Identification of a short form of ubiquitin-specific protease 3 that is a repressor of rat glutathione S-transferase gene expression.

The transcription rate and protein expression from both GSTA2 (glutathione S-transferase A2) and albumin genes decrease in rat liver after IL-6 (interleukin 6) plus DEX (dexamethasone) treatment of primary hepatocytes or after LPS (lipopolysaccharide)-induced acute-phase response in animals. The down-regulation is associated with the induced expression of a nuclear protein (termed IL6DEX-NP for IL-6/DEX-induced nuclear protein) that binds to a specific site on the promoter of GSTA2, leading to a decrease in transcriptional activity. IL6DEX-NP is not similar to other transcription factors, and, for identification, we functionally cloned it from a rat liver library using a yeast one-hybrid screen based on DNA-binding activity. The cloned sequence was a truncated form of USP3 (ubiquitin-specific protease 3) and the truncated USP3 protein in a yeast extract bound to DNA containing the IL6DEX-NP recognition sequence. Using 5'- and 3'-RACE (rapid amplification of cDNA ends), the complete sequence of USP3 was found in liver from LPS-treated rats. However, using Western blot analysis, only truncated forms of USP3 could be identified in nuclear extracts from LPS-treated rat livers. A GSTA2 promoter-reporter gene plasmid and USP3-expressing plasmids were transfected into rat hepatoma cells. Expression of the short form of USP3, but not the full-length protein, abolished expression from the reporter gene. Chromatin immunoprecipitation localized USP3 to the GSTA2 promoter in rat hepatocytes in vivo. We believe that the short form of USP3 is IL6DEX-NP and that it may play an important role in the negative regulation of proteins during the acute-phase response.

Characterization of the molecular forms of glutathione S-transferase P1 in human gastric cancer cells (Kato III) and in normal human erythrocytes.

GSTP1 (glutathione S-transferase pi) is involved in stress responses and in cellular proliferation pathways as an inhibitor of JNK (c-Jun N-terminal kinase). It has been proposed that monomeric GSTP1 functions as a JNK inhibitor. All of the studies to date have been performed using rodent cells, and it is unclear if monomeric GSTP1 exists in human cells. Monomeric GSTP1 was sought in human gastric cancer cells (Kato III) and in normal human erythrocytes using gel filtration, ELISA and Western blots. Monomeric GSTP1 was found in conditioned medium, in cytosol of Kato III cells and in cytosol of erythrocytes. GSTP1 subunits from Kato III cells and erythrocytes were heterogeneous when analysed by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, suggesting that there were post-translational modifications to GSTP1. One post-translational modification, phosphorylation of a serine residue in the C-terminal portion of GSTP1 where JNK binds, was identified in GSTP1 purified from Kato III cells, but not in GSTP1 purified from human erythrocytes. Therefore normal and malignant human cells contain GSTP1 monomers with post-translational modifications, and it is likely that GSTP1 monomers regulate JNK activity in human cells in the same manner as in rodent cells.

Decreased expression levels of rat liver glutathione S-transferase A2 and albumin during the acute phase response are mediated by HNF1 (hepatic nuclear factor 1) and IL6DEX-NP.

The acute phase response is characterized by positive and negative regulation of many liver proteins including GSTs (glutathione S-transferases) and albumin. The expression of albumin and some GSTs are dependent on HNF1 (hepatic nuclear factor 1). Interleukin 6 plus dexamethasone induce a nuclear protein (IL6DEX-NP) in rat hepatocytes in vitro that binds to a promoter element adjacent to the HNF1 site of rGSTA2 and decreases its expression. We determined how HNF1 and IL6DEX-NP regulate rGSTA2 and albumin expression in rats during the acute phase response after LPS (lipopolysaccharide) treatment. Expression of rGSTA2 and albumin mRNA decreased 3 h after LPS treatment and remained low for 48 h. Transcription rates showed a similar pattern but albumin transcription was less affected. HNF1 and IL6DEX-NP binding to the rGSTA2 promoter was present in control livers but was absent at 3 and 6 h after LPS. By 12 h, HNF1 and IL6DEX-NP binding to the rGSTA2 promoter reappeared and increased to above normal at 48 h. The patterns of HNF1 and IL6DEX-NP binding to the albumin promoter were similar. Affinity of IL6DEX-NP for the albumin promoter was less than that for the rGSTA2 promoter and changes in the transcription rates were consistent with the difference. Early decreases in rGSTA2 and albumin during the acute phase response are due to decreased binding of HNF1. Later persistent decreases in transcriptional rate of rGSTA2 and to a lesser extent albumin are due to increased IL6DEX-NP binding. IL6DEX-NP appears to be an important negative regulator of gene expression in vitro and in vivo.

Mechanism of negative regulation of rat glutathione S-transferase A2 by the cytokine interleukin 6.

A decrease in concentration of some liver proteins, including the detoxification enzyme glutathione S-transferase A2 (rGSTA2), occurs during the acute-phase response. Interleukin 6 (IL-6) with dexamethasone (DEX) decreases transcription of rGSTA2 in rat hepatocytes. The promoter region that mediates suppression of rGSTA2 was localized to 150 bp. These 150 bp were divided and used for electrophoretic mobility-shift assays. Induction of a protein that specifically bound to an oligonucleotide from this region required new protein synthesis and IL-6 with DEX in the culture media. The protein bound to part of the hepatocyte nuclear factor 1 (HNF1) site but was different from and did not displace HNF1. A core sequence, TGATT, was required for binding. The protein also bound to an HNF1 site in the albumin promoter. We hypothesize that IL-6 along with DEX induced a novel protein that decreased transcription of rGSTA2 and possibly albumin by interfering with the transactivating function of HNF1. The protein may be an important negative regulator of transcription during the acute-phase response.