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Dysfunctional glucocorticoid receptor with a single point mutation ablates the CCAAT/enhancer binding
R A Ramos1, W J Meilandt, E C Wang
1Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California at Berkeley, Berkeley, California 94720, USA.
Abstract:
We used glucocorticoid-resistant and -sensitive hepatoma cell variants to characterize the mechanism of hepatoma cell resistance to the growth inhibitory effects of glucocorticoids. BDS1 hepatoma cells express transcriptionally active glucocorticoid receptors and undergo a stringent G1 cell cycle arrest in response to glucocorticoids that is dependent on the induced expression of the CCAAT/enhancer binding protein alpha (C/EBPalpha) transcription factor. In contrast, EDR1 hepatoma cells, which express normal levels of glucocorticoid receptors, fail to growth arrest or express C/EBPalpha when treated with glucocorticoids. Ectopic expression of wild-type rat glucocorticoid receptors into EDR1 cells restored the growth suppression response, suggesting a defect in the EDR1 receptor. DNA sequence analysis revealed a single point mutation causing a cysteine-to-tyrosine substitution at amino acid position 457 (C457Y-GR) in the zinc finger region of the glucocorticoid receptor that mediates both receptor-DNA and receptor-protein interactions. Glucocorticoid activation of the alpha1-acid glycoprotein (AGP) promoter, a liver acute-phase response gene, requires receptor-DNA binding as well as an interaction with C/EBPalpha. In contrast to the wild-type glucocorticoid receptor, ectopic expression of C/EBPalpha in EDR1 cells, or coexpression of C/EBPalpha along with the C457Y-GR into receptor-deficient EDR3 cells was required to partially restore glucocorticoid responsiveness of the AGP promoter by the EDR1 glucocorticoid receptor. Constitutive expression of the wild-type glucocorticoid receptor, but not the C457Y-GR mutant, was sufficient to restore the glucocorticoid growth suppression response to receptor-deficient EDR3 cells. Thus, we have identified a glucocorticoid-resistant hepatoma cell variant with a single point mutation in the zinc finger region of the glucocorticoid receptor gene that ablates the glucocorticoid growth suppression response and attenuates transcriptional activation of the AGP promoter.
Insights
A specific mutation in the glucocorticoid receptor (GR) gene causes hepatoma cell resistance to glucocorticoids. This C457Y-GR mutation impairs growth suppression and gene activation, highlighting GR’s role in liver cancer progression.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Glucocorticoids inhibit hepatoma cell growth via glucocorticoid receptors (GR).
- Hepatoma cell variants exhibit differential sensitivity to glucocorticoid-induced growth arrest.
- CCAAT/enhancer binding protein alpha (C/EBPalpha) is crucial for GR-mediated growth suppression.
Purpose of the Study:
- To elucidate the molecular mechanism of glucocorticoid resistance in hepatoma cells.
- To identify genetic defects in GR-resistant hepatoma cell variants.
- To understand the functional consequences of GR mutations on gene regulation and cell growth.
Main Methods:
- Comparison of glucocorticoid-sensitive (BDS1) and -resistant (EDR1) hepatoma cell lines.
- Analysis of GR expression, DNA binding, and protein interactions.
- Site-directed mutagenesis to create and test the C457Y-GR mutant.
- Ectopic expression of wild-type and mutant GR in receptor-deficient cells.
- Assessment of cell cycle arrest and alpha1-acid glycoprotein (AGP) promoter activity.
Main Results:
- EDR1 cells, unlike BDS1 cells, resist glucocorticoid-induced G1 cell cycle arrest and C/EBPalpha induction.
- A single point mutation (C457Y-GR) in the GR zinc finger region was identified in EDR1 cells, impairing GR function.
- The C457Y-GR mutation disrupts GR-DNA binding and GR-C/EBPalpha interaction, affecting AGP promoter activation.
- Restoration of wild-type GR, but not C457Y-GR, rescued glucocorticoid-mediated growth suppression in EDR3 cells.
Conclusions:
- A specific GR mutation (C457Y-GR) underlies glucocorticoid resistance in a hepatoma cell variant.
- This mutation ablates GR-mediated growth suppression and attenuates transcriptional activation of target genes like AGP.
- The findings identify a critical role for the GR zinc finger region in mediating both growth inhibition and gene regulation in liver cancer.