P120和IQGAP1在气道上皮损伤修复过程中的作用研究
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摘要
实验背景
气道上皮是呼吸系统对抗有害刺激的第一道防线,各种病原微生物在机体抵抗力低下时均可引起肺部感染性疾病。其中以革兰阴性菌细胞壁的主要成分脂多糖(lipopolysaccharide, LPS)为主要致病因素,不仅引起肺部炎症反应,还可使原有肺部疾病发展和加重。气道上皮在致病因素的攻击下受到损伤,随后启动自身修复机制。LPS致气道上皮细胞损伤后,可促使细胞发生坏死、凋亡和分泌前炎症因子等反应,这些反应共同推动肺损伤的发展过程。大量研究证明,核因子-κB (nuclear factor, NF-κB)信号途径参与调节细胞生长、分化、增殖、存活、凋亡及炎症反应过程中多种基因的转录,与LPS引起的肺损伤密切相关。然而,LPS导致肺部炎症反应的分子机制还远未阐明。
NF-κB在正常情况下与其抑制蛋白ⅠκB结合以无活性的形式存在于静息细胞胞浆中。外界刺激可引起ⅠκB被迅速磷酸化、泛素化而降解,从而释放NF-κB活性亚单位转位入核,激活下游靶基因转录。P120-catenin(p120)是连环素家族成员,可在细胞膜上与上皮钙粘蛋白(E-Cadherin, E-Cad)相互结合而调节细胞粘附。近年来有研究表明,p120通过调节NF-κB活性参与表皮炎症反应。我们推测在气道炎症反应中,p120也可能通过NF-κB信号途径发挥调节作用。
实验目的
本实验使用脂多糖刺激建立体外气道炎症反应模型,初步探讨脂多糖刺激后气道上皮细胞中p120的表达变化及其对NF-κB信号通路的影响,从而进一步分析气道炎症发生发展的机制。
实验方法
使用脂多糖刺激建立体外气道炎症反应模型；采用免疫荧光共聚焦成像、Western blot、核浆分离方法检测p120、NF-κB、IκBα等蛋白表达及定位变化；采用荧光定量PCR、定量酶联免疫吸附实验(ELISA)检测IL-8表达变化；采用荧光素酶报告基因分析的方法检测NF-κB的活性；通过瞬时转染及小干扰RNA技术改变细胞中p120的表达量再检测p120对NF-κB信号途径的影响。
实验结果
(1)细胞瞬时转染NF-κB报告质粒后,报告基因分析结果显示,LPS刺激可引起气道上皮细胞中NF-κB信号的活化；同时荧光定量PCR和ELISA结果显示NF-κB靶基因IL-8--一种前炎症因子的表达亦有明显增多。
(2)p120在正常气道上皮细胞中含量丰富,而LPS刺激后,其表达迅速下调,早在15 min时即可表现出来。基于以上两点,我们推测在LPS所致气道炎症反应中,p120与NF-κB信号途径可能存在某种联系。接下来,我们先探讨NF-κB信号活化的机制。
(3)实验发现,在LPS刺激15 min-30 min时,NF-κB的抑制蛋白IκBα迅速降解至几近消失；其磷酸化形式在正常细胞中几乎检测不到,但在LPS处理15 min-30 min这段时间内含量显著增加,在IκBα恢复正常水平后,p-IKBa又再次消失不见。
(4)核浆分离试验及免疫荧光共聚焦结果均显示LPS促使NF-κB活性亚单位p65发生核内转位。
结果(3)和(4)表明,LPS在气道上皮细胞中引起的NF-κB活化是通过使IκBa发生磷酸化降解,从而导致p65由NF-κB/IκB复合体中释放并转位入核激活相应靶基因转录。接下来,我们通过对细胞转染p120质粒或小干扰RNA,研究p120对NF-κB信号活化的影响。
(5)实验结果显示,过表达p120可以部分抑制NF-κB的活性及IL-8的表达,但免疫荧光及核浆分离结果依然可以观测到p65核转位。
(6)小干扰RNA敲低p120的表达后,免疫荧光结果显示,LPS可引起p65更强烈的核染色；同时核浆分离产物的免疫印迹结果也证实阻断p120表达可进一步促进LPS诱导的p65核转位。
(7)阻断p120的表达可大大提升NF-κB的转录活性及其靶基因IL-8的表达。
实验结论
本实验表明：
(1)LPS可导致气道上皮细胞中p120表达下调。
(2)LPS可诱导气道上皮细胞NF-κB信号的活化及其靶基因IL-8的表达。
(3) NF-κB信号途径的活化是通过IκBα的磷酸化降解释放p65入核实现的。
(4)过表达p120可部分抑制NF-κB信号的活化；敲低p120能显著促进NF-κB信号的活化。
本实验提示：
在LPS引起的气道炎症反应中,p120可能负性调节NF-κB信号途径的活化,
从而抑制气道炎症反应。
实验背景
大量研究表明,吸烟可从多个方面损伤气道上皮细胞,更是导致慢性阻塞性肺疾病(COPD)和支气管源性肺癌发生发展的确切因素。然而,吸烟致气道上皮损伤的具体机制仍未完全阐明。上皮细胞损伤后,立即启动自我修复过程。这是一个步骤繁杂的过程,包括细胞延伸、迁移和增殖。而细胞间粘附的改变表现在损伤修复的初始阶段,因此对细胞粘附的研究显得非常有意义。
已有研究证实,IQGAP1参与许多生命活动,是一个多功能蛋白。作为Rho家族GTPases成员Racl和Cdc42的一个重要的效应因子,IQGAP1不仅参与调节细胞骨架,影响细胞粘附、极化和迁移,还可能介导β-catenin/TCF信号的转录活化,从而引起其下游细胞增殖相关基因的表达。有研究显示IQGAP1可能通过P-catenin调节细胞粘附,然而具体机制并不明了。由于IQGAP1可与作为细胞粘附成分和Wnt信号通路成员的β-catenin结合,因此研究IQGAP1在损伤修复中的作用就显得尤为重要。有报导指出,IQGAP1在肺组织中高表达。我们推测IQGAP1在气道上皮损伤修复中可能发挥作用,并探讨其可能机制。
实验目的
通过建立香烟烟雾提取物(CSE)损伤气道上皮的体外模型,观察IQGAP1在损伤修复过程中的表达及定位变化及其对细胞粘附复合体成员β-catenin的影响,初步研究其对气道上皮损伤修复中细胞粘附改变的影响,并为进一步探讨细胞增殖修复提供依据。
实验方法
使用CSE建立体外气道上皮损伤模型,首先使用相差显微镜观察气道上皮细胞在CSE刺激下的形态学变化,同时采用MTT方法检测不同浓度下细胞活力的变化。接着应用Western blot、免疫荧光共聚焦成像、免疫沉淀等技术观察CSE刺激后IQGAP1的表达、定位变化及其对细胞粘附复合体的影响。最后通过瞬时转染野生型IQGAP1和小干扰RNA,采用核浆分离等方法检测过表达或敲低IQGAP1对β-catenin的影响。
实验结果
(1)在相差显微镜下观察,可见培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态,立体感强,微凸起,细胞间连接紧密。在CSE损伤后,细胞伸展扁平,细胞间间隙增宽,伸出突起相接触,更高浓度的CSE致使细胞皱缩变圆,死亡增加。
(2)MTT结果显示,低浓度CSE可促进细胞增殖,而高浓度则引起细胞活力降低。
(3)采用Western blot和免疫荧光技术,我们发现,CSE促使气道上皮细胞中IQGAP1呈现时间依赖性过表达,且其定位信号由细胞连接处扩散至整个胞浆。更为有意义的是,我们通过免疫荧光和免疫印迹发现IQGAP1可与β-catenin结合,CSE刺激后,这种结合变得更强,而β-catenin与a-catenin的结合锐减。
(4)进一步通过使用瞬时转染技术,我们发现IQGAP1过表达不仅可以增加β-catenin在细胞内聚集,还可以促使积聚的β-catenin转位入核。而用干扰RNA阻断IQGAP1的表达后,β-catenin在胞浆内的聚集减少,其入核也受到影响。
实验结论本实验表明：
(1)CSE可在体外诱导气道上皮细胞过表达IQGAP1。
(2)CSE减弱β-catenin与粘附复合体中a-catenin的结合,但加强其与IQGAP1的结合
(3)过表达IQGAP1促使β-catenin在胞浆中积聚并进而转位入核。本实验提示：
IQGAP1通过与α-catenin竞争结合β-catenin,破坏细胞粘附复合体,从而减弱细胞粘附,启动CSE损伤的气道上皮细胞修复,并促进β-catenin转位入核,为下一步增殖修复损伤做准备。
Background
As the first line to defence harmful stimuli, the airway is frequently injured because of its exposure to the external environment. Some pathogenic factors may cause airway inflammation, such as lipopolysaccharide (LPS), which is the main component of the cell wall of Gram-negative bacteria. LPS may promotes several cellular processes including necrosis, apoptosis, and secretion of pro-inflammatory cytokines, which contribute to the development of lung injury. LPS is a potent activator that induces inflammatory gene expression through nuclear factor-KB (NF-κB) activation. NF-κB is a transcription factor expressed ubiquitously, which could be activated by LPS in various cells.
NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor of NF-κB (IκB) and remains transcriptionally inactive. Stimulation by triggers such as LPS induces the ubiquitylation and degradation of IκB. The loss of IκB exposes the nuclear localization signal sequence on NF-κB, resulting in the nuclear translocation of NF-κB and transcriptional activation of its target gene promoters. P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, which is involved in maintaining the stability and regulating the turnover of E-cadherin. Recent studies revealed that loss of p120 was associated with NF-κB activation and inflammation in p120 null epidermal cells. This discovery raised an interest in the pathophysiology of the diseases in which NF-κB activation is involved. Although airway inflammation has been extensively studied, it is uncertain whether p120 partcipates in airway inflammation through NF-κB signaling pathway. Therefore, our present studies focused on the effects of p120 on NF-κB signaling during the lung injury induced by LPS.
Objective
To investigate the changes of p120 expression and the effects of p120 on NF-κB signaling pathway during the inflammatory response induced by LPS.
Methods
In this study, we treated cells with LPS to establish a lung inflammation model in vitro. Using confocal immunofluorescence imaging, Western blot, isolation of cytoplasmic and nuclear proteins, we observed and examined the localizations and expressions of p120, NF-κB and IκBα. Then we detected the expressions of IL-8 by fluorescence quantitative PCR and enzyme-linked immunosorbent assay. Luciferase reporter analysis was used to detect the activity of NF-κB. Finally, transient transfection and small interfering RNA were used to over-expression or knock down p120, and then the effects of p120 on NF-κB signaling pathway were detected.
Results
(1) LPS induced the activation of NF-κB signaling in bronchial epithelial cells (BECs) by transient transfection and luciferase reporter assay. Meanwhile, IL-8, a proinflammatory factor, which is the target gene of NF-κB was also increased significantly after LPS treatment.
(2) Western blot showed that p120 was rich in BECs, but rapidly reduced by LPS as early as 15 min.
(3) It was found that IκBαwas rapidly phosphorylated and degradated by LPS from 15 to 30 min.
(4) The translocation of p65 from cytoplasm to nucleus after LPS treatment was confirmed by western blot and immunofluorescence.
(5) After cells transfected with pEGFPp120 followed by LPS treatment, we found the activaity of NF-κB induced by LPS was partially blocked. Data also showed that IL-8 production in response to LPS was partly, but not completely down-regulated by over-expression of p120.
(6) Inhibition of p120 by siRNA significantly enhanced the LPS-induced NF-κB activity, promoted LPS-induced p65 nuclear translocation and elevated IL-8 production.
Conclusions
(1) LPS induced the up-regulation of p120 in BECs.
(2) LPS induced NF-κB activation and IL-8 production.
(3) The activation of NF-κB induced by LPS was accompanied with IκBαdegradation and p65 nuclear translocation in BECs.
(4) The activation of NF-κB signaling was partially inhibited by over-expression of p120, but could be significantly promoted by knocking down of p 120.
These data strongly suggest that NF-κB activation is one of the cytoplasmic-nuclear signaling pathways involved in airway epithelial cells in response to LPS, and p120 is able to inhibit this activation.
Background
Numerous studies showed that cigarette smoke have various injurious effects on epithelial cells, which may contribute to the development of lung diseases including chronic obstructive pulmonary disease (COPD) and bronchogenic lung cancer. After injury, the airway epithelium initiates a wound repair process to keep normal function, which requires spreading, migration, and eventually proliferation into the injured area. However, the specific molecular mechanisms involved in the injury and repair of airway epithelium induced by smoking have not been fully understood. Studies for cell adhesion are very meaningful due to its performance in the initial stage of wound healing.
IQ domain GTPase-activating protein 1 (IQGAP1) is a multifunctional protein and could interacts with cytoskeleton, cell adhesion complex and microtubule associated proteins (MAPs). Therefore, IQGAP1 was considered to play roles in cell adhesion, polarization and migration. It has also been confirmed that IQGAP1 mediated the transcriptional activation ofβ-catenin/Tcf signaling and the expression of downstream target gene involved in cell proliferation. All the studies showed an important role of IQGAP1 in injury repair. Due to its highly expression in lung tissue, we speculated that IQGAP1 may regulate the process of airway epithelial injury and repair, and we tried to explore its possible mechanism.
Objective
To determine the dynamic expression of IQGAP1 and the effects of IQGAP1 on cell adhesion complex during the injury and repair process following CSE exposure, and to provide more evidences for further exploring the mechanism of injury and repair.
Methods
In this study, we established a model of injury and repair of airway epithelia by CSE in vitro. Firstly, we used the phase contrast microscope to observe the morphological changes of bronchial epithelial cells (BECs) after CSE treatment. Furthermore, MTT analysis was used to reveal the changes of cell viability after CSE treatment. Then, the following experiments were performed:Western blot, confocal laser scanning, co-immunoprecipitation. We aimed to observe the expression and localization of IQGAP1, and its impact on cell adhesion complex. Finally, transient transfection and isolation of cytoplasmic and nuclear proteins was used to detect the effects of IQGAP1 onβ-catenin.
Results
(1) Under phase contrast microscope, cells display morphological changes after CSE treatment, including widened cell-cell interspaces, more widely flattened appearance and more dead cells, compared with control cells, which showed a classic cobblestone-like epithelial morphology that was three-dimensional, slightly raised and closely adherent.
(2) MTT results showed that low concentrations of CSE can promote cell proliferation, while high concentrations lead to a dose-dependent reduction of cell viability.
(3) Western blot and immunofluoresence showed that CSE induced a time-dependent up-regulation of IQGAP1 in the airway epithelial cells and its positioning signal translocated from the cell-cell junctions to the whole cytoplasm, respectively. More meaningfully, we discovered that IQGAP1 combined withβ-catenin by immunofluorescence and Western blot. Such integration had become stronger after CSE stimulation, as well as the combination betweenβ-catenin and a-catenin was sharply reduced.
(4) Over-expression of IQGAP1 by transient transfection not only increased the accumulation ofβ-catenin in the cell, but also promoted the process of its nuclear translocation. After the expression of IQGAP1 was knocked down by small interfering RNA(siRNA), the accumulation and nuclear translocation of P-catenin were reduced.
Conclusions
(1) CSE induced up-regulation of IQGAP1 in vitro of airway epithelial cells.
(2) CSE attenuated the combination betweenβ-catenin and a-catenin, but enhanced the association between P-catenin and IQGAP1.
(3) over-expression of IQGAP1 promoted the accumulation and nuclear translocation ofβ-catenin.
The results suggest that IQGAP1 may weakens cell adhesion by binding to P-catenin and leading to the release of a-catenin from adhesion complex. Moreover, IQGAP1 may plays roles in cell proliferation by promoting the nuclear translocation ofβ-catenin.
气道上皮是呼吸系统对抗有害刺激的第一道防线,各种病原微生物在机体抵抗力低下时均可引起肺部感染性疾病。其中以革兰阴性菌细胞壁的主要成分脂多糖(lipopolysaccharide, LPS)为主要致病因素,不仅引起肺部炎症反应,还可使原有肺部疾病发展和加重。气道上皮在致病因素的攻击下受到损伤,随后启动自身修复机制。LPS致气道上皮细胞损伤后,可促使细胞发生坏死、凋亡和分泌前炎症因子等反应,这些反应共同推动肺损伤的发展过程。大量研究证明,核因子-κB (nuclear factor, NF-κB)信号途径参与调节细胞生长、分化、增殖、存活、凋亡及炎症反应过程中多种基因的转录,与LPS引起的肺损伤密切相关。然而,LPS导致肺部炎症反应的分子机制还远未阐明。
NF-κB在正常情况下与其抑制蛋白ⅠκB结合以无活性的形式存在于静息细胞胞浆中。外界刺激可引起ⅠκB被迅速磷酸化、泛素化而降解,从而释放NF-κB活性亚单位转位入核,激活下游靶基因转录。P120-catenin(p120)是连环素家族成员,可在细胞膜上与上皮钙粘蛋白(E-Cadherin, E-Cad)相互结合而调节细胞粘附。近年来有研究表明,p120通过调节NF-κB活性参与表皮炎症反应。我们推测在气道炎症反应中,p120也可能通过NF-κB信号途径发挥调节作用。
实验目的
本实验使用脂多糖刺激建立体外气道炎症反应模型,初步探讨脂多糖刺激后气道上皮细胞中p120的表达变化及其对NF-κB信号通路的影响,从而进一步分析气道炎症发生发展的机制。
实验方法
使用脂多糖刺激建立体外气道炎症反应模型；采用免疫荧光共聚焦成像、Western blot、核浆分离方法检测p120、NF-κB、IκBα等蛋白表达及定位变化；采用荧光定量PCR、定量酶联免疫吸附实验(ELISA)检测IL-8表达变化；采用荧光素酶报告基因分析的方法检测NF-κB的活性；通过瞬时转染及小干扰RNA技术改变细胞中p120的表达量再检测p120对NF-κB信号途径的影响。
实验结果
(1)细胞瞬时转染NF-κB报告质粒后,报告基因分析结果显示,LPS刺激可引起气道上皮细胞中NF-κB信号的活化；同时荧光定量PCR和ELISA结果显示NF-κB靶基因IL-8--一种前炎症因子的表达亦有明显增多。
(2)p120在正常气道上皮细胞中含量丰富,而LPS刺激后,其表达迅速下调,早在15 min时即可表现出来。基于以上两点,我们推测在LPS所致气道炎症反应中,p120与NF-κB信号途径可能存在某种联系。接下来,我们先探讨NF-κB信号活化的机制。
(3)实验发现,在LPS刺激15 min-30 min时,NF-κB的抑制蛋白IκBα迅速降解至几近消失；其磷酸化形式在正常细胞中几乎检测不到,但在LPS处理15 min-30 min这段时间内含量显著增加,在IκBα恢复正常水平后,p-IKBa又再次消失不见。
(4)核浆分离试验及免疫荧光共聚焦结果均显示LPS促使NF-κB活性亚单位p65发生核内转位。
结果(3)和(4)表明,LPS在气道上皮细胞中引起的NF-κB活化是通过使IκBa发生磷酸化降解,从而导致p65由NF-κB/IκB复合体中释放并转位入核激活相应靶基因转录。接下来,我们通过对细胞转染p120质粒或小干扰RNA,研究p120对NF-κB信号活化的影响。
(5)实验结果显示,过表达p120可以部分抑制NF-κB的活性及IL-8的表达,但免疫荧光及核浆分离结果依然可以观测到p65核转位。
(6)小干扰RNA敲低p120的表达后,免疫荧光结果显示,LPS可引起p65更强烈的核染色；同时核浆分离产物的免疫印迹结果也证实阻断p120表达可进一步促进LPS诱导的p65核转位。
(7)阻断p120的表达可大大提升NF-κB的转录活性及其靶基因IL-8的表达。
实验结论
本实验表明：
(1)LPS可导致气道上皮细胞中p120表达下调。
(2)LPS可诱导气道上皮细胞NF-κB信号的活化及其靶基因IL-8的表达。
(3) NF-κB信号途径的活化是通过IκBα的磷酸化降解释放p65入核实现的。
(4)过表达p120可部分抑制NF-κB信号的活化；敲低p120能显著促进NF-κB信号的活化。
本实验提示：
在LPS引起的气道炎症反应中,p120可能负性调节NF-κB信号途径的活化,
从而抑制气道炎症反应。
实验背景
大量研究表明,吸烟可从多个方面损伤气道上皮细胞,更是导致慢性阻塞性肺疾病(COPD)和支气管源性肺癌发生发展的确切因素。然而,吸烟致气道上皮损伤的具体机制仍未完全阐明。上皮细胞损伤后,立即启动自我修复过程。这是一个步骤繁杂的过程,包括细胞延伸、迁移和增殖。而细胞间粘附的改变表现在损伤修复的初始阶段,因此对细胞粘附的研究显得非常有意义。
已有研究证实,IQGAP1参与许多生命活动,是一个多功能蛋白。作为Rho家族GTPases成员Racl和Cdc42的一个重要的效应因子,IQGAP1不仅参与调节细胞骨架,影响细胞粘附、极化和迁移,还可能介导β-catenin/TCF信号的转录活化,从而引起其下游细胞增殖相关基因的表达。有研究显示IQGAP1可能通过P-catenin调节细胞粘附,然而具体机制并不明了。由于IQGAP1可与作为细胞粘附成分和Wnt信号通路成员的β-catenin结合,因此研究IQGAP1在损伤修复中的作用就显得尤为重要。有报导指出,IQGAP1在肺组织中高表达。我们推测IQGAP1在气道上皮损伤修复中可能发挥作用,并探讨其可能机制。
实验目的
通过建立香烟烟雾提取物(CSE)损伤气道上皮的体外模型,观察IQGAP1在损伤修复过程中的表达及定位变化及其对细胞粘附复合体成员β-catenin的影响,初步研究其对气道上皮损伤修复中细胞粘附改变的影响,并为进一步探讨细胞增殖修复提供依据。
实验方法
使用CSE建立体外气道上皮损伤模型,首先使用相差显微镜观察气道上皮细胞在CSE刺激下的形态学变化,同时采用MTT方法检测不同浓度下细胞活力的变化。接着应用Western blot、免疫荧光共聚焦成像、免疫沉淀等技术观察CSE刺激后IQGAP1的表达、定位变化及其对细胞粘附复合体的影响。最后通过瞬时转染野生型IQGAP1和小干扰RNA,采用核浆分离等方法检测过表达或敲低IQGAP1对β-catenin的影响。
实验结果
(1)在相差显微镜下观察,可见培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态,立体感强,微凸起,细胞间连接紧密。在CSE损伤后,细胞伸展扁平,细胞间间隙增宽,伸出突起相接触,更高浓度的CSE致使细胞皱缩变圆,死亡增加。
(2)MTT结果显示,低浓度CSE可促进细胞增殖,而高浓度则引起细胞活力降低。
(3)采用Western blot和免疫荧光技术,我们发现,CSE促使气道上皮细胞中IQGAP1呈现时间依赖性过表达,且其定位信号由细胞连接处扩散至整个胞浆。更为有意义的是,我们通过免疫荧光和免疫印迹发现IQGAP1可与β-catenin结合,CSE刺激后,这种结合变得更强,而β-catenin与a-catenin的结合锐减。
(4)进一步通过使用瞬时转染技术,我们发现IQGAP1过表达不仅可以增加β-catenin在细胞内聚集,还可以促使积聚的β-catenin转位入核。而用干扰RNA阻断IQGAP1的表达后,β-catenin在胞浆内的聚集减少,其入核也受到影响。
实验结论本实验表明：
(1)CSE可在体外诱导气道上皮细胞过表达IQGAP1。
(2)CSE减弱β-catenin与粘附复合体中a-catenin的结合,但加强其与IQGAP1的结合
(3)过表达IQGAP1促使β-catenin在胞浆中积聚并进而转位入核。本实验提示：
IQGAP1通过与α-catenin竞争结合β-catenin,破坏细胞粘附复合体,从而减弱细胞粘附,启动CSE损伤的气道上皮细胞修复,并促进β-catenin转位入核,为下一步增殖修复损伤做准备。
Background
As the first line to defence harmful stimuli, the airway is frequently injured because of its exposure to the external environment. Some pathogenic factors may cause airway inflammation, such as lipopolysaccharide (LPS), which is the main component of the cell wall of Gram-negative bacteria. LPS may promotes several cellular processes including necrosis, apoptosis, and secretion of pro-inflammatory cytokines, which contribute to the development of lung injury. LPS is a potent activator that induces inflammatory gene expression through nuclear factor-KB (NF-κB) activation. NF-κB is a transcription factor expressed ubiquitously, which could be activated by LPS in various cells.
NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor of NF-κB (IκB) and remains transcriptionally inactive. Stimulation by triggers such as LPS induces the ubiquitylation and degradation of IκB. The loss of IκB exposes the nuclear localization signal sequence on NF-κB, resulting in the nuclear translocation of NF-κB and transcriptional activation of its target gene promoters. P120-catenin (p120), a prototypic member of a subfamily of Armadillo repeat domain (Arm domain) proteins, which is involved in maintaining the stability and regulating the turnover of E-cadherin. Recent studies revealed that loss of p120 was associated with NF-κB activation and inflammation in p120 null epidermal cells. This discovery raised an interest in the pathophysiology of the diseases in which NF-κB activation is involved. Although airway inflammation has been extensively studied, it is uncertain whether p120 partcipates in airway inflammation through NF-κB signaling pathway. Therefore, our present studies focused on the effects of p120 on NF-κB signaling during the lung injury induced by LPS.
Objective
To investigate the changes of p120 expression and the effects of p120 on NF-κB signaling pathway during the inflammatory response induced by LPS.
Methods
In this study, we treated cells with LPS to establish a lung inflammation model in vitro. Using confocal immunofluorescence imaging, Western blot, isolation of cytoplasmic and nuclear proteins, we observed and examined the localizations and expressions of p120, NF-κB and IκBα. Then we detected the expressions of IL-8 by fluorescence quantitative PCR and enzyme-linked immunosorbent assay. Luciferase reporter analysis was used to detect the activity of NF-κB. Finally, transient transfection and small interfering RNA were used to over-expression or knock down p120, and then the effects of p120 on NF-κB signaling pathway were detected.
Results
(1) LPS induced the activation of NF-κB signaling in bronchial epithelial cells (BECs) by transient transfection and luciferase reporter assay. Meanwhile, IL-8, a proinflammatory factor, which is the target gene of NF-κB was also increased significantly after LPS treatment.
(2) Western blot showed that p120 was rich in BECs, but rapidly reduced by LPS as early as 15 min.
(3) It was found that IκBαwas rapidly phosphorylated and degradated by LPS from 15 to 30 min.
(4) The translocation of p65 from cytoplasm to nucleus after LPS treatment was confirmed by western blot and immunofluorescence.
(5) After cells transfected with pEGFPp120 followed by LPS treatment, we found the activaity of NF-κB induced by LPS was partially blocked. Data also showed that IL-8 production in response to LPS was partly, but not completely down-regulated by over-expression of p120.
(6) Inhibition of p120 by siRNA significantly enhanced the LPS-induced NF-κB activity, promoted LPS-induced p65 nuclear translocation and elevated IL-8 production.
Conclusions
(1) LPS induced the up-regulation of p120 in BECs.
(2) LPS induced NF-κB activation and IL-8 production.
(3) The activation of NF-κB induced by LPS was accompanied with IκBαdegradation and p65 nuclear translocation in BECs.
(4) The activation of NF-κB signaling was partially inhibited by over-expression of p120, but could be significantly promoted by knocking down of p 120.
These data strongly suggest that NF-κB activation is one of the cytoplasmic-nuclear signaling pathways involved in airway epithelial cells in response to LPS, and p120 is able to inhibit this activation.
Background
Numerous studies showed that cigarette smoke have various injurious effects on epithelial cells, which may contribute to the development of lung diseases including chronic obstructive pulmonary disease (COPD) and bronchogenic lung cancer. After injury, the airway epithelium initiates a wound repair process to keep normal function, which requires spreading, migration, and eventually proliferation into the injured area. However, the specific molecular mechanisms involved in the injury and repair of airway epithelium induced by smoking have not been fully understood. Studies for cell adhesion are very meaningful due to its performance in the initial stage of wound healing.
IQ domain GTPase-activating protein 1 (IQGAP1) is a multifunctional protein and could interacts with cytoskeleton, cell adhesion complex and microtubule associated proteins (MAPs). Therefore, IQGAP1 was considered to play roles in cell adhesion, polarization and migration. It has also been confirmed that IQGAP1 mediated the transcriptional activation ofβ-catenin/Tcf signaling and the expression of downstream target gene involved in cell proliferation. All the studies showed an important role of IQGAP1 in injury repair. Due to its highly expression in lung tissue, we speculated that IQGAP1 may regulate the process of airway epithelial injury and repair, and we tried to explore its possible mechanism.
Objective
To determine the dynamic expression of IQGAP1 and the effects of IQGAP1 on cell adhesion complex during the injury and repair process following CSE exposure, and to provide more evidences for further exploring the mechanism of injury and repair.
Methods
In this study, we established a model of injury and repair of airway epithelia by CSE in vitro. Firstly, we used the phase contrast microscope to observe the morphological changes of bronchial epithelial cells (BECs) after CSE treatment. Furthermore, MTT analysis was used to reveal the changes of cell viability after CSE treatment. Then, the following experiments were performed:Western blot, confocal laser scanning, co-immunoprecipitation. We aimed to observe the expression and localization of IQGAP1, and its impact on cell adhesion complex. Finally, transient transfection and isolation of cytoplasmic and nuclear proteins was used to detect the effects of IQGAP1 onβ-catenin.
Results
(1) Under phase contrast microscope, cells display morphological changes after CSE treatment, including widened cell-cell interspaces, more widely flattened appearance and more dead cells, compared with control cells, which showed a classic cobblestone-like epithelial morphology that was three-dimensional, slightly raised and closely adherent.
(2) MTT results showed that low concentrations of CSE can promote cell proliferation, while high concentrations lead to a dose-dependent reduction of cell viability.
(3) Western blot and immunofluoresence showed that CSE induced a time-dependent up-regulation of IQGAP1 in the airway epithelial cells and its positioning signal translocated from the cell-cell junctions to the whole cytoplasm, respectively. More meaningfully, we discovered that IQGAP1 combined withβ-catenin by immunofluorescence and Western blot. Such integration had become stronger after CSE stimulation, as well as the combination betweenβ-catenin and a-catenin was sharply reduced.
(4) Over-expression of IQGAP1 by transient transfection not only increased the accumulation ofβ-catenin in the cell, but also promoted the process of its nuclear translocation. After the expression of IQGAP1 was knocked down by small interfering RNA(siRNA), the accumulation and nuclear translocation of P-catenin were reduced.
Conclusions
(1) CSE induced up-regulation of IQGAP1 in vitro of airway epithelial cells.
(2) CSE attenuated the combination betweenβ-catenin and a-catenin, but enhanced the association between P-catenin and IQGAP1.
(3) over-expression of IQGAP1 promoted the accumulation and nuclear translocation ofβ-catenin.
The results suggest that IQGAP1 may weakens cell adhesion by binding to P-catenin and leading to the release of a-catenin from adhesion complex. Moreover, IQGAP1 may plays roles in cell proliferation by promoting the nuclear translocation ofβ-catenin.
引文
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