Novel isoforms of NADPH oxidase in vascular physiology and pathophysiology.

1. Vascular cells have evolved to use reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, as signalling molecules. Under physiological conditions, ROS are important regulators of cell cycle, protein kinase activity and gene expression. However, in vascular disease states, such as hypertension and hypercholesterolaemia, excessive production of ROS may overwhelm the anti-oxidant defence mechanisms of cells, resulting in 'oxidative stress', damage to the artery wall and, ultimately, development of atherosclerotic plaques. 2. The primary source of ROS in the vasculature is NADPH oxidase. There appear to be at least three isoforms of NADPH oxidase expressed in the vascular wall, each differing with respect to the flavin-containing catalytic subunit it uses to transfer electrons from NADPH to molecular oxygen. Thus, although endothelial cells and adventitial fibroblasts express a gp91phox-containing NADPH oxidase similar to that originally identified in phagocytes, vascular smooth muscle cells may rely on novel homologues of gp91phox, namely Nox1 and Nox4, to produce superoxide. 3. Controversy remains over which isoform(s) of NADPH oxidase is responsible for the oxidative stress associated with vascular diseases. We and others have shown that although gp91phox mRNA expression is upregulated during atherogenesis in human and animal models, expression of the Nox4 subunit remains unchanged. Nox1 expression is also likely to be increased in diseased arteries; however, its relative level of expression, at least at the mRNA level, appears to be markedly lower than that of the other gp91phox homologues, even after upregulation. 4. Whether these findings suggest that a gp91phox-containing NADPH oxidase is more important than either Nox4 or Nox1 in vascular disease awaits studies examining relative protein expression and enzyme kinetics of each subunit, as well as the effects of targeted gene deletion of each of these gp91phox homologues on atherogenesis.

Transcriptional regulation of the human carboxyl ester lipase gene in THP-1 monocytes: an E-box required for activation binds upstream stimulatory factors 1 and 2.

The bile salt-stimulated carboxyl ester lipase (CEL) is important for the digestion and absorption of dietary lipids, and is expressed at high levels by the exocrine pancreas and the lactating mammary gland. However, the presence of CEL in human plasma suggests that the role of CEL in lipid metabolism may stretch beyond its function in the intestinal lumen, and possibly include interactions with cholesterol and oxidized lipoproteins to modulate the progression of atherosclerosis. We have used the CEL-expressing human monocytic cell line THP-1 to investigate the transcriptional regulation of the human CEL in monocytes. Analyses of the promoter region revealed that an E-box located at -47/-52 is necessary for CEL expression. Point mutations in the E-box almost completely abolish the transcriptional activity. Electrophoretic mobility-shift assay analyses reveal that the E-box binds the upstream stimulatory factors 1 and 2, and the binding of an upstream stimulatory factor-containing complex in THP-1 cells also requires the presence of a putative nuclear receptor-binding site at -60/-66. Furthermore, we demonstrate that the E-box is also necessary for CEL expression in the pancreas and the mammary gland, although there are tissue-specific requirements for additional activating elements.