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Suppression of ANP gene transcription by liganded vitamin D receptor: involvement of specific receptor domains
1Metabolic Research Unit, University of California at San Francisco, 94143, USA.
Abstract:
We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.
Insights
Liganded vitamin D receptor (VDR) suppresses human atrial natriuretic peptide (hANP) gene promoter activity. This suppression requires VDR
Area of Science:
- Molecular Endocrinology
- Gene Regulation
- Cardiovascular Physiology
Background:
- The vitamin D receptor (VDR) plays a crucial role in regulating gene expression, including the human atrial natriuretic peptide (hANP) gene.
- Previous studies indicated that liganded VDR suppresses hANP gene promoter activity in cultured neonatal rat atrial myocytes.
Purpose of the Study:
- To identify the specific structural domains of the VDR responsible for mediating the suppression of hANP gene promoter activity.
- To investigate the role of VDR DNA binding and activation functions in this suppressive mechanism.
Main Methods:
- Utilized a series of VDR mutants, including deletions and point mutations, to assess their impact on hANP promoter activity.
- Employed a chloramphenicol acetyltransferase (CAT) reporter gene assay to quantify promoter activity.
- Examined the effects of VDR mutants on a positively regulated promoter (DR3-CAT) to confirm activity profiles.
Main Results:
- The VDR DNA binding region (Delta134 mutant) and a mutant defective in retinoid X receptor (RXR) heterodimer formation (L254G) effectively suppressed hANP promoter activity.
- A deletant containing only the hormone-binding domain (HBD) and a mutant defective in activation function (K246G) did not attenuate reporter activity.
- Similar activity patterns were observed for a positively regulated promoter, indicating the functional significance of the identified VDR domains.
Conclusions:
- Inhibition of hANP promoter activity by liganded VDR necessitates both DNA binding and activation functions.
- The suppressive mechanism does not appear to rely on the formation of a classic RXR alpha-VDR heterodimer.
- Nonhypercalcemic vitamin D3 analogues exhibit similar inhibitory activity, suggesting potential therapeutic applications.