Transcription of the human microsomal epoxide hydrolase gene (EPHX1) is regulated by an HNF-4α/CAR/RXR/PSF complex

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα–NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.     

Osmotic stress blocks NF-kappaB-dependent inflammatory responses by inhibiting ubiquitination of IkappaB

The inhibitory effects of hypertonic conditions on immune responses have been described in clinical studies; however, the molecular mechanism underlying this phenomenon has yet to be defined. Here we investigate osmotic stress-mediated modification of the NF-kappaB pathway, a central signaling pathway in inflammation. We unexpectedly found that osmotic stress could activate IkappaBalpha kinase but did not activate NF-kappaB. Osmotic stress-induced phosphorylated IkappaBalpha was not ubiquitinated, and osmotic stress inhibited interleukin 1-induced ubiquitination of IkappaBalpha and ultimately blocked expression of cytokine/chemokines. Thus, blockage of IkappaBalpha ubiquitination is likely to be a major mechanism for inhibition of inflammation by hypertonic conditions.     

IL-4/Stat6 activities correlate with apoptosis and metastasis in colon cancer cells

IL-4-induced Stat6 signaling is active in a variety of cell types and plays a role in cell proliferation/growth and resistance to apoptosis. Using EMSA, we identified differential IL-4/Stat6 activities in colorectal cancer cell lines, HT-29 being active Stat6^high phenotype and Caco-2 being defective Stat6^null phenotype, respectively. Active Stat6^high HT-29 cells exhibited resistance to apoptosis by flowcytometry and aggressive metastasis by Transwell assay compared with defective Stat6^null Caco-2 cells. Comparing one another using RT-PCR, Stat6^high HT-29 cells expressed more mRNA of anti-apoptotic and pro-metastatic genes Survivin, MDM2, and TMPRSS4, while Stat6^null Caco-2 cells expressed more mRNA of pro-apoptotic and anti-metastatic genes BAX, CAV1, and P53, respectively. This is the first study describing correlations of IL-4/Stat6 activities with apoptosis and metastasis in colon cancer. These findings, together with the observation of constitutive Stat6 activation in many human malignancies, suggest that Stat6 activities could be a biomarker for cancer cell’s invasive/metastatic capability.