Allergy Asthma Immunol Res.  2015 Jul;7(4):367-375. 10.4168/aair.2015.7.4.367.

TMEM16A-Mediated Mucin Secretion in IL-13-Induced Nasal Epithelial Cells From Chronic Rhinosinusitis Patients

Affiliations
  • 1Department of Otolaryngology, Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing, China. dr.luozhang@gmail.com
  • 2Key Laboratory of Otolaryngology, Head and Neck Surgery, Ministry of Education, Beijing Institute of Otolaryngology, Beijing, China.
  • 3Upper Airways Research Laboratory, Department of Oto-Rhino-Laryngology, Ghent University Hospital, Ghent, Belgium.

Abstract

PURPOSE
Chronic rhinosinusitis with nasal polyps (CRSwNP), a mainly Th2 cytokine-mediated disease, often involves mucus secretion. Recent evidence suggests that transmembrane protein 16A (TMEM16A), a calcium-activated Cl- channel (CaCC), can regulate mucus secretion from airway epithelium by transepithelial electrolyte transport and hydration. However, the role of TMEM16A in mucin production/secretion in the airway epithelium is not clear. This study was conducted to determine the role of TMEM16A in mediating mucin secretion in human nasal polyp epithelial cells (HNPECs) induced by IL-13.
METHODS
Human sinonasal mucosa tissue and dissociated sinonasal epithelium from control subjects and patients with CRSwNP were assessed for the expression of TMEM16A and the secretion of human mucin 5AC (MUC5AC) by immunohistochemistry, Western blot analysis, and enzyme-linked immuno-sorbent assay (ELISA). A model of the Th2 inflammatory environment was created by exposure of primary air-liquid interface (ALI)-cultured HNPECs to interleukin-13 (IL-13) for 14 days, with subsequent assessment of TMEM16A expression in cell lysates by Western blotting and MUC5AC secretion in apical washings of cells by ELISA.
RESULTS
The expressions of TMEM16A and MUC5AC were increased in human nasal polyp tissue and dissociated nasal polyp epithelium. TMEM16A was detected in IL-13-treated HNPECs, specifically in MUC5AC-positive cells but not in ciliated cells. IL-13 treatment increased percentages of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing TMEM16A/MUC5AC, the expression of TMEM16A protein, and the secretion of MUC5AC. T16Ainh-A01, a TMEM16A inhibitor, attenuated these IL-13-induced effects.
CONCLUSIONS
The expression of TMEM16A and MUC5AC are increased in CRSwNP, which might be a direct effect of Th2 cytokines present in the sinonasal mucosa in CRSwNP. Down-regulation of TMEM16A expression and MUC5AC secretion in HNPECs by T16Ainh-A01 indicates that TMEM16A might play an important role in mucin secretion in upper airway inflammatory diseases.

Keyword

Chronic rhinosinusitis with nasal polyps; MUC5AC; mucin secretion; nasal epithelial cells; TMEM16A

MeSH Terms

Blotting, Western
Cytokines
Down-Regulation
Enzyme-Linked Immunosorbent Assay
Epithelial Cells*
Epithelium
Humans
Immunohistochemistry
Interleukin-13
Mucin 5AC
Mucins*
Mucous Membrane
Mucus
Nasal Polyps
Negotiating
Cytokines
Interleukin-13
Mucin 5AC
Mucins

Figure

  • Fig. 1 Immunohistochemical staining for TMEM16A and MUC5AC in human sinonasal epithelium: TMEM16A and MUC5AC proteins in human nasal polyp epithelium (HNPE group) (A, B) and normal sinonasal epithelium (control group) (C, D). Tissues were stained with anti-TMEM16A (1:200) and anti-MUC5AC (1:500). Arrowheads show TMEM16A-positive cells, and arrows show MUC5AC-positive cells. The images were observed with a 40× objective.

  • Fig. 2 Expressions of TMEM16A and MUC5AC proteins in dissociated human sinonasal epithelium. (A) The expression of TMEM16A protein was detected by Western blotting in dissociated normal sinonasal epithelium (control group) and nasal polyp epithelium (HNPE group). (B) Comparison of the expression of TMEM16A protein by densitometric analysis between the control and HNPE groups. Each bar represents the relative density of TMEM16A band normalized to β-actin band for each sample (n=6, ***P<0.001). (C) Measurement of MUC5AC by ELISA from supernatants of nasal tissue homogenate in control and HNPE groups (n=6, ***P<0.001).

  • Fig. 3 Coexpression of TMEM16A and MUC5AC in ALI-cultured HNPECs. Confocal microscopy xy images of ALI-cultured HNPECs without IL-13 (A) and with IL-13 treatment (10 ng/mL, 14 days) (B) showing over lapping immunoreactivity for TMEM16A (red) and MUC5AC (green). Nuclei stained with DAPI appear blue. The images were taken with a 40× objective. Scale bars: 50 µm.

  • Fig. 4 Expression of TMEM16A in non-ciliated cells. Confocal microscopy xy images of ALI-cultured HNPECs without IL-13 (A) and with IL-13 (10 ng/mL, 14 days) (B) showing immunoreactivity for TMEM16A (red) and acetylated-tubulin (green). Nuclei stained with DAPI appear blue. The 2 images show that TMEM16A and acetylated-tubulin staining appears on different epithelial cells. The images were taken with a 40× objective. Scale bars: 50 µm.

  • Fig. 5 Increased expression of TMEM16A and secretion of MUC5AC in ALI-cultured HNPECs with IL-13. (A) The percentage of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing MUC5AC and TMEM16A in ALI-cultured HSNECs (control group) and HNPECs incubated with or without IL-13 (10 ng/ml) for 14 days (n=6, ***P<0.001). (B) Detection of TMEM16A protein by Western blot in lysates from HNPECs and control cells incubated with or without IL-13 (10 ng/mL) for 14 days. (C) Comparison of the expression of TMEM16A protein by densitometric analysis. Each bar reports the relative density of TMEM16A band normalized to β-actin band for each sample (n=6, ***P<0.001). (D) Detection of MUC5AC by ELISA in apical washings from HNPECs and control cells incubated with or without IL-13 (10 ng/mL) for 14 days (n=6, ***P<0.001).

  • Fig. 6 Decreased expression of TMEM16A and secretion of MUC5AC in ALI-cultured HNPECs by the TMEM16A inhibitor T16Ainh-A01. (A) The percentage of TMEM16A-positive cells, MUC5AC-positive cells, and cells coexpressing MUC5AC and TMEM16A in ALI-cultured HNPECs incubated with or without IL-13 (10 ng/mL) + T16Ainh-A01(10 µM) for 14 days (n=6, *P<0.05, ***P<0.001). (B) Western blot analysis of TMEM16A protein in lysates from HNPECs incubated with or without IL-13 (10 ng/mL) + T16Ainh-A01(10 µM) for 14 days. (C) Comparison of the expression of TMEM16A protein by densitometric analysis. Each bar shows the relative density of TMEM16A band normalized to β-actin band for each sample (n=6, ***P<0.001). (D) Detection of MUC5AC by ELISA in apical washings from HNPECs incubated with or without IL-13 (10 ng/mL) + T16Ainh-A01 (10 µM) for 14 days (n=6, *P<0.05, **P<0.01, ***P<0.001).


Reference

1. Slavin RG. Nasal polyps and sinusitis. JAMA. 1997; 278:1849–1854. PMID: 9396646.
Article
2. Drake-Lee AB, Lowe D, Swanston A, Grace A. Clinical profile and recurrence of nasal polyps. J Laryngol Otol. 1984; 98:783–793. PMID: 6470574.
Article
3. Settipane GA, Klein DE, Settipane RJ. Nasal polyps. State of the art. Rhinol Suppl. 1991; 11:33–36. PMID: 1888555.
4. Ramanathan M Jr, Lee WK, Spannhake EW, Lane AP. Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells. Am J Rhinol. 2008; 22:115–121. PMID: 18416964.
Article
5. Park SJ, Kim TH, Jun YJ, Lee SH, Ryu HY, Jung KJ, et al. Chronic rhinosinusitis with polyps and without polyps is associated with increased expression of suppressors of cytokine signaling 1 and 3. J Allergy Clin Immunol. 2013; 131:772–780. PMID: 23375208.
Article
6. Van Zele T, Claeys S, Gevaert P, Van Maele G, Holtappels G, Van Cauwenberge P, et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. Allergy. 2006; 61:1280–1289. PMID: 17002703.
Article
7. Van Bruaene N, Pérez-Novo CA, Basinski TM, Van Zele T, Holtappels G, De Ruyck N, et al. T-cell regulation in chronic paranasal sinus disease. J Allergy Clin Immunol. 2008; 121:1435–1441. 1441.e1–1441.e3. PMID: 18423831.
Article
8. Lu X, Wang N, Long XB, You XJ, Cui YH, Liu Z. The cytokine-driven regulation of secretoglobins in normal human upper airway and their expression, particularly that of uteroglobin-related protein 1, in chronic rhinosinusitis. Respir Res. 2011; 12:28. PMID: 21385388.
Article
9. Ocampo C, Suh L, Kern R, Kato A, Conley D, Chandra R, et al. Levels of the cytokines IL-5, IL-13 and Rantes in nasal lavage fluids parallel the cytokine content of nasal polyps in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). J Allergy Clin Immunol. 2013; 131:AB237.
Article
10. Nabavi M, Arshi S, Bahrami A, Aryan Z, Bemanian MH, Esmaeilzadeh H, et al. Increased level of interleukin-13, but not interleukin-4 and interferon-γ in chronic rhinosinusitis with nasal polyps. Allergol Immunopathol (Madr). 2014; 42:465–471. PMID: 23969075.
Article
11. Bachert C, Zhang N, Holtappels G, De Lobel L, van Cauwenberge P, Liu S, et al. Presence of IL-5 protein and IgE antibodies to staphylococcal enterotoxins in nasal polyps is associated with comorbid asthma. J Allergy Clin Immunol. 2010; 126:962–968. 968.e1–968.e6. PMID: 20810157.
Article
12. Iwashita H, Fujimoto K, Morita S, Nakanishi A, Kubo K. Increased human Ca2+-activated Cl- channel 1 expression and mucus overproduction in airway epithelia of smokers and chronic obstructive pulmonary disease patients. Respir Res. 2012; 13:55. PMID: 22731784.
Article
13. Huang F, Wong X, Jan LY. International Union of Basic and Clinical Pharmacology. LXXXV: calcium-activated chloride channels. Pharmacol Rev. 2012; 64:1–15. PMID: 22090471.
Article
14. Cunningham SA, Awayda MS, Bubien JK, Ismailov II, Arrate MP, Berdiev BK, et al. Cloning of an epithelial chloride channel from bovine trachea. J Biol Chem. 1995; 270:31016–31026. PMID: 8537359.
Article
15. Sun H, Tsunenari T, Yau KW, Nathans J. The vitelliform macular dystrophy protein defines a new family of chloride channels. Proc Natl Acad Sci U S A. 2002; 99:4008–4013. PMID: 11904445.
Article
16. Qu Z, Wei RW, Mann W, Hartzell HC. Two bestrophins cloned from Xenopus laevis oocytes express Ca2+-activated Cl- currents. J Biol Chem. 2003; 278:49563–49572. PMID: 12939260.
Article
17. Suzuki M, Mizuno A. A novel human Cl(-) channel family related to Drosophila flightless locus. J Biol Chem. 2004; 279:22461–22468. PMID: 15010458.
Article
18. Galietta LJ. The TMEM16 protein family: a new class of chloride channels? Biophys J. 2009; 97:3047–3053. PMID: 20006941.
Article
19. Schroeder BC, Cheng T, Jan YN, Jan LY. Expression cloning of TMEM16A as a calcium-activated chloride channel subunit. Cell. 2008; 134:1019–1029. PMID: 18805094.
Article
20. Yang YD, Cho H, Koo JY, Tak MH, Cho Y, Shim WS, et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance. Nature. 2008; 455:1210–1215. PMID: 18724360.
Article
21. Caputo A, Caci E, Ferrera L, Pedemonte N, Barsanti C, Sondo E, et al. TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity. Science. 2008; 322:590–594. PMID: 18772398.
Article
22. Tian Y, Kongsuphol P, Hug M, Ousingsawat J, Witzgall R, Schreiber R, et al. Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A. FASEB J. 2011; 25:1058–1068. PMID: 21115851.
Article
23. Rock JR, O'Neal WK, Gabriel SE, Randell SH, Harfe BD, Boucher RC, et al. Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl- secretory channel in mouse airways. J Biol Chem. 2009; 284:14875–14880. PMID: 19363029.
Article
24. Huang F, Zhang H, Wu M, Yang H, Kudo M, Peters CJ, et al. Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction. Proc Natl Acad Sci U S A. 2012; 109:16354–16359. PMID: 22988107.
Article
25. Namkung W, Phuan PW, Verkman AS. TMEM16A inhibitors reveal TMEM16A as a minor component of calcium-activated chloride channel conductance in airway and intestinal epithelial cells. J Biol Chem. 2011; 286:2365–2374. PMID: 21084298.
Article
26. Fokkens WJ, Lund VJ, Mullol J, Bachert C, Alobid I, Baroody F, et al. EPOS 2012: European position paper on rhinosinusitis and nasal polyps 2012. A summary for otorhinolaryngologists. Rhinology. 2012; 50:1–12. PMID: 22469599.
Article
27. Kondo M, Tamaoki J, Takeyama K, Nakata J, Nagai A. Interleukin-13 induces goblet cell differentiation in primary cell culture from Guinea pig tracheal epithelium. Am J Respir Cell Mol Biol. 2002; 27:536–541. PMID: 12397012.
Article
28. Kanoh S, Tanabe T, Rubin BK. IL-13-induced MUC5AC production and goblet cell differentiation is steroid resistant in human airway cells. Clin Exp Allergy. 2011; 41:1747–1756. PMID: 22092504.
Article
29. Patou J, Gevaert P, Van Zele T, Holtappels G, van Cauwenberge P, Bachert C. Staphylococcus aureus enterotoxin B, protein A, and lipoteichoic acid stimulations in nasal polyps. J Allergy Clin Immunol. 2008; 121:110–115. PMID: 17980412.
Article
30. Scudieri P, Caci E, Bruno S, Ferrera L, Schiavon M, Sondo E, et al. Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia. J Physiol. 2012; 590:6141–6155. PMID: 22988141.
Article
31. Ousingsawat J, Martins JR, Schreiber R, Rock JR, Harfe BD, Kunzelmann K. Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport. J Biol Chem. 2009; 284:28698–28703. PMID: 19679661.
Article
32. Kondo M, Tamaoki J, Takeyama K, Isono K, Kawatani K, Izumo T, et al. Elimination of IL-13 reverses established goblet cell metaplasia into ciliated epithelia in airway epithelial cell culture. Allergol Int. 2006; 55:329–336. PMID: 17075276.
Article
33. Nakano T, Inoue H, Fukuyama S, Matsumoto K, Matsumura M, Tsuda M, et al. Niflumic acid suppresses interleukin-13-induced asthma phenotypes. Am J Respir Crit Care Med. 2006; 173:1216–1221. PMID: 16528019.
Article
34. Rogers DF. The airway goblet cell. Int J Biochem Cell Biol. 2003; 35:1–6. PMID: 12467641.
Article
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