Activation of NADPH oxidase 1 in tumour colon epithelial cells

Abstract

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.

Reactive Oxygen Species, Vascular Noxs, and Hypertension: Focus on Translational and Clinical Research

“…In a recent comprehensive descriptive study, Pandey et al (157) reported that Nox5 is present in human saphenous vein and internal mammary artery, cultured human VSMCs, and endothelium, but not in fibroblasts, with the a and b isoforms being most abundant in vascular cells. Adenovirus-mediated overexpression of Nox5 promoted phosphorylation of MAPK in ECs and VSMCs (195), which is similar to our studies in Ang II-stimulated Nox5-containing cells (151,228). These studies, although informative of human Nox expression, have limitations, as they are essentially descriptive and do not reveal much insight into the mechanisms of oxidative stress.…”

“…Nox1 is abundant in colon epithelium, but is also found in endothelium, smooth muscle, fibroblasts, cardiomyocytes, and microglia (151). It requires p22phox, p47phox (or its homologue NoxO1 [Nox organizer 1]), and p67phox (or its homologue NoxA1 [Nox activator 1]) for its activity.…”

Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-βI and prooxidant enzyme NADPH oxidase

“…Despite constituting the main Nox isoform in colon epithelial cells, Nox1 is also associated with production of low levels of O 2 •- in vasculature [25,26]. However, through a complex reaction involving concomitant induction of PKC-, MAPK- and PKA-dependent mechanisms, the vascular pathologies appear to elevate Nox1-mediated release of O 2 •- [27–29] which in turn may trigger BMEC apoptosis to elicit barrier permeability.…”

Reactive oxygen species and vascular biology: implications in human hypertension

“…78,79 Nox1 requires p22phox, p47phox and p67phox for its activity. It is regulated by the redox chaperone protein disulfide isomerase in vascular smooth muscle cells 80 and has been implicated in vascular smooth muscle cell migration, proliferation and extracellular matrix production, effects mediated by cofilin.…”