Cortactin在应力诱导的气道黏液分泌过程中的介导作用
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摘要
目的:探讨皮层肌动蛋白结合肽(Cortactin)对剪应力(SS)诱导的气道黏液高分泌的影响及其关键靶点。方法:体外培养气道上皮细胞Beas-2B,并随机分为6组:空白对照组、SS组、SS+Wild-Cortactin组、SS+pc DNA3-Cortactin~(421)-MU组、SS+pcDNA3-Cortactin~(466)-MU组、SS+pcDNA3-Cortactin~(482)-MU组。用Western blot法检测Cortactin蛋白、p-Cortactin蛋白表达水平,RT-PCR、ELISA法分别检测MUC5AC转录水平和MUC5AC的分泌量。结果:转染Cortactin各型重组表达载体后细胞Cortactin蛋白明显增加,提示转染成功。转染Wild-Cortactin后再予剪应力处理,细胞的p-Cortactin蛋白、MUC5AC mRNA及蛋白表达显著增加。而转染pc DNA3-Cortactin~(466)-MU后,细胞p-Cortactin蛋白、细胞培养上清液MUC5AC的相对分泌量显著下降,但MUC5AC的转录水平无明显影响。结论:在剪应力刺激诱导的气道上皮细胞MUC5AC高分泌过程中,Cortactin具有重要的介导作用,且Y466为其介导该过程的潜在关键作用靶点。
Objective: To explore the effects and pivotal site of Cortactin on secretion of mucin( MUC) 5 AC induced by the shear stress( SS) in airway epithelial cells. Methods: The airway epithelial cells( Beas-2 B) were cultured in vitro,and divided into 6 groups: Blank control group,SS group,SS + Wild-Cortactin group,SS + pc DNA3-Cortactin421-MU group,SS + pc DNA3-Cortactin~(466)-MU group,SS + pc DNA3-Cortactin~(482)-MU group. The relative content of Cortactin and phosphorylated Cortactin( p-Cortactin) protein was detected with Western blot,the content of MUC5 AC protein and transcription level of MUC5 AC were observed by ELISA and RT-PCR respectively. Results: After transfecting recombinant expression vectors of Cortactin,the levels of Cortactin were increased significantly,Which confirm Beas-2 B cells were transfected with each type of Cortactin successfully. After transfecting recombinant expression vectors of Wild-Cortactin,and then stimulated with shear stress( SS),the contents of p-Cortactin,MUC5 AC mRNA and MUC5 AC protein were evidently higher. Whereas transfecting pc DNA3-Cortactin~(466)-MU,the relative contents of p-Cortactin and MUC5 AC protein were obviously decreased,however,there was no significant effect in the transcriptional level of MUC5 AC mRNA. Conclusion: Cortactin plays a critical role in the process of MUC5 AC hypersecretion induced by shear stress( SS) in airway epithelial cells,and the phosphorylation site Y466 of Cortactin may be a potential key target for the process.
Objective: To explore the effects and pivotal site of Cortactin on secretion of mucin( MUC) 5 AC induced by the shear stress( SS) in airway epithelial cells. Methods: The airway epithelial cells( Beas-2 B) were cultured in vitro,and divided into 6 groups: Blank control group,SS group,SS + Wild-Cortactin group,SS + pc DNA3-Cortactin421-MU group,SS + pc DNA3-Cortactin~(466)-MU group,SS + pc DNA3-Cortactin~(482)-MU group. The relative content of Cortactin and phosphorylated Cortactin( p-Cortactin) protein was detected with Western blot,the content of MUC5 AC protein and transcription level of MUC5 AC were observed by ELISA and RT-PCR respectively. Results: After transfecting recombinant expression vectors of Cortactin,the levels of Cortactin were increased significantly,Which confirm Beas-2 B cells were transfected with each type of Cortactin successfully. After transfecting recombinant expression vectors of Wild-Cortactin,and then stimulated with shear stress( SS),the contents of p-Cortactin,MUC5 AC mRNA and MUC5 AC protein were evidently higher. Whereas transfecting pc DNA3-Cortactin~(466)-MU,the relative contents of p-Cortactin and MUC5 AC protein were obviously decreased,however,there was no significant effect in the transcriptional level of MUC5 AC mRNA. Conclusion: Cortactin plays a critical role in the process of MUC5 AC hypersecretion induced by shear stress( SS) in airway epithelial cells,and the phosphorylation site Y466 of Cortactin may be a potential key target for the process.
引文
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[9] Boyle SN,Michaud GA,Schweitzer B,et al. A critical role for cortactin phosphorylation by abl-family kinases in PDGF-induced dorsal-wave formation[J]. Curr Biol,2007,17(5):445-451.
[10] Tehrani S,Tomasevic N,Weed S,et al. Src phosphorylation of cortactin enhances actin assembly[J]. Proc Natl Acad Sci U S A,2007,104(29):11933-11938.
[11] Siton O,Ideses Y,Albeck S,et al. Cortactin releases the brakes in actin-based motility by enhancing WASP-VCA detachment from Arp2/3 branches[J]. Curr Biol,2011,21:2092-2097.
[2] Mac Grath SM,Koleske AJ. Cortactin in cell migration and cancer at a glance[J]. J Cell Sci,2012,125:1621-1626.
[3]王鹏,周向东,尤列.皮尔曼,等.气道剪应力作用下黏蛋白5AC胞外极向分泌的主要介导分子[J].中华医学杂志,2013,93(32):2587-2591.Wang P,Zhou XD,Perelman JM,et al. Main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion[J]. Chin Med J,2013,93(32):2587-2591.
[4]江德鹏,Kolosov VP,周向东,等.白介素13通过FOXA2调控气道上皮细胞黏液分泌[J].中国免疫学杂志,2011,27(2):99-102.Jiang DP,Kolosov VP,Zhou XD,et al. IL-13 regulates mucus secretion via FOXA2 in vitro[J]. Chin J Immunol,2011,27(2):99-102.
[5] Evans JV,Ammer AG,Jett JE,et al. Src binds cortactin through an SH2 domain cystine-mediated linkage[J]. J Cell Sci,2012,125(24):6185-6197.
[6] Mc Niven MA,Kim I,Krueger EW,et al. Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape[J]. J Cell Biol,2000,151(1):187-198.
[7] Shimada A,Niwa H,Tsujita K,et al. Curved EFC/F-BAR-domain dimers are joined end to end into a filament for membrane invagination in endocytosis[J]. Cell,2007,129(4):761-772.
[8] Tarran R,Button B,Boucher RC. Regulation of normal and cysticfibrosis airway surface liquid volume by phasic shear stress[J]. Annu Rev Physiol,2006,68(1):543-561.
[9] Boyle SN,Michaud GA,Schweitzer B,et al. A critical role for cortactin phosphorylation by abl-family kinases in PDGF-induced dorsal-wave formation[J]. Curr Biol,2007,17(5):445-451.
[10] Tehrani S,Tomasevic N,Weed S,et al. Src phosphorylation of cortactin enhances actin assembly[J]. Proc Natl Acad Sci U S A,2007,104(29):11933-11938.
[11] Siton O,Ideses Y,Albeck S,et al. Cortactin releases the brakes in actin-based motility by enhancing WASP-VCA detachment from Arp2/3 branches[J]. Curr Biol,2011,21:2092-2097.