γ-Glutamyl transpeptidase (GGT) plays critical roles in glutathione homeostasis and metabolism.

γ-Glutamyl transpeptidase (GGT) plays critical roles in glutathione homeostasis and metabolism. EpRE binding complexes contained nuclear factor erythroid 2-related factor (Nrf) 1 Nrf2 JunB c-Jun FosB c-Fos Fra1 and Fra2. HNE-induced binding of Nrf2 and c-Jun in GP5 FK-506 EpRE was confirmed by chromatin immunoprecipitation assays. Using reporter assays and specific inhibitors we found that HNE induction of rat GGT mRNA V-2 was dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) but not protein kinase C or phosphatidylinositol 3-kinase. Pretreatment with FK-506 ERK and p38MAPK inhibitors also blocked HNE-increased EpRE binding. HNE-increased nuclear content of Nrf1 Nrf2 and c-Jun in L2 cells was partially blocked by inhibition of either ERK1/2 or p38MAPK and completely blocked by simultaneous inhibition of both MAPKs. In conclusion HNE induces GGT mRNA V-2 through altered EpRE transcription factor binding mediated by both ERK and p38MAPK. for 5 min. The supernatant was then used for determination of the activity of luciferase and β-galactosidase. To determine the β-galactosidase activity 25 μl of supernatant was added to a reaction mixture containing 300 μM 4-methyllumbelliferyl β-D-galactoside. After incubation at room temperature for 20 min with shaking β-galactosidase activity was determined in a fluorescence microplate reader (Molecular Device Corp. Sunnyvale CA) at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. For the luciferase assay a luciferase FK-506 assay kit (Promega) was used. Briefly 20 μl of cell lysate was added to the reaction mixture provided in the kit and luciferase activity was determined in a luminometor (Berthold Detection Systems Pforzheim Germany). The final luciferase activity was normalized with the activity of cotransfected β-galactosidase. Electrophoretic Mobility Shift Assay Electrophoretic mobility shift assay (EMSA) was performed as described previously (47). Briefly nuclear extracts were prepared from L2 cells treated with or without HNE using NE-PER nuclear extraction reagent (Pierce). A total of 8 μg nuclear extract was preincubated in a gel shift binding reaction containing 4% glycerol 1 mM MgCl2 0.5 mM EDTA 4 mM DTT 50 mM NaCl 10 mM Tris-HCl (pH 7.5) and 0.2 μg poly (dI-dC) at room temperature for 10 min before 32P-γ-ATP end-labeled double-stranded oligonucleotides were added. Samples were then incubated for an additional 20 min at room temperature. The samples were electrophoresed in 6% DNA retardation gel at 150 V for 2-3 h. Gels were dried and scanned with the Cyclone Storage Phosphor System and the total counts were quantified with OptiQuant Image analysis software (Packark Instrument Co. Meriden CT). The sequence of the sense oligonucleotide used was 5′-GTAC CCACAAT GACACAGCAAGAAAGCCT-3′. Immunodepletion EMSA Assay To determine the EpRE binding proteins 4 μg of antibodies against specific proteins were added to the EMSA reaction mixture and incubated for 1 h at room temperature before radiolabeled oligonucleotides were added. Because the addition of antibodies produced bands that have principally decreased intensity of DNA binding (immunodepletion) rather than producing a clear shift the decrease in the intensity of the bands was used for quantitation (47 48 An SGK antibody to the p65 component of NF-κB was used to demonstrate that the decrease in binding was not a result of nonspecific interaction. ChIP Assay ChIP assays were performed by following a protocol provided with the kit from Upstate. Briefly cells were incubated with formaldehyde by directly adding it into the medium (1% final concentration) at room temperature (49) for 10 min. FK-506 The cell pellet was then lysed on ice for 10 min and sonicated under conditions that cause DNA to be broken into 200- to 800-bp fragments. Sonicated cell lysate was precleared with 75 μl of salmon sperm DNA/agarose and the supernatant was used for immunoprecipitation with antibodies to specific transcription factors overnight at 4°C. The protein/DNA complex was eluted from agarose in elution buffer; the DNA/protein complex was reversed by adding 5 M NaCl and incubating the mixture at 65°C for 4 h. The DNA was extracted with phenol:chloroform:isoamyl alcohol (25:24:1). Primers used for PCR in the ChIP assay were forward 5 TTATCA-3′ and reverse 5 ATAGAGTGGGAGCAT-3′. Statistical Analysis SigmaStat software (SPSS Science Chicago IL) was used.