Protein Kinase CK2 Acts as a Molecular Brake to Control NADPH Oxidase 1 Activation and Colon Inflammation.

BACKGROUND & AIMS: NADPH oxidase 1 (NOX1) has emerged as a prime regulator of intestinal mucosa immunity and homeostasis. Dysregulation of NOX1 may cause inflammatory bowel disease (IBD). It is not clear how NOX1 is regulated in vivo under inflammatory conditions. We studied the role of CK2 in this process. METHODS: The NOX1 organizer subunit, NADPH oxidase organizer 1 (NOXO1), was immunoprecipitated from cytokine-treated colon epithelial cells, and bound proteins were identified by mass spectrometry analysis. Sites on NOXO1 phosphorylated by CK2 were identified by nanoscale liquid chromatography coupled to tandem mass spectrometry. NOX1 activity was determined in colon epithelial cells and colonoids in the presence or absence of CX-4945, a CK2 specific inhibitor. Acute colitis was induced by administration of trinitrobenzenesulfonic acid in mice treated or not with CX-4945. Colon tissues were analyzed by histologic examination, quantitative polymerase chain reaction, and Western blots. CK2 activity, markers of inflammation, and oxidative stress were assessed. RESULTS: We identified CK2 as a major partner of NOXO1 in colon epithelial cells under inflammatory conditions. CK2 directly binds NOXO1 at the C-terminus containing the Phox homology domain and phosphorylates NOXO1 on several sites. CX-4945 increased ROS generation by NOX1 in human colon epithelial cells and organoids. Strikingly, CK2 activity was reduced in trinitrobenzenesulfonic acid-induced acute colitis, and CX-4945 exacerbated colitis inflammation as shown by increased levels of CXCL1, ROS generation, lipid peroxidation, and colon damage. CONCLUSIONS: The ubiquitous protein kinase CK2 limits NOX1 activity via NOXO1 binding and phosphorylation in colonic epithelial cells and lessens experimental colitis. Loss of CK2 activity during acute colitis results in excessive ROS production, contributing to the pathogenesis. Strategies to activate CK2 could be an effective novel therapeutic approach in IBD.

Thymoquinone strongly inhibits fMLF-induced neutrophil functions and exhibits anti-inflammatory properties in vivo.

Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47(PHOX), a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91(PHOX) and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47(PHOX) phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential.