Inhibition of CREB-Mediated ZO-1 and Activation of NF-κB-Induced IL-6 by Colonic Epithelial MCT4 Destroys Intestinal Barrier Function

Abstract

Background: Inflammatory bowel disease(IBD) is characterized by intestinal inflammation and impaired barrier function, associated with increased epithelial expression of monocarboxylate Transporter 4 (MCT4). Methods: we performed qPCR, IF, WB and utilized luciferase, and ChIP to analyze MCT4 on NF-κB and CREB activity and DNA binding in IECs. IP was used to analyze the effect of MCT4 on the interaction NF-κB-CBP or CREBCBP. In vivo assay were detected by IF, WB and to analyze the role and mechanism of MCT4 in IBD. Findings: Functional studies further showed that ectopic expression of MCT4 disrupted tight junction protein 1(ZO-1) expression and increased pro-inflammatory factors expression, leading to destroy intestinal epithelial barrier function in vitro and in vivo. Mechanistically, one hand, MCT4 led to nuclear translocation of p65 and increased the binding of NF-κB p65 to the promoter of IL-6, on the other hand, MCT4 suppressed cAMP-response element binding protein (CREB) activity and reduced CREBmediated ZO-1 expression. these effect were attributed to MCT4 contributed to NF-κB-CBP interaction, while dissolved CREB-CBP complex. Treatment of experimental colitis with MCT4 inhibitor by α-cyano-4- hydroxycinnamate (CHC) significantly ameliorated mucosal intestinal barrier function in vivo, which was attributed to attenuation of proinflammation factors expression and enhancement of ZO-1 expression. Interpretation: These findings suggested a novel MCT4 role in controlling development of IBD, which could serve as a new therapeutic target of IBD. Funding Statement: This work was supported by National Natural Science Foundation of China (No.81770552, No.81860101), Natural Science Foundation of Guangdong (No.2017A030313838, No.2018A0303130175), Medical Science and Technology Foundation of Guangdong (No.A2018395), Science and Technology plan program of Guangzhou (No.201804010148), General program of Guangzhou Health and plan commission (No.805150202068), Postdoctoral Research Funding of Guangzhou Women and Children’s Medical Center (NO.5001-3001075), Funding of Guangzhou Institute of Pediatrics/Guangzhou Women and Children’s Medical Center (NO.IP-2016-005, NO.IP2018-009). Declaration of Interests: The authors declared that they have no competing interests. Ethics Approval Statement: Human study: Based on the declaration of Helsinki as reflected in a prior approval by the institution’s human research committee, this study was conducted in a cohort of 54 patients with inflammatory bowel disease (IBD) and 16 healthy control in Guangzhou Women and Children’s Medical Center from 2016 to 2018 approved by the Medical Ethical Review Board(2017030602). Animal study: All animal experiments were approved by the Guangzhou Women and Children’s Medical Center animal care and use committee.