Relationship Between Inactivation Of C674 In The SERCA2 And Bronchial Asthma And Its Mechanism

Bronchial asthma（asthma）is a heterogeneous disease characterized by airway inflammation and airway remodeling,which is involved in a variety of cells and cellular components.With the increasing environmental pollution in the world,the incidence of asthma is increasing rapidly worldwide.The pathogenesis of asthma mainly includes genetic factors,airway inflammation,airway remodeling and oxidative stress.Airway inflammation runs through the entire process of asthma.Inflammatory cells secrete a variety of cytokines that folding into the airway and interact with the airway epithelium and smooth muscle layer,resulting in airway changes.These changes include:proliferation and activation of goblet cells in the epithelial layer,increased mucus secretion in the mucosal layer,and abnormal contraction of smooth muscle.The persistent inflammatory response causes remodeling of airway wall.Airway remodeling includes:smooth muscle thickening,submucosal neovascularization,subepithelial collagen deposition,fibrosis,mucous gland hypertrophy,etc.Airway inflammation and airway remodeling are complementary to each other and both are involved in the pathogenesis of asthma.Among them,airway smooth muscle cells（ASMC）have always been the focus of asthma research.Sarcoplasmic/endoplasmic reticulum calcium ATPase（SERCA）is a key protein for the transport of Ca²⁺on the sarcoplasmic/endoplasmic reticulum,and its main function is to take up cytoplasmic Ca²⁺into the sarcoplasm/endoplasmic reticulum,which is essential for maintaining the calcium homeostasis of cytoplasm and sarcoplasmic/endoplasmic reticulum.SERCA2 is the main subtype of SERCA in the airway,which mainly includes two genotypes,SERCA2a and SERCA2b,and SERCA2b is the dominant one in ASMC.In ASMC of asthmatic patients,the expression of SERCA2 is reduced,the regulation of Ca²⁺is unbalanced,and the capacity of cell proliferation and secretion is enhanced,suggested that SERCA2 dysfunction is involved in airway remodeling.Cysteine 674（C674）of SERCA2 is a key redox site regulating SERCA2activity.It is irreversibly oxidized and inactivated with increased ROS under many pathological conditions.Whether C674 inactivation in SERCA2 is involved in the process of asthma is unclear.In order to explore whether C674 inactivation in SERCA2is involved in bronchial asthma progression and its related mechanism,we mutated C674 into serine（C674→S674）to simulate the irreversible oxidative inactivation of C674 under pathological conditions.SERCA2 C674S gene mutation knock-in（SKI）mice were constructed under the regulation of SERCA2 promoter.Homozygous SKI mice are embryonic lethal,indicating that the redox state of C674 is very important for maintaining cell homeostasis.Heterozygous SKI mice（herein referred to as SKI mice）were used to study the role and mechanism of C674 inactivation in SERCA2 in bronchial asthma.Compared with the littermate control wild type（WT）mice,SKI mice showed pathological phenotypes of bronchial asthma,including:increased inflammatory cells in alveolar lavage fluid（BALF）,presence of large number of inflammatory cells surrounding the smooth muscle and fibroblasts layer increased airway epithelial goblet cell metaplasia and mucus secretion,thickening of airway epithelial layer and smooth muscle layer,and elevated collagen deposition under airway epithelium and around the airway,indicating that C674 inactivation is involved in the process of bronchial asthma.We performed the detection of bronchial asthma-related proteins and cell function analysis.In lung tissues,compared with WT mice,SKI mice:1)Increased expression of inflammation-related proteins increased,including phospho-p65-nuclear factor kappa B（p-p65NF-κB）,interleukin 5（IL-5）,IL-6,IL-1β,tumor necrosis factorα（TNF-α）,monocyte chemotactic factor 1（MCP-1）,intercellular adhesion factor 1（ICAM-1）and vascular cell adhesion factor 1（VCAM-1）;2)Increased expression of airway remodeling and cellular phenotypic transformation related proteins,includingα-smooth muscle actin（α-SMA）,calmodulin 1（calponin1）,transforming growth factorβ1（TGF-β1）,typeⅢcollagen（collagenⅢ）,matrix metalloproteinase 2（MMP2）and MMP9.In ASMC,compared with WT mice,SKI mice:1)The expression of SERCA2was not affected,but the concentration of cytoplasmic Ca²⁺was increased,and nuclear factor 4（NFAT4）and p65NF-κB nucleus entry,which are regulated by cytoplasmic Ca²⁺,was increased.2)Cell proliferation and migration capacity was increased;3)expression of airway inflammation,remodeling,and cell phenotype transformation related proteins were increased,including p-p65NFκB,IL-5,IL-6,IL-1β,TNF-α,MCP-1,ICAM-1,VCAM-1 and MMP2.In addition,we found that ROS production increased in ASMC and bronchus of SKI mice.These results indicate that the redox state of C674 in SERCA2 is critical for maintaining bronchial homeostasis,and its irreversible oxidative inactivation under pathological conditions induces the pathological phenotype of bronchial asthma,which is related to the regulation of airway inflammation and airway remodeling.Protecting C674 against oxidation or activation of SERCA2 is expected to improve bronchial asthma.