No abstract

Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1␣ and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1-dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired‐end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories’. Some factories further specialize in transcribing responsive genes encoding micro‐RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

Objective-Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme degradation, has recently been considered to have protective roles against various pathophysiological conditions. Since we demonstrated that HO-1 overexpression inhibits atherosclerotic formation in animal models, we examined the effect of HO modulation on proinflammatory cytokine production, endothelial NO synthase (eNOS) expression, and endothelium-dependent vascular relaxation responses. Methods and Results-After HO-1 induction by heme arginate (HA), vascular endothelial cell cultures were exposed to oxidized low-density lipoprotein (oxLDL) or tumor necrosis factor-␣ (TNF-␣). HA pretreatment significantly attenuated the production of vascular cell adhesion molecule-1, monocyte chemotactic protein-1, and macrophage colonystimulating factor, suggesting that HO-1 induction attenuates proinflammatory responses. In addition, HO-1 overexpression also alleviated endothelial dysfunction as judged by restoration of attenuated eNOS expression after exposure to oxLDL and TNF-␣. Importantly, impaired endothelium-dependent vascular relaxation responses in thoracic aortic rings from high-fat-fed LDL receptor knockout mice were also improved. These effects were observed by treatment with bilirubin not by carbon monoxide. Key Words: heme oxygenase Ⅲ oxidized LDL Ⅲ endothelial nitric oxide synthase Ⅲ bilirubin Ⅲ carbon monoxide V ascular endothelial cell activation by oxidized LDL (oxLDL) and cytokines such as tumor necrosis factor-␣ (TNF-␣) is considered to play an essential role in the development of atherosclerotic lesions. 1 Activated endothelial cells produce adhesion molecules, chemokines, and growth factors such as vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and macrophage colony-stimulating factor (MCSF). [2][3][4] Numerous studies have shown that these molecules promote multiple steps in the formation of atherosclerotic lesion. 1,3,4 Endothelial dysfunction, which is associated with decreased bioavailability of NO from endothelial NO synthase (eNOS), is also considered to play an important role in atherogenesis. 1,5 NO formed by eNOS has been shown to contribute to vascular smooth muscle cell relaxation and inhibition of platelet aggregation. 1 Heme oxygenase (HO) catalyzes the rate-limiting step of heme degradation in mammals. 6 The products of the reaction are biliverdin, carbon monoxide (CO), and free iron. It has been suggested that biliverdin and CO have cytoprotective effects against various cellular stresses. [7][8][9] We demonstrated that the inducible form of HO (HO-1) is induced in cultured vascular endothelial cells, smooth muscle cells, and macrophages by oxidized low-density lipoprotein (oxLDL), and that high expression of HO-1 results in attenuation of monocyte chemotaxis by oxLDL. 9 In fact, HO-1 is expressed in atherosclerotic lesions. 10,11 We also demonstrated that overexpression of HO-1 inhibits the formation of atherosclerotic lesions by inhibiting lipid peroxidation and by affecting NO meta...

In order to characterize the monocytic cell line THP-1 and its mature, macrophagelike form treated with phorbol 12-myristate 13-acetate (PMA), we have conducted an oligonucleotide microarray assay and compared the results with those from an assay of human monocytes and macrophages. We found that early THP-1 cells have a pattern of gene expression distinct from monocytes, and when treated with PMA, certain genes which are induced in macrophages, such as apolipoprotein-E, matrix metalloproteinase 9 and α2 macroglobulin are also induced in the PMA-treated THP-1 cells (THP1PMA cells). However, these were some genes which are conversely regulated among macrophages and THP1PMA cells such as interleukin-1-β and the overall correlation coefficient was not very high. It is shown that, although certain morphological and other characteristics of PMA-differentiated THP-1 cells are similar to macrophages, from a transcriptomic view, the two are different. This suggests a need for careful recognition of and allowance for this difference when interpreting the results of experiments done with THP-1 cells in which it is otherwise assumed they are representative of the macrophage. J Atheroscler Thromb, 2004; 11: 88-97.