Special Issue Secreted Mucins on the Ocular Surface

Yuichi Hori Department of Ophthalmology, Toho University Graduate School of Medicine, Tokyo, Japan

Correspondence: Yuichi Hori, De- Mucins, which play important roles on the ocular surface in wettability, lubrication, and partment of Ophthalmology, Toho barrier function, are classified into two categories: secreted mucins and membrane-associated University Graduate School of Medi- mucins. The most important secreted mucin on the ocular surface is MUC5AC, which is cine, 6-11-1, Omori-Nishi, Ota-ku, secreted by the conjunctival goblet cells. In the human conjunctiva, goblet cells are present in Tokyo 143-8541, Japan; [email protected]. higher concentrations in the fornix, inferior nasal bulbar, and the lid wiper on the lid margin. The number of conjunctival goblet cells and MUC5AC expression/secretion are decreased in a Submitted: December 12, 2017 patient with dry eye. In Japan, drugs that stimulate mucin secretion or increase the number of Accepted: February 14, 2018 conjunctival goblet cells are commercially available. A P2Y2 receptor, diquafosol, stimulates Citation: Hori Y. Secreted mucins on tear fluid secretion from conjunctival epithelial cells and promotes mucin secretion from the ocular surface. Invest Ophthalmol conjunctival goblet cells. Rebamipide was marketed originally as an oral therapeutic drug to Vis Sci. 2018;59:DES151–DES156. treat gastritis in Japan. Topical rebamipide increases numbers of goblet cells in the bulbar https://doi.org/10.1167/iovs.17-23623 conjunctiva and the lid wiper area of palpebral conjunctiva. Many researchers have reported decreases in the ocular surface mucin expression including MUC5AC secreted by goblet cells in patients with dry eye. However, it is unknown whether changes in mucin expression on the ocular surface cause or result from dry eye. Further study is needed to determine the true mechanism of dry eye disease. Keywords: MUC5AC, goblet cell, Osaka study, diquafosol, rebamipide

ucins are heavy molecular glycoproteins formed by The conjunctival goblet cells that secrete MUC5AC are M numerous sugar chains linked to a core protein called scattered on the conjunctival epithelium (Figs. 1A, 1B). Eyelid apomucin, with 50% to 80% of their mass comprised of movement during blinking evenly spreads MUC5AC secreted carbohydrate.1 In mucins, N-acetylgalactosamine is linked to from the goblet cells into the lacrimal fluid over the entire serine or threonine, which are two of the amino acids of the ocular surface to maintain its wettability and lubrication (Fig. 2,20 protein core, in what is called an O-type glycosylation. The 2). The distribution of goblet cells varies depending on the mucin structure is characterized by the area of the mucin anatomic location. In the human conjunctiva, goblet cells are protein core called the tandem repeat (repeated sequence present in higher concentrations in the fornix, and in particular, domain), which is rich in amino acids such as serine and in the inferior nasal fornix near the exit of the tear drainage 11,20,21 threonine that link to the sugar chains via O-glycosylation.1,2 system. Knop et al. recently reported that goblet cells are Mucin can be classified broadly by its structural character- abundant in the lid wiper portion of the lid margins to facilitate lubrication of the ocular surface during blinking.22,23 Kase et istics as secreted mucins and membrane-associated mucins. The 24 former can be classified further into gel-forming mucins and al. also found significantly more goblet cells in the lid wiper soluble mucins. Gel-forming mucins are, in turn, classified than the palpebral conjunctiva in a study of 30 eyelid tissues surgically removed due to involutional entropion. Goblet cell further into the following subtypes: MUC2, MUC5AC, MUC5B, expression likely increases in the area subjected to more MUC6, and MUC19,3–8 and soluble mucins are classified into friction from blinking between the eyelid and cornea and subtypes MUC7 and MUC9.9,10 between the eyelid and bulbar conjunctiva. As such, goblet Mucin has many important roles on the ocular surface (i.e., cells are abundant on the ocular surface, and the MUC5AC maintenance of lacrimal fluid, lubrication of the ocular surface secreted from them contribute to maintaining the homeostasis to facilitate smooth blinking, formation of a smooth spherical of the ocular surface. surface for good vision, provision of a barrier for the ocular 2,11 The conjunctiva is innerved by sympathetic and parasym- surface, and trapping and removing pathogens and debris). pathetic nerves, the nerve endings of which surround the At least four subtypes of secreted mucins (MUC5AC, MUC7, 25 12–15 goblet cells at the level of the secretory granules. Goblet cells MUC2, and MUC19) and four types of the membrane- have muscarinic receptors of M3 and M2 subtypes for 16–19 associated mucins (MUC1, MUC4, MUC16, and MUC20) neurotransmitters released from parasympathetic nerves in- are expressed on the ocular surface in the mRNA and/or cluding acetylcholine (Ach) and vasoactive intestinal peptide protein levels. Of the secreted mucins, gel-forming mucin (VIP).25,26 The parasympathetic nerves release Ach and VIP, MUC5AC is believed to be expressed most prevalently on the which stimulate goblet cell secretion through M3 and M2 ocular surface and is secreted from the conjunctival goblet cells muscarinic receptors.25–27 into the tear fluid. MUC7, a soluble mucin with a small Goblet cells are associated with epidermal growth factor molecular mass, is expressed in the acinar cells of the lacrimal receptor (EGFR). Several studies have reported that the EGFR- glands.13 MUC2 and MUC19 are thought to have lower signaling pathway serves an important role in goblet cell expression levels on the ocular surface.14,15 proliferation and mucin secretion in rat conjunctiva.27–29

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FIGURE 1. Impression cytology of human conjunctiva. After topical anesthesia is applied, cellulose acetate filters (4 3 6 mm) are pressed onto the bulbar conjunctiva (A). PAS staining of the impression cytology sample (bulbar conjunctiva) (B). Positive cells represent goblet cells.

Kanno et al.27 reported that carbachol, a cholinergic agonist, transactivated the EGFR to activate mitogen-activated protein FIGURE 2. MUC5AC expression on the human conjunctiva. A cross section of human conjunctiva is labeled with fluorescein-labeled kinase (MAPK), leading to conjunctival goblet cell secretion. MUC5AC (antibody 781 provided by Ilene Gipson) to demonstrate This indicates that the mucin secretion from conjunctival mucin secreted from goblet cells. goblet cells is under neural control. Secreted mucin is thought to contribute to tear fluid on the ocular surface increased IFN-c expression, which stabilization because of its ability to promote water retention consequently reduced the goblet cell count and MUC5AC on the ocular surface for long periods by binding to and gelling 37 2,11 expression. with a large number of water molecules. In addition, Changes in MUC5AC expression also have been reported in secreted mucin might suppress epithelial damage resulting allergic eye diseases. Kunert et al. reported a significant from friction when the eyelid comes into contact with the decrease in the conjunctival goblet cell count and Muc5AC epithelium during blinking, because it acts as a lubricating expression in the conjunctiva in mouse allergic conjunctivitis agent to reduce ocular surface friction. Furthermore, secreted models.38 Dogru et al.39 reported that the MUC16 and MUC5AC mucin is also thought to trap and remove debris and harmful 2,11 expression levels decreased significantly in the conjunctival pathogens as a barrier function. However, the inherent epithelial cells in patients with atopic keratoconjunctivitis. function of secreted mucin MUC5AC on the ocular surface is As described previously, conjunctival goblet cells and mucin unknown, because of the difficulty of using animal model expression decrease in ocular surface diseases, particularly in testing to demonstrate secreted mucin. Only a few studies of 30,31 dry eye, which emphasizes the need to develop drugs that the ocular surface have used Muc5ac-knockout mice. increase mucin expression and secretion. Marko et al. reported that Muc5b expression increased as a compensatory mechanism in the Muc5ac-knockout mice, and no findings indicated signs of dry eye, such as inflammatory DRY EYE THERAPIES AND THEIR EFFECT ON MUCIN change, keratoconjunctival epithelial disorder, or decreased lacrimal fluid in the Muc5ac-knockout mice.31 The SAM PRODUCTION pointed domain-containing ETS transcription factor (SPDEF) To date, several drugs or agents have been reported to induce promotes differentiation of conjunctival goblet cells.32 Marko mucin expression or secretion by the ocular surface epithelia. et al. conducted an experiment with SPDEF-knockout mice and These are good treatments for dry eye, especially for the mucin reported that, although those mice lacked conjunctival goblet deficiency type of dry eye. cells, lacrimal secretion increased in a compensatory manner, The 2007 International Dry Eye Workshop report, ‘‘Tear thereby preventing dry eye.32 Further studies should elucidate stimulation: Secretagogues,’’ which stimulate aqueous or the functions of MUC5AC on the ocular surface. mucous secretion or both, described the drugs as future potential topical pharmacologic agents for treating dry eye, including diquafosol, rebamipide, gefarnate, ecabet sodium HANGES IN UMBERS OF OBLET ELLS AND C N G C (mucous secretion stimulants), and 15(S)-HETE (an MUC1 MUC5AC EXPRESSION IN OCULAR SURFACE DISEASE stimulant).40 In 2017, the Tear Film & Ocular Surface Society launched the Dry Eye Workshop II, which reported several Several studies have reported decreased MUC5AC secretion by topical pharmacologic agents that stimulate aqueous, mucin, goblet cells in patients with dry eye.33,34 Argueso et al.33 and/or lipid secretion as secretagogues, including diquafosol, reported that MUC5AC expression was significantly lower in lacritin, rebamipide, galectin-3, mycophenolate mofetil, trefoil the tear fluid and conjunctival epithelial cells in patients with 41 Sj¨ogrens syndrome compared with normal individuals. Shima- factors, and nerve growth factor. In Japan, drugs are zaki-Den et al.34 also reported that MUC5AC expression levels commercially available that stimulate mucin secretion or in the conjunctival epithelium were significantly lower in normalize the ocular surface mucosa to increase the number patients with aqueous deficiency–type dry eye and dry eye of conjunctival goblet cells and used as the first-choice with a short tear film breakup time. treatments for dry eye, including diquafosol and rebamipide. In a murine experiment, Muc5AC expression increased in response to interleukin 13, a Th2 cytokine, and decreased in response to IFN-c, a Th1 cytokine.35,36 Pflugfelder et al. further RECENT PROGRESS IN JAPAN REGARDING SECRETED compared patients with aqueous tear deficiency or meibomian MUCINS:SECRETED MUCIN STUDY IN EPIDEMIOLOGIC gland dysfunction and a control group and discovered that IFN- SURVEY IN JAPAN c, goblet cell density, and MUC5AC expression decreased significantly in the conjunctiva of patients with aqueous tear To investigate the prevalence of dry eye in patients who use deficiency.37 The authors concluded that decreased tear fluid visual display terminals (VDTs), a large epidemiologic survey

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was performed at a company in Osaka, Japan (Osaka Study, see gastric mucosal disorders and gastritis. To date, rebamipide for section on epidemiology).42 Among many published manu- gastritis have been approved in several countries in Asia, scripts associated with the Osaka Study, two papers have including Japan, South Korea, China, Russia, and India. The reported alterations in tear MUC5AC levels in office workers agent improves the quality of gastric ulcer healing and reduces who use VDTs.43,44 Uchino et al. reported that office workers the recurrence rate of ulcers.60 The major properties of with prolonged daily use of VDTs (>7 hours) had significantly rebamipide are the stimulation of prostaglandin and mucus low MUC5AC concentrations in their tears.43 They also glycoprotein synthesis and inhibition of reactive oxygen reported in the other manuscript that cigarette smoking in species, inflammatory cytokines, chemokines, and neutrophil office workers decreased goblet cell density and tear MUC5AC activation.60 concentrations significantly.44 Urashima et al. reported that rebamipide increased the number of conjunctival goblet cells in normal rabbits61 and the presence of mucin-like substance contents on the ocular NEW THERAPEUTIC APPROACH FOR TREATMENT OF surface in the N-acetylcysteine–treated rabbit model.62 Biolog- DRY EYE ically, goblet cells are associated with the EGFR signaling pathway, the activation of which leads to goblet cell metaplasia Regarding secretory mucins, other areas of interest and and mucin secretion from the cells.28 Rios et al.29 reported that progress in research in Japan are in the mucin production rebamipide activated the EGFR-signaling pathway and induced therapies (i.e., the secretagogues, which stimulate mucous proliferation of cultured rat conjunctival goblet cells. Ohguchi secretion) for treating dry eye.40 In Japan, two new secreta- et al.63 showed that rebamipide increased the expression of gogue eyedrops were launched in December 2010 and January muc5 mRNA on the ocular surface using superoxide dismutase- 2012, respectively, to treat dry eye: diquafosol and rebamipide. 1 knockout mice. Both drugs induce expression of MUC5AC secreted from In human conjunctiva, Kase et al.64 reported that topical conjunctival goblet cells. Several investigators have reported rebamipide administration for 3 months increased markedly the effects of these drugs in basic and clinical research. the number of goblet cells histologically in patients with Diquafosol (Diquas Ophthalmic Solution 3%; Santen Phar- conjunctival hyperemia and that topical rebamipide induced maceutical, Co., Ltd., Osaka, Japan) is a potent, purinergic the number of EGFR-positive cells and goblet cells in the lid 24 P2Y2 receptor agonist. Generally speaking, P2Y2 receptor wiper of human conjunctiva and bulbar conjunctiva. These agonists act on P2Y2 receptors in cellular membranes and data indicated that topical rebamipide might decrease the activate phospholipase C via G proteins to produce inositol friction during blinking by increasing the mucin levels on the 2þ triphosphate. Thus, calcium ion (Ca ) release is induced from ocular surface. the cell endoplasmic reticulum, which elevates intracellular In addition to increasing the number of conjunctival goblet 2þ Ca concentrations and induces various physiologic respons- cells and MUC5AC expression, rebamipide affects the human 45 es. P2Y2 receptors have been found at a number of ocular corneal epithelial cells. RT-PCR and Western blot analysis sites (i.e., the palpebral and bulbar conjunctival epithelium, showed that rebamipide increased membrane-associated mu- conjunctival goblet cells, corneal epithelium, and meibomian cins in mRNA and protein levels in the cultivated human 45,46 glands). Regarding the mechanism of action, after binding corneal epithelial cells.65,66 Uchino et al.66 reported that to P2Y2 receptors, diquafosol stimulates tear fluid secretion rebamipide increased MUC16 protein biosynthesis in the from conjunctival epithelial cells and promotes mucin secre- cultured human corneal epithelial, but not MUC1, MUC4, or 47 tion from conjunctival goblet cells. Diquafosol decreased the MUC20. Two Japanese groups reported that rebamipide periodic acid Schiff (PAS)-positive staining area in a dose- 48 increased transmembrane electric resistance and protected dependent manner immediately after instillation. When TNF-a–induced disruption of the barrier function in cultured diquafosol was applied at concentrations exceeding 0.1%, the human corneal cells.67,68 PAS-positive staining area was maximally decreased and 45 Generally speaking, the goblet cell density decreased reached nearly 40%. These results suggested that diquafosol significantly after ocular surgery and/or application of the stimulated mucin secretion from the conjunctival goblet cells. postoperative eyedrops, such as the topical nonsteroidal anti- A similar stimulatory effect on mucin secretion was observed inflammatory drug diclofenac. In a clinical study, Kato et al.69 in evaluations of the PAS-positive staining areas using 49 reported that rebamipide significantly increased the number of conjunctival histopathologic samples from normal rats. Hori conjunctival goblet cells 14 days after vitrectomy. They also et al. reported that the MUC5AC level in rabbit tears increased reported the results of a randomized clinical trial that 15 minutes after instillation of 3% diquafosol tetrasodium 50 compared the goblet cell density after cataract surgery with eyedrops. In addition to MUC5AC, Takaoka-Shichijo and diclofenac versus diclofenac and rebamipide.70 The reduction Nakamura reported that diquafosol increased the mRNA of the goblet cell density was significantly suppressed by the expression of membrane-associated mucins, MUC1, MUC4, concomitant use of topical rebamipide.70 and MUC16, after a 3-hour incubation with 100 lM diquafosol in cultured human corneal epithelial cells.51 To date, many studies have reported the effectiveness of 3% COMPARISON OF DIQUAFOSOL AND REBAMIPIDE diquafosol ophthalmic solution for treating many types of dry eye, including the aqueous deficient type,52,53 short tear film As secretagogues, both diquafosol and rebamipide are useful breakup time type,54–56 laser in situ keratomileusis–associated for treating dry eye in Japan (see section on TFOT and TFOD). dry eye,57,58 and obstructive meibomian gland dysfunction.59 To clarify the difference between diquafosol and rebamipide, Nowadays, this product has been approved in several Asian we compared the short-term changes in tear volume after countries, including Japan, South Korea, Thailand, and instilling these eyedrops in normal rabbits.71 The tear meniscus Vietnam. area increased significantly up to 30 minutes after instillation of Rebamipide ophthalmic suspension (Mucosta Ophthalmic diquafosol compared with rebamipide.71 We also compared Suspension UD2% ; Otsuka Pharmaceutical, Co., Ltd., Tokyo, the short-term effects on the MUC5AC level in rabbit tear fluid Japan) was approved for treating dry eye at the end of 2011 and conjunctival goblet cells after instilling both eyedrops.50 and was launched in Japan in 2012. Rebamipide was marketed Only diquafosol increased the MUC5AC level 15 minutes after originally in 1990 in Japan as an oral therapeutic drug to treat one instillation.50 These results suggested that diquafosol

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