Gloria J.-F. Ding

Characterization and Quantitation of NF-κB Nuclear Translocation Induced by Interleukin-1 and Tumor Necrosis Factor-α DEVELOPMENT AND USE OF A HIGH CAPACITY FLUORESCENCE CYTOMETRIC SYSTEM

A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-κB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-α (TNFα). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-κB was found to move to the nucleus with a half-time of 7–8 min for HeLa and 12–13 min for chondrocytes, a rate in each case about 4–5 min slower than that of IκBα degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFα and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFα stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFα stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent IκBα degradation and subsequent NF-κB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 μm, while clastolactacystin β-lactone was a complete inhibitor with an IC50 of 10 μm. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFα stimulation in both cell types with an IC50 of 0.4 μm. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFα concentration measured after 17 h of stimulation. These results suggest that the ArrayScan™ technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-κB.

11β-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11β-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11β-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11β-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11β-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11β-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11β-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11β-HSD1 inhibition. Taken together, our data suggest 11β-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Sequence analysis and regulation of rat liver glutathione S-transferase mRNAs

We have utilized polysomal immunoadsorption techniques to purify the rat liver glutathione S-transferase mRNAs. Using the purified mRNAs as template, cDNA clones complementary to the Ya, Yb1, and Yc mRNAs have been constructed. The cDNA clones have been utilized in RNA blot hybridization and nuclear run-off assays to demonstrate that the Ya and Yb mRNAs are elevated 8 and 5-fold, respectively by phenobarbital; whereas the Yc mRNA is elevated only 2.0-fold. The elevation in glutathione S-transferase mRNAs is due in part to transcriptional activation of the corresponding genes. Nucleotide sequence analysis of the three glutathione S-transferase clones suggest that the Ya and Yc genes represent one rat liver glutathione S-transferase gene family whereas the Yb genes represent a second distinct glutathione S-transferase gene family. The construction of these cDNA clones will allow identification and characterization of the glutathione S-transferase structural genes as well as aid in the identification of regulatory elements that are responsible for transcriptional activation of the genes by xenobiotics.

Expression and Sequence Analysis of Rat Liver Glutathione S-Transferase Genes

The rat liver glutathione S-transferases are a family of enzymes which catalyze the conjugation of the reduced sulfhydryl group of glutathione with various electrophiles. In addition, the transferases bind with high affinity various exogenous hydrophobic compounds as well as potentially toxic endogenous compounds such as bilirubin and heme (1–3). The enzymes are comprised of binary combinations of at least six major subunits, Yα, Ya, Ybl, Yb2, Yc and Yn, which can be separated by onedimensional SDS-polyacrylamide gel electrophoresis (4–6).