Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase

Lianxin Li; Jinge Xin; Hesong Wang; Yadong Wang; Weiqi Peng; Ning Sun; Haonan Huang; Yanxi Zhou; Xingmei Liu; Yu Lin; Jing Fang; Bo Jing; Kangcheng Pan; Yan Zeng; Dong Zeng; Xiang Qin; Yang Bai; Xueqin Ni

Ecotoxicology and Environmental Safety (Jun 2023)

Fluoride disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated RhoA/ROCK signaling and myosin light chain kinase

Lianxin Li,
Jinge Xin,
Hesong Wang,
Yadong Wang,
Weiqi Peng,
Ning Sun,
Haonan Huang,
Yanxi Zhou,
Xingmei Liu,
Yu Lin,
Jing Fang,
Bo Jing,
Kangcheng Pan,
Yan Zeng,
Dong Zeng,
Xiang Qin,
Yang Bai,
Xueqin Ni

Affiliations

Lianxin Li: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Jinge Xin: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Hesong Wang: Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China
Yadong Wang: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
Weiqi Peng: Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China
Ning Sun: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Haonan Huang: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Yanxi Zhou: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Xingmei Liu: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Yu Lin: Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China
Jing Fang: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Bo Jing: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Kangcheng Pan: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Yan Zeng: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Dong Zeng: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China
Xiang Qin: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, China; Corresponding authors.
Yang Bai: Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China; Guangdong Provincial Key Laboratory of Gastroenterology, Department of Gastroenterology, Institute of Gastroenterology of Guangdong Province, Nanfang Hospital, Southern Medical University, Guangzhou, China; Corresponding author at: Baiyun Branch, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Xueqin Ni: Animal Microecology Institute, College of Veterinary, Sichuan Agricultural University, Chengdu, Sichuan, China; Corresponding authors.

Journal volume & issue: Vol. 257
p. 114940

Abstract

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Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Published in Ecotoxicology and Environmental Safety

ISSN: 0147-6513 (Print); 1090-2414 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Technology: Environmental technology. Sanitary engineering: Environmental pollution; Geography. Anthropology. Recreation: Environmental sciences
Website: https://www.journals.elsevier.com/ecotoxicology-and-environmental-safety

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