Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation

Yasuda‑Onozawa,Yuriko; Handa,Osamu; Naito,Yuji; Ushiroda,Chihiro; Suyama,Yosuke; Toyokawa,Yuki; Murakami,Takaaki; Yasuda,Tomoyo; Ueda,Tomohiro; Majima,Atsushi; Hotta,Yuma; Doi,Toshifumi; Tanaka,Makoto; Horii,Yusuke; Higashimura,Yasuki; Mizushima,Katsura; Morita,Mayuko; Uehara,Yukiko; Horie,Hideki; Fukui,Akifumi; Dohi,Osamu; Okayama,Tetsuya; Yoshida,Naohisa; Kamada,Kazuhiro; Katada,Kazuhiro; Uchiyama,Kazuhiko; Ishikawa,Takeshi; Takagi,Tomohisa; Konishi,Hideyuki; Itoh,Yoshito

doi:10.3892/mmr.2017.7647

Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation

Authors:
- Yuriko Yasuda‑Onozawa
- Osamu Handa
- Yuji Naito
- Chihiro Ushiroda
- Yosuke Suyama
- Yuki Toyokawa
- Takaaki Murakami
- Tomoyo Yasuda
- Tomohiro Ueda
- Atsushi Majima
- Yuma Hotta
- Toshifumi Doi
- Makoto Tanaka
- Yusuke Horii
- Yasuki Higashimura
- Katsura Mizushima
- Mayuko Morita
- Yukiko Uehara
- Hideki Horie
- Akifumi Fukui
- Osamu Dohi
- Tetsuya Okayama
- Naohisa Yoshida
- Kazuhiro Kamada
- Kazuhiro Katada
- Kazuhiko Uchiyama
- Takeshi Ishikawa
- Tomohisa Takagi
- Hideyuki Konishi
- Yoshito Itoh
View Affiliations

Affiliations: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
Published online on: September 28, 2017 https://doi.org/10.3892/mmr.2017.7647
Pages: 8216-8222

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Mucin is produced and secreted by epithelial goblet cells and is a key component of the innate immune system, acting as a barrier in the intestinal tract. However, no studies have been conducted investigating the increase in mucin secretion to enhance the intestinal barrier function. The present study investigated whether rebamipide (Reb) acts as a secretagogue of intestinal mucin and the underlying mechanisms involved, thereby focusing on the effect on goblet cells. The LS174T cell line was used as goblet cell‑like cells. Using Reb‑treated LS174T cells, the level of mucin content was assessed by periodic acid‑Schiff (PAS) staining, and mucin 2, oligomeric mucus/gel‑forming (MUC2) mRNA expression was assessed using quantitative polymerase chain reaction (PCR). Furthermore, MUC2 secretion in the supernatant was quantified by the dot blot method. The present study additionally investigated the involvement of the epidermal growth factor receptor/Akt serine/threonine kinase 1 (Akt) pathway in mucin secretion by western blotting. The results suggested that Reb strongly enhanced the positivity of PAS staining in LS174T cells, thereby suggesting increased intracellular mucin production. The PCR results indicated that Reb significantly increased MUC2 mRNA in whole cell lysate of LS174T cells. In order to assess the subsequent secretion of mucin by LS174T, MUC2 protein expression in the supernatant was assessed using the dot blot method and it was demonstrated that Reb significantly increased the secretion of MUC2 in a concentration‑dependent manner. The p‑Akt was significantly increased by Reb treatment, and an Akt inhibitor specifically suppressed MUC2 secretion. Overall, Reb increased mucin secretion directly via p‑Akt. Reb‑increased mucin may act as a strong non‑specific barrier against pathogenic stimulants in various intestinal diseases.

View Figures

View References

1	Wang L, Cao H, Liu L, Wang B, Walker WA, Acra SA and Yan F: Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein. J Biol Chem. 289:20234–20244. 2014. View Article : Google Scholar : PubMed/NCBI
2	McElroy SJ, Prince LS, Weitkamp JH, Reese J, Slaughter JC and Polk DB: Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: A potential role in neonatal necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol. 301:G656–G666. 2011. View Article : Google Scholar : PubMed/NCBI
3	Iwashita J, Sato Y, Sugaya H, Takahashi N, Sasaki H and Abe T: mRNA of MUC2 is stimulated by IL-4, IL-13 or TNF-alpha through a mitogen-activated protein kinase pathway in human colon cancer cells. Immunol Cell Biol. 81:275–282. 2003. View Article : Google Scholar : PubMed/NCBI
4	Corfield AP: Mucins: A biologically relevant glycan barrier in mucosal protection. Biochim Biophys Acta. 1850:236–252. 2015. View Article : Google Scholar : PubMed/NCBI
5	Deplancke B and Gaskins HR: Microbial modulation of innate defense: Goblet cells and the intestinal mucus layer. Am J Clin Nutr. 73:1131S–1141S. 2001.PubMed/NCBI
6	Elamin E, Masclee A, Troost F, Dekker J and Jonkers D: Cytotoxicity and metabolic stress induced by acetaldehyde in human intestinal LS174T goblet-like cells. Am J Physiol Gastrointest Liver Physiol. 307:G286–G294. 2014. View Article : Google Scholar : PubMed/NCBI
7	Satoh H, Amagase K and Takeuchi K: Mucosal protective agents prevent exacerbation of NSAID-induced small intestinal lesions caused by antisecretory drugs in rats. J Pharmacol Exp Ther. 348:227–235. 2014. View Article : Google Scholar : PubMed/NCBI
8	Takeuchi T, Ensrud ER and Steggerda FR: The effects of large doses of aspirin and cortisone on the goblet cells and the mucosal membranes in the small and large intestine. Am J Dig Dis. 17:49–53. 1972. View Article : Google Scholar : PubMed/NCBI
9	Kim YS and Ho SB: Intestinal goblet cells and mucins in health and disease: Recent insights and progress. Curr Gastroenterol Rep. 12:319–330. 2010. View Article : Google Scholar : PubMed/NCBI
10	Jacobs LR and Huber PW: Regional distribution and alterations of lectin binding to colorectal mucin in mucosal biopsies from controls and subjects with inflammatory bowel diseases. J Clin Invest. 75:112–118. 1985. View Article : Google Scholar : PubMed/NCBI
11	Theodossi A, Spiegelhalter DJ, Jass J, Firth J, Dixon M, Leader M, Levison DA, Lindley R, Filipe I, Price A, et al: Observer variation and discriminatory value of biopsy features in inflammatory bowel disease. Gut. 35:961–968. 1994. View Article : Google Scholar : PubMed/NCBI
12	Naito Y and Yoshikawa T: Rebamipide: A gastrointestinal protective drug with pleiotropic activities. Expert Rev Gastroenterol Hepatol. 4:261–270. 2010. View Article : Google Scholar : PubMed/NCBI
13	Kim JH, Park SH, Cho CS, Lee ST, Yoo WH, Kim SK, Kang YM, Rew JS, Park YW, Lee SK, et al: Preventive efficacy and safety of rebamipide in nonsteroidal anti-inflammatory drug-induced mucosal toxicity. Gut Liver. 8:371–379. 2014. View Article : Google Scholar : PubMed/NCBI
14	Ríos JD, Shatos MA, Urashima H and Dartt DA: Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells. Exp Eye Res. 86:629–636. 2008. View Article : Google Scholar : PubMed/NCBI
15	Iijima K, Ichikawa T, Okada S, Ogawa M, Koike T, Ohara S and Shimosegawa T: Rebamipide, a cytoprotective drug, increases gastric mucus secretion in human: Evaluations with endoscopic gastrin test. Dig Dis Sci. 54:1500–1507. 2009. View Article : Google Scholar : PubMed/NCBI
16	Mizukami K, Murakami K, Abe T, Inoue K, Uchida M, Okimoto T, Kodama M and Fujioka T: Aspirin-induced small bowel injuries and the preventive effect of rebamipide. World J Gastroenterol. 17:5117–5122. 2011. View Article : Google Scholar : PubMed/NCBI
17	Watanabe T, Takeuchi T, Handa O, Sakata Y, Tanigawa T, Shiba M, Naito Y, Higuchi K, Fujimoto K, Yoshikawa T and Arakawa T: A multicenter, randomized, double-blind, placebo-controlled trial of high-dose rebamipide treatment for low-dose aspirin-induced moderate-to-severe small intestinal damage. PLoS One. 10:e01223302015. View Article : Google Scholar : PubMed/NCBI
18	Lai Y, Zhong W, Yu T, Xia ZS, Li JY, Ouyang H, Shan TD, Yang HS and Chen QK: Rebamipide promotes the regeneration of aspirin-induced small-intestine mucosal injury through accumulation of β-catenin. PLoS One. 10:e01320312015. View Article : Google Scholar : PubMed/NCBI
19	van Klinken BJ, Oussoren E, Weenink JJ, Strous GJ, Büller HA, Dekker J and Einerhand AW: The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression. Glycoconj J. 13:757–768. 1996. View Article : Google Scholar : PubMed/NCBI
20	Akamatsu T, Nagaya T, Ichikawa S, Sudo T, Takeda R, Takenaka K, Kodama R, Ito T, Arakura N and Tanaka E: Small bowel tissue concentration of rebamipide: Study of two dosages in healthy subjects. J Clin Biochem Nutr. 47:256–260. 2010. View Article : Google Scholar : PubMed/NCBI
21	Ishihara K, Komuro Y, Nishiyama N, Yamasaki K and Hotta K: Effect of rebamipide on mucus secretion by endogenous prostaglandin-independent mechanism in rat gastric mucosa. Arzneimittelforschung. 42:1462–1466. 1992.PubMed/NCBI
22	Urashima H, Okamoto T, Takeji Y, Shinohara H and Fujisawa S: Rebamipide increases the amount of mucin-like substances on the conjunctiva and cornea in the N-acetylcysteine-treated in vivo model. Cornea. 23:613–619. 2004. View Article : Google Scholar : PubMed/NCBI
23	Itoh S, Itoh K and Shinohara H: Regulation of human corneal epithelial mucins by rebamipide. Curr Eye Res. 39:133–141. 2014. View Article : Google Scholar : PubMed/NCBI
24	Zhang X, Nagahara H, Mimori K, Inoue H, Sawada T, Ohira M, Hirakawa K and Mori M: Mutations of epidermal growth factor receptor in colon cancer indicate susceptibility or resistance to gefitinib. Oncol Rep. 19:1541–1544. 2008.PubMed/NCBI
25	Liu Z, Tabakman S, Sherlock S, Li X, Chen Z, Jiang K, Fan S and Dai H: Multiplexed five-color molecular imaging of cancer cells and tumor tissues with carbon nanotube raman tags in the near-infrared. Nano Res. 3:222–233. 2010. View Article : Google Scholar : PubMed/NCBI
26	Fukui A, Naito Y, Handa O, Kugai M, Tsuji T, Yoriki H, Qin Y, Adachi S, Higashimura Y, Mizushima K, et al: Acetyl salicylic acid induces damage to intestinal epithelial cells by oxidation-related modifications of ZO-1. Am J Physiol Gastrointest Liver Physiol. 303:G927–G936. 2012. View Article : Google Scholar : PubMed/NCBI
27	Yamasaki K, Kanbe T, Chijiwa T, Ishiyama H and Morita S: Gastric mucosal protection by OPC-12759, a novel antiulcer compound, in the rat. Eur J Pharmacol. 142:23–29. 1987. View Article : Google Scholar : PubMed/NCBI
28	Kleine A, Kluge S and Peskar BM: Stimulation of prostaglandin biosynthesis mediates gastroprotective effect of rebamipide in rats. Dig Dis Sci. 38:1441–1449. 1993. View Article : Google Scholar : PubMed/NCBI
29	Cassidy MM and Lightfoot FG: Effects of prostaglandin E1, administered by gastric intubation, on mucus secretory patterns in rat small intestine. Adv Prostaglandin Thromboxane Res. 8:1589–1593. 1980.PubMed/NCBI