Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: crosstalk through C/EBPs

Abstract

Regulation of the rat glutathione S-transferase A2 (GSTA2) gene by glucocorticoids is biphasic in its concentration dependence to glucocorticoids, with concentrations of 10-100 nM repressing gene activity (GR-dependent), and concentrations above 1 microM increasing transactivation (PXR-dependent) in adult rat hepatocytes or transient transfection assays. Over-expression of either C/EBP alpha or beta negatively regulates basal and inducible expression of a 1.65 Kb GSTA2 luciferase reporter, and synergizes the response to glucocorticoids (GC). C/EBP responsive elements have been identified in the GSTA2 5'-flanking sequence, associated with the palindrominic Glucocorticoid Responsive Element (GRE), the Ah receptor response elements, and the antioxidant response element. In reporters lacking the palindromic GRE, negative regulation by GC is observed only when C/EBP alpha is co-expressed. Co-transfection of C/EBP alpha/beta induced gene expression of the GSTA2 XRE reporter, but negatively regulated the GSTA2 ARE-reporter. In contrast, the ARE from the rat NAD(P)H quinone oxidoreductase gene was induced by co-transfection of C/EBPs, but was still negatively regulated by GC. PXR-induction of the GSTA2 reporter was partially ablated by co-transfection of C/EBP alpha and enhanced by co-transfection of C/EBPbeta. We conclude that C/EBP alpha and beta are involved in GC-dependent repression of GSTA2 gene expression and ARE sequences that bind C/EBPs appears to be critical for these responses.

Figure 1

Schematic Map of the responsive elements on the 5’-flanking region of the rat glutathione S-transferase gene and reporter constructs prepared.

Figure 2

Effect of co-transfection of either C/EBPα or β on the GR-dependent repression of GSTA2 reporter construct activities in HepG2 cells transfected with either p1.65YaLuc. Luciferase and β-galactosidase assays were performed on lysed HepG2 cells that had been transfected with p1.65Ya-Luc, a control vector pCMV-β, an expression vector for the glucocorticoid receptor pRSV-GR, and pMEX expression vectors for either pC/EBPα or β, as described in materials and methods. Cells were treated with either 0.1 μM DEX, 50 μM BA, or a combination of both compounds (BA+DEX). Control cells (CON) received DMSO alone. The normalized luciferase activity is expressed as the relative light units divided by β-galactosidase activity and is the average activity ± SD for three independent samples. *P < 0.05 statistically significant difference from control cells, **P < 0.05 statistically significant different from basal or BA-induced cells.

Figure 3

Effects of DEX and RU 38486 on luciferase activity of HepG2 cells transfected with plasmid p1.65Ya-Luc and expression vectors for C/EBPα and β. Luciferase and β-galactosidase assays were performed on lysed HepG2 cells that had been transfected with p1.65Ya-Luc, a control vector pCMV-β, an expression vector for the glucocorticoid receptor pRSV-GR, and pMEX expression vectors for either pC/EBPα or β, as described in materials and methods. Cells were treated with either 0.1 μM DEX, 1 μM RU 38486 or a combination of the two compounds. The normalized luciferase activity is expressed as the relative light units divided by β-galactosidase activity and is the mean ± SD of three wells. *P < 0.05 statistically significant decrease from control cells. **P < 0.05 statistically significant increase from control cells.

Figure 4

Effects of DEX on the expression of luciferase activity in HepG2 cells transfected with deletion constructs of GSTA2 genes co-transfected with either C/EBPα or β. Deletions constructs are shown in Figure 1. Luciferase and β-galactosidase assays were performed on lysed HepG2 cells that had been transfected with deletion constructs, 0.914−0.638Ya-Luc (Construct B), XRE-Luc (Construct C), 0.721−0.681Ya-Luc (Construct D), or 0.164YaLuc (Construct E). All cells were co-transfected with a control vector pCMV-β, an expression vector for the glucocorticoid receptor pRSV-GR, and pMEX expression vectors for either pC/EBPα or β, as described in materials and methods. Cells were treated with either DMSO alone or 0.1 μM DEX. The normalized luciferase activity is expressed as the relative light units divided by β-galactosidase activity and is the average normalized luciferase ± SD of three wells. *P < 0.05 statistically significant difference from control cells.

Figure 5

Effect of co-transfection of either C/EBPα or β on the GR-dependent repression of a rat NAD(P)H:quinone oxidoreductase ARE luciferase reporter construct activities in HepG2 cells. Luciferase and β-galactosidase assays were performed on lysed HepG2 cells that had been transfected with pQARE-Luc, a control vector pCMV-β, an expression vector for the glucocorticoid receptor pRSV-GR, and pMEX expression vectors for either pC/EBPα or β, as described in Materials and Methods. Cells were treated with either DMS0 or 0.1 μM DEX. The normalized luciferase activity is expressed as the relative light units divided by β-galactosidase activity and is the average activity ± SD for three independent samples. *P < 0.05 statistically significant decrease by DEX from control cells, **P < 0.05statistically significant induction by C/EBP.

Figure 6

Effect of co-transfection of either C/EBPα or β on the GR-dependent repression of a rat GSTA2 ARE luciferase reporter construct activities in HepG2 cells. Luciferase and β-galactosidase assays were performed on lysed HepG2 cells that had been transfected with pARE-Luc, a control vector pCMV-β, an expression vector for the glucocorticoid receptor pRSV-GR, and pMEX expression vectors for either pC/EBPα or β as described in Materials and Methods. Cells were treated with either DMS0 or 50 μM t-butyl-DEX. The normalized luciferase activity is expressed as the relative light units divided by β-galactosidase activity and is the average activity ±SD for three independent samples. *P < 0.05 statistically significant induction from control cells.