Akashi Izumi

A wave of nascent transcription on activated human genes.

Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-α (TNFα) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at ≈3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.

Histone Deacetylase Inhibitor Reduces Monocyte Adhesion to Endothelium Through the Suppression of Vascular Cell Adhesion Molecule-1 Expression

Objective—Tumor necrosis factor (TNF)-&agr; initiates numerous changes in endothelial cell (EC) gene expression that contributes to the pathology of various diseases including inflammation. We hypothesized that TNF-&agr;–mediated gene induction involves multiple signaling pathways, and that inhibition of one or more of these pathways may selectively target subsets of TNF-&agr;–responsive genes and functions. Methods and Results—Human umbilical vein endothelial cells (ECs) were preincubated with inhibitors of PI3 kinase (LY294002), histone deacetylases (HDAC) (trichostatin A [TSA]), de novo protein synthesis (CHX), proteasome (MG-132), and GATA factors (K-11430) before exposure to TNF-&agr; at 4 hours and analyzed by microarray. TNF-&agr;–mediated induction of vascular cell adhesion molecule-1 (VCAM-1) was attenuated by all of these inhibitors, whereas in contrast, stimulation of intercellular adhesion molecule-1 (ICAM-1) was blocked by MG-132 alone. Moreover TSA blocked TNF-&agr;–mediated induction of monocyte adhesion both in vitro and in vivo through the suppression of VCAM-1. Further analysis demonstrated that HDAC3 plays a significant role in the regulation of TNF-&agr;–mediated VCAM-1 expression. Conclusions—TNF-&agr; activates ECs via multiple signaling pathways, and these pathways may be selectively targeted to modulate EC function. Moreover, TSA treatment reduced monocyte adhesion via VCAM-1 suppression in vitro and in vivo, suggesting that TSA might be useful for the attenuation of the inflammatory response in EC.

Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray.

Backgound. Interferon (IFN)-&ggr; and the IFN-&ggr;-dependent pathway are prominent in vascularized allograft during acute rejection. However, IFN-&ggr; deficient (IFN-&ggr;−/−) mice can rapidly reject cardiac allografts. To bring the alternative pathway during allograft rejection into more precise focus, we investigated the gene expression profile in murine cardiac allografts in IFN-&ggr;−/− mice by means of DNA microarray. Material and Method. We screened for gene expression changes in murine cardiac allografts of BALB/c H-2d into both wild-type C57BL/6 H-2b (n=3) and IFN-&ggr;−/− C57BL/6 H-2b(IFN-&ggr;−/−, n=4) using Affymetrix oligonucleotide arrays to monitor more than 11,000 genes and expressed sequence tag (ESTs). The heart was heterotopically transplanted. Transplanted hearts were harvested on day 5. As a control, isografts (C57BL/6 to C57BL/6) were also harvested on day 5. Results. On day 5, 64 of the 84 genes induced in the allografts in wild-type mice were not up-regulated in IFN-&ggr;−/− mice. We identified a group of 54 genes that were up-regulated in allografts in IFN-&ggr;−/− mice. Several chemokine genes, including monocyte chemoattractant protein=1 and macrophage inflammatory protein, were induced in the allografts in both wild-type and IFN-&ggr;−/− mice. Interestingly, a group of genes, including C10-like chemokine and platelet factor 4, were specifically induced in the IFN-&ggr;−/− mice. Conclusion. DNA microarray analysis reveals a unique pattern of mRNA expression in allografts in IFN-&ggr;−/− mice as well as a group of genes induced in cardiac allografts in both wild-type and IFN-&ggr;−/− mice, including monocyte chemoattractant protein-1 and monocyte chemoattractant protein-1.

Lipid Accumulation in Smooth Muscle Cells Under LDL Loading Is Independent of LDL Receptor Pathway and Enhanced by Hypoxic Conditions

Objective—The effect of a variety of hypoxic conditions on lipid accumulation in smooth muscle cells (SMCs) was studied in an arterial wall coculture and monocultivation model. Methods and Results—Low density lipoprotein (LDL) was loaded under various levels of oxygen tension. Oil red O staining of rabbit and human SMCs revealed that lipid accumulation was greater under lower oxygen tension. Cholesterol esters were shown to accumulate in an oxygen tension–dependent manner by high-performance liquid chromatographic analysis. Autoradiograms using radiolabeled LDL indicated that LDL uptake was more pronounced under hypoxia. This result holds in the case of LDL receptor–deficient rabbit SMCs. However, cholesterol biosynthesis and cellular cholesterol release were unaffected by oxygen tension. Conclusions—Hypoxia significantly increases LDL uptake and enhances lipid accumulation in arterial SMCs, exclusive of LDL receptor activity. Although the molecular mechanism is not clear, the model is useful for studying lipid accumulation in arterial wall cells and the difficult-to-elucidate events in the initial stage of atherogenesis.

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRα) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRα protein level in the cell is low and the LXRα protein itself is very hard to detect. We have previously reported that the mRNA for LXRα is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRα protein in the human macrophage, we have established a monoclonal antibody against LXRα, K-8607. The binding of mAb K-8607 to the human LXRα protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRα antibody should prove to be a useful tool in the analysis of the human LXRα protein.