Allergy Asthma Immunol Res.  2015 Sep;7(5):458-466. 10.4168/aair.2015.7.5.458.

Expression and Regulation of Transcription Factor FoxA2 in Chronic Rhinosinusitis With and Without Nasal Polyps

• Luo Q
• Zhang J
• Wang H
• Chen F
• Luo X
• Miao B
• Wu X
• Ma R
• Luo X
• Xu G
• Shi J
• Li H

• KMID: 2147954
• DOI: http://doi.org/10.4168/aair.2015.7.5.458

Abstract

PURPOSE
Chronic rhinosinusitis (CRS) is characterized by the excessive production of mucus. However, the molecular mechanism underlying mucin overproduction in CRS with or without nasal polyps (CRSwNP and CRSsNP, respectively) is poorly understood. This study was conducted to assess the importance of the transcription factor FoxA2 in mucin production and to investigate the targeting of FoxA2 as a potential therapeutic strategy for mucus hypersecretion in CRS patients.
METHODS
We enrolled 15 CRSwNP patients, 15 CRSsNP patients, and 10 normal controls in this study. The expression levels of FoxA2, MUC5AC, and MUC5B in inflamed and healthy nasal tissues were examined via immunohistochemistry and quantitative reverse transcription-polymerase chain reaction, and the levels of several proinflammatory cytokines in nasal secretions were measured via FlowCytomix analysis. In addition, the expression of MUC5AC and FoxA2 was determined in polyp-derived epithelial cells and NCI-H292 cells after in vitro stimulation.
RESULTS
FoxA2 was significantly down-regulated, and MUC5AC and MUC5B were significantly up-regulated in both the CRSwNP and CRSsNP patients compared to the controls (P<0.05), and the protein level of FoxA2 was negatively associated with the IL-6 level in the CRS patients (P<0.05). IL-6 significantly increased MUC5AC expression but inhibited FoxA2 expression in vitro (P<0.05). Transfection with a FoxA2 expression plasmid significantly decreased MUC5AC promoter activity (P<0.05) and inhibited IL-6-induced MUC5AC production (P<0.05). In addition, clarithromycin significantly alleviated IL-6-induced FoxA2 suppression and decreased MUC5AC expression in vitro (P<0.05).
CONCLUSIONS
Our findings suggest that FoxA2 may be considered a therapeutic target for the modulation of mucus hypersecretion in CRS patients.

Keyword

Chronic rhinosinusitis; nasal polyps; FoxA2; mucin; MUC5AC; MUC5B; IL-6; macrolide

MeSH Terms

Clarithromycin
Cytokines
Epithelial Cells
Humans
Immunohistochemistry
Interleukin-6
Mucins
Mucus
Nasal Polyps*
Plasmids
Transcription Factors*
Transfection
Clarithromycin
Cytokines
Interleukin-6
Mucins
Transcription Factors

Figure

• 

  Fig. 1 Immunoreactivity of MUC5AC, MUC5B, and FoxA2 in the nasal tissues from the CRSsNP and CRSwNP patients and the normal controls. PAS staining shows that mucin is highly expressed in the epithelial and glandular mucous cells of CRS patients (A); IHC staining shows that MUC5AC is highly expressed in the epithelial mucous cells and mildly expressed in the glandular mucous cells (B) and that MUC5B is moderately expressed in the epithelial mucous cells and highly expressed in the glandular mucous cells (C). Both MUC5AC and MUC5B are mildly expressed in the normal controls. FoxA2 is highly expressed in the epithelial and glandular mucous cells of the normal controls and mildly detected in the cytoplasm and the nuclei of these cell types (D) (magnification, 200×).

• 

  Fig. 2 The mRNA and protein levels of MUC5AC, MUC5B, and FoxA2 in the nasal tissues from the CRSsNP and CRSwNP patients and the normal controls. The relative mRNA levels of MUC5AC (A) and MUC5B (B) increase in the CRSsNP and CRSwNP patients compared to the normal controls. The relative FoxA2 mRNA level decreases in the CRSsNP and CRSwNP patients compared to the normal controls (C). The protein levels of MUC5AC (D) and MUC5B (E) significantly increase in the CRSsNP and CRSwNP patients compared to the normal controls. A representative Western blot of FoxA2 in the CRSsNP and CRSwNP patients and the normal controls is shown (F). The relative FoxA2 protein level significantly decreases in the CRSsNP and CRSwNP patients compared to the normal controls (G). *P<0.05.

• 

  Fig. 3 The levels of IFN-γ, IL-4, IL-5, IL-6, and IL-17A in the nasal secretions of the CRSsNP and CRSwNP patients and the normal controls. The levels of IFN-γ (A), IL-4 (B), IL-5(C), IL-6 (D), and IL-17A (E) in the nasal secretions are significantly higher in both the CRSsNP and CRSwNP patients than in the normal controls. The relative FoxA2 protein level in the inflamed nasal tissues is negatively associated with the levels of IL-6 in the nasal secretions of the CRSsNP and CRSwNP patients. *P<0.05; **P<0.01.

• 

  Fig. 4 FoxA2 negatively regulates IL-6-induced MUC5AC production in NCI-H292 cells. DNA microarray analysis of NCI-H292 cells in response to IL-6 (10 ng/mL) stimulation. A heatmap of the genes that were up-regulated or down-regulated in the IL-6-induced NCI-H292 cells is shown (A). IL-6 (0-20 ng/mL)-induced MUC5AC mRNA expression in a dose-dependent manner (B). IL-6 (0-20 ng/mL) inhibits FoxA2 mRNA and protein expression in a dose-dependent manner (C, D). Representative Western blot for FoxA2 in NCI-H292 cells after transfection with the FoxA2 expression plasmid (E). Transfection with the FoxA2 expression plasmid significantly inhibits MUC5AC promoter activity in the presence of IL-6, as suggested by a luciferase activity assay (F). Transfection with the FoxA2 expression plasmid significantly inhibits IL-6-induced MUC5AC production (G). Transfection with FoxA2 siRNA significantly increases IL-6-induced MUC5AC production (H). The data are expressed as the means (SEM) of the 3 independent experiments. *P<0.05.

• 

  Fig. 5 CAM promotes FoxA2 expression in CRSwNP patients in vivo and in vitro. Individual nasal symptom scores (rhinorrhea and nasal congestion) before and after oral administration of CAM (250 mg, twice daily) for 2 weeks (A, B). Representative immunoreactivity of FoxA2 and MUC5AC in polyp tissues before and after oral administration of CAM for 2 weeks (C). FoxA2 mRNA expression in polyp tissues before and after oral administration of CAM for 2 weeks (D). CAM (10 ng/mL) significantly attenuates the IL-6-induced suppression of FoxA2 mRNA and protein expression in PECs (E-G). The in vitro data are expressed as the means (SEM) of the 3 independent experiments. *P<0.05; **P<0.01.

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