Lyn regulates mucus secretion and MUC5AC via the STAT6 signaling pathway during allergic airway inflammation
Min Wu、Guoping Li
Hypersecretion of mucus is an important component of airway remodeling and contributes to the mucus plugs and airflow obstruction associated with severe asthma phenotypes. Lyn has been shown to down-regulate allergen-induced airway inflammation. However, the role of Lyn in mucin gene expression remains unresolved. In this study, we first demonstrate that Lyn overexpression decreased the mucus hypersecretion and levels of the muc5ac transcript in mice exposed to ovalbumin (OVA). Lyn overexpression also decreased the infiltration of inflammatory cells and the levels of IL-13 and IL-4 in OVA-challenged airways. Whereas Lyn knockdown increased the IL-4 or IL-13-induced MUC5AC transcript and protein levels in the human bronchial epithelial cell line, 16HBE, Lyn overexpression decreased IL-4- or IL-13-induced MUC5AC transcript and protein levels. Overexpression of Lyn also decreased the expression and phosphorylation of STAT6 in OVA-exposed mice, whereas Lyn knockdown increased STAT6 and MUC5AC levels in 16HBE cells. Finally, chromatin immunoprecipitation analysis confirmed that Lyn overexpression decreased the binding of STAT6 to the promoter region of Muc5ac in mice exposed to OVA. Collectively, these findings demonstrated that Lyn overexpression ameliorated airway mucus hypersecretion by down-regulating STAT6 and its binding to the MUC5AC promoter.A Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibodies for histology were purchased from Santa Cruz Biotechnology: MUC5AC (mucin 5AC, Clone # K-20; Cat # sc-16903), STAT6 (Clone # S-20, Cat # sc-621), β-actin (Clone # N-21, Cat # sc-130656), and phospho-STAT6 (Clone # Tyr641, Cat # sc-11762). The ELISA kits for IL-4, and IL-13 were purchased from Beijing Yonghui biotechnology Co. Ltd (Beijing, China). The MUC5AC-pGL3 luciferase vector was constructed by our laboratory based on the pGL3 luciferase reporter vector (Promega) following the manufacturer’s instructions.Based on our previous findings showing mucus hypersecretion during exposure of Lyn−/− (Lyn knockout) mice to HDM24, we investigated mucus secretion and muc5ac expression using a transgenic approach. To more thoroughly elucidate the critical role of Lyn in asthmatic pathology, we generated Lyn transgenic mice (Lyntg mice) and successfully verified the overexpression of Lyn in these mice by western-blot. We then backcrossed these mice with C57BL/6 J for 2 generations and challenged them as well as control mice with OVA. We employed an initial sensitization using an intraperitoneal (i.p.) injection of OVA followed by repeated intranasal instillations to induce chronic airway inflammation in the Lyntg and WT mice (Fig. 1A). We further confirmed that Lyn kinase regulates mucus secretion and muc5ac expression in a murine model of allergic airway inflammation. The lungs of the WT and Lyntg mice exposed to PBS remained normal, with few mucin-positive goblet cells and trace amounts of muc5ac (Fig. 1B,D,F). The lungs of the WT and Lyntg mice exposed to OVA (at 3, 6 and 8 weeks) showed an increased number of mucin-positive goblet cells (Fig. 1B,C; Supplementary Figure S1A). At 8 weeks, we observed a robust decrease in the number of mucin-positive goblet cells in the Lyntg mice exposed to OVA compared to the WT mice (2.0-fold decrease; Fig. 1C; P < 0.001). Immunofluorescent staining of lung tissue showed that expression of muc5ac in Lyntg mice exposed to OVA was less than WT mice exposed to OVA (Fig. 1D,E; Supplementary Figure S1B; P < 0.05. In Supplementary Figure S1B, “I&II” both refer to the negative control of immunohistochemistry staining in Fig. 1D. “I” indicates the sample’s autofluorescence. “II” shows non-specific fluorescence.). To examine the transcript levels of mucous secretion genes, RT-PCR was utilized to detect the mRNA levels of muc1, muc2, muc3, muc4, muc5ac, muc5b and muc13 in lung tissue. The transcript levels for muc1, muc2, muc3, muc4, muc5b and muc13 in lung tissue were unaltered, whereas the Muc5ac transcript was significantly increased in the WT mice exposed to OVA (Fig. 1F; Supplementary Figure S1C). We also observed a robust decrease in muc5ac transcript levels in the Lyntg mice exposed to OVA (at 8 weeks) compared with WT mice (Fig. 1F; P < 0.001). Taken together, these findings indicate that Lyn overexpression decreased mucus secretion and muc5ac transcript levels in mice exposed to OVA.This project was supported by the Chinese National Science Foundation (81170032).The authors declare no competing financial interests.Author Contributions Conceived and designed the study: Guoping Li, Min Wu and Nanshan Zhong. Performed the experiments: Xiaoyun Wang, Yin Li, Deyu Luo, Xing Wang, and Yun Zhang. Analyzed the data: Xiaoyun Wang, Yin Li, Deyu Luo, Zhigang Liu, and Xing Wang. Wrote the paper: Guoping Li, Yin Li, and Min Wu. All authors read and approved the final manuscript.