EBioMedicine PMID: 28024734(2017) 

Lyn kinase represses mucus hypersecretion by regulating IL-13-induced endoplasmic reticulum stress in asthma



In asthma, mucus hypersecretion is thought to be a prominent pathological feature associated with widespread mucus plugging. However, the current treatments for mucus hypersecretion are often ineffective or temporary. The potential therapeutic targets of mucus hypersecretion in asthma remain unknown. Here, we show that Lyn is a central effector of endoplasmic reticulum stress (ER stress) and mucous hypersecretion in asthma. In Lyn-transgenic mice (Lyn-TG) and wild-type (WT) C57BL/6J mice exposed to ovalbumin (OVA), Lyn overexpression attenuates mucus hypersecretion and ER stress. Interleukin 13 (IL-13) induced MUC5AC expression by enhancing ER stress in vitro. Lyn serves as a negative regulator of IL-13-induced ER stress and MUC5AC expression. We further find that an inhibitor of ER stress, which is likely involved in the PI3K p85α/Akt pathway and NFκB activity, blocked MUC5AC expression in Lyn-knockdown cells. Furthermore, PI3K/Akt signaling is required for IL-13-induced ER stress and MUC5AC expression in airway epithelial cells. The ER stress regulation of MUC5AC expression depends on NFκB in Lyn-knockdown airway epithelial cells. Our studies indicate not only a concept of mucus hypersecretion in asthma that involves Lyn kinase but also an important therapeutic candidate for asthma.The following antibodies for histology and cell staining were purchased from Santa Cruz Biotechnology: anti-MUC5AC (Santa Cruz, CA, sc-16903, AB_649616), anti-phospho-PI3K p85α (Tyr467: Santa Cruz, CA, sc-293115, AB_10844180), anti-PI3K p85α (Santa Cruz, CA, sc-31970, AB_2268186), anti-phospho-Akt1 (Thr308: Santa Cruz, CA, sc-135650, AB_2224730), anti-Akt1 (Santa Cruz, CA, sc-1618, AB_630849), anti-phospho-NFκB p65 (Ser536: Santa Cruz, CA, sc-33020, AB_2179018), anti-NFκB p65 (Santa Cruz, CA, sc-109, AB_632039), anti-Lyn (Santa Cruz, CA, sc-15, AB_2281450), and anti-β-actin (Santa Cruz, CA, sc-130656, AB_2223228). The following antibodies for histology were purchased from Abcam Biotechnology: anti-BIP (Abcam, ab21685, AB_2119834), anti-CHOP (Abcam, ab11419, AB_298023), anti-histone H3 (Abcam, ab1791, AB_302613), and anti-IL-13 (Abcam, ab133353, AB_11157609). Anti-phospho-Lyn (Tyr416: Cell Signaling Technology, #2101, AB_331697) was purchased from Cell Signaling Technology. Anti-IL13 (R&D Systems, AF-413-NA, and AB_2124173) and IL-13 ELISA reagents (R&D Systems, M1300CB) were purchased from R&D Systems. IL-13 (PeproTech, #200-13), 4-Phenylbutyric acid (4-PBA, Sigma-Aldrich, P21005), PI3K Inhibitor PI-103 (Selleck, S1038) were purchased as indicated. A nonsilencing siRNA control and a Lyn-specific siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Previous studies have shown that ER stress may be critical for the pathogenesis of bronchial asthma, especially the steroid-resistant neutrophilic asthma (Kim et al., 2013). Nonetheless, we found that Lyn modulates the development of airway remodeling (Li et al., 2013), raising the question of whether Lyn may affect the ER stress implicated in asthma. To examine the effects of Lyn on ER stress, Lyn-TG mice and WT mice were sensitized using an intraperitoneal (i.p.) injection and challenged by repeated OVA intranasal administrations to induce ER stress (Fig. 1a). 4-Phenylbutyric acid (4-PBA) was evaluated as a positive control for ER stress in the WT mouse model of OVA-induced chronic airway inflammation. In present studies, immunohistochemical staining showed that Lyn-TG mice showed increased expression of Lyn the airway epithelium, alveolar epithelial cell and inflammatory cells in lung compared with WT mice (Supplementary Fig. S1 online). OVA induced the characteristic features of allergic airway inflammation: an increased number of total cells, neutrophils and eosinophils in the bronchoalveolar lavage fluid (BALF); lung inflammation, and increased inflammatory index. In OVA-induced Lyn-TG mice, the features of allergic airway inflammation were mitigated: the number of total cells, neutrophils and eosinophils in the BALF were decreased, and lung inflammation and the inflammatory index were both decreased. As expected, 4-PBA also significantly suppressed the characteristic features of OVA-induced chronic airway inflammation shown above in positive control mice compared with that in normal WT mice (Fig. 1b–f).To investigate the effect of Lyn on ER stress in airway epithelial cells in the mouse model of allergic airway inflammation, the localization of ER stress markers expressed in lung tissue was determined by immunofluorescence staining. Confocal microscope analyses revealed that OVA induced the features of ER stress, increasing the expression of CHOP and BIP in the lung tissue of OVA-induced WT mice. The expression of BIP and CHOP predominantly occurred in the cytoplasmic areas of the airway epithelium in OVA-induced WT mice. OVA-induced mice exhibited lower fluorescence intensities corresponding to BIP and CHOP in the lung tissue than OVA-induced WT mice (Fig. 1h–i). 4-PBA also substantially suppressed BIP and CHOP in OVA-induced WT mice (Fig. 1g). The immunofluorescence intensities of BIP and CHOP in 4-PBA-treated positive control WT mice were significantly decreased by 39.2 (14.63/40.13) and 34.5 (19.66/57.03) percent, respectively, compared with those in OVA-induced WT mice (Fig. 1h–i, P < 0.05). Taken together, these results indicated that Lyn overexpression repressed the OVA-induced chronic airway inflammation and ER stress in mice exposed to OVA. The amelioration of airway inflammation caused by Lyn overexpression may be associated with the inhibition of ER stress.This project was supported by grant from the Chinese National Science Foundation (81170032, 81600024) and project grants from National Institute of Health (AI109317-01A1 and AI101973-01).Appendix ASupplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ebiom.2016.12.010.Supplementary content
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