Nox1 downstream of 12‐lipoxygenase controls cell proliferation but not cell spreading of colon cancer cells

Abstract

The catalytic subunit of the NADPH oxidase complex, Nox1 (homologue of gp91phox/Nox2), expressed mainly in intestinal epithelial and vascular smooth muscle cells, functions in innate immune defense and cell proliferation. The molecular mechanisms underlying these functions, however, are not completely understood. We measured Nox1-dependent O 2 2 production during cell spreading on Collagen IV (Coll IV) in colon carcinoma cell lines. Knocking down Nox1 by shRNA, we showed that Nox1-dependent O 2 2 production is activated during cell spreading after 4 hr of adhesion on Collagen IV. Nox1 activation during cell spreading relies on Rac1 activation and arachidonic metabolism. Our results showed that manoalide (a secreted phospholipase A2 inhibitor) and cinnamyl-3,4-dihydroxy-a-cyanocinnamate (a 12-lipoxygenase inhibitor) inhibit O 2 2 production, cell spreading and cell proliferation in these colonic epithelial cells. 12-Lipoxygenase inhibition of ROS production and cell spreading can be reversed by adding 12-HETE, a 12-lipoxygenase product, supporting the specific effect observed with cinnamyl-3,4-dihydroxy-a-cyanocinnamate. In contrast, Nox1 shRNA and DPI (NADPH oxidase inhibitor) weakly affect cell spreading while inhibiting O 2 2 production and cell proliferation. These results suggest that the 12-lipoxygenase pathway is upstream of Nox1 activation and controls cell spreading and proliferation, while Nox1 specifically affects cell proliferation. ' 2007 Wiley-Liss, Inc.Key words: NADPH oxidase; Nox1; colon; integrins; arachidonic acidThe phagocytic NADPH oxidase is composed of 5 subunits, including p22phox and gp91 phox that constitute the membranebound flavocytochrome b 558 , and p40 phox , p47phox and p67 phox that translocate from the cytoplasm to the membrane upon cell activation.1 In addition, the small Rac GTPase have emerged as important functional components that are indispensable for NADPH oxidase activity. Since the initial identification of Nox1 (gp91 phox homolog) in human Caco-2 colon carcinoma cells, the biological significance of superoxide production by NAPDH oxidase in nonphagocytic cells has garnered much interest.2 In contrast to gp91phox/Nox2, which catalyzes a massive and explosive production of superoxide involved in host defense, Nox1 controls a lower superoxide production that acts as mediator of cell signaling. 2Nox1 is expressed mainly in normal and cancerous intestinal epithelial cells and vascular smooth muscle cells. Although being the subject of intense investigation, the role and the endogenous regulation of Nox1 in colon epithelial cells is still unclear.3,4 In colon carcinoma cells, homologs of p47 phox and p67 phox , respectively called Noxo1 (for Nox organizer 1) and Noxa1 (for Nox activator 1), have recently been identified. 5,6 In contrast to p47phox , Noxo1 appears to be constitutively associated with the NADPH oxidase complex.Activation of NADPH oxidase is dependent upon lipid mediators, such as phosphatidic and arachidonic acids and phosphatidylinositol. Arachidonic acid (AA)...

NADPH Oxidase 1 Controls the Persistence of Directed Cell Migration by a Rho-Dependent Switch of α2/α3 Integrins

“…We previously showed that ROS produced after 4 h of adhesion to Col-I in HT29-D4 cells are inhibited by DPI, the flavoprotein inhibitor classically used to inhibit NADPH oxidase. We and others have shown that Nox1 is the only Nox homologue detected in HT29 cells (6,12). By using shRNA directed against Nox1, we confirmed that the DPI effect relies on Nox1 inhibition in our experimental model (6).…”

“…Control plasmid pRk5 and plasmid pRk5-RhoA V14 were kindly given by Alan Hall (Sloan-Kettering Cancer Center). Nox1 short hairpin RNA (shRNA) was previously validated and described (6,38). Transfected cells were serum depleted for 48 h, detached with 0.25% trypsin, and seeded onto Col-I-precoated plates (10 g/ml).…”

Cytosolic phospholipase A2, lipoxygenase metabolites, and reactive oxygen species

“…During LOX-catalyzed metabolism, unstable byproducts of AA hydroperoxidation can function as ROS (15,17,18). In addition, the metabolism of 5-, 12-, and 15-LOX produce leukotrienes, 12(S)-HETE, and 15(S)-HETE, which can induce NADPH oxides to stimulate ROS production (11,13). For example, a 5-LOX metabolite [i.e., leukotriene B4 (LTB4)] has been shown to activate NADPH oxidase in non-phagocytes (19)(20)(21)(22).…”

“…De Carvalho et al identified a signaling connection between 12-LOX and NADPH oxidase, using a mouse knockout model. For example, in the pathogenesis of colon cancer, 12-LOX regulates Nox1-dependent ROS production to enhance the spread and proliferation of colon epithelial cells (13). However, the exact role of 12-LOX in the activation of Nox1 remains to be clarified in further studies (40).…”

“…In addition to the oxidative metabolic processes of AA by COX and LOX, AA itself has also been reported to activate NADPH oxidase directly, thereby inducing ROS generation (10)(11)(12). Over the past few years, LOX-and COX-generated AA metabolites have been demonstrated to stimulate the generation of ROS by NOXs (11,13), thus revealing the existence of an inter-connecting signaling system between eicosanoids and NOXs. The generation of ROS by AA metabolites may also perform pivotal roles in various cell signaling pathways, although the mechanisms by which AA metabolites mediate ROS generation remain to be clearly elucidated.…”