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Quinone Oxidoreductase-1 in the Tight Junctions of Colonic Epithelial Cells

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Quinone Oxidoreductase-1 in the Tight Junctions of Colonic Epithelial Cells BMB Rep. 2014; 47(9): 494-499 BMB www.bmbreports.org Reports Role of NADH: quinone oxidoreductase-1 in the tight junctions of colonic epithelial cells Seung Taek Nam1,#, Jung Hwan Hwang2,#, Dae Hong Kim1, Mi Jung Park1, Ik Hwan Lee1, Hyo Jung Nam1, Jin Ku Kang1, Sung Kuk Kim1, Jae Sam Hwang3, Hyo Kyun Chung4, Minho Shong4, Chul-Ho Lee2,* & Ho Kim1,* 1Department of Life Science, College of Natural Science, Daejin University, Pocheon 487-711, 2Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, 3Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon 441-707, 4Department of Internal Medicine, Chungnam National University, Daejon 301-721, Korea NADH:quinone oxidoreductase 1 (NQO1) is known to be lating the intracellular ratio of NAD and NADH (two funda- involved in the regulation of energy synthesis and metabolism, mental mediators of energy metabolism) in various cell and the functional studies of NQO1 have largely focused on systems. NQO1 is also known as an antioxidant flavoprotein metabolic disorders. Here, we show for the first time that that scavenges reactive oxygen species (ROS) (3). Having pre- compared to NQO1-WT mice, NQO1-KO mice exhibited a viously shown that NQO1 activity is associated with cancer (4) marked increase of permeability and spontaneous inflammation and metabolic disorders, including diabetes and obesity (5), in the gut. In the DSS-induced colitis model, NQO1-KO mice we herein focused on the possible role of NQO1 in the gastro- showed more severe inflammatory responses than NQO1-WT intestinal tract. We report for the first time that the expression mice. Interestingly, the transcript levels of claudin and levels of NQO1 are much higher in the intestine and colon occludin, the major tight junction molecules of gut epithelial than in other tissues. NQO1-KO (knockout) mice exhibited cells, were significantly decreased in NQO1-KO mice. The spontaneous gut inflammation, and in the DSS-induced colitis colons of NQO1-KO mice also showed high levels of reactive model, NQO1-KO mice showed more severe inflammatory oxygen species (ROS) and histone deacetylase (HDAC) activity, symptoms than NQO1-WT (wild-type) mice. which are known to affect transcriptional regulation. Taken Our results suggested that NQO1 could affect integrity of together, these novel findings indicate that NQO1 contributes tight junction. Epithelial cells in the intestine and colon are to the barrier function of gut epithelial cells by regulating the known to form intercellular junctional complexes that act as a transcription of tight junction molecules. [BMB Reports 2014; barrier, separating the lumenal contents from the human body 47(9): 494-499] (6, 7) and limiting the paracellular movement of various lume- nal agents. Defects in this epithelial barrier have been pro- posed as a first step in a number of inflammatory bowel dis- INTRODUCTION eases (8). However, the molecular mechanisms responsible for regulating tight junction molecules are poorly understood. NADH:quinone oxidoreductase 1 (NQO1) catalyzes the re- Here, we report for the first time that a macromolecular per- duction of quinone metabolites by using nicotinamide adenine meability study revealed that the tight junctions of epithelial dinucleotide (NADH) as an electron donor (1, 2), thereby regu- cells were severely defective in NQO1-KO mice. This was due to the transcriptional downregulation of the tight junction pro- *Corresponding authors. Ho Kim, Tel: +82-31-539-1855; Fax: teins, claudin and occludin, and resulted in gut inflammation. +82-31-539-1850; E-mail: [email protected], Chul-Ho Lee, Tel: Our results collectively suggest that NQO1 may be a key mol- +82-42-860-4637; Fax: +82-42-860-4609; E-mail: chullee@kribb. ecule responsible for regulating the epithelial cell tight junc- re.kr tions that form the physical barrier of the gut. #Nam, S. T., Hwang, J. H. contributed equally to this work as a first author. RESULTS AND DISCUSSION http://dx.doi.org/10.5483/BMBRep.2014.47.9.196 NQO1 deficiency markedly increases gut permeability and Received 28 August 2013, Revised 4 October 2013, inflammation Accepted 19 December 2013 In the present study, we observed that the expression levels of NQO1 in the intestine and colon were relatively higher than Keywords: Barrier dysfunction of epithelial cells, Chromosome those in other organs, including brain, spleen, liver and lung in condensation, Claudin-1, Gut epithelial cell tight junction, Gut NQO1-WT mice (Fig. 1A). Marked expression of NQO1 was inflammation, Histone acetylation/deacetylation, NQO1 knockout mice, Occludin, Transcription also detected in the kidneys of NQO1-WT mice. This is con- ISSN: 1976-670X (electronic edition) Copyright ⓒ 2014 by the The Korean Society for Biochemistry and Molecular Biology This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/li- censes/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Regulation of colonic epithelial cell tight junctions by NQO1 Seung Taek Nam, et al. sistent with the notion that NQO1 plays a critical role in the the lack of NQO1 affected epithelial cell tight junctions. We kidney, as suggested by the observation that pharmacological used a gut macromolecular permeability test to measure epi- activation of NQO1 by β-lapachone can attenuate kidney in- thelial cell tight junctions (12). As shown in Fig. 1C (left pan- jury (9). RT-PCR experiments confirmed that the transcript lev- el), we observed significantly higher gut permeability in els of NQO1 in the intestine and colon were significantly high- NQO1-KO mice compared to NQO1-WT mice (∼3-fold). er compared to those in the other tested organs (Fig. 1B). These Unlike other proteins, Cox-2 and tubulin, NQO1 protein was results suggest that NQO1 may play a critical role in the gut. not detected in NQO1-KO mice, proving specific NQO1 gene Since one of the essential roles of gut epithelial cells is to knockout (Fig. 1C, right panel). The concentrations of the form a physical barrier against lumenal contents, such as bac- proinflammatory cytokine, TNF-α, in the intestine (left panel) teria and orally ingested foods (10, 11), we assessed whether and colon (right panel) were significantly higher in NQO1-KO mice than in NQO1-WT (Fig. 1D), suggesting the presence of increased inflammation in NQO1-KO mice. These results sug- gest that NQO1 deficiency causes a marked increase in the macromolecular permeability of the gut, and that this may be due to mild inflammation of the intestine and colon. NQO1 deficiency causes transcriptional down-regulation of claudin and occludin Given that NQO1-KO mice showed increased gut perme- Fig. 1. NQO1 is abundant in gut epithelial cells, and NQO1 knockdown causes increased epithelial permeability in the mouse gut. (A) The organ distribution of NQO1 was elucidated by im- munoblot analysis with tissue extracts of NQO1-WT mice. (B) The organ distribution of NQO1 was elucidated by RT-PCR with NQO1-specific primers. (C) Left panel: Ileal loops of NQO1-WT Fig. 2. Decreased expression of the tight junction molecules, oc- and NQO1-KO mice were lumenally injected with fluorescein-la- cludin and claudin, in the colons of NQO1-KO mice. (A) Colonic beled dextran, and blood fluorescence was determined. *P ＜ tissue extracts were obtained from NQO1-WT and NQO1-KO 0.05 vs. NQO1-WT mice (n=12 per group). Right panel: Colon mice respectively (n=8 mice per group). The presented results are tissues from near the cecum were isolated from NQO1-WT and representative of three independent experiments. (B) The mRNA NQO1-KO mice, total protein extracts were resolved on poly- expressions levels of claudin-1, claudin-6 and occludin were eval- acrylamide gels, and the blotted membrane was probed with anti- uated by real-time reverse transcription-PCR (RT-PCR). (C) Light bodies against NQO1, Cox2, tubulin, and β-actin. (D) The con- micrographs of mouse colon samples subjected to immunohisto- centrations of TNF-α were measured in the intestine (left panel) chemistry for occludin (IHC stating; original magnification, ×200). and colon (right panel). The bars represent the mean ± SEM of Data are representative of three independent samples (arrows in- three independent experiments (*P ＜ 0.005 vs. NQO1-WT mice). dicate occludin proteins in the apical area of colonocytes). http://bmbreports.org BMB Reports 495 Regulation of colonic epithelial cell tight junctions by NQO1 Seung Taek Nam, et al. ability, we next assessed whether NQO1 deficiency affects the those of NQO1-WT mice (Fig. 2B). These results suggest that expression levels of tight junction molecules, such as claudin colonic NQO1 may be associated with the transcriptional reg- and occludin. Protein extracts were isolated from the colons of ulation of tight junction molecules, thereby affecting epithelial NQO1-WT and NQO1-KO mice, and the expression levels of barrier function. Immunohistochemistry confirmed that the claudin and occludin were determined by immunoblot protein levels of occludin were lower in the colonic epithelial analysis. As shown in Fig. 2A, the expression levels of clau- cells of NQO1-KO mice versus NQO1-WT mice (Fig. 2C). din-1 and occludin in the colons of NQO1-KO mice were sig- nificantly lower than those in NQO1-WT colons. In contrast, NQO1 deficiency increases various symptoms of inflammation the expression of E-cadherin, a major protein component of in the DSS-induced colitis model desmosomal junctions for cell-cell adhesion (12), did not differ Next, we evaluated the potential pathophysiological role of between NQO-WT and NQO1-KO mice. RT-PCR experiments NQO1 in compromised colons. To induce colitis in mice, showed that the colons of NQO1-KO mice had reduced tran- NQO1-WT and NQO1-KO mice were treated with DSS (3%) script levels of claudin-1, claudin-6 and occludin compared to in their drinking water, and several clinical parameters of colitis (e.g., mortality and weight loss) were measured daily for the in- dicated durations.
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