Mechanisms Of Neddylation In Regulating Epithelial Injury And Emphysema In COPD

BackgroundChronic obstructive pulmonary disease(COPD),which contributes significantly to chronic morbidity and mortality,is responsible for six percent of all death worldwide in 2012 and is the fourth leading cause of death now.The disease is characterized by longterm breathing problems and poor airflow.The poor airflow is the result of breakdown of lung tissue(known as emphysema),and small airways disease known as obstructive bronchiolitis.The relative contributions of these two factors vary between people.COPD is a progressive disease,meaning it typically worsens over time.The most common cause of COPD is cigarette smoking,with a smaller number due to factors such as air pollution and genetics.COPD develops as a significant and chronic inflammatory response to inhaled irritants.Chronic bacterial infections may also add to this inflammatory state.The inflammatory cells involved include neutrophil granulocytes and macrophages,two types of white blood cells.Those who smoke additionally have Tc1/Th1/Th17 lymphocyte involvement.Part of this cell response is brought on by inflammatory mediators such as chemotactic factors.Other processes involved with lung damage include oxidative stress produced by high concentrations of free radicals in cigarette smoke and released by inflammatory cells,and breakdown of the connective tissue of the lungs by proteases that are insufficiently inhibited by protease inhibitors.The destruction of the connective tissue of the lungs leads to emphysema,which then contributes to the poor airflow,and finally,poor absorption and release of respiratory gases.Neddylation is the process by which the ubiquitin-like protein NEDD8 is conjugated to its target proteins.This process is analogous to ubiquitination,although it relies on its own E1 and E2 enzymes.Few NEDD8-specific E3 has yet been identified and it is possible that the Neddylation system relies on ubiquitination E3 ligases with dual specificity.Evidences shows that increased NEDD8 conjugation in human lung carcinoma cells led to abnormal higher degrees of proliferation likely due to its important role in the regulation of the cell cycle.Pevonedistat(MLN4924),the novel selective NEDD8 inhibitor,is being investigated as a cancer treatment,including clinical trials against relapsed/refractory multiple myeloma or lymphoma,metastatic melanoma,and advanced solid tumors.Inflammatory responses play decisive roles at different stages of tumor development,including initiation,promotion,malignant conversion,invasion,and metastasis.Studies show that chronic inflammation including COPD,tuberculosis and pneumonia could promote lung cancer development while long-term usage of nonsteroidal antiinflammatory,like aspirin,could reduce the risks.Based on the above fact,we propose that neddylation-cullin-ring-ligase might have also been involved in certain chronic inflammatory disease including COPD.This study thus is aimed to investigate the role and mechanism of neddylation in regulating epithelial injury and emphysema in COPD.Part ? Role and mechanism of neddylation E2 UBE2 M in regulating pulmonary epithelial injury in COPD ObjectiveTo investigate the role and mechanisms of neddylation E2 UBE2 M in cigarette smoke-induced epithelial injury and mucus hyper-production.Methods1.Animal models/Human sampleSix to ten-week-old mice were exposed to cigarette smoke in a designed chamber to establish classic COPD mouse model.Lungs were harvested and neddylation pathway proteins were assayed using WB/IHC;Lung tissues of human COPD patients and health volunteers were obtained to assay the pathway;Airway specific inducible UBE2 M knockout mice were generated to test its response in a novel COPD bronchitis model.2.In vitro experimentsHBE were exposed to CSE to mimic cigarette smoke exposure in vitro;An NEDD8-selective inhibitor MLN4924 and small interfering RNAs were applied to impair neddylation pathway,and c-JUN-si RNA was used to defect AP-1 signaling.IL6 and MUC5 AC expression were assayed upon above manipulation to assess the potential roles of above pathways in CSE-induced epithelial injury.Results1.Nedd8 is widely expressed in lung tissue,and lung sections of COPD patients and cigarette smoke-exposed mice should an elevated expression of neddylation pathway proteins;2.Inhibited by MLN4924 or silenced by si RNA of neddylation pathway blocked CSE-induced inflammation and mucus hyper-production in HBE;3.Impairment of AP-1 signaling protected CSE-induced epithelial injury;4.Airway specific knockout of UBE2 M mice showed resistance to epithelial injury in a novel mouse model of COPD.ConclusionCigarette smoke upregulated the expression of neddylation pathway especially the level of UBE2 M.Neddylation promote CS/CSE-induced epithelial injury likely through activation of AP-1 signaling?Part ? Role and mechanism of neddylation E2 UBE2 M in regulating emphysema in COPDObjective To investigate the role and mechanisms of neddylation E2 UBE2 M in pulmonary emphysema development.Methods 1.RNA sequencing Myeloid-specific knockout of UBE2 M mice were generated by crossing Ube2mflox/flox with Lys MCre mice,bone marrow derived-macrophages(BMDMs)were cultured and then harvested to perform RNA sequencing to figure out COPD pathogenesis-related genes.2.Animal models/Human samples Six to ten-week-old mice were exposed to cigarette smoke in a designed chamber to establish classic COPD mouse model.Bronchoalveolar lavage fluid(BALF)were retrieved to harvest alveolar macrophages(AMs),and NEDD8 and UBE2 M protein levels were measured using WB/IF.BALF cells of human COPD patients and health volunteers were obtained to assay the pathway.3.In vitro experiments BMDMs,peritoneal macrophages(p-MACs)from myeloid-specific UBE2 Mdeficient mice were obtained;MLN4924 or PT2385 treated mouse alveolar macrophage cell line MH-S and human monocyte derived macrophage cell line THP-1 were used along with BMDMs and p-MACs to test the regulatory axis among UBE2 M,Hypoxia inducible factor(HIF)-2? and Matrix metalloproteinase(MMP)12.Results 1.RNA sequencing revels an up-regulation of MMP12,which is tightly related to COPD pathogenesis,upon UBE2M-depletion.2.Myeloid-specific UBE2M-deficient mice exhibited spontaneous pulmonary emphysema.3.BALF cells from both COPD patients and cigarette smoke-exposed mice present a decreased UBE2 M expression compared to healthy volunteers and air controls.4.Neddylation regulated MMP12 likely through its downstream substrate HIF-2? to maintain homeostasis.