antagonizing AP-1 activity
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G Salbert1, A Fanjul, F J Piedrafita
1La Jolla Cancer Research Foundation, California 92037.
Abstract:
Overexpression of the multifunctional growth factor transforming growth factor-beta 1 (TGF beta 1) has been connected to numerous diseases in human. TGF beta 1 expression is largely governed by three AP-1 binding sites located in two different promoters of this gene. We have examined the ability of retinoid receptors to inhibit the activity of the two promoters (especially the promoter 1) by cotransfection assays in the hepatocellular carcinoma cell line HepG2. When the TGF beta 1 promoter activity is induced by 12-O-tetradecanoyl phorbol13-acetate (an activator of AP-1-controlled gene transcription), this activity can be strongly repressed by retinoic acid receptor-alpha (RAR alpha), RAR beta, or retinoid X receptor-alpha (RXR alpha) as well as other members of the nuclear receptor family. Repression was hormone dependent and a function of receptor concentration. Heterodimerization of RAR alpha or RAR beta with RXR alpha did not modify the inhibition activities of these receptors, indicating that heterodimer formation is not required for antagonizing of AP-1 activity. On further examining the anti-AP-1 activity of RXR alpha we observed that three different AP-1-controlled promoters (TGF beta 1, collagenase, and cFos) can be inhibited. Using gel shift assays, we demonstrated that RXR alpha inhibits Jun and Fos DNA binding and that 9-cis RA enhances this inhibition, suggesting that a mechanism involving direct protein-protein interaction between RXR and AP-1 components mediates the inhibitory effect observed in vivo. Transfection analyses with RXR alpha point mutations revealed that residues L422, C432, and, to a lesser extent, residues L418 and L430, are involved in ligand-induced anti-AP1 activity of RXR alpha in vivo. Thus both types of retinoid receptors can inhibit AP-1-activated promoters, including the TGF beta 1 gene promoter, via a mechanism that involves protein-protein interaction.
Insights
Retinoid receptors, including retinoic acid receptor-alpha (RAR alpha) and retinoid X receptor-alpha (RXR alpha), can inhibit the expression of transforming growth factor-beta 1 (TGF beta 1). This inhibition occurs through protein-protein interactions with AP-1 components, affecting TGF beta 1 gene promoters.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Transforming growth factor-beta 1 (TGF beta 1) overexpression is linked to various human diseases.
- TGF beta 1 gene expression is regulated by AP-1 binding sites within its promoters.
Purpose of the Study:
- To investigate the inhibitory effects of retinoid receptors on TGF beta 1 promoter activity.
- To elucidate the mechanism by which retinoid receptors repress AP-1-driven transcription.
Main Methods:
- Cotransfection assays in HepG2 cells to assess promoter activity.
- Hormone-dependent repression studies with varying receptor concentrations.
- Gel shift assays to analyze DNA-protein interactions.
- Point mutation analyses of retinoid X receptor-alpha (RXR alpha).
Main Results:
- Retinoid receptors (RAR alpha, RAR beta, RXR alpha) strongly repressed TGF beta 1 promoter activity induced by AP-1 activators.
- Repression was hormone-dependent and dose-dependent.
- RXR alpha inhibited AP-1 activity on multiple promoters (TGF beta 1, collagenase, cFos) by interfering with Jun and Fos DNA binding.
- Protein-protein interaction between RXR alpha and AP-1 components was implicated in the inhibitory mechanism.
- Specific residues in RXR alpha were identified as crucial for ligand-induced anti-AP-1 activity.
Conclusions:
- Retinoid receptors effectively inhibit AP-1-activated promoters, including the TGF beta 1 gene.
- The inhibitory mechanism involves direct protein-protein interactions between retinoid receptors and AP-1 components, independent of heterodimerization.
- This finding offers potential therapeutic targets for diseases associated with TGF beta 1 overexpression.