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. 2019 Jun;60(6):695-704.
doi: 10.1165/rcmb.2018-0199OC.

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma

Lingxiang Zhu  1 , Lingling An  2   3   4 , Di Ran  2 , Rosa Lizarraga  1 , Cheryl Bondy  1 , Xu Zhou  1 , Richart W Harper  5 , Shu-Yi Liao  5 , Yin Chen  1   6
Affiliations

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma

Lingxiang Zhu et al. Am J Respir Cell Mol Biol. 2019 Jun.

Abstract

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Keywords: CC10; FOXA2; asthma; rhinovirus; secretoglobin.

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Figures

Figure 1.
Figure 1.
Differential gene expression in an ovalbumin (OVA)-induced mouse model of asthma. Mice were challenged with OVA or PBS control. As described in Methods, after completion of the experiment, the mice were killed and lung tissues were harvested. Total RNA was extracted and subjected to qPCR analysis. Lung tissues were also preserved, fixed, and subjected to immunofluorescence staining. (A) Scgb1a1 mRNA was measured by qPCR analysis in mouse lung. (B) Scgb1a1 protein was measured by immunofluorescence in mouse airway tissue sections using a specific anti-Scgb1a1 antibody (green). Blue: DAPI staining for the cell nuclei. (C) FoxA2 mRNA was measured by qPCR analysis in mouse lung. (D) Muc5ac mRNA was measured by qPCR analysis in mouse lung. Data shown are mean ± SEM; *P < 0.05 and #P < 0.05; OVA versus control (Con); n = 6.
Figure 2.
Figure 2.
Exacerbated inflammation and airway hyperreactivity in Scgb1a1 knockout (KO) mice. Both wild-type (WT) and Scgb1a1 KO mice were challenged with OVA or with PBS control. (A) Lung resistance in response to increased doses of methacholine challenge was measured using the flexiVent system. (B) A differential cell count was performed to quantify BAL cells. *P < 0.05 WT OVA versus WT PBS. $P < 0.05 Scgb1a1 KO OVA versus WT OVA. n = 6.
Figure 3.
Figure 3.
Differential epithelial gene expression in response to IL-4 or IL-13 treatment. Human bronchial epithelial cells (HBECs) were treated with IL-4, IL-13, or solvent Con for 3 days. Total RNA was collected and subjected to qPCR analysis for (A) SCGB1A1, (B) FOXA2, (C) MUC5AC, and (D) FOXA3. Data shown are mean ± SD; *P < 0.05 and #P < 0.05; treatment versus Con; n = 4.
Figure 4.
Figure 4.
Differential epithelial protein production in response to IL-13 treatment. (A–F) HBECs were treated with IL-13 (D–F) or solvent control (A–C) for 3 days. Cells were fixed and subjected to immunofluorescence imaging for MUC5AC (A and D, green) and SCGB1A1 (B and E, red). C and F are merged images. Blue: nuclear staining with DAPI. (G) Image quantification from six biological replicates of MUC5AC (A and D) or SCGB1A1 (B and E) expression in response to IL-13 treatment. *P < 0.05 and #P < 0.05; n = 6. (H) Representative image from Western blot analysis of FOXA2 protein after IL-13 treatment. Actin was used as a loading control.
Figure 5.
Figure 5.
Differential epithelial gene expression in response to rhinovirus 16 (RV16) infection. HBECs were infected with RV16 or sham Con for 24 hours. Total RNA was collected and subjected to qPCR analysis for (A) SCGB1A1, (B) FOXA2, (C) MUC5AC, and (D) FOXA3. Data shown are mean ± SD. *P < 0.05 and #P < 0.05; RV versus Con; n = 4.
Figure 6.
Figure 6.
Genetic manipulation of FOXA2 altered SCGB1A1 expression. (A) FOXA2 expression plasmid or its vector control was delivered into HBECs via electroporation, and the cells were then analyzed for FOXA2 expression by qPCR. (B) Endogenous SCGB1A1 expression was measured in the cells with FOXA2 overexpression or with vector control. (C) WT or mutated (mutant) SCGB1A1 promoter luciferase reporter was delivered into HBECs via electroporation with FOXA2 or with its vector control. Promoter activity was analyzed by luciferase assay. *P < 0.05. Cells electroporated with WT promoter + FOXA2 versus cells electroporated with WT promoter + vector. n = 4. #P < 0.05. Cells electroporated with WT promoter + FOXA2 versus cells electroporated with mutant promoter + FOXA2. n = 4. (D) Western blot analysis of FOXA2 expression in the cells transfected with control siRNA (siC) or siRNA against FOXA2 (siFOXA2). (E) Endogenous SCGB1A1 expression was measured by real-time PCR; #P < 0.05; cells transfected with siFOXA2 versus cells transfected with siC; n = 4. (F) HBECs with FOXA2 overexpression or vector control were treated with or without IL-13. The percentage of repression was calculated as the magnitude of IL-13–induced SCGB1A1 repression. (G) HBECs with FOXA2 overexpression or vector control were infected with or without RV. The percentage of repression was calculated as the magnitude of RV-induced SCGB1A1 repression. Data shown are mean ± SD; #P < 0.05; cells electroporated with FOXA2 versus cells electroporated with the vector; n = 4.
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