The effect of GSTA1-1 (glutathione S-transferase Alpha 1-1) on JNK (c-Jun N-terminal kinase) activation was investigated in Caco-2 cells in which GSTA1 expression increases with degree of confluency, and in MEF3T3 cells with Tet-Off-inducible GSTA1 expression. Comparison of GSTA1 expression in pre-confluent, confluent and 8-day post-confluent Caco-2 cells revealed progressively increasing mRNA and protein levels at later stages of confluency. Exposure of pre-confluent cells to stress conditions including IL-1β (interleukin-1β), H2O2 or UV irradiation resulted in marked increases in JNK activity as indicated by c-Jun phosphorylation. However, JNK activation was significantly reduced in post-confluent cells exposed to the same stresses. Western-blot analysis of GSTA1-1 protein bound to JNK protein pulled down from cellular extracts showed approx. 4-fold higher GSTA1-1–JNK complex formation in post-confluent cells compared with pre-confluent cells. However, stress conditions did not alter the amount of GSTA1-1 bound to JNK. The role of GSTA1-1 in JNK suppression was more specifically revealed in Tet-Off-inducible MEF3T3-GSTA1-1 cells in which GSTA1 overexpression significantly reduced phosphorylation of c-Jun following exposure to IL-1β, H2O2 and UV irradiation. Finally, the incidence of tumour necrosis factor α/butyrate-induced apoptosis was significantly higher in pre-confluent Caco-2 cells expressing low levels of GSTA1 compared with post-confluent cells. These results indicate that GSTA1 suppresses activation of JNK signalling by a pro-inflammatory cytokine and oxidative stress and suggests a protective role for GSTA1-1 in JNK-associated apoptosis.

Human cytochrome P450 2A6 (CYP2A6) metabolizes various clinically relevant compounds, including nicotine- and tobacco-specific procarcinogens; however, transcriptional regulation of this gene is poorly understood. We investigated the role of the glucocorticoid receptor (GR) in transcriptional regulation of CYP2A6. Dexamethasone (DEX) increased CYP2A6 mRNA and protein levels in human hepatocytes in primary culture. This effect was attenuated by the GR receptor antagonist mifepristone (RU486; 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one), suggesting that induction of CYP2A6 by DEX was mediated by the GR. In gene reporter assays, DEX caused dose-dependent increases in luciferase activity that was also prevented by RU486 and progressive truncations of the CYP2A6 promoter delineated DEX-responsiveness to a -95 to +12 region containing an hepatic nuclear factor 4 (HNF4) α response element (HNF4-RE). Mutation of the HNF4-RE abrogated HNF4α- and DEX-mediated transactivation of CYP2A6. In addition, overexpression of HNF4α increased CYP2A6 transcriptional activity by 3-fold. DEX increased HNF4α mRNA levels by 4-fold; however, the amount of HNF4α nuclear protein was unaltered. Electrophoretic mobility shift, chromatin immunoprecipitation (ChIP), and streptavidin DNA binding assays revealed that DEX increased binding of HNF4α to the HNF4-RE and that an interaction of GR and HNF4α occurred at this site. Moreover, ChIP assays indicated that histone H4 acetylation of the CYP2A6 proximal promoter chromatin was increased by DEX that may allow for increased binding of HNF4α to the HNF4-RE in human hepatocytes. These findings indicate that increased expression of CYP2A6 by DEX is mediated by the GR via a nonconventional transcriptional mechanism involving interaction of HNF4α with an HNF4-RE rather than a glucocorticoid response element.

Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

Unlike most cytochrome P450 (CYP) enzymes, murine hepatic CYP2A5 is induced during pathological conditions that result in liver injury including hepatotoxicity mediated by xenobiotics, hepatitis caused by various microbial agents and liver neoplasia. Since CYP2A5 metabolizes various important xenobiotics including nicotine and pro-carcinogens such as nitrosamines and aflatoxin B1, altered gene expression could affect tobacco addiction, hepatotoxicity and hepatocarcinogenesis. This article synthesizes the current knowledge concerning hepatic expression of Cyp2a5 including the transcriptional and post-transcriptional regulatory mechanisms, pathophysiological conditions associated with enzyme induction such as oxidative and endoplasmic reticulum stress and altered lipid and energy homeostasis as well as the known exogenous and putative endogenous substrates. Knowledge of the stimuli responsible for the unique overexpression of CYP2A5 during liver injury may provide clues to a functional role for this enzyme and the impact of variable CYP2A5 expression on xenobiotic metabolism and toxicity, disease development and the adaptive response to hepatocellular stress.