Activator Protein-1 Regulation of Murine Aldehyde Dehydrogenase 1a1

Abstract

Previously we demonstrated that aldehyde dehydrogenase (ALDH) 1a1 is the major ALDH expressed in mouse liver and is an effective catalyst in metabolism of lipid aldehydes. Quantitative real-time polymerase chain reaction analysis revealed a Ϸ2.5-to 3-fold induction of the hepatic ALDH1A1 mRNA in mice administered either acrolein (5 mg/kg acrolein p.o.) or butylated hydroxylanisole (BHA) (0.45% in the diet) and of cytosolic NAD ϩ -dependent ALDH activity. We observed Ϸ2-fold increases in ALDH1A1 mRNA levels in both Nrf2(ϩ/ϩ) and Nrf2(Ϫ/Ϫ) mice treated with BHA compared with controls, suggesting that BHA-induced expression is independent of nuclear factor E2-related factor 2 (Nrf2). The levels of activator protein-1 (AP-1) mRNA and protein, as well as the amount of phosphorylated c-Jun were significantly increased in mouse liver or Hepa1c1c7 cells treated with either BHA or acrolein. With use of luciferase reporters containing the 5Ј-flanking sequence of Aldh1a1 (Ϫ1963/ϩ27), overexpression of c-Jun resulted in an Ϸ4-fold induction in luciferase activity, suggesting that c-Jun transactivates the Aldh1a1 promoter as a homodimer and not as a c-Jun/c-Fos heterodimer. Promoter deletion and mutagenesis analyses demonstrated that the AP-1 site at position Ϫ758 and possibly Ϫ1069 relative to the transcription start site was responsible for c-Jun-mediated transactivation. Electrophoretic mobility shift assay analysis with antibodies against c-Jun and c-Fos showed that c-Jun binds to the proximal AP-1 site at position Ϫ758 but not at Ϫ1069. Recruitment of c-Jun to this proximal AP-1 site by BHA was confirmed by chromatin immunoprecipitation analysis, indicating that recruitment of c-Jun to the mouse Aldh1a1 gene promoter results in increased transcription. This mode of regulation of an ALDH has not been described before.

Regulation of Human CYP2C9 Expression by Electrophilic Stress Involves Activator Protein 1 Activation and DNA Looping

“…Total cell and nuclear extracts were prepared from HepG2 cells, as described previously (Makia et al, 2012). The extracts were separated on 4-20% SDS-PAGE gels and transferred onto nitrocellulose membranes.…”

“…Each cDNA (100 ng) was mixed with 1Â TaqMan Universal PCR master mix (Applied Biosystems). All quantitative real-time PCR experiments were performed in triplicate with cDNA samples from independent samples, as described previously (Makia et al, 2012). mRNA levels were normalized with GAPDH as the endogenous control.…”

“…Chromatin immunoprecipitation (ChIP) assays were performed, as described previously (Makia et al, 2012), using the MAGnify Chromatin-Immunoprecipitation System (Life Technologies), according to the manufacturer's protocol, Regulation of CYP2C9 by AP-1 Activation and DNA Looping with minor modifications. Briefly, HepG2 cells in 10-cm dishes treated with either DMSO (control) or tBHQ (50 mM) were fixed in 1% formaldehyde at room temperature for 10 minutes to cross-link the nuclear proteins to DNA, and the reaction was stopped by incubation with 125 mM glycine for 10 minutes.…”

“…Nuclear extracts were prepared from HepG2 cells, as described previously (Makia et al, 2012). The sequences of the complementary oligonucleotides were as follows: AP-1 control (forward, 59-CGCTTGATGAGTCAGCCGGAA-39 and reverse, 59-TTCCGGCTGACTCATCAAGCG-39); distal CYP2C9 AP-1 site (forward, 59-GTGATACTTTGTCTCACTGAGTCAATAATTGCTCATTTCT-39 and reverse, 59-AGAAATGAGCAATTATTGACTCAGTGAGACAAAGTAT-CAC-39); and proximal CYP2C9 AP-1 site (forward, 59-GAGTTTA-GAGTTTCATGAGTCAGGGACCAAGTTATTGCT-39 and reverse, 59-AGCAATAACTTGGTCCCTGACTCATGAAACTCTAAACTC-39).…”

CYP2C8 Is a Novel Target of Peroxisome Proliferator-Activated Receptor α in Human Liver

“…Total cell and nuclear extracts were prepared from HepG2 cells as previously described (Makia et al, 2012). The extracts were separated on 4-20% SDS-PAGE gels and transferred onto nitrocellulose membranes.…”