皮层肌动结合蛋白不同位点磷酸化对人气道上皮细胞黏蛋白5AC分泌的影响
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摘要
目的:了解皮层肌动结合蛋白(cortical actin-binding protein,又称cortactin)在人气道上皮细胞黏蛋白(mucin,MUC)5AC分泌中的作用及不同磷酸化位点对其的影响。方法:培养人气道上皮细胞HBE16,以皮层肌动结合蛋白不同磷酸化位点突变载体转染细胞,并分为空白对照组、剪应力刺激组、剪应力+增强绿色荧光蛋白质粒(enhanced greenfluorescentproteinplasmid,p EGFP)-N1组、剪应力+p EGFP-N1-cortactin(Cort)组、剪应力+p EGFP-N1-Cort-Y421A组、剪应力+p EGFP-N1-Cort-Y470A组及剪应力+p EGFP-N1-Cort-Y486A组,剪应力设定为4 dynes/cm~2。采用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)、real-time PCR法测定转染前后各组细胞及培养上清中MUC5AC蛋白和m RNA水平,Western印迹检测各组皮层肌动结合蛋白及磷酸化皮层肌动结合蛋白含量;异硫氰酸荧光素(fl uorescein isothiocyanate,FITC)标记的鬼笔环肽染色观察丝状肌动蛋白(fi lamentous actin,F-actin)的分布情况。结果:4 dynes/cm~2剪应力刺激30 min后,细胞中磷酸化皮层肌动结合蛋白表达增加,在2 h时表达水平最高;剪应力刺激2 h后,MUC5AC m RNA表达增强,细胞及培养上清中MUC5AC的含量与空白对照组相比,各组均显著增加(均P<0.05);与剪应力刺激组相比,剪应力+p EGFP-N1-cortactin(Cort)组的细胞培养上清液中MUC5AC蛋白含量显著增加,F-actin在胞膜的分布也明显增多(均P<0.05),而胞质内MUC5AC蛋白及MUC5AC m RNA水平变化不大。与剪应力+p EGFP-N1-Cort组相比,剪应力+p EGFP-N1-Cort-Y421A组、剪应力+p EGFP-N1-Cort-Y470A组培养上清液中的MUC5AC蛋白含量有明显降低,F-actin在胞膜的聚集也有所减少(均P<0.05),而剪应力+p EGFP-N1-Cort-Y486A组中MUC5AC含量变化不明显(P>0.05)。结论:皮层肌动结合蛋白参与了剪应力诱导产生的人气道上皮细胞MUC5AC蛋白分泌,Tyr421和Tyr470位点磷酸化在其中可能起重要的作用。
Objective: To explore the role of cortical actin-binding protein(cortactin) in shear stress-induced mucin(MUC) 5 AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin(Cort), pEGFP-N1-Cort-Y421 A, pEGFP-N1-Cort-Y470 A and p EGFP-N1-Cort-Y486 A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFPN1-Cort-Y421 A group, a shear stress + pEGFP-N1-Cort-Y470 A group, and a shear stress + pEGFPN1-Cort-Y486 A group. The shear stress were set at 4 dynes/cm~2. The levels of MUC5 AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay(ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate(FITC)-phalloidin.Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm~2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5 AC protein production and mRNA expression in the different experiment groups(all P<0.05). Compared with single shear stress-stimulated group, MUC5 AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group(both P<0.05), while there were no changes in the MUC5 AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5 AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421 A group and the shear stress + pEGFPN1-Cort-Y470 A group(both P<0.05), while there was no significant effect in the shear stress + p EGFP-N1-Cort-Y486 A group(P>0.05).Conclusion: Cortactin is involved in shear stress-mediated MUC5 AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.
Objective: To explore the role of cortical actin-binding protein(cortactin) in shear stress-induced mucin(MUC) 5 AC secretion in human airway epithelial cells and the effect of phosphorylation of cortactin at different sites.Methods: HBE16 airway epithelial cells were cultured, and then transfected with mutation carrier, such as pEGFP-N1-cortactin(Cort), pEGFP-N1-Cort-Y421 A, pEGFP-N1-Cort-Y470 A and p EGFP-N1-Cort-Y486 A. The cells were divided into a normal control group, a shear stress group, a shear stress + pEGFP-N1 group, a shear stress + PEGFP-N1-Cort group, a shear stress + pEGFPN1-Cort-Y421 A group, a shear stress + pEGFP-N1-Cort-Y470 A group, and a shear stress + pEGFPN1-Cort-Y486 A group. The shear stress were set at 4 dynes/cm~2. The levels of MUC5 AC protein and mRNA in cells and culture supernatant were assayed with enzyme-linked immunosorbent assay(ELISA) and real-time PCR. The cortactin and phosphorylated cortactin were detected by Western blot. F-actin was stained by fluorescein isothiocyanate(FITC)-phalloidin.Results: There was an obvious increase of phosphorylated cortactin in cells exposed to 4 dynes/cm~2 of shear stress for 30 min, which reached climax at 2 hours concomitant with elevation of MUC5 AC protein production and mRNA expression in the different experiment groups(all P<0.05). Compared with single shear stress-stimulated group, MUC5 AC in supernatant was increased obviously, and the distribution of F-actin in cytomembrane was also increased in the pEGFP-N1-Cort group(both P<0.05), while there were no changes in the MUC5 AC protein and mRNA levels in cytoplasm. Compared with the shear stress+pEGFP-N1-Cort group, the MUC5 AC protein in the culture supernatant was decreased, and the polymerization of F-actin at cell membranes were also attenuated in the shear stress+pEGFP-N1-Cort-Y421 A group and the shear stress + pEGFPN1-Cort-Y470 A group(both P<0.05), while there was no significant effect in the shear stress + p EGFP-N1-Cort-Y486 A group(P>0.05).Conclusion: Cortactin is involved in shear stress-mediated MUC5 AC secretion in human airway epithelial cells, and the phosphorylated site of Tyr421 and Tyr470 may play an important role in it.
引文
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[7]Yin M,Ma WQ,An LG.Cortactin in cancer cell migration and invasion[J].Oncotarget,2017,8(50):88232-88243.
[8]Tarran R,Button B,Picher M,et al.Normal and cystic fibrosis airway surface liquid homeostasis.The effects of phasic shear stress and viral infections[J].J Biol Chem,2005,280(42):35751-35759.
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[12]Molladavoodi S,Robichaud M,WulffD,et al.Corneal epithelial cells exposed to shear stress show altered cytoskeleton and migratory behaviour[J].PLoS One,2017,12(6):e0178981.
[13]Sidhaye VK,Schweitzer KS,Caterina MJ,et al.Shear stress regulates aquaporin-5 and airway epithelial barrier function[J].Proc Natl Acad Sci USA,2008,105(9):3345-3350.
[14]Friedrich C,Endlich N,Kriz W,et al.Podocytes are sensitive to fluid shear stress in vitro[J].Am J Physiol Renal Physiol,2006,291(4):856-865.
[15]Tarran R,Button B,Picher M,et al.Normal and cystic fibrosis airway surface liquid homeostasis.The effects of phasic shear stress and viral infections[J].J Biol Chem,2005,280(42):35751-35759.
[16]Schnoor M,Stradal TE,Rottner K.Cortactin:Cell functions of amultifaceted actin-binding protein[J].Trends Cell Biol,2018,28(2):79-98.
[17]Kellye CK,Bong HS,Seema S,et al.Cortactin:A multifunctional regulator of cellular invasiveness[J].Cell Adh Migr,2011,5(2):187-198.
[18]Baumer Y,Drenckhahn D,Waschke J.AMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium[J].Histochem Cell Biol,2008,129(6):765-778.
[19]Muriel O,Tomas A,Scott CC,et al.Moesin and cortactin control actin-dependent multivesicular endosome biogenesis[J].Mol Biol Cell,2016,27(21):3305-3316.
[20]Huang C,Liu J,Haudenschild CC,et al.The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells[J].J Biol Chem,1998,273(40):25770-25776.
[21]Buday L,Downward J.Roles of cortactin in tumor pathogenesis[J].Biochim Biophys Acta,2007,1775(2):263-273.
[22]Head JA,Jiang D,Li M,et al.Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton[J].Mol Biol Cell,2003,14(8):3216-3229.
[23]Hammer A,Laghate S,Diakonova M.Src tyrosyl phosphorylates cortactin in response to prolactin[J].Biochem Biophys Res Commun,2015,463(4):644-649.
[24]Evans JV,Ammer AG,Jett JE,et al.Src binds cortactin through an SH2domain cystine-mediated linkage[J].J Cell Sci,2012,125(Pt24):6185-6197.
[25]Rosenberg BJ,Gil-Henn H,Mader CC,et al.Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells[J].Mol Biol Cell,2017,289(10):1347-1360.
[26]Martin C,Frija-MassonJ,Burgel PR.Targeting mucus hypersecretion:new therapeutic opportunities for COPD?[J].Drugs,2014,74(10):1073-1089.
[27]Saco TV,Breitzig MT,Lockey RF,et al.Epigenetics of mucus hypersecretion in chronic respiratory diseases[J].Am J Respir Cell Mol Biol,2018,58(3):299-309.
[28]李升锦,周向东.人MUC5AC基因启动子荧光素酶报告基因载体的构建及其转录活性分析(英文)[J].中南大学学报(医学版),2010,35(8):792-799.LI Shengjin,ZHOU Xiangdong.Construction of luciferase reporter gene vector for human MUC5AC gene promoter and analysis of its transcriptional activity[J].Journal of Central South Unvierstiy.Medical Science,2010,35(8):792-799.
[2]Hauber HP,Foley SC,Hamid Q.Mucin overproduction in chronic inflammatory lung disease[J].Can Respir J,2006,13(6):327-335.
[3]Ridley C,Thornton DJ.Mucins:the frontline defence of the lung[J].Biochem Soc Trans,2018,46(5):1099-1106.
[4]Weed SA,Parsons JT.Cortactin:coupling membrane dynamics to cortical actin assembly[J].Oncogene,2001,20(44):6418-6434.
[5]Kirkbride KC,Sung BH,Sinha S,et al.Cortactin:a multifunctional regulator of cellular invasiveness[J].Cell Adh Migr,2011,5(2):187-198.
[6]Liu CY,Li Q,Zhou XD,et al.Cortactin mediates elevated shear stress-induced mucin hypersecretion via actin polymerization in human airway epithelial cells[J].Int J Biochem Cell Biol,2013,45(12):2756-2763.
[7]Yin M,Ma WQ,An LG.Cortactin in cancer cell migration and invasion[J].Oncotarget,2017,8(50):88232-88243.
[8]Tarran R,Button B,Picher M,et al.Normal and cystic fibrosis airway surface liquid homeostasis.The effects of phasic shear stress and viral infections[J].J Biol Chem,2005,280(42):35751-35759.
[9]Evans DJ,Green AS,Thomas NK.Wall shear stress distributions in a model of normal and constricted small airways[J].Proc Inst Mech Eng H,2014,228(4):362-370.
[10]Green AS.Modelling of peak-flow wall shear stress in major airways of the lung[J].J Biomech,2004,37(5):661-667.
[11]Birukov KG,Birukova AA,Dudek SM,et al.Shear stress-mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells[J].Am J Respir Cell Mol Biol,2002,26(4):453-464.
[12]Molladavoodi S,Robichaud M,WulffD,et al.Corneal epithelial cells exposed to shear stress show altered cytoskeleton and migratory behaviour[J].PLoS One,2017,12(6):e0178981.
[13]Sidhaye VK,Schweitzer KS,Caterina MJ,et al.Shear stress regulates aquaporin-5 and airway epithelial barrier function[J].Proc Natl Acad Sci USA,2008,105(9):3345-3350.
[14]Friedrich C,Endlich N,Kriz W,et al.Podocytes are sensitive to fluid shear stress in vitro[J].Am J Physiol Renal Physiol,2006,291(4):856-865.
[15]Tarran R,Button B,Picher M,et al.Normal and cystic fibrosis airway surface liquid homeostasis.The effects of phasic shear stress and viral infections[J].J Biol Chem,2005,280(42):35751-35759.
[16]Schnoor M,Stradal TE,Rottner K.Cortactin:Cell functions of amultifaceted actin-binding protein[J].Trends Cell Biol,2018,28(2):79-98.
[17]Kellye CK,Bong HS,Seema S,et al.Cortactin:A multifunctional regulator of cellular invasiveness[J].Cell Adh Migr,2011,5(2):187-198.
[18]Baumer Y,Drenckhahn D,Waschke J.AMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium[J].Histochem Cell Biol,2008,129(6):765-778.
[19]Muriel O,Tomas A,Scott CC,et al.Moesin and cortactin control actin-dependent multivesicular endosome biogenesis[J].Mol Biol Cell,2016,27(21):3305-3316.
[20]Huang C,Liu J,Haudenschild CC,et al.The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells[J].J Biol Chem,1998,273(40):25770-25776.
[21]Buday L,Downward J.Roles of cortactin in tumor pathogenesis[J].Biochim Biophys Acta,2007,1775(2):263-273.
[22]Head JA,Jiang D,Li M,et al.Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton[J].Mol Biol Cell,2003,14(8):3216-3229.
[23]Hammer A,Laghate S,Diakonova M.Src tyrosyl phosphorylates cortactin in response to prolactin[J].Biochem Biophys Res Commun,2015,463(4):644-649.
[24]Evans JV,Ammer AG,Jett JE,et al.Src binds cortactin through an SH2domain cystine-mediated linkage[J].J Cell Sci,2012,125(Pt24):6185-6197.
[25]Rosenberg BJ,Gil-Henn H,Mader CC,et al.Phosphorylated cortactin recruits Vav2 guanine nucleotide exchange factor to activate Rac3 and promote invadopodial function in invasive breast cancer cells[J].Mol Biol Cell,2017,289(10):1347-1360.
[26]Martin C,Frija-MassonJ,Burgel PR.Targeting mucus hypersecretion:new therapeutic opportunities for COPD?[J].Drugs,2014,74(10):1073-1089.
[27]Saco TV,Breitzig MT,Lockey RF,et al.Epigenetics of mucus hypersecretion in chronic respiratory diseases[J].Am J Respir Cell Mol Biol,2018,58(3):299-309.
[28]李升锦,周向东.人MUC5AC基因启动子荧光素酶报告基因载体的构建及其转录活性分析(英文)[J].中南大学学报(医学版),2010,35(8):792-799.LI Shengjin,ZHOU Xiangdong.Construction of luciferase reporter gene vector for human MUC5AC gene promoter and analysis of its transcriptional activity[J].Journal of Central South Unvierstiy.Medical Science,2010,35(8):792-799.