| Objective:Milk fat globule epidermal growth factor 8（MFG-E8）participates in a range of cellular processes,including reducing apoptosis and oxidative stress.However,its protective activity against cigarette smoke-induced ferroptosis in the pathogenesis of chronic obstructive pulmonary disease（COPD）and the modulation of MFG-E8 remain unclear.This study has three objectives:1.To explore the expression of MFG-E8 in lung tissues of COPD patients and cigarette smoke extract（CSE）exposed mice and human bronchial epithelial cell line,and explore whether MFG-E8participates in the pathogenesis of COPD induced by CSE;2.To clarify the role of MFG-E8 in CSE-induced ferroptosis of bronchial epithelial cells;3.To explore the specific molecular biological mechanism of MFG-E8degradation via the ubiquitin-proteasome pathway involved in CSE-induced bronchial epithelial cell ferroptosis.Methods:1.Observe the protein expression and distribution of MFG-E8:Immunohistochemical staining（IHC）and Western blot（WB）were used to detect the expression and distribution of MFG-E8 in lung tissues of COPD patients and CSE exposed mouse model and human bronchial epithelial cell lines（BEAS-2B and HBE）.2.Study the effect of MFG-E8 knockout on lung tissues of CSE exposed mice:MFG-E8 knockout mice(KO,MFG-E8-/-)and wild-type mice(WT,MFG-E8+/+)were bred and were genotyped by agarose gel electrophoresis.The experimental mice were divided into four groups:wild-type mice（WT-Ctrl）,wild-type mice exposed to CSE（WT-CSE）,MFG-E8 knockout mice（KO-Ctrl）and MFG-E8 knockout mice exposed to CSE（KO-CSE）.The WT-CSE group and KO-CSE group were injected intraperitoneally with CSE at days 1,12,and 23,while the WT-Ctrl group and KO-Ctrl group were injected with PBS at days 1,12,and 23intraperitoneally.All mice were sacrificed to collect lung tissues on day 29.Hematoxylin eosin（HE）staining was used to observe the pathomorphological changes of lung tissues of mice in each group,and the mean linear intercept（MLI）and alveolar destructive index（DI）were measured.3.Study the effect of silencing MFG-E8 on ferroptosis of bronchial epithelial cells:Bronchial epithelial cell line stably silencing MFG-E8 was established to analyze the levels of oxidative stress and ferroptosis of cells in the control group and MFG-E8 silenced group.WB was used to detect the expression levels of antioxidant factors nuclear factor erythroid 2related factor 2（Nrf2）,NAD（P）H:quinone oxidoreductase 1（NQO1）and heme oxygenase-1（HO-1）,and antiferroptotic molecules glutathione peroxidase 4（GPx4）and solute carrier family 7 member 11（SLC7A11）.Real-time quantitative polymerase chain reaction（RT-q PCR）was used to detect the m RNA levels of GPx4 and SLC7A11.Superoxide dismutase（SOD）,malonaldehyde（MDA）and reactive oxygen species（ROS）assay kits were used to detect the activity of SOD and the levels of MDA and ROS in cells.The morphological changes of cells and mitochondria were observed with a transmission electron microscope.Cell counting kit-8（CCK-8）was used to detect the cell viability after the intervention of ferroptosis inducer RSL3.The level of intracellular lipid oxidation was detected by flow cytometry.4.Study the effect of recombinant human MFG-E8（rh MFG-E8）on CSE-induced ferroptosis of bronchial epithelial cells:We used rh MFG-E8to treat cells directly and the levels of oxidative stress and ferroptosis of cells in the control group,CSE group and CSE+rh MFG-E8 group were analyzed.WB was used to detect the expression levels of antioxidant factors Nrf2,NQO1 and HO-1,and antiferroptotic molecules GPx4 and SLC7A11.RT-q PCR was used to detect the m RNA levels of GPx4 and SLC7A11.SOD,MDA and ROS assay kits were used to detect the activity of SOD and the levels of MDA and ROS in cells.The morphological changes of cells and mitochondria were observed with a transmission electron microscope.CCK-8 was used to detect the effect of CSE,ferroptosis inhibitor Fer-1 and rh MFG-E8 alone or in combination on the cell viability.The intracellular iron content was measured with the iron assay kit.The level of intracellular lipid oxidation was detected by flow cytometry.5.Study the effect of MFG-E8 knockout on CSE-induced ferroptosis of bronchial epithelial cells in mice:The levels of oxidative stress and ferroptosis of lung tissues in the WT-Ctrl,WT-CSE,KO-Ctrl and KO-CSE groups were analyzed.WB was used to detect the expression levels of antioxidant factors Nrf2,NQO1 and HO-1,and antiferroptotic molecules GPx4 and SLC7A11.RT-q PCR was used to detect the m RNA levels of GPx4 and SLC7A11.IHC was used to further observe the expression and distribution of GPx4 and SLC7A11 in bronchial epithelial cells of lung tissue.SOD,MDA and myeloperoxidase（MPO）assay kits were used to detect the activities of SOD and MPO in lung tissue and the level of MDA.The morphological changes of mitochondria in cells of lung tissue were observed with a transmission electron microscope.Prussian blue staining was used to assess the iron accumulation in mouse bronchial epithelial cells.6.Study MFG-E8 deubiquitinated by ubiquitin-specific protease 14（USP14）in bronchial epithelial cells:The level of MFG-E8 RNA in COPD patients was analyzed by searching the GEO Profiles database,and the levels of MFG-E8 m RNA in lung tissues of COPD patients and CSE exposed mice and human bronchial epithelial cell lines were further detected by RT-q PCR to explore the effect of cigarette smoke on the level of MFG-E8 m RNA.After intervention with protein synthesis inhibitor cycloheximide（CHX）or proteasome inhibitor MG132,the levels of MFG-E8 protein at different time points were detected by WB.The expression of MFG-E8 was detected by WB after transfection of different Flag-tagged deubiquitinase overexpression plasmids into cells to screen the deubiquitinase that may target MFG-E8.USP14 overexpression cells and USP14 silenced cells were constructed by plasmid transfection.The stability of MFG-E8 was analyzed by WB detection of MFG-E8 protein expression at different time points after CHX intervention in the control cells and cells overexpressing/silencing USP14.Immunofluorescence（IF）staining and co-localization analyses and immunoprecipitation（IP）were used to investigate the protein interaction between USP14 and MFG-E8.The IP reaction of endogenous poly-ubiquitinated MFG-E8 protein was carried out by using the cells overexpressing/silencing USP14.Then,the ubiquitination of MFG-E8 protein was detected by using anti-Ub antibody to explore whether USP14 directly regulated MFG-E8 through deubiquitination.7.Study the effect of MFG-E8 stabilized by USP14 on CSE-induced ferroptosis of bronchial epithelial cells:WB and RT-q PCR were used to detect the effect of CSE on the expression of USP14 in mouse lung tissue and bronchial epithelial cells.To explore whether CSE could regulate ferroptosis by regulating MFG-E8 proteasomal degradation,WB was used to detect the expression of MFG-E8 and its downstream antiferroptotic molecules GPx4 and SLC7A11 in bronchial epithelial cells after intervention with CSE and/or MG132.To explore whether USP14 could regulate CSE-induced MFG-E8 proteasomal degradation and ferroptosis,the cells overexpressing/silencing USP14 were treated with CSE,and MG132 was used on the basis of CSE+USP14 si RNA treatment before the WB detection of MFG-E8,GPx4 and SLC7A11.To explore whether USP14 could regulate ferroptosis by regulating MFG-E8 after CSE exposure,USP14 si RNA and rh MFG-E8 were used to treat cells at the same time before the WB detection of GPx4 and SLC7A11.Results:1.MFG-E8 involved in the development of COPD induced by cigarette smoke:（1）IHC showed that MFG-E8 was widely distributed in bronchial epithelial cells,and the expression of MFG-E8 protein in lung tissues of COPD patients and mice exposed to CSE was significantly reduced.（2）WB showed that MFG-E8 protein levels were aberrantly decreased in the lung tissues of COPD patients and mice exposed to CSE.In human bronchial epithelial cell lines,CSE decreased MFG-E8 protein levels in a concentration-dependent manner.（3）HE staining showed that compared with the WT-Ctrl group,the MLI and DI values were significantly increased in the WT-CSE group,suggesting successful modeling of emphysema mice.These values were dramatically increased in the KO-CSE group compared with the WT-CSE group.2.Silencing MFG-E8 induced ferroptosis of bronchial epithelial cells:（1）WB and RT-q PCR showed that in BEAS-2B and HBE cells,the protein expression levels of Nrf2,NQO1,HO-1,GPx4 and SLC7A11 and the m RNA expression levels of GPx4 and SLC7A11 were significantly decreased after MFG-E8 silencing.（2）Silencing MFG-E8 significantly decreased the activity of SOD in bronchial epithelial cells and induced the increase of MDA and ROS levels.（3）The transmission electron microscope showed that MFG-E8knockdown led to mitochondrial shrinkage,membrane density increasing,and cristae rupture in BEAS-2B and HBE cells.（4）CCK-8 detection showed that MFG-E8 knockdown cells exhibited higher sensitivity to RSL3-induced ferroptosis.（5）Flow cytometry showed that MFG-E8 silencing apparently increased the lipid peroxidation in BEAS-2B and HBE cells.3.Rh MFG-E8 suppressed CSE-induced ferroptosis of bronchial epithelial cells:（1）WB and RT-q PCR showed that CSE significantly suppressed Nrf2,NQO1,HO-1,GPx4 and SLC7A11 expressions,which were dramatically reversed by pretreatment with rh MFG-E8.（2）CSE significantly decreased the activity of SOD in bronchial epithelial cells and induced the increase of MDA and ROS levels.After pretreatment with rh MFG-E8,the activity of SOD increased and the accumulation of peroxide products caused by CSE was also effectively reduced.（3）The transmission electron microscope showed that deformation and vacuolization of mitochondria with an increased membrane density were observed in CSE-treated cells,and these morphological alterations were markedly ameliorated in rh MFG-E8-pretreated cells.（4）CCK-8 detection showed that CSE significantly reduced cell viability,but the administration of ferroptosis inhibitor Fer-1 or rh MFG-E8decreased CSE-induced growth inhibition.（5）The measurement of iron showed that CSE increased the intracellular concentrations of iron,which could be reversed by pretreatment with rh MFG-E8.（6）Flow cytometry showed that pretreatment with rh MFG-E8 notably attenuated the lipid peroxidation induced by CSE.4.MFG-E8 knockout promoted CSE-induced ferroptosis in mouse lung tissues:（1）WB and RT-q PCR showed that both CSE exposure and MFG-E8knockout significantly inhibited Nrf2,NQO1,HO-1,GPx4 and SLC7A11expressions in the lung tissues of mice.The levels of these molecules were dramatically decreased in the KO-CSE group compared with the WT-CSE group.（2）IHC showed a lower expression of GPx4 and SLC7A11 in the KO-CSE group than that in the WT-CSE group.（3）CSE significantly reduced the activity of SOD,enhanced the activity of MPO,and induced the increase of MDA level in the lung tissues of mice.MFG-E8 knockout aggravated the imbalance between oxidation and antioxidation caused by CSE.（4）The transmission electron microscope showed that obvious shrinkage of mitochondria with an increased membrane density and reduction of mitochondrial cristae were observed in the WT-CSE group.MFG-E8 deficiency aggravated these morphological changes in CSE-exposed mice,accompanied by more rupture of mitochondrial cristae and membrane.（5）Prussian blue staining disclosed that the strongest staining of iron deposition,along with more stained areas in lung sections,were detected in the KO-CSE group.5.USP14 deubiquitinated MFG-E8 and stabilized MFG-E8:（1）Cigarette smoke did not alter MFG-E8 m RNA levels in vivo and in vitro:RNA expression data were downloaded from the GEO Profiles.MFG-E8 expression levels in small airway epithelium were similar between non-smokers and smokers.MFG-E8 expression levels in human lung tissues were similar between subjects with no or mild emphysema and those with severe emphysema.MFG-E8 RNA levels in sputum had no association with the severity of COPD.RT-q PCR further showed that there was no significant difference in the expression of MFG-E8 m RNA between COPD patients and the controls.In addition,CSE exposure showed no significant effect on MFG-E8 m RNA level in mouse lung tissues and two bronchial epithelial cell lines.These indicated that CSE probably regulated MFG-E8 expression at the post-transcriptional level.（2）MFG-E8 was degraded by the proteasome:In BEAS-2B cells,CHX diminished MFG-E8 expression in a time-dependent manner.MFG-E8 degradation could be rescued by the proteasome inhibitor MG132.Similar phenomena were also observed in HBE cells.（3）Deubiquitinase screen for the regulator of MFG-E8:Several deubiquitinases were overexpressed in BEAS-2B cells and the screen showed that USP14 remarkably upregulated the MFG-E8 level.USP14overexpression resulted in MFG-E8 elevation in a dose-dependent manner in both BEAS-2B and HBE cells.（4）USP14 maintained MFG-E8 stability:Using CHX to treat BEAS-2B and HBE cells,the MFG-E8 protein level decreased more slowly in USP14-overexpressed cells than in the control cells and the MFG-E8protein level reduced faster after silencing USP14.（5）USP14 interacted with MFG-E8:IF staining showed that USP14and MFG-E8 coexisted in the cytoplasm,suggesting that USP14-mediated MFG-E8 protein upregulation might mainly occur in the cytoplasm.IP showed that MFG-E8 and USP14 were readily co-immunoprecipitated with each other.MFG-E8 was detected in the anti-USP14immunoprecipitates,and in turn,USP14 was detected in the anti-MFG-E8immunoprecipitates.（6）USP14 deubiquitinated MFG-E8:Overexpression of USP14substantially decreased the ubiquitination of endogenous MFG-E8 in BEAS-2B and HBE cells.Conversely,depletion of endogenous USP14 in BEAS-2B and HBE cells dramatically elevated endogenous MFG-E8ubiquitination.6.MFG-E8 stabilized by USP14 suppressed CSE-induced ferroptosis in bronchial epithelial cells:（1）USP14 expression was downregulated after CSE exposure:WB and RT-q PCR showed that CSE significantly decreased USP14 m RNA and protein levels,both in a concentration-dependent manner.In the lung tissue of mice exposed to CSE,USP14 protein levels were also aberrantly decreased.（2）CSE induced the proteasomal degradation of MFG-E8:WB showed that the proteasome inhibitor MG132 prevented MFG-E8 proteins from CSE-induced degradation.（3）USP14 inhibited CSE-induced MFG-E8 proteasomal degradation:In both BEAS-2B and HBE cells,overexpression of USP14 significantly inhibited the reduction in MFG-E8 protein induced by CSE.Silencing of USP14 promoted the protein reduction of MFG-E8 in CSE-treated cells.Furthermore,the decreased MFG-E8 level in USP14 silenced cells under CSE treatment could be rescued by MG132.（4）USP14/MFG-E8 axis involved in CSE-induced ferroptosis in human bronchial epithelial cells:The CSE-regulated MFG-E8 level and its downstream signals were modulated by USP14.USP14 overexpression enhanced the promoting effects of MFG-E8 on GPx4 and SLC7A11expressions in bronchial epithelial cells treated with CSE.Suppression of USP14 enhanced the reduction in GPx4 and SLC7A11 levels induced by CSE.MG132 could also significantly reverse the decreased levels of MFG-E8 downstream signals in USP14 silenced cells exposed to CSE.USP14 si RNA and rh MFG-E8 were simultaneously applied to treat bronchial epithelial cells.WB showed that the decreased levels of GPx4and SLC7A11 induced by the depletion of USP14 could be rescued by the administration of rh MFG-E8 in CSE-treated cells,suggesting that USP14could regulate ferroptosis by regulating MFG-E8 after CSE exposure.Conclusions:1.MFG-E8 deficiency aggravates cigarette smoke-induced emphysema in the pathogenesis of COPD.2.MFG-E8 suppresses CSE-induced ferroptosis of bronchial epithelial cells.3.USP14 downregulated by CSE enhances MFG-E8 degradation via the ubiquitin-proteasome pathway and finally leads to bronchial epithelial cell ferroptosis. |
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