The role of ORMDL3 in cigarette smoke-induced airway injury

Abstract

Background: Orosomucoid 1-Like Protein 3 (ORMDL3) is a transmembrane protein located in the endoplasmic reticulum (ER) which is proved to be strongly linked with childhood-onset asthma in genome-wide association studies. Given finding that ORMDL3 is also associated with chronic obstructive pulmonary disease (COPD), in which cigarette smoke (CS) is known to be the major risk factor, we aimed to investigate whether ORMDL3 might play a crucial role in the cigarette smoke-induced airway injury. Methods: In the in vivo study, equal numbers (n=8) of male Sprague-Dawley rats were randomized to sham air (SA) as controls or CS for 7 and 56 days respectively. Lung tissues were collected after sacrifice for Western blot analysis and immunofluorescence (IF) staining. In the in vitro study, primary normal human bronchial epithelial (NHBE) cells were stimulated with cigarette smoke medium (CSM) for 24 hours (n=4). The expression of ORMDL3, sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and ER stress markers (Binding immunoglobulin protein, BiP and calnexin) were assessed by qPCR and Western blot analysis. Results: Localization of ORMDL3 and macrophage marker (F4/80) using IF staining showed that ORMDL3 expression was colocalized to alveolar macrophages. CS elevated the ORMDL3 expression due to increased number of macrophages in lungs of both CS-exposed groups in comparison to SA-exposed group. Furthermore, upregulation of ORMDL3 expression was also observed in airway epithelium and smooth muscle in CS-exposed groups. In support, ORMDL3 expression was increased after CS exposure in rat lungs using Western blot analysis. The expression of SERCA2 was also elevated in rat lungs of CS-exposed group. In the in vitro study, CSM caused upregulation of ORMDL3 expression at both mRNA and protein level in a dose-dependent manner (mRNA: 1.108 ± 0.206 for control vs. 3.615 ± 0.615 for 2% CSM, p<0.01; protein: 0.998 ± 0.002 for control vs. 1.867 ± 0.286 for 2% CSM, p<0.05). SERCA2 protein expression was also upregulated at 2% CSM (p<0.001). CSM caused significant elevation of protein expressions of BiP, a master regulator of the unfolded protein response (UPR) (p<0.001), and calnexin, a key calcium binding chaperone in the ER membrane (p<0.001).Conclusion: Our current data suggested that cigarette smoke could induce the expression of ORMDL3 leading to ER stress via SERCA2. Therefore, ORMDL3 might play a significant role in cigarette smoke-induced airway injury.