Knockdown of dual oxidase 1 suppresses activin A-induced fibrosis in cardiomyocytes via the reactive oxygen species-dependent pyroptotic pathway.

Fibrotic diseases account for more than 8 million deaths worldwide annually. Reactive oxygen species (ROS) has been shown to activate pyroptosis and promote the production of interleukin (IL)-1ß and IL-18, leading to fibrosis development. However, the role of dual oxidase 1 (DUOX1)-induced ROS production and pyroptosis in cardiac fibrosis remains largely unknown. Activin A was used to induce ROS and pyroptosis in cardiomyocytes. ROS level, pyroptosis, and cytokine production were detected using Active Oxygen Detection Kit, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Western blotting analysis was used to measure expression changes of proteins. DUOX1 was silenced or overexpressed to investigate its role in fibrosis. We found that activin A induced ROS production and pyroptosis in cardiomyocytes, which was blocked by the ROS scavenger, N-acetyl-L-cysteine (NAC). Knockdown of DUOX1 reversed activin A-induced ROS production, pyroptosis, cytokine release, and the upregulation of proinflammatory proteins. Overexpression of DUOX1 resulted in opposite effects of knockdown DUOX1. Administration of an ROS scavenger blocked the effect of DUOX1 overexpression. Supplementation of IL-1ß and IL-18 caused significant fibrosis in human cardiac fibroblasts (hCFs). The knockdown of DUOX1 protected cardiomyocytes against activin A-induced fibrosis via the inhibition of ROS, cytokine release, and pyroptosis.

Inactivation of the PtdIns(4)P phosphatase Sac1 at the Golgi by H2O2 produced via Ca2+-dependent Duox in EGF-stimulated cells.

Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H2O2, which serves as an intracellular messenger. We have shown that exogenous H2O2 reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca2+ concentration and on H2O2. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca2+-dependent members of the NADPH oxidase family as the major source of H2O2 for Sac1 oxidation. Expression of a Golgi-targeted H2O2 probe revealed transient EGF-induced H2O2 production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca2+ mobilization to events at the Golgi including Duox activation, H2O2 production, Sac1 oxidation, and PtdIns(4)P accumulation.

Dual oxidases participate in the regulation of hemolymph microbiota homeostasis in mud crab Scylla paramamosain.

Dual oxidases (DUOXs) were originally identified as NADPH oxidases (NOXs), found to be associated with the reactive oxygen species (ROS) hydrogen peroxide (H2O2) production at the plasma membrane and crucial in host biological processes. In this study, SpDUOX1 and SpDUOX2 of mud crab (Scylla paramamosain) were identified and studied. Both SpDUOX1 and SpDUOX2 are transmembrane proteins, including an N-signal peptide region and a peroxidase homology domain in the extracellular region, transmembrane regions, and three EF (calcium-binding region) domains, a FAD-binding domain, and a NAD binding domain in the intracellular region. The SpDUOXs were expressed in all tissues examined, but mainly in hepatopancreas, heart, and mid-intestine. The expression of the SpDUOXs in the hemolymph of mud crabs was up-regulated after challenge with Vibrio parahemolyticus or LPS. RNA interference (RNAi) of the SpDUOXs resulted in reduced ROS production in hemolymph. The bacterial count increased in the hemolymph of mud crabs injected with SpDUOX1 or SpDUOX2-RNAi, while the bacterial clearance ability of hemolymph significantly reduced. At the phylum level, the phyla Bacteroidetes and Actinobacteria were significantly increased, while Proteobacteria were significantly reduced following SpDUOX2 knockdown. There was a significant increase in the relative abundance of the genera Marinomonas, Pseudoalteromonas, Shewanella, and Hydrogenoph in SpDUOX2 depleted mud crabs compared with the controls. Our current findings therefore indicated that SpDUOXs might play important roles in maintaining the homeostasis in the hemolymph microbiota of mud crab.

Dual oxidase 2 and pancreatic adenocarcinoma: IFN-γ-mediated dual oxidase 2 overexpression results in H2O2-induced, ERK-associated up-regulation of HIF-1α and VEGF-A.

Several NADPH oxidase family members, including dual oxidase 2 [DUOX2], are expressed in human tumors, particularly gastrointestinal cancers associated with long-standing chronic inflammation. We found previously that exposure of pancreatic ductal adenocarcinoma cells to the pro-inflammatory cytokine IFN-γ increased DUOX2 expression (but not other NADPH oxidases) leading to long-lived H2O2 production. To elucidate the pathophysiology of DUOX2-mediated H2O2 formation in the pancreas further, we demonstrate here that IFN-γ-treated BxPC-3 and CFPAC-1 pancreatic cancer cells (known to increase DUOX2 expression) produce significant levels of intracellular oxidants and extracellular H2O2 which correlate with concomitant up-regulation of VEGF-A and HIF-1α transcription. These changes are not observed in the PANC-1 line that does not increase DUOX2 expression following IFN-γ treatment. DUOX2 knockdown with short interfering RNA significantly decreased IFN-γ-induced VEGF-A or HIF-1α up-regulation, as did treatment of pancreatic cancer cells with the NADPH oxidase inhibitor diphenylene iodonium, the multifunctional reduced thiol N-acetylcysteine, and the polyethylene glycol-modified form of the hydrogen peroxide detoxifying enzyme catalase. Increased DUOX2-related VEGF-A expression appears to result from reactive oxygen-mediated activation of ERK signaling that is responsible for AP-1-related transcriptional effects on the VEGF-A promoter. To clarify the relevance of these observations in vivo, we demonstrate that many human pre-malignant pancreatic intraepithelial neoplasms and frank pancreatic cancers express substantial levels of DUOX protein compared to histologically normal pancreatic tissues, and that expression of both DUOX2 and VEGF-A mRNAs is significantly increased in surgically-resected pancreatic cancers compared to the adjacent normal pancreas.