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

• Zhang Y
• Wang X
• Wang H
• Jiao J
• Li Y
• Fan E
• Zhang L
• Bachert C

• KMID: 2166684
• DOI: http://doi.org/10.4168/aair.2015.7.4.367

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).

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