Hematologically Important Mutations: X-Linked Chronic Granulomatous Disease

Abstract

The mRNA expression of p22-phox, a membrane-bound component of NAD(P)H oxidase, is elevated in proliferating compared to quiescent rat coronary microvascular endothelial cells (CMEC). However, the underlying molecular mechanisms of this increase and its relevance to cell confluence remain unknown. This study shows greater levels of p22-phox mRNA/protein expression, NAD(P)H oxidase activity, and superoxide anion (O2−) production in proliferating versus fully confluent and growth-arrested 50% confluent CMEC. Both enhanced transcription of the p22-phox gene and enhanced mRNA half-life were shown to contribute to the increase in p22-phox mRNA levels as demonstrated by nuclear run-on studies and Northern analyses after actinomycin D transcriptional arrest, respectively. Suppression of O2− availability by a cell-permeable superoxide dismutase mimetic or inhibition of NAD(P)H oxidase activity via its specific inhibitors (apocynin, diphenyleneiodinium, phenylarsine oxide, and 4-(2-aminoethyl)bezenesulfonyl fluoride) or antisense p22-phox cDNA attenuated cell growth independent of changes in intracellular antioxidant glutathione levels. Inhibition of nascent protein synthesis by a translation inhibitor, cycloheximide, significantly reduced p22-phox mRNA stability in proliferating but not in confluent CMEC. These findings suggest that expression of p22-phox mRNA in CMEC is upregulated by growth through a mechanism that involves increases in transcriptional activity of the p22-phox gene and stability of the p22-phox message after its formation.