The GI-GPx gene is a target for Nrf2

Abstract

The gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenoprotein that was suggested to act as barrier against hydroperoxide absorption but has also been implicated in the control of inflammation and malignant growth. In CaCo-2 cells, GI-GPx was induced by t-butyl hydroquinone (tBHQ) and sulforaphane (SFN), i.e., "antioxidants" known to activate the "antioxidant response element" (ARE) via electrophilic thiol modification of Keap1 in the Nrf2/Keap1 system. The functional significance of a putative ARE in the GI-GPx promoter was validated by transcriptional activation of reporter gene constructs upon exposure to electrophiles (tBHQ, SFN, and curcumin) or overexpression of Nrf2 and by reversal of these effects by mutation of the ARE in the promoter and by overexpressed Keap1. Binding of Nrf2 to the ARE sequence in authentic gpx2 was corroborated by chromatin immunoprecipitation. Thus, the presumed natural antioxidants sulforaphane and curcumin may exert their anti-inflammatory and anticarcinogenic effects not only by induction of phase 2 enzymes but also by the up-regulation of the selenoprotein GI-GPx.

FIG. 1.

Localization of potential AREs within the GI-GPx promoter. The promoter of human GI-GPx contains two putative AREs. The element GI-ARE-1 differs in 1 base (underlined and bold) from the consensus sequence, whereas GI-ARE-2 matches the consensus sequence completely. Numbers indicate the position starting from the ATG.

FIG. 2.

Nrf2 translocates to the nucleus and binds to GI-ARE-2 in response to tBHQ or SFN exposure. Nuclear extracts were prepared from HepG2 cells treated with tBHQ (20, 200 μM; 16 h) or SFN (5, 15 μM; 4 h). EMSAs were performed as described in Materials and Methods. (A) HO-ARE (lanes 1 to 5), GI-ARE-1 (lanes 6 to 10), and GI-ARE-2 (lanes 11 to 15) probes were incubated with nuclear extracts from stimulated cells as indicated. For control, a 100-fold molar excess of the respective unlabeled specific (lanes 4, 9, 14) or unspecific (κB; lanes 5, 10, 15) oligonucleotide was added during the binding procedure. (B and C) GI-ARE-2 was incubated with nuclear extracts of tBHQ-treated (B) or SFN-treated (C) cells. For supershift, nuclear extracts were incubated with anti-Nrf2 (lane 4). Results are representative of three independent experiments. (D) HepG2 cells were stimulated with tBHQ (200 μM, 16 h) or SFN (15 μM, 4 h). Nuclear extracts were analyzed for Nrf2 by Western blotting as described in Materials and Methods. Results are representative of three independent experiments.

FIG. 3.

Nrf2 binds to the GI-ARE-2 in the GI-GPx promoter. Chromatin immunoprecipitation was carried out in HepG2 cells, as described in Materials and Methods. Protein-DNA complexes of cells treated with either tBHQ (200 μM, 16 h) or SFN (5 to 15 μM, 4 h) and of untreated cells were cross-linked with formaldehyde. Sheared complexes were precipitated with an antibody against Nrf2 or with a nonspecific immunoglobulin (IgG), and the thus coprecipitated fragmented genomic DNA was analyzed by PCR with primers specific for a fragment of the GI-GPx promoter (A) or the HO-1 promoter (B) containing the specific Nrf2 binding site(s). For control of equal sample amounts, input DNA (sheared DNA prior to immunoprecipitation) was PCR amplified. ctrl, control. Results are representative of three independent experiments.

FIG. 4.

GI-ARE-2 is responsive to Nrf2, tBHQ, SFN, and CUR. (A) HepG2 cells were transfected with the reporter gene construct HO-ARE-pGL, GI-ARE-1-pGL, GI-ARE-2-pGL, or pGL3-promoter in combination with either pcDNA3-mNrf2 or empty pcDNA3. Cells were harvested 48 h after transfection, and luciferase and β-galactosidase activities were analyzed. Relative luciferase activities (luciferase activity divided by β-galactosidase activity) were normalized to that obtained with the plasmid pGL3-promoter without responsive elements. The respective pcDNA3-transfected sample was set as 1. Values are means of three experiments measured in triplicate ± standard deviation. #, P < 0.05 versus pcDNA3. (B) HepG2 cells were transfected with the reporter gene construct HO-ARE-pGL, GI-ARE-2-pGL, or pGL3-promoter. Twenty-four hours after transfection, cells were exposed to tBHQ (20 μM), SFN (5 μM), or CUR (25 μM) for 24 h. Relative luciferase activity was normalized to the activity of the empty plasmid pGL3-promoter. The respective values obtained in untreated cells (control) were set to 1. Values are means of three experiments measured in triplicate ± standard deviation. #, P < 0.05 versus the respective control.

FIG. 5.

Nrf2-dependent induction of GI-GPx promoter activity is reversed by Keap1. (A) The GI-GPx promoter constructs GI-prom-1 to -6 and GI-prom-1-mut were cotransfected in HepG2 cells together with pcDNA3-mNrf2 or with empty pcDNA3. The factor of induction by Nrf2 is shown at the right side. Relative luciferase activity of GI-prom-1 pcDNA3 was set as 1. Potential AREs are indicated by boxes (mutated ARE in black). Values are means of three experiments measured in triplicate ± standard deviation. #, P < 0.05 versus pcDNA3. (B) The GI-GPx promoter construct GI-prom-1 was cotransfected with 10 ng pcDNA3-mKeap1 or pcDNA3 plasmid. Twenty-four hours after transfection, cells were exposed to tBHQ (20 μM), SFN (5 μM), or CUR (25 μM) for 24 h. Relative luciferase activity of the untreated construct without Keap1 was set as 1. Values are means of three experiments measured in triplicate ± standard deviation. #, P ≤ 0.05 versus respective control.

FIG. 6.

Dose-dependent regulation of GI-GPx-promoter activity by Nrf2 and Keap1. Repression of promoter activity by Keap1 (A) and reversal of the Keap1 effect by Nrf2 (B). HepG2 cells were transfected with the reporter construct GI-prom-1 and either 150 ng pcDNA3-mNrf2 or 150 ng pcDNA3-mKeap1 and mutually transfected with increasing amounts of Keap1 or Nrf2 expression plasmid, respectively. Empty pcDNA3 was used to equalize the amount of cotransfected expression plasmid. Relative luciferase activity in cells transfected with GI-prom-1 and pcDNA3 was set as 1. Values are means of two experiments measured in triplicate ± standard deviation.

FIG. 7.

Induction of endogenous GI-GPx in CaCo-2 cells. (A) GI-GPx promoter activation. CaCo-2 cells were transfected with the GI-GPx promoter construct GI-prom-1 together with 150 ng pcDNA3-mNrf2, pcDNA3-mKeap1, or empty pcDNA3. Twenty-four hours after transfection, cells were incubated with tBHQ (20 μM), SFN (5 μM), or CUR (25 μM) for 24 h. Relative luciferase activity of the untreated pcDNA3-construct was set as 1. Values are means of three experiments measured in triplicate ± standard deviation. #, P < 0.05 versus control. (B) GI-GPx mRNA expression in CaCo-2 cells. Selenium-supplemented (50 nM sodium selenite) CaCo-2 cells were grown to confluence for 3 days and stimulated with tBHQ (20 μM) or SFN (5 μM) for 8 h. RNA was extracted, reverse transcribed, and amplified by PCR. β-Actin was taken as reference and was not influenced by Nrf2 activators. PCR products were separated on agarose gels and quantified densitometrically. Values are means of three experiments measured in duplicate ± standard deviation. #, P < 0.05 versus control. (C) Expression of endogenous GI-GPx. CaCo-2 cells were grown as in panel B and stimulated with either tBHQ (20 μM) or SFN (5 μM) for 48 h. Cell lysates (75 μg protein per lane) were analyzed for GI-GPx by Western blotting. Results are representative of three independent experiments.