多功能蛋白GSK3β与IQGAP1在气道上皮毒性损伤过程中的作用研究
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摘要
第一部分
博莱霉素致气道上皮细胞毒性损伤中GSK3β的活性变化对NF-κB信号通路的影响
实验背景
博莱霉素是一种有效的化疗药物，但所引起的急性肺损伤与肺纤维化的副作用却不容忽视。气道上皮是呼吸系统对抗毒性刺激的第一道防线，因此气道上皮细胞通常被认为是博莱霉素肺毒性的主要靶点。暴露于博莱霉素的细胞可发生各种细胞反应，包括细胞凋亡、细胞坏死及前炎症因子分泌等，这些细胞反应共同促进了博莱霉素导致的肺损伤与肺纤维化的发展过程。大量研究显示，核因子-κB(nuclear factor-κB，NF-κB)信号参与调节细胞存活、细胞分化、增殖、凋亡及炎症过程的多种基因的转录，与博莱霉素引起的肺毒性密切相关。
正常情况下，NF-κB与抑制蛋白IκB相结合并隐匿于静息细胞的胞浆，保持无活性的状态。外界刺激可引起IκB进入泛素化降解途径从而使NF-κB被释放并转位入核，激活下游靶基因的转录。糖原合成酶激酶3(glycogen synthase kinase 3，GSK3)是一种多功能的丝／苏氨酸蛋白激酶，与NF-κB不同，GSK3β在静息细胞内呈组成性激活的状态，其N末端丝氨酸残基可发生PI3K(phosphatidylinositol3-kinase)／Akt(also called protein kinase B，PKB)依赖性的磷酸化并导致其活性抑制。近年来的研究显示，GSK3β可能参与调节与NF-κB信号相关的多种疾病的病理生理过程，但具体机制并不明确。因此，本研究针对GSK3β在博莱霉素肺毒性中的活性变化及其对NF-κB信号通路的影响进行了初步探讨。
实验目的
本实验使用博莱霉素刺激建立体外肺毒性损伤模型，初步探讨在博莱霉素作用下气道上皮细胞中GSK3β的活性变化对NF-κB信号通路的影响，从而进一步分析肺纤维化发生发展的机制。
实验方法
使用博莱霉素刺激建立体外肺毒性损伤模型，首先采用相差显微镜及细胞荧光染色观察气道上皮细胞在博莱霉素刺激下的形态变化，并进一步使用MTT方法检测不同浓度下细胞活力的变化。然后使用Western blot、免疫荧光共聚焦成像、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测博莱霉素处理后NF-κB信号的活化状态、Akt／GSK3β通路的活性变化及其对NF-κB信号的影响。
实验结果
相差显微镜下观察显示培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态，其特点是立体的，稍微凸起的，细胞间紧密连接。经博莱霉素毒性刺激后，细胞间隙增宽，伸展扁平，立体感消失，死细胞增多，在荧光显微镜下可见大量细胞核呈现皱缩变圆、亮蓝色的凋亡形态。MTT结果显示博莱霉素可引起浓度依赖性的细胞活力降低。
然后，我们使用瞬时转染及报告基因分析方法发现博莱霉素可引起气道上皮细胞中NF-κB信号的活化，而且在博莱霉素刺激2 h时，NF-κB的抑制蛋白IκBα迅速发生降解并几近消失，同时核浆分离实验及免疫荧光共聚焦结果均显示NF-κB亚单位p65发生了向核内的转位，与文献所报道的体内实验的结果相符。接着我们发现，博莱霉素刺激以后，Akt与GSK3β发生了明显的磷酸化，而使用抑制剂LY294002抑制二者的磷酸化以后，NF-κB信号的活性也随之降低。因此我们推测，博莱霉素所引起的NF-κB信号的活化必与GSK3β的活化状态有着密切的关系。于是我们进一步使用持续激活型GSK3βS9A质粒转染入细胞后再使用博莱霉素刺激，结果发现GSK3β的持续活化抑制了博莱霉素诱导的NF-κB信号活化，并保护内源性IκBα不受降解，继而阻止了NF-κB亚单位p65转位入核。为了更深入的探究这种现象的可能原因所在，我们使用免疫共沉淀的方法将GSK3β与IκBα互相沉淀，发现二者可以共同沉淀下来，并在博莱霉素刺激后共同沉淀的蛋白减少，而在沉淀的IκBα蛋白中并未检测到磷酸化的GSK3β，提示GSK3β与IκBα存在于同一个复合物中，并且很有可能对IκBα有着某种机制的保护作用，进一步说明GSK3β可能通过与IκBα的这种相互作用从而影响了博莱霉素诱导的NF-κB信号活化。
实验结论
本实验表明：(1)博莱霉素可导致气道上皮细胞发生形态学改变及细胞活力降低。(2)博莱霉素可诱导气道上皮细胞NF-κB信号的活化。(3)博莱霉素诱导的气道上皮细胞中Akt及GSK3β的磷酸化可能参与了NF-κB信号的活化。(4)GSK3β的活性改变可能通过与NF-κB抑制蛋白IκBα的相互作用从而负向调节博莱霉素诱导的气道上皮细胞中NF-κB信号的活化过程。
第二部分
甲醛暴露致气道上皮毒性损伤中IQGAP1的表达变化对APC蛋白及β-catenin／Tcf信号通路的影响
实验背景
甲醛(formaldehyde，FA)是一种常见的化学污染物，不论是动物还是人体，长期低剂量的接触均可引起慢性中毒性呼吸道疾病，甚至诱导基因突变并提高机体罹患各种恶性肿瘤的几率。
目前已证实，IQGAP1在多种疾病及肿瘤组织中均存在过表达的现象，但其中具体机制并不明确。IQGAP1是Rho家族GTPases成员Rac1和Cdc42的一个重要的效应因子，具有多个蛋白结合位点，不仅参与调节细胞骨架，影响细胞黏附，而且还可能介导β-连环素／Tcf信号的转录活化，从而引起其下游细胞增殖相关基因(如cyclin D1和c-myc等)的表达。另一方面，活化的Rac1和Cdc42可与IQGAP1相结合并促使结肠腺瘤性息肉病蛋白(adenomatous polyposis coli，APC)在迁移细胞前缘(leading edge)稳定微管正极末端，从而参与细胞极化和定向迁移。然而近年大量研究发现，APC也可存在于核内，不仅作用于纺锤体，还可以通过抑制β-连环素／Tcf信号进而抑制细胞增殖。因此，本研究通过建立甲醛毒性暴露的体内外模型以观察IQGAP1在气道上皮损伤修复中的动态变化，并使用转染及RNA干扰等技术进一步在体外实验中研究IQGAP1对APC蛋白及β-连环素／Tcf信号的影响，以期了解甲醛暴露后气道上皮损伤修复的过程，为阐明甲醛可能的致瘤性机制提供一个新的思路。
实验目的
建立甲醛毒性暴露的体内、外模型，观察IQGAP1在气道上皮损伤修复中的动态变化，并使用免疫荧光、免疫共沉淀、转染及RNA干扰等技术进一步在体外实验中研究IQGAP1对APC蛋白及β-连环素／Tcf信号的影响，以期了解甲醛暴露后气道上皮损伤修复的过程，为阐明甲醛可能的致瘤性机制提供一个新的思路。
实验方法
使用甲醛毒性暴露建立体内、外气道上皮损伤模型，首先采用HE染色在普通显微镜下观察小鼠气道上皮染毒后的病理变化，并同时使用免疫荧光共聚焦及免疫印迹技术检测IQGAP1在小鼠肺组织内的定位与表达变化。体外实验中，相差显微镜及MTT方法观察气道上皮细胞在甲醛暴露下的形态变化及IQGAP1对细胞增殖的影响，pull down方法检测Rac1及Cdc42的活性，免疫荧光共聚焦分析APC蛋白的亚细胞定位，使用RNA干扰、Western blot、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测甲醛暴露后IQGAP1的表达变化及其对APC蛋白和β-连环素／Tcf信号的影响。
实验结果
在甲醛吸入染毒过程中，小鼠气道上皮经历了从细胞坏死脱落、纤毛倒伏、炎症细胞浸润到纤毛重现、基底细胞增生、细胞层次增加的损伤修复的动态过程。IQGAP1的表达随着损伤修复过程的进展呈现先升高后降低的趋势，并在损伤严重时大量聚集于胞浆中，而在修复末期重新定位于浆膜面，暗示IQGAP1在小鼠经历甲醛毒性刺激时发挥了重要作用。
另外，在相差显微镜下观察可见培养的气道上皮细胞在甲醛暴露后失去其特有的铺路石状形态，细胞间隙增宽，伸展扁平，更高浓度时细胞皱缩变圆，死细胞明显增多。MTT结果显示低浓度甲醛可促进细胞增殖，而高浓度甲醛则引起细胞活力降低并呈浓度依赖性。接着，我们使用pull down方法检测Rho家族蛋白Rac1及Cdc42的活性，发现二者在甲醛暴露后的活性存在不一致的变化趋势(Cdc42活性降低，Rac1活性先升后降)，可能在损伤修复过程中发挥着不同的作用。然后，我们进一步检测其效应因子IQGAP1在细胞内的表达与定位，Western blot结果显示甲醛可诱导气道上皮细胞中IQGAP1呈时间-浓度依赖性的过表达，其定位信号也从细胞连接处及核周扩散至整个胞浆。最重要的是，我们发现IQGAP1过表达可明显促进细胞增殖，提升细胞活力，而这一切是通过增加β-连环素在胞浆内的聚集继而促进后者的核转位过程来实现的。使用RNA干扰敲低IQGAP1的表达以后，不仅严重影响了甲醛诱导的β-连环素核转位过程，而且也显著降低了β-连环素／Tcf信号的转录活性及其靶基因cyclin D1的表达，更加证明了IQGAP1在细胞增殖过程中的重要作用。
最后，我们在检测微管相关蛋白APC在气道上皮细胞内的表达与定位时惊人地发现，甲醛暴露不仅引起APC蛋白表达上调，而且还诱导了原本弥散分布于胞浆的APC蛋白聚集于胞浆一侧及胞核之中。为了解这一现象出现的原因，我们使用免疫共沉淀方法将IQGAP1与APC蛋白互相沉淀，发现二者在甲醛暴露后结合加强，于是我们结合文献推论，APC蛋白在胞浆中聚集的一侧可能预示着细胞迁移的方向，而IQGAP1可能通过与APC蛋白相结合参与细胞极化与迁移，更重要的是，机制未明的APC蛋白转位入核可能起着抑制β-连环素／Tcf信号过度活化的作用，在如此精细调节的蛋白网络中作为一个终止信号避免上皮修复后的过度增殖。
实验结论
本实验表明：(1)甲醛暴露能在体内、外诱导IQGAP1过表达。(2)IQGAP1过表达可通过增加β-连环素在细胞内的聚集及向核内转位，从而促进β-连环素／Tcf信号活化及增殖相关cyclin D1的表达，最终在细胞增殖中起重要作用。(3)甲醛暴露后，IQGAP1与APC蛋白、β-连环素结合增加，可能与细胞迁移及细胞增殖有关。(4)甲醛诱导APC蛋白聚集于胞浆一侧及胞核，可能预示了细胞迁移的方向及抑制细胞过度增殖。
PartⅠ
Bleomycin-induced nuclear factor-κB activation in humanbronchial epithelial cells involves the phosphorylation ofglycogen synthase kinase 3β
Background:
Bleomycin(BLM) is an effective chemotherapeutic antibiotic which has beenused successfully to treat a wide variety of malignancies such as squamous cellcarcinoma,sarcoma and lymphoma.However,the clinical application of the drug islimited by its adverse side effects,especially severe lung injury and fibrosis.Epithelialcells are known to be a main target of BLM lung toxicity.Exposure of cells to BLMresults in diverse cellular responses including apoptosis,cell death and secretion ofpro-inflammatory cytokines,which contribute to the development of lung injury andfibrosis induced by BLM.Nuclear factor-κB(NF-κB) regulates a large number of genesinvolved in cell survival,differentiation,proliferation,apoptosis and inflammation andis also considered to be closely correlated with BLM lung toxicity.
NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor ofNF-κB(IκB) and remains transcriptionally inactive.Stimulation by cytotoxic triggerssuch as BLM and tumor necrosis factorα(TNFα) induces the ubiquitylation anddegradation of IκB.The loss of IκB exposes the nuclear localization signal sequence onNF-κB,resulting in the nuclear translocation of NF-κB and transcriptional activation ofNF-κB target gene promoters.Glycogen synthase kinase 3β(GSK3β) is a ubiquitousand important serine/threonine kinase.In contrast to most other kinases,GSK3βisconstitutively active in resting cells and can be inactivated by phosphorylation on anN-terminal serine residue(Ser~9) through a PI3K(phosphatidylinositol 3-kinase)/Akt(also called protein kinase B,PKB)-dependent mechanism.Recent studies revealed that GSK3βmight be involved in the control of various signaling pathways that activateNF-κB,and the effect of GSK3βon the regulation of NF-κB raised an interest in thepathophysiology of the diseases in which NF-κB activation is involved.However,theexact mechanisms are still unclear.Therefore,our present studies focus on the changesof GSK3βactivity and the effects of GSK3βon NF-κB signaling during the toxic lunginjury induced by BLM.
Objective
To investigate the changes of GSK3βactivity and the effects of GSK3βon NF-κBsignaling during the toxic lung injury induced by BLM.
Methods
In this study,we treated the cells with BLM to establish the lung toxic injury modelin vitro.Firstly,we used the phase contrast microscope and fluorescence staining toobserve the morphological changes of bronchial epithelial cells(BECs) after BLMtreatment.Furthermore,MTT analysis was used to reveal the changes of cell viabilityafter BLM treatment.Then the following experiments were performed:Western blot,confocal laser scanning,co-immunoprecipitation,transient transfection,luciferasereporter assay and so on.We aimed to study the active status of NF-κB signaling,theactivity changes of Akt/GSK3βpathway and its effects on NF-κB signaling after BLMtreatment.
Results
Under phase contrast microscope,treatment with BLM made cells displaymorphological changes,including widened cell-cell interspaces,more widely flattenedappearance and numerous dead cells,compared with control cells,which showed aclassic cobblestone-like epithelial morphology that was three-dimensional,slightlyraised and closely adherent.In addition,nuclear morphological changes were observed under fluorescence microscope by Hoechst 33258 staining.Results showed that BLMcaused nuclear condensation and cell apoptosis.MTT analysis revealed adose-dependent reduction of cell viability after BLM treatment.
Then,we further confirmed that BLM induced the activation of NF-κB signaling inBECs by transient transfection and luciferase reporter assay.Furthermore,theexpression of IκBαreduced and almost disappeared within 2 h after BLM treatment.Inthe same time,both the data from immunofluorescence staining and Western blot ofnuclear extract revealed that incubation with BLM in BECs caused a striking shift ofp65(the subunit of NF-κB) towards the nucleus.These results are consistent with theprevious experiments in vivo which have been published.Subsequently,we found thatphosphorylation levels of Akt and GSK3βrapidly increased in 0.5 h after BLMtreatment and became higher at 2 h.However,pretreated with LY294002,whichinhibited BLM-induced phosphorylation of Akt and GSK3β,the transcriptionalactivation of NF-κB in response to BLM was also significantly reduced in adose-dependent manner.Thus we suppose that BLM-induced NF-κB activation must beclosely associated with the active status of GSK3β.So cells were transiently transfectedwith a stable mutant of GSK3β(S9A) which is constitutively active followed by BLMtreatment.The results showed that continuous activation of GSK3βled to a partial butsignificant inhibition of BLM-induced NF-κB activation,protected endogenous IκBαfrom degradation and blocked the nuclear translocation of p65 subunit.To investigatethe possible cause of this phenomenon,a co-immunoprecipitation assay was used andthe results showed that GSK3βwas indeed able to be co-precipitated with IκBαin BECs.Moreover,the co-precipitated proteins decreased after BLM treatment.Importantly,there was no phosphorylated GSK3βto be detected in IκBαprecipitates,indicating thatIκBαmight be preserved by a mechanism that is dependent on direct GSK3βbinding.These data further demonstrated that GSK3βaffected BLM-induced NF-κB activationthrough an interaction with IκBα.
Conclusions
(1) BLM induced the morphological changes and reduction of cell viability in BECs.
(2) BLM induced NF-κB activation,accompanied with IκBαdegradation and p65nuclear translocation in BECs.
(3) BLM induced the phosphorylation of Akt and GSK3βin BECs.
(4) GSK3βmight negatively regulate BLM-induced NF-κB activation in BECs by amechanism that is dependent on the interaction of GSK3βand IκBα.
PartⅡ
Effects of IQ domain GTPase-activating protein 1 on APCprotein andβ-catenin/Tcf signaling during the toxic injury ofairway epithelia after formaldehyde exposure
Background:
Formaldehyde(FA) is a ubiquitous chemical pollutant,which can induce chronictoxic respiratory disease of animals or human after long-term and low-concentrationtouch.It even leads to gene mutation and increases the probability of some malignanttumours in human.
It has been confirmed that IQ domain GTPase-activating protein 1(IQGAP1) isalways over-expressed in most tumours with unclear mechanism.IQGAP1,animportant effector of Rho GTPase family including Cdc42 and Racl,has many proteinbinding sites,not only regulating cell skeleton and affecting cell adhesion,but alsopossibly mediating the transcriptional activation ofβ-catenin/Tcf signaling and theexpression of downstream target gene involved in cell proliferation,such as cyclin D1and c-myc.On the other hand,active Cdc42 and Racl can promote APC proteinstabilizing the plus end of microtubule in the leading edge of migrating cells throughbinding with IQGAP1 and play key roles in cell polarization and migration.Nevertheless,a great quantity of recent studies demonstrated that APC protein alsolocated in the nuclei where it functions in regulating the spindle and inhibits cellproliferation by interfering with the activation ofβ-catenin/Tcf signaling.Therefore,inthe present study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro,and observed that the dynamic changes ofIQGAP1 during the process of injury and repair.We further studied the effects ofIQGAP1 on APC protein andβ-catenin/Tcf signaling in vitro by using transfection andsmall interfering RNA technique.We aim to understand the injury and repair process of airway epithelia following formaldehyde exposure,providing a novel idea in elucidatingthe potential tumourigenicity mechanism of formaldehyde.
Objective
To determine the dynamic changes of IQGAP1 and the effects of IQGAP1 onAPC protein andβ-catenin/Tcf signaling during the toxic injury of airway epitheliafollowing formaldehyde exposure.
Methods
In this study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro.We first observed the pathological changesof murine airway epithelia following formaldehyde inhalation under light microscopeafter H&E staining.Then we detected the location and expression of IQGAP 1 by usingimmunofluorescence staining and Western blot.In our experiment in vitro,we used thephase contrast microscope and MTT method to observe the morphological changes ofbronchial epithelial cells(BECs) after formaldehyde exposure and the effect of IQGAP 1over-expression on cell proliferation.Pull down assay was used to detect the activity ofCdc42 and Racl.Immunofluorescence staining and confocal laser scanning microscopewere used to determine the subcellular location of APC andβ-catenin protein.Thefollowing experiments were performed:Western blot,co-immunoprecipitation assay,transient transfection,small interfering RNA,luciferase reporter assay and so on.Weaim to study the effects of IQGAP1 on APC protein andβ-catenin/Tcf signaling,understand the injury and repair process of airway epithelia following formaldehydeexposure,providing a novel idea in elucidating the potential tumourigenicitymechanism of formaldehyde.
Results
In the process of formaldehyde inhalation,the murine airway epithelia experiencedthe dynamic course from increased number of goblet cells,lodgings and losses of cilia, loose contacts between cells,infiltrations of inflammatory cells,shedding of necroticcells into the airway lumen to the re-appearance of cilia,tighter cell-cell junction,hyperplasia of basal layer cells,increased cellular layer and infiltration of chronicinflammatory cells.The expression of IQGAP 1 showed a corresponding tendency withthe progression of injury and repair.A strong signal of IQGAP 1 diffused in the wholecytoplasm in the early stage of severe injury and located around the plasma membraneagain in the end stage of repair,implicating that IQGAP 1 plays an important role in theinjury and repair of airway epithelia following the toxic stimulation from formaldehyde.
Under phase contrast microscope,formaldehyde exposure made cells lose itsnatural classic cobblestone-like epithelial morphology and display widened cell-cellinterspaces,more widely flattened appearance,even cellular condensation andnumerous dead cells when exposed to higher concentration.MTT analysis revealed thatlow-concentration formaldehyde promoted cell proliferation and high-concentrationformaldehyde induced a dose-dependent reduction of cell viability.Then,pull downassay was used to detect the activity of Cdc42 and Racl and the results showed adifferent alteration after formaldehyde exposure.After that,we further determined theexpression and location of their effectors,IQGAP 1.The data from Western blot analysisshowed that formaldehyde induced the time-and concentration-dependentover-expression of IQGAP1 in the airway epithelial cells.The fluorescent signal alsotranslocated from the cell-cell boundary and circum-nuclei to the whole cytoplasm.Importantly,we found that IQGAP1 over-expression stimulated cell proliferation andelevated the cell viability significantly,which might be achieved through increasing theaccumulation ofβ-catenin in the cell and promoted the process of its nucleartranslocation.After the expression of IQGAP1 was knocked down by small interferingRNA(siRNA),not only formaldehyde-inducedβ-catenin translocation,but also theactivation ofβ-catenin/Tcf signal and the expression of its downstream target genecyclin D 1 were inhibited,further confirming a key role of IQGAP 1 in cell proliferation.
Finally,when we detected the expression and location of APC protein in airway epithelial cells,we surprisingly found that formaldehyde not only induced the increaseof APC protein but also resulted in a striking translocation from diffusion in the wholecytoplasm to accumulation in the nuclei and one side of cytoplasm.To understand thecause of this phenomenon,co-immunoprecipitation assay was used to detect the relationof IQGAP 1 and APC.The results showed that they could be precipitated each other andthe interaction was enhanced after formaldehyde exposure.So we proposed that the sideof APC accumulation might signify the direction of cell migration and IQGAP1participated in cell migration through binding with APC.Importantly,the nucleartranslocation of APC protein with unknown mechanism might function as a terminationsignal in the protein network under complicate regulations to inhibit the activation ofβ-catenin/Tcf signaling and prevent the excess proliferation of epithelial cells in therepair process after injury.
Conclusions
(1) Formaldehyde induced the over-expression of IQGAP 1 in vivo and in vitro.
(2) Over-expression of IQGAP1 resulted inβ-catenin accumulation and nucleartranslocation,activatedβ-catenin/Tcf signaling,elevated expression levels of cyclinD 1 and promoted cell proliferation in airway epithelial cells.
(3) The enhanced interaction of IQGAP1 andβ-catenin and APC after formaldehydeexposure might be correlated with cell migration and cell adhesion.
(4) Formaldehyde exposure up-regulated the expression of APC protein in airwayepithelial cells and made it accumulate in one side of cytoplasm and the nuclei withthe tighter interaction of IQGAP1 and APC in the cytoplasm,which could playimportant roles in signifying the direction of cell migration and preventing theexcess proliferation of epithelial cells.
博莱霉素致气道上皮细胞毒性损伤中GSK3β的活性变化对NF-κB信号通路的影响
实验背景
博莱霉素是一种有效的化疗药物，但所引起的急性肺损伤与肺纤维化的副作用却不容忽视。气道上皮是呼吸系统对抗毒性刺激的第一道防线，因此气道上皮细胞通常被认为是博莱霉素肺毒性的主要靶点。暴露于博莱霉素的细胞可发生各种细胞反应，包括细胞凋亡、细胞坏死及前炎症因子分泌等，这些细胞反应共同促进了博莱霉素导致的肺损伤与肺纤维化的发展过程。大量研究显示，核因子-κB(nuclear factor-κB，NF-κB)信号参与调节细胞存活、细胞分化、增殖、凋亡及炎症过程的多种基因的转录，与博莱霉素引起的肺毒性密切相关。
正常情况下，NF-κB与抑制蛋白IκB相结合并隐匿于静息细胞的胞浆，保持无活性的状态。外界刺激可引起IκB进入泛素化降解途径从而使NF-κB被释放并转位入核，激活下游靶基因的转录。糖原合成酶激酶3(glycogen synthase kinase 3，GSK3)是一种多功能的丝／苏氨酸蛋白激酶，与NF-κB不同，GSK3β在静息细胞内呈组成性激活的状态，其N末端丝氨酸残基可发生PI3K(phosphatidylinositol3-kinase)／Akt(also called protein kinase B，PKB)依赖性的磷酸化并导致其活性抑制。近年来的研究显示，GSK3β可能参与调节与NF-κB信号相关的多种疾病的病理生理过程，但具体机制并不明确。因此，本研究针对GSK3β在博莱霉素肺毒性中的活性变化及其对NF-κB信号通路的影响进行了初步探讨。
实验目的
本实验使用博莱霉素刺激建立体外肺毒性损伤模型，初步探讨在博莱霉素作用下气道上皮细胞中GSK3β的活性变化对NF-κB信号通路的影响，从而进一步分析肺纤维化发生发展的机制。
实验方法
使用博莱霉素刺激建立体外肺毒性损伤模型，首先采用相差显微镜及细胞荧光染色观察气道上皮细胞在博莱霉素刺激下的形态变化，并进一步使用MTT方法检测不同浓度下细胞活力的变化。然后使用Western blot、免疫荧光共聚焦成像、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测博莱霉素处理后NF-κB信号的活化状态、Akt／GSK3β通路的活性变化及其对NF-κB信号的影响。
实验结果
相差显微镜下观察显示培养的气道上皮细胞呈现上皮细胞典型的铺路石状形态，其特点是立体的，稍微凸起的，细胞间紧密连接。经博莱霉素毒性刺激后，细胞间隙增宽，伸展扁平，立体感消失，死细胞增多，在荧光显微镜下可见大量细胞核呈现皱缩变圆、亮蓝色的凋亡形态。MTT结果显示博莱霉素可引起浓度依赖性的细胞活力降低。
然后，我们使用瞬时转染及报告基因分析方法发现博莱霉素可引起气道上皮细胞中NF-κB信号的活化，而且在博莱霉素刺激2 h时，NF-κB的抑制蛋白IκBα迅速发生降解并几近消失，同时核浆分离实验及免疫荧光共聚焦结果均显示NF-κB亚单位p65发生了向核内的转位，与文献所报道的体内实验的结果相符。接着我们发现，博莱霉素刺激以后，Akt与GSK3β发生了明显的磷酸化，而使用抑制剂LY294002抑制二者的磷酸化以后，NF-κB信号的活性也随之降低。因此我们推测，博莱霉素所引起的NF-κB信号的活化必与GSK3β的活化状态有着密切的关系。于是我们进一步使用持续激活型GSK3βS9A质粒转染入细胞后再使用博莱霉素刺激，结果发现GSK3β的持续活化抑制了博莱霉素诱导的NF-κB信号活化，并保护内源性IκBα不受降解，继而阻止了NF-κB亚单位p65转位入核。为了更深入的探究这种现象的可能原因所在，我们使用免疫共沉淀的方法将GSK3β与IκBα互相沉淀，发现二者可以共同沉淀下来，并在博莱霉素刺激后共同沉淀的蛋白减少，而在沉淀的IκBα蛋白中并未检测到磷酸化的GSK3β，提示GSK3β与IκBα存在于同一个复合物中，并且很有可能对IκBα有着某种机制的保护作用，进一步说明GSK3β可能通过与IκBα的这种相互作用从而影响了博莱霉素诱导的NF-κB信号活化。
实验结论
本实验表明：(1)博莱霉素可导致气道上皮细胞发生形态学改变及细胞活力降低。(2)博莱霉素可诱导气道上皮细胞NF-κB信号的活化。(3)博莱霉素诱导的气道上皮细胞中Akt及GSK3β的磷酸化可能参与了NF-κB信号的活化。(4)GSK3β的活性改变可能通过与NF-κB抑制蛋白IκBα的相互作用从而负向调节博莱霉素诱导的气道上皮细胞中NF-κB信号的活化过程。
第二部分
甲醛暴露致气道上皮毒性损伤中IQGAP1的表达变化对APC蛋白及β-catenin／Tcf信号通路的影响
实验背景
甲醛(formaldehyde，FA)是一种常见的化学污染物，不论是动物还是人体，长期低剂量的接触均可引起慢性中毒性呼吸道疾病，甚至诱导基因突变并提高机体罹患各种恶性肿瘤的几率。
目前已证实，IQGAP1在多种疾病及肿瘤组织中均存在过表达的现象，但其中具体机制并不明确。IQGAP1是Rho家族GTPases成员Rac1和Cdc42的一个重要的效应因子，具有多个蛋白结合位点，不仅参与调节细胞骨架，影响细胞黏附，而且还可能介导β-连环素／Tcf信号的转录活化，从而引起其下游细胞增殖相关基因(如cyclin D1和c-myc等)的表达。另一方面，活化的Rac1和Cdc42可与IQGAP1相结合并促使结肠腺瘤性息肉病蛋白(adenomatous polyposis coli，APC)在迁移细胞前缘(leading edge)稳定微管正极末端，从而参与细胞极化和定向迁移。然而近年大量研究发现，APC也可存在于核内，不仅作用于纺锤体，还可以通过抑制β-连环素／Tcf信号进而抑制细胞增殖。因此，本研究通过建立甲醛毒性暴露的体内外模型以观察IQGAP1在气道上皮损伤修复中的动态变化，并使用转染及RNA干扰等技术进一步在体外实验中研究IQGAP1对APC蛋白及β-连环素／Tcf信号的影响，以期了解甲醛暴露后气道上皮损伤修复的过程，为阐明甲醛可能的致瘤性机制提供一个新的思路。
实验目的
建立甲醛毒性暴露的体内、外模型，观察IQGAP1在气道上皮损伤修复中的动态变化，并使用免疫荧光、免疫共沉淀、转染及RNA干扰等技术进一步在体外实验中研究IQGAP1对APC蛋白及β-连环素／Tcf信号的影响，以期了解甲醛暴露后气道上皮损伤修复的过程，为阐明甲醛可能的致瘤性机制提供一个新的思路。
实验方法
使用甲醛毒性暴露建立体内、外气道上皮损伤模型，首先采用HE染色在普通显微镜下观察小鼠气道上皮染毒后的病理变化，并同时使用免疫荧光共聚焦及免疫印迹技术检测IQGAP1在小鼠肺组织内的定位与表达变化。体外实验中，相差显微镜及MTT方法观察气道上皮细胞在甲醛暴露下的形态变化及IQGAP1对细胞增殖的影响，pull down方法检测Rac1及Cdc42的活性，免疫荧光共聚焦分析APC蛋白的亚细胞定位，使用RNA干扰、Western blot、核浆分离、免疫共沉淀、瞬时转染及报告基因分析等方法观测甲醛暴露后IQGAP1的表达变化及其对APC蛋白和β-连环素／Tcf信号的影响。
实验结果
在甲醛吸入染毒过程中，小鼠气道上皮经历了从细胞坏死脱落、纤毛倒伏、炎症细胞浸润到纤毛重现、基底细胞增生、细胞层次增加的损伤修复的动态过程。IQGAP1的表达随着损伤修复过程的进展呈现先升高后降低的趋势，并在损伤严重时大量聚集于胞浆中，而在修复末期重新定位于浆膜面，暗示IQGAP1在小鼠经历甲醛毒性刺激时发挥了重要作用。
另外，在相差显微镜下观察可见培养的气道上皮细胞在甲醛暴露后失去其特有的铺路石状形态，细胞间隙增宽，伸展扁平，更高浓度时细胞皱缩变圆，死细胞明显增多。MTT结果显示低浓度甲醛可促进细胞增殖，而高浓度甲醛则引起细胞活力降低并呈浓度依赖性。接着，我们使用pull down方法检测Rho家族蛋白Rac1及Cdc42的活性，发现二者在甲醛暴露后的活性存在不一致的变化趋势(Cdc42活性降低，Rac1活性先升后降)，可能在损伤修复过程中发挥着不同的作用。然后，我们进一步检测其效应因子IQGAP1在细胞内的表达与定位，Western blot结果显示甲醛可诱导气道上皮细胞中IQGAP1呈时间-浓度依赖性的过表达，其定位信号也从细胞连接处及核周扩散至整个胞浆。最重要的是，我们发现IQGAP1过表达可明显促进细胞增殖，提升细胞活力，而这一切是通过增加β-连环素在胞浆内的聚集继而促进后者的核转位过程来实现的。使用RNA干扰敲低IQGAP1的表达以后，不仅严重影响了甲醛诱导的β-连环素核转位过程，而且也显著降低了β-连环素／Tcf信号的转录活性及其靶基因cyclin D1的表达，更加证明了IQGAP1在细胞增殖过程中的重要作用。
最后，我们在检测微管相关蛋白APC在气道上皮细胞内的表达与定位时惊人地发现，甲醛暴露不仅引起APC蛋白表达上调，而且还诱导了原本弥散分布于胞浆的APC蛋白聚集于胞浆一侧及胞核之中。为了解这一现象出现的原因，我们使用免疫共沉淀方法将IQGAP1与APC蛋白互相沉淀，发现二者在甲醛暴露后结合加强，于是我们结合文献推论，APC蛋白在胞浆中聚集的一侧可能预示着细胞迁移的方向，而IQGAP1可能通过与APC蛋白相结合参与细胞极化与迁移，更重要的是，机制未明的APC蛋白转位入核可能起着抑制β-连环素／Tcf信号过度活化的作用，在如此精细调节的蛋白网络中作为一个终止信号避免上皮修复后的过度增殖。
实验结论
本实验表明：(1)甲醛暴露能在体内、外诱导IQGAP1过表达。(2)IQGAP1过表达可通过增加β-连环素在细胞内的聚集及向核内转位，从而促进β-连环素／Tcf信号活化及增殖相关cyclin D1的表达，最终在细胞增殖中起重要作用。(3)甲醛暴露后，IQGAP1与APC蛋白、β-连环素结合增加，可能与细胞迁移及细胞增殖有关。(4)甲醛诱导APC蛋白聚集于胞浆一侧及胞核，可能预示了细胞迁移的方向及抑制细胞过度增殖。
PartⅠ
Bleomycin-induced nuclear factor-κB activation in humanbronchial epithelial cells involves the phosphorylation ofglycogen synthase kinase 3β
Background:
Bleomycin(BLM) is an effective chemotherapeutic antibiotic which has beenused successfully to treat a wide variety of malignancies such as squamous cellcarcinoma,sarcoma and lymphoma.However,the clinical application of the drug islimited by its adverse side effects,especially severe lung injury and fibrosis.Epithelialcells are known to be a main target of BLM lung toxicity.Exposure of cells to BLMresults in diverse cellular responses including apoptosis,cell death and secretion ofpro-inflammatory cytokines,which contribute to the development of lung injury andfibrosis induced by BLM.Nuclear factor-κB(NF-κB) regulates a large number of genesinvolved in cell survival,differentiation,proliferation,apoptosis and inflammation andis also considered to be closely correlated with BLM lung toxicity.
NF-κB is normally sequestered in the cytoplasm of resting cells by inhibitor ofNF-κB(IκB) and remains transcriptionally inactive.Stimulation by cytotoxic triggerssuch as BLM and tumor necrosis factorα(TNFα) induces the ubiquitylation anddegradation of IκB.The loss of IκB exposes the nuclear localization signal sequence onNF-κB,resulting in the nuclear translocation of NF-κB and transcriptional activation ofNF-κB target gene promoters.Glycogen synthase kinase 3β(GSK3β) is a ubiquitousand important serine/threonine kinase.In contrast to most other kinases,GSK3βisconstitutively active in resting cells and can be inactivated by phosphorylation on anN-terminal serine residue(Ser~9) through a PI3K(phosphatidylinositol 3-kinase)/Akt(also called protein kinase B,PKB)-dependent mechanism.Recent studies revealed that GSK3βmight be involved in the control of various signaling pathways that activateNF-κB,and the effect of GSK3βon the regulation of NF-κB raised an interest in thepathophysiology of the diseases in which NF-κB activation is involved.However,theexact mechanisms are still unclear.Therefore,our present studies focus on the changesof GSK3βactivity and the effects of GSK3βon NF-κB signaling during the toxic lunginjury induced by BLM.
Objective
To investigate the changes of GSK3βactivity and the effects of GSK3βon NF-κBsignaling during the toxic lung injury induced by BLM.
Methods
In this study,we treated the cells with BLM to establish the lung toxic injury modelin vitro.Firstly,we used the phase contrast microscope and fluorescence staining toobserve the morphological changes of bronchial epithelial cells(BECs) after BLMtreatment.Furthermore,MTT analysis was used to reveal the changes of cell viabilityafter BLM treatment.Then the following experiments were performed:Western blot,confocal laser scanning,co-immunoprecipitation,transient transfection,luciferasereporter assay and so on.We aimed to study the active status of NF-κB signaling,theactivity changes of Akt/GSK3βpathway and its effects on NF-κB signaling after BLMtreatment.
Results
Under phase contrast microscope,treatment with BLM made cells displaymorphological changes,including widened cell-cell interspaces,more widely flattenedappearance and numerous dead cells,compared with control cells,which showed aclassic cobblestone-like epithelial morphology that was three-dimensional,slightlyraised and closely adherent.In addition,nuclear morphological changes were observed under fluorescence microscope by Hoechst 33258 staining.Results showed that BLMcaused nuclear condensation and cell apoptosis.MTT analysis revealed adose-dependent reduction of cell viability after BLM treatment.
Then,we further confirmed that BLM induced the activation of NF-κB signaling inBECs by transient transfection and luciferase reporter assay.Furthermore,theexpression of IκBαreduced and almost disappeared within 2 h after BLM treatment.Inthe same time,both the data from immunofluorescence staining and Western blot ofnuclear extract revealed that incubation with BLM in BECs caused a striking shift ofp65(the subunit of NF-κB) towards the nucleus.These results are consistent with theprevious experiments in vivo which have been published.Subsequently,we found thatphosphorylation levels of Akt and GSK3βrapidly increased in 0.5 h after BLMtreatment and became higher at 2 h.However,pretreated with LY294002,whichinhibited BLM-induced phosphorylation of Akt and GSK3β,the transcriptionalactivation of NF-κB in response to BLM was also significantly reduced in adose-dependent manner.Thus we suppose that BLM-induced NF-κB activation must beclosely associated with the active status of GSK3β.So cells were transiently transfectedwith a stable mutant of GSK3β(S9A) which is constitutively active followed by BLMtreatment.The results showed that continuous activation of GSK3βled to a partial butsignificant inhibition of BLM-induced NF-κB activation,protected endogenous IκBαfrom degradation and blocked the nuclear translocation of p65 subunit.To investigatethe possible cause of this phenomenon,a co-immunoprecipitation assay was used andthe results showed that GSK3βwas indeed able to be co-precipitated with IκBαin BECs.Moreover,the co-precipitated proteins decreased after BLM treatment.Importantly,there was no phosphorylated GSK3βto be detected in IκBαprecipitates,indicating thatIκBαmight be preserved by a mechanism that is dependent on direct GSK3βbinding.These data further demonstrated that GSK3βaffected BLM-induced NF-κB activationthrough an interaction with IκBα.
Conclusions
(1) BLM induced the morphological changes and reduction of cell viability in BECs.
(2) BLM induced NF-κB activation,accompanied with IκBαdegradation and p65nuclear translocation in BECs.
(3) BLM induced the phosphorylation of Akt and GSK3βin BECs.
(4) GSK3βmight negatively regulate BLM-induced NF-κB activation in BECs by amechanism that is dependent on the interaction of GSK3βand IκBα.
PartⅡ
Effects of IQ domain GTPase-activating protein 1 on APCprotein andβ-catenin/Tcf signaling during the toxic injury ofairway epithelia after formaldehyde exposure
Background:
Formaldehyde(FA) is a ubiquitous chemical pollutant,which can induce chronictoxic respiratory disease of animals or human after long-term and low-concentrationtouch.It even leads to gene mutation and increases the probability of some malignanttumours in human.
It has been confirmed that IQ domain GTPase-activating protein 1(IQGAP1) isalways over-expressed in most tumours with unclear mechanism.IQGAP1,animportant effector of Rho GTPase family including Cdc42 and Racl,has many proteinbinding sites,not only regulating cell skeleton and affecting cell adhesion,but alsopossibly mediating the transcriptional activation ofβ-catenin/Tcf signaling and theexpression of downstream target gene involved in cell proliferation,such as cyclin D1and c-myc.On the other hand,active Cdc42 and Racl can promote APC proteinstabilizing the plus end of microtubule in the leading edge of migrating cells throughbinding with IQGAP1 and play key roles in cell polarization and migration.Nevertheless,a great quantity of recent studies demonstrated that APC protein alsolocated in the nuclei where it functions in regulating the spindle and inhibits cellproliferation by interfering with the activation ofβ-catenin/Tcf signaling.Therefore,inthe present study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro,and observed that the dynamic changes ofIQGAP1 during the process of injury and repair.We further studied the effects ofIQGAP1 on APC protein andβ-catenin/Tcf signaling in vitro by using transfection andsmall interfering RNA technique.We aim to understand the injury and repair process of airway epithelia following formaldehyde exposure,providing a novel idea in elucidatingthe potential tumourigenicity mechanism of formaldehyde.
Objective
To determine the dynamic changes of IQGAP1 and the effects of IQGAP1 onAPC protein andβ-catenin/Tcf signaling during the toxic injury of airway epitheliafollowing formaldehyde exposure.
Methods
In this study,we established the toxic injury model of airway epithelia afterformaldehyde exposure in vivo and in vitro.We first observed the pathological changesof murine airway epithelia following formaldehyde inhalation under light microscopeafter H&E staining.Then we detected the location and expression of IQGAP 1 by usingimmunofluorescence staining and Western blot.In our experiment in vitro,we used thephase contrast microscope and MTT method to observe the morphological changes ofbronchial epithelial cells(BECs) after formaldehyde exposure and the effect of IQGAP 1over-expression on cell proliferation.Pull down assay was used to detect the activity ofCdc42 and Racl.Immunofluorescence staining and confocal laser scanning microscopewere used to determine the subcellular location of APC andβ-catenin protein.Thefollowing experiments were performed:Western blot,co-immunoprecipitation assay,transient transfection,small interfering RNA,luciferase reporter assay and so on.Weaim to study the effects of IQGAP1 on APC protein andβ-catenin/Tcf signaling,understand the injury and repair process of airway epithelia following formaldehydeexposure,providing a novel idea in elucidating the potential tumourigenicitymechanism of formaldehyde.
Results
In the process of formaldehyde inhalation,the murine airway epithelia experiencedthe dynamic course from increased number of goblet cells,lodgings and losses of cilia, loose contacts between cells,infiltrations of inflammatory cells,shedding of necroticcells into the airway lumen to the re-appearance of cilia,tighter cell-cell junction,hyperplasia of basal layer cells,increased cellular layer and infiltration of chronicinflammatory cells.The expression of IQGAP 1 showed a corresponding tendency withthe progression of injury and repair.A strong signal of IQGAP 1 diffused in the wholecytoplasm in the early stage of severe injury and located around the plasma membraneagain in the end stage of repair,implicating that IQGAP 1 plays an important role in theinjury and repair of airway epithelia following the toxic stimulation from formaldehyde.
Under phase contrast microscope,formaldehyde exposure made cells lose itsnatural classic cobblestone-like epithelial morphology and display widened cell-cellinterspaces,more widely flattened appearance,even cellular condensation andnumerous dead cells when exposed to higher concentration.MTT analysis revealed thatlow-concentration formaldehyde promoted cell proliferation and high-concentrationformaldehyde induced a dose-dependent reduction of cell viability.Then,pull downassay was used to detect the activity of Cdc42 and Racl and the results showed adifferent alteration after formaldehyde exposure.After that,we further determined theexpression and location of their effectors,IQGAP 1.The data from Western blot analysisshowed that formaldehyde induced the time-and concentration-dependentover-expression of IQGAP1 in the airway epithelial cells.The fluorescent signal alsotranslocated from the cell-cell boundary and circum-nuclei to the whole cytoplasm.Importantly,we found that IQGAP1 over-expression stimulated cell proliferation andelevated the cell viability significantly,which might be achieved through increasing theaccumulation ofβ-catenin in the cell and promoted the process of its nucleartranslocation.After the expression of IQGAP1 was knocked down by small interferingRNA(siRNA),not only formaldehyde-inducedβ-catenin translocation,but also theactivation ofβ-catenin/Tcf signal and the expression of its downstream target genecyclin D 1 were inhibited,further confirming a key role of IQGAP 1 in cell proliferation.
Finally,when we detected the expression and location of APC protein in airway epithelial cells,we surprisingly found that formaldehyde not only induced the increaseof APC protein but also resulted in a striking translocation from diffusion in the wholecytoplasm to accumulation in the nuclei and one side of cytoplasm.To understand thecause of this phenomenon,co-immunoprecipitation assay was used to detect the relationof IQGAP 1 and APC.The results showed that they could be precipitated each other andthe interaction was enhanced after formaldehyde exposure.So we proposed that the sideof APC accumulation might signify the direction of cell migration and IQGAP1participated in cell migration through binding with APC.Importantly,the nucleartranslocation of APC protein with unknown mechanism might function as a terminationsignal in the protein network under complicate regulations to inhibit the activation ofβ-catenin/Tcf signaling and prevent the excess proliferation of epithelial cells in therepair process after injury.
Conclusions
(1) Formaldehyde induced the over-expression of IQGAP 1 in vivo and in vitro.
(2) Over-expression of IQGAP1 resulted inβ-catenin accumulation and nucleartranslocation,activatedβ-catenin/Tcf signaling,elevated expression levels of cyclinD 1 and promoted cell proliferation in airway epithelial cells.
(3) The enhanced interaction of IQGAP1 andβ-catenin and APC after formaldehydeexposure might be correlated with cell migration and cell adhesion.
(4) Formaldehyde exposure up-regulated the expression of APC protein in airwayepithelial cells and made it accumulate in one side of cytoplasm and the nuclei withthe tighter interaction of IQGAP1 and APC in the cytoplasm,which could playimportant roles in signifying the direction of cell migration and preventing theexcess proliferation of epithelial cells.
引文
1.Jules-Elyse K and White DA.Bleomycin-induced pulmonary toxicity.Clin.Chest Med.1990,11:1-20.
2.Wallach-Dayan SB,Izbicki G,Cohen PY,et al.Bleomycin initiates apoptosis of
lung epithelial cells by ROS but not by Fas/FasL pathway.Am.J.Physiol.Lung Cell Mol.Physiol.2006,290:L790-L796.
3.Hong JS,Ko HH,Han ES,et al.Inhibition of bleomycin-induced cell death in rat alveolar macrophages and human lung epithelial cells by ambroxol.Biochem.Pharmacol.2003,66:1297-1306.
4.Gon Y,Hashimoto S,Nakayama T,et al.N-acetyl-L-cysteine inhibits bleomycin-induced interleukin-8 secretion by bronchial epithelial cells.Respirology 2000,5:309-313.
5.Clemo NK,Collard TJ,Southern SL,et al.BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-κB activity.Carcinogenesis 2008,29:849-857.
6.Guttridge DC,Albanese C,Reuther JY,et al.NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1.Mol.Cell.Biol.1999,19:5785-5799.
7.Sethi G,Ahn KS,Sung B,et al.Pinitol targets NF-κB activation pathway leading to inhibition of gene products associated with proliferation,apoptosis,invasion,and angiogenesis.Mol.Cancer Ther.2008,7:1604-1614.
8.Tak PP and Firestein GS.NF-κB:a key role in inflammatory diseases.J.Clin.Invest.2001,107:7-11.
9.Gurujeyalakshmi G,Wang Y,Girl SN.Taurine and niacin block lung injury and fibrosis by down-regulating bleomycin-induced activation of transcription NF-κB in mice.J.Phannacol.Exp.Ther.2000,293:82-90.
10．张晓晔，周妍，刘卫青，等．核因子-κB在博莱霉素致小鼠肺纤维化病理过程中的动态表达．中国医科大学学报 2007，36：505-510．
11．张晓晔，周妍，刘卫青，等．核因子-κB在博莱霉素损伤小鼠肺组织培养细胞中的表达及对白细胞介素4的影响．中国现代医学杂志 2008，18：142-146．
12.Zhang XY,Shimura S,Masuda T,et al.Antisense oligonucleotides to NF-κB improve survival in bleomycin-induced pneumopathy of the mouse.Am.J.Respir.Crit.Care Med.2000,162:1561-1568.
13.Verma IM,Stevenson JK,Schwarz EM,et al.Rel/NF-κB/IκB family:intimate tales of association and dissociation.Genes Dev.1995,9:2723-2735.
14.Schmitz ML and Baeuerle PA.The p65 subunit is responsible for the strong transcription activating potential of NF-κB.EMBO J.1991,10:3805-3817.
15.Sanchez JF,Sniderhan LF,Williamson AL,et al.Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of NF-κB signaling.Mol.Cell Biol.2003,23:4649-4662.
16.Henkel T,Mechleidt T,Alkalay I,et al.Rapid proteolysis of IκBα is necessary for activation of transcription factor NF-κB.Nature 1993,365:182-185.
17.Grimes CA and Jope RS.The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling.Prog.Neurobiol.2001,65:391-426.
18.Tian D,Zhu M,Chen WS,et al.Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.Food Chem.Toxicol.2006,44:1590-1596.
19．朱敏，马燕，李娜萍，等．GSK3β在几种气道上皮损伤模型中的表达．中国组织化学与细胞化学杂志．2007，16：369-377．
20.Zhu M,Tian D,Li JS,et al.Glycogen synthase kinase 3β and β-catenin are involved in the injury and repair of bronchial epithelial cells induced by scratching.Exp.Mol.Pathol.2007,83:30-38.
21.Hoeflich KP,Luo J,Rubie EA,et al.Requirement for glycogen synthase kinase-3beta in cell survival and NF-κB activation.Nature 2000,406:86-90.
22.Bournat JC,Brown AM,Soler AP.Wnt-1 dependent activation of the survival factor NF-κB in PC12 cells.J.Neurosci.Res.2000,61:21-32.
23.Buss H,D(?)rrie A,Schmitz ML,et al.Phosphorylation of serine 468 by GSK3β negatively regulates basal p65 NF-κB activity.J.Biol.Chem.2004,279:49571-49574.
24.Steinbrecher KA,Wilson W,Cogswell PC,et al.Glycogen synthase kinase 3β functions to specify gene-specific,NF-κB-dependent transcription.Mol.Cell.Biol.2005,25:8444-8455.
25.Kane LP,Shapiro VS,Stokoe D,et al.Induction of NF-κB by the Akt/PKB kinase.Curr.Biol.1999,9:601-604.
26.Yoshimi M,Maeyama T,Yamada M,et al.Recombinant human erythropoietin reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice.Respirology 2008,13:639-645.
27.Li LF,Liao SK,Huang CC,et al.Serine/threonine kinase-protein kinase B and extracellular signal-regulated kinase regulate ventilator-induced pulmonary fibrosis after bleomycin-induced acute lung injury:a prospective,controlled animal experiment.Crit.Care 2008,12:R 103.[Epub ahead of print]
28.Vittal R,Horowitz JC,Moore BB,et al.Modulation of prosurvival signaling in fibroblasts by a protein kinase inhibitor protects against fibrotic tissue injury.Am.J.Pathol.2005,166:367-375.
29.Wang C,Jiang Z,Yao J,et al.Participation of cathepsin B in emodin-induced apoptosis in HK-2 cells.Toxicol.Lett.2008,181:196-204.
30．E．哈洛，D．莱恩．抗体实验指南．沈关心，龚非力等译．北京：科学出版社，2002．
31.Etienne-Manneville S and Hall A.Cdc42 regulates GSK3β and adenomatous polyposis coli to control cell polarity.Nature 2003,421:753-756.
32.Stambolic V and Woodgett JR.Mitogen inactivation of synthase kinase-3β in intact cells via serine 9 phosphorylation.Biochem J 1994,303:701-704.
33.Etienne-Manneville S and Hall A.Integrin-mediated activation of cdc42 controls cell polarity in migrating astrocytes through PKCξ.Cell 2001,106:489-498.
34．J．萨姆布鲁克，D．W．拉塞尔．分子克隆实验指南，第3版，黄培堂等译．北京：科学出版社，2003：1-146．
35．D．L．斯佩克特，R．D．戈德曼，莱因万德．细胞实验指南，黄培堂等译．北京：科 学出版社，2001：10-12．
36.Tucker TA,Varga K,Bebok Z,et al.Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipid.Am.J.Physiol.Cell Physiol.2003,284:C791-C804.
37.Alessi DR,James SR,Downes CP,et al.Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha.Curr.Biol.1997,7:261-269.
38.Kim SD,Yang SI,Kim HC,et al.Inhibition of GSK3β mediates expression of MMP-9 through ERK1/2 activation and translocation of NF-κB in rat primary astrocyte.Brain Res.2007,1186:12-20.
39.Shukla A,Timblin CR,Hubbard AK,et al.Silica-induced activation of c-Jun-NH2-terminal amino kinases,protracted expression of the activator protein-1 proto-oncogene,fra-1,and S-phase alterations are mediated via oxidative stress.Cancer Res.2001,61:1791-1795.
40.Simon AR,Rai U,Fanburg BL,et al.Activation of the JAK-STAT pathway by reactive oxygen species.Am.J.Physiol.1998,275:C1640-C1652.
41．张晓晔，焦光宇，朱敏，等．NF-κB反义寡核苷酸对博莱霉素致小鼠急性肺损伤的保护作用．中国医科大学学报2008，37：217-220．
42.Sun SC,Ganchi PA,Ballard DW,et al.NF-κB controls expression of inhibitor IκBα:evidence for an inducible auto-regulatory pathway.Science 1993,259:1912-1915.
43.Woodgett JR.Molecular cloning and expression of glycogen synthase kinase-3/factor A.EMBO J 1990,9:2431-2418.
44.Lau KF,Miller CC,Anderton BH,et al.Expression analysis of glycogen synthase kinase-3 in human tissues.J.Peptide Res.1999,54:85-91.
45.Kwon O,Kim KA,He L,et al.Ionizing radiation can induce GSK3β phosphorylation and NF-κB transcriptional transactivation in ATM-deficient fibroblasts.Cell Signal 2008,20:602-612.
46.Dugo L,Collin M,Thiemermann C.Glycogen synthase kinase 3beta as a target for the therapy of shock and inflammation.Shock 2007,27:113-123.
1. Flyvholm MA and Andersen P. Identification of FA releasers and occurrence of FA and FA releasers in registered chemical products. Am. J. Ind. Med. 1993, 24:533-552.
2. Baker RR. The generation of FA in cigarettes - Overview and recent experiments.Food Chem.Toxicol. 2006, 44: 1799-1822.
3. Casset A, Marchand C, Purohit A, et al.Inhaled FA exposure: effect on bronchial response to mite allergen in sensitized asthma patients. Allergy 2006, 61:1344-1350.
4. Kita T, Fujimura M, Myou S, et al. Potentiation of allergic bronchoconstriction by repeated exposure to formaldehyde in guinea-pigs in vivo. Clin. Exp. Allergy 2003,33: 1747-1753.
5. Arts JH, Rennen MA, de Heer C. Inhaled FA: evaluation of sensory irritation in relation to carcinogenicity. Exp. Anim.2006, 46: 279-286.
6. The International Agency for Research on Cancer (IARC) 1995,62:217-362.
7. Weissbach L, Settleman J, Kalady MF, et al. Identification of a human rasGAP-related protein containing calmodulin-binding motifs. J. Biol.Chem. 1994,269:20517-20521.
8. Futaka M, Nakagawa M,Kaibuchi K. Roles of Rho-family GTPases in cell polarization and directional migration. Curr. Opin. Cell Biol. 2003, 15: 590-597.
9. Watanabe T, Wang S, Noritake J, et al. Interaction with IQGAP1 links APC to Racl,Cdc42 and actin filaments during cell polarization and migration. Dev. Cell 2004, 7:871-883.
10. Erickson JW, Cerione RA, Hart MJ. Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase. J. Biol. Chem. 1997, 272:24443-24447.
11. Roy M, Li Z, Sacks DB. IQGAP1 binds ERK2 and modulates its activity. J. Biol.Chem. 2004, 279: 17329-17337.
12. Roy M, Li Z, Sack DB. 1QGAP1 is a scaffold for mitogen-activated protein kinase signaling. Mol Cell Biol 2005, 25: 7940-7952.
13. Hart MJ, Callow MG, Souza B, et al.IQGAP1,a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs. EMBO J. 1996, 15:2997-3005.
14. Kuroda S, Fukata M, Nakagawa M, et al. Role of IQGAP1,a target of the small GTPases Cdc42 and Racl,in regulation of E-cadherin-mediated cell-cell adhesion.Science 1998, 281: 832-835.
15. Li Z, Kim SH, Higgins JM, et al. IQGAP1 and calmodulin modulate E-cadherin function. J. Biol. Chem.1999, 274: 37885-37892.
16. Fukata M, Watanabe T, Noritake J, et al. Racl and Cdc42 capture microtubules through IQGAP1 and CLIP-170.Cell 2002, 109: 873-885.
17. Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by P-catenin is modulated by calmodulin. J. Biol. Chem. 2002, 277:7453-7465.
18. Suqimoto N, Imoto I, Fukuda Y, et al. IQGAP1,a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A. J. Hum. Genet. 2001,46:21-25.
19. Sun W, Zhang K, Zhang X, et al. Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.Cancer Lett. 2004, 212: 83-93.
20. Nabeshima K, Shimao Y, Inoue T, et al. Immunohistochemical analysis of IQGAP1 expression in human colorectal carcinomas: its overexpression in carcinomas and association with invasion fronts. Cancer Lett. 2002, 176: 101-109.
21. Dong P, Nabeshima K, Nishimura N, et al. Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent prognostic parameters in ovarian carcinomas. Cancer Lett. 2006, 243: 120-127.
22. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000, 406: 532-535.
23.Miyoshi T,Shirakusa T,Ishikawa Y,et al.Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern.Pathol.Int.2005,55:419-424.
24.Jadeski L,Mataraza JM,Jeong HW,et al.IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells.J.Biol.Chem.2008,283:1008-1017.
25.Dong PX,Jia N,Xu ZJ,et al.Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro.J.Exp.Clin.Cancer Res.2008,27:77-85.
26.Wang Y,Wang M,Wang F,et al.IQGAP1 promotes cell proliferation and is involved in a phosphorylation-dependent manner in wound closure of bronchial epithelial cells.Int.J.Mol.Med.2008,22:79-87.
27.Wang Y,Wang A,Wang F,et al.IQGAP1 activates Tcf signal independent of Racl and Cdc42 in injury and repair of bronchial epithelial cells.Exp.Mol.Pathol.2008,85:122-128.
28.Hatsell S,Rowlands T,Hiremath M,et al.Beta-catenin and Tcfs in mammary development and cancer.J.Mammary Gland Biol.Neoplasia 2003,8:145-158.
29．许三林，吴人亮，陈春莲，等．上皮钙黏附素在吸烟小鼠呼吸道上皮损伤修复中表达的影响．中华结核和呼吸杂志1999，22：417-419．
30．陈芳，吴人亮，王曦，等．香烟烟雾提取物对培养猪气道上皮细胞β-连环素及酪氨酸磷酸化的影响．中华医学杂志2001，81：406-410．
31.Chen WS,Wu RL,Tian D,et al.Role of glycogen synthase kinase 3 in squamous differentiation of pig airway epithelial cells:A primary study.Acta Physiologica Sinica 2005,57:467-472.
32.Tian D,Zhu M,Chen WS,et al.Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.Food Chem.Toxicol.2006,44:1590-1596.
33.Zhu Min,Tian Dan,Li Jiansha,et al.Glycogen synthase kinase 3beta and beta-catenin are involved in the injury and repair of bronchial epithelial cells induced by scratching.Exp.Mol.Pathol.2007,83:30-38.
34.Tian Dan,Zhu Min,Li Jiansha,et al.Cigarette smoke extract induces activation of β-catenin/TCF signaling through inhibiting GSK3β in human alveolar epithelial cell line.Toxicol.Lett.2009,187:58-62.
35．刘明阁，李娜萍，吴人亮，等．APC蛋白、GSK3β在吸烟小鼠气道上皮损伤修复中的动态变化．生理学报2006，58：255-261．
36．朱敏，李建莎，田丹，等．GSK3β和APC蛋白在气道上皮细胞机械损伤修复过程的时空分布．生理学报．2007，59：197-203．
37.Sena P,Saviano M,Monni S,et al.Subcellular localization of beta-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions.Cancer Lett.2006,241:203-212.
38.Neufeld KL and White RL.Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein.Proc.Natl.Acad.Sci.USA 1997,94:3034-3039.
39.Neufeld KL,Nix DA,Bogerd H,et al.Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm.Proc.Natl.Acad.Sci.USA 2000,97:12085-12090.
40.Rosin-Arbesfeld R,Cliffe A,Brabletz T,et al.Nuclear export of the APC tumour suppressor controls β-catenin function in transcription.EMBO J.2003,22:1101-1113.
41.Yang YH,Xi ZG,Chao FH,et al.Effects of FA inhalation on lung of rats.Biomed.Environ.Sci.2005,18:164-168.
42.Fukata M,Nakagawa M,Itoh N,et al.Involvement of IQGAP1,an effector of Racl and Cdc42 GTPases,in cell-cell dissociation during cell scattering.Mol.Cell.Biol.2001,21:2165-2183.
43.Korinek V,Barker N,Morin P J,et al.Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science 1997,275:1784-1787.
44.Etienne-Manneville S and Hall A.Rho GTPases in cell biology.Nature 2002,420:629-635.
45. Roemer E, Anton HJ, Kindt R. Cell proliferation in the respiratory tract of the rat after acute inhalation of formaldehyde or acrolein. J. Appl.Toxicol. 1993, 13:103-107.
46. Hsu SC, Gakceran J, Grosschedl R. Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin. Mol. Cell Biol. 1998,18:4807-4818.
47. Cheon SS, Nadesan P, Poon R, et al. Growth factors regulate Beta-catenin-mediated TCF- dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing. Exp. Cell Res. 2004, 293: 267-274.
48. Tetsu O and McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999, 398: 422-426.
49. Roose J and Clevers H. TCF transcription factors: molecular switches in carcinogenesis. Biochim. Biophys. Acta. 1999, 1424: M23-M37.
50. Monticello TM, Morgan KT, Everitt JI,et al. Effects of formaldehyde gas on the respiratory tract of rhesus monkeys. Pathology and cell proliferation. Am. J. Pahtol.1989,134:515-527.
51. Sacco O, Silvestri M, Sabatini F, et al. Epithelial cells and fibroblasts: structural repair and remodeling in the airways. Paediatr. Respir. Rev. 2004, 5: S35-40.
52. Raftopoulou M and Hall A. Cell migration: Rho GTPases lead the way. Dev Biol 2004, 265: 23-32.
53. Eaton S, Auvinen P, Luo L, et al. Cdc42 and Racl control different actin-dependent processes in the Drosophila wing disc epithelium. J. Cell Biol. 1995, 131:151-164.
54. Wu X, Tu X, Joeng KS, et al. Racl activation controls nuclear localization of beta-catenin during canonical Wnt signaling. Cell 2008, 133: 340-353.
55. Willert K and Nusse R. Beta-catenin: a key mediator of Wnt signaling. Curr. Opin.Gene Dev.1998,8:95-102.
56. Natsume H, Sasaki S, Kitagawa M, et al.β-Catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone. Biochem.Biophys. Res. Comm.2003, 309: 408-413.
1.Weissbach L,Settleman J,Kalady MF,et al.Identification of a human rasGAP-related protein containing calmodulin-binding motifs.J.Biol.Chem.1994,269:20517-20521.
2.Noritake J,Watanabe T,Sato K,et al.IQGAP1:a key regulator of adhesion and migration.J.Cell Sci.2005,118:2085-2092.
3.Brown MD and Sacks DB.IQGAP1 in cellular signaling:bridging the GAP.Trends Cell Biol.2006,16:242-249.
4.Suqimoto N,Imoto I,Fukuda Y,et al.IQGAP1,a negative regulator of cell-cell adhesion,is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A.J.Hum.Genet.2001,46:21-25.
5.Sun W,Zhang K,Zhang X,et al.Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.Cancer Lett.2004,212:83-93.
6.Nabeshima K,Shimao Y,Inoue T,et al.Immunohistochemical analysis of IQGAP1 expression in human colorectal carcinomas:its overexpression in carcinomas and association with invasion fronts.Cancer Lett.2002,176:101-109.
7.Dong P,Nabeshima K,Nishimura N,et al.Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent prognostic parameters in ovarian carcinomas.Cancer Lett. 2006,243:120-127.
8. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC.Nature 2000,406: 532-535.
9. Miyoshi T, Shirakusa T, Ishikawa Y, et al. Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern. Pathol. Int. 2005, 55: 419-424.
10. McCallum SJ, Wu WJ, Cerione RA. Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAPl and a Cdc42Hs binding partner with similarity to IQGAP2. J. Biol. Chem. 1996, 271:21732-21737.
11. Briggs MW and Sacks DB. IQGAPl as signal integrator: Ca~(2+),calmodulin, Cdc42 and the cytoskeleton. FEBS Lett. 2003, 542: 7-11.
12. Fukata M,Kuroda S, Nakagawa M, et al. Cdc42 and Rac1 regulate the interaction of IQGAP1 with #####-catenin. J. Biol. Chem. 1999, 274: 26044-26050.
13. Kuroda S, Fukata M, Nakagawa M, et al. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin-mediated cell-cell adhesion.Science 1998,281: 832-835.
14. Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by #######-catenin is modulated by calmodulin. J. Biol. Chem. 2002, 277:7453-7465.
15. Wang Y, Wang A, Wang F, et al. IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells. Exp. Mol. Pathol. 2008,85: 122-128.
16. Wang Y, Wang M, Wang F, et al. IQGAP1 promotes cell proliferation and is involved in a phosphorylation-dependent manner in wound closure of bronchial epithelial cells. Int. J. Mol. Med. 2008, 22: 79-87.
17. Akhmanova A and Hoogenroad CC. Microtubule plus-end-tracking proteins:mechanisms and function. Curr. Opin. Cell Biol. 2005, 17:47-54.
18. Fukata M, Watanabe T, Noritake J, et al. Racl and Cdc42 capture microtubules through IQGAP1 and CLIP-170.Cell 2002, 109: 873-885.
19. Watanabe T, Wang S, Noritake J, et al. Interaction with IQGAP1 links APC to Rac1,Cdc42 and actin filaments during cell polarization and migration. Dev. Cell 2004, 7:871-883.
20. Walch A, Seidl S, Hermannstadter C, et al. Combined analysis of Racl, IQGAP1,Tiam1 and E-cadherin expression in gastric cancer. Mod. Pathol. 2008, 21:544-552.
21. Takemoto H, Doki Y, Shiozaki H, et al. Localization of IQGAP1 is inversely correlated with intercellular adhesion mediated by E-cadherin in gastric cancers. Int.J.Cancer 2001, 91: 783-788.
22. Morris LE, Bloom GS, Frierson HF Jr, et al. Nucleotide variants within the IQGAP1 gene in diffuse-type gastric cancers. Genes Chromosomes Cancer 2005,42: 280-286.
23. Li S, Wang Q, Chakladar A, et al. Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1. Mol.Cell Biol.2000, 20: 697-701.
24. Schmidt VA, Chiariello CS, Capilla E, et al. Development of hepatocellular carcinoma in Iqgap2-deficient mice is IQGAP1 dependent. Mol. Cell Biol. 2008, 28:1489-1502.
25. Jin SH, Akiyama Y, Fukamachi H, et al. IQGAP2 inactivation through aberrant promoter methylation and promotion of invasion in gastric cancer cells. Int. J.Cancer 2008, 122:1040-1046.
26. Nakamura H, Fujita K, Nakagawa H, et al. Expression pattern of the scaffold protein IQGAP1 in lung cancer. Oncol. Rep. 2005,13: 427-431.
27. McDonald KL, O'Sullivan MG, Parkinson JF, et al. IQGAP1 and IGFBP2: valuable biomarkers for determining prognosis in glioma patients. J. Neuropathol. Exp.Neurol. 2007, 66:405-417.
28. Balenci L, Clarke ID, Dirks PB, et al. IQGAP1 protein specifies amplifying cancer cells in glioblastoma multiforme. Cancer Res. 2006, 66: 9074-9082.
29.Jadeski L,Mataraza JM,Jeong HW,et al.IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells.J.Biol.Chem.2008,283:1008-1017.
30.Bourguignon LY,Gilad E,Rothman K,et al.Hyaluronan-CD44 interaction with IQGAP1 promotes Cdc42 and ERK signaling,leading to actin binding,Elk-1/estrogen receptor transcriptional activation,and ovarian cancer progression.J.Biol.Chem.2005,280:11961-11972.
31.Dong PX,Jia N,Xu Z J,et al.Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro.J.Exp.Clin.Cancer Res.2008,27:77.
2.Wallach-Dayan SB,Izbicki G,Cohen PY,et al.Bleomycin initiates apoptosis of
lung epithelial cells by ROS but not by Fas/FasL pathway.Am.J.Physiol.Lung Cell Mol.Physiol.2006,290:L790-L796.
3.Hong JS,Ko HH,Han ES,et al.Inhibition of bleomycin-induced cell death in rat alveolar macrophages and human lung epithelial cells by ambroxol.Biochem.Pharmacol.2003,66:1297-1306.
4.Gon Y,Hashimoto S,Nakayama T,et al.N-acetyl-L-cysteine inhibits bleomycin-induced interleukin-8 secretion by bronchial epithelial cells.Respirology 2000,5:309-313.
5.Clemo NK,Collard TJ,Southern SL,et al.BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-κB activity.Carcinogenesis 2008,29:849-857.
6.Guttridge DC,Albanese C,Reuther JY,et al.NF-κB controls cell growth and differentiation through transcriptional regulation of cyclin D1.Mol.Cell.Biol.1999,19:5785-5799.
7.Sethi G,Ahn KS,Sung B,et al.Pinitol targets NF-κB activation pathway leading to inhibition of gene products associated with proliferation,apoptosis,invasion,and angiogenesis.Mol.Cancer Ther.2008,7:1604-1614.
8.Tak PP and Firestein GS.NF-κB:a key role in inflammatory diseases.J.Clin.Invest.2001,107:7-11.
9.Gurujeyalakshmi G,Wang Y,Girl SN.Taurine and niacin block lung injury and fibrosis by down-regulating bleomycin-induced activation of transcription NF-κB in mice.J.Phannacol.Exp.Ther.2000,293:82-90.
10．张晓晔，周妍，刘卫青，等．核因子-κB在博莱霉素致小鼠肺纤维化病理过程中的动态表达．中国医科大学学报 2007，36：505-510．
11．张晓晔，周妍，刘卫青，等．核因子-κB在博莱霉素损伤小鼠肺组织培养细胞中的表达及对白细胞介素4的影响．中国现代医学杂志 2008，18：142-146．
12.Zhang XY,Shimura S,Masuda T,et al.Antisense oligonucleotides to NF-κB improve survival in bleomycin-induced pneumopathy of the mouse.Am.J.Respir.Crit.Care Med.2000,162:1561-1568.
13.Verma IM,Stevenson JK,Schwarz EM,et al.Rel/NF-κB/IκB family:intimate tales of association and dissociation.Genes Dev.1995,9:2723-2735.
14.Schmitz ML and Baeuerle PA.The p65 subunit is responsible for the strong transcription activating potential of NF-κB.EMBO J.1991,10:3805-3817.
15.Sanchez JF,Sniderhan LF,Williamson AL,et al.Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of NF-κB signaling.Mol.Cell Biol.2003,23:4649-4662.
16.Henkel T,Mechleidt T,Alkalay I,et al.Rapid proteolysis of IκBα is necessary for activation of transcription factor NF-κB.Nature 1993,365:182-185.
17.Grimes CA and Jope RS.The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling.Prog.Neurobiol.2001,65:391-426.
18.Tian D,Zhu M,Chen WS,et al.Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.Food Chem.Toxicol.2006,44:1590-1596.
19．朱敏，马燕，李娜萍，等．GSK3β在几种气道上皮损伤模型中的表达．中国组织化学与细胞化学杂志．2007，16：369-377．
20.Zhu M,Tian D,Li JS,et al.Glycogen synthase kinase 3β and β-catenin are involved in the injury and repair of bronchial epithelial cells induced by scratching.Exp.Mol.Pathol.2007,83:30-38.
21.Hoeflich KP,Luo J,Rubie EA,et al.Requirement for glycogen synthase kinase-3beta in cell survival and NF-κB activation.Nature 2000,406:86-90.
22.Bournat JC,Brown AM,Soler AP.Wnt-1 dependent activation of the survival factor NF-κB in PC12 cells.J.Neurosci.Res.2000,61:21-32.
23.Buss H,D(?)rrie A,Schmitz ML,et al.Phosphorylation of serine 468 by GSK3β negatively regulates basal p65 NF-κB activity.J.Biol.Chem.2004,279:49571-49574.
24.Steinbrecher KA,Wilson W,Cogswell PC,et al.Glycogen synthase kinase 3β functions to specify gene-specific,NF-κB-dependent transcription.Mol.Cell.Biol.2005,25:8444-8455.
25.Kane LP,Shapiro VS,Stokoe D,et al.Induction of NF-κB by the Akt/PKB kinase.Curr.Biol.1999,9:601-604.
26.Yoshimi M,Maeyama T,Yamada M,et al.Recombinant human erythropoietin reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice.Respirology 2008,13:639-645.
27.Li LF,Liao SK,Huang CC,et al.Serine/threonine kinase-protein kinase B and extracellular signal-regulated kinase regulate ventilator-induced pulmonary fibrosis after bleomycin-induced acute lung injury:a prospective,controlled animal experiment.Crit.Care 2008,12:R 103.[Epub ahead of print]
28.Vittal R,Horowitz JC,Moore BB,et al.Modulation of prosurvival signaling in fibroblasts by a protein kinase inhibitor protects against fibrotic tissue injury.Am.J.Pathol.2005,166:367-375.
29.Wang C,Jiang Z,Yao J,et al.Participation of cathepsin B in emodin-induced apoptosis in HK-2 cells.Toxicol.Lett.2008,181:196-204.
30．E．哈洛，D．莱恩．抗体实验指南．沈关心，龚非力等译．北京：科学出版社，2002．
31.Etienne-Manneville S and Hall A.Cdc42 regulates GSK3β and adenomatous polyposis coli to control cell polarity.Nature 2003,421:753-756.
32.Stambolic V and Woodgett JR.Mitogen inactivation of synthase kinase-3β in intact cells via serine 9 phosphorylation.Biochem J 1994,303:701-704.
33.Etienne-Manneville S and Hall A.Integrin-mediated activation of cdc42 controls cell polarity in migrating astrocytes through PKCξ.Cell 2001,106:489-498.
34．J．萨姆布鲁克，D．W．拉塞尔．分子克隆实验指南，第3版，黄培堂等译．北京：科学出版社，2003：1-146．
35．D．L．斯佩克特，R．D．戈德曼，莱因万德．细胞实验指南，黄培堂等译．北京：科 学出版社，2001：10-12．
36.Tucker TA,Varga K,Bebok Z,et al.Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipid.Am.J.Physiol.Cell Physiol.2003,284:C791-C804.
37.Alessi DR,James SR,Downes CP,et al.Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha.Curr.Biol.1997,7:261-269.
38.Kim SD,Yang SI,Kim HC,et al.Inhibition of GSK3β mediates expression of MMP-9 through ERK1/2 activation and translocation of NF-κB in rat primary astrocyte.Brain Res.2007,1186:12-20.
39.Shukla A,Timblin CR,Hubbard AK,et al.Silica-induced activation of c-Jun-NH2-terminal amino kinases,protracted expression of the activator protein-1 proto-oncogene,fra-1,and S-phase alterations are mediated via oxidative stress.Cancer Res.2001,61:1791-1795.
40.Simon AR,Rai U,Fanburg BL,et al.Activation of the JAK-STAT pathway by reactive oxygen species.Am.J.Physiol.1998,275:C1640-C1652.
41．张晓晔，焦光宇，朱敏，等．NF-κB反义寡核苷酸对博莱霉素致小鼠急性肺损伤的保护作用．中国医科大学学报2008，37：217-220．
42.Sun SC,Ganchi PA,Ballard DW,et al.NF-κB controls expression of inhibitor IκBα:evidence for an inducible auto-regulatory pathway.Science 1993,259:1912-1915.
43.Woodgett JR.Molecular cloning and expression of glycogen synthase kinase-3/factor A.EMBO J 1990,9:2431-2418.
44.Lau KF,Miller CC,Anderton BH,et al.Expression analysis of glycogen synthase kinase-3 in human tissues.J.Peptide Res.1999,54:85-91.
45.Kwon O,Kim KA,He L,et al.Ionizing radiation can induce GSK3β phosphorylation and NF-κB transcriptional transactivation in ATM-deficient fibroblasts.Cell Signal 2008,20:602-612.
46.Dugo L,Collin M,Thiemermann C.Glycogen synthase kinase 3beta as a target for the therapy of shock and inflammation.Shock 2007,27:113-123.
1. Flyvholm MA and Andersen P. Identification of FA releasers and occurrence of FA and FA releasers in registered chemical products. Am. J. Ind. Med. 1993, 24:533-552.
2. Baker RR. The generation of FA in cigarettes - Overview and recent experiments.Food Chem.Toxicol. 2006, 44: 1799-1822.
3. Casset A, Marchand C, Purohit A, et al.Inhaled FA exposure: effect on bronchial response to mite allergen in sensitized asthma patients. Allergy 2006, 61:1344-1350.
4. Kita T, Fujimura M, Myou S, et al. Potentiation of allergic bronchoconstriction by repeated exposure to formaldehyde in guinea-pigs in vivo. Clin. Exp. Allergy 2003,33: 1747-1753.
5. Arts JH, Rennen MA, de Heer C. Inhaled FA: evaluation of sensory irritation in relation to carcinogenicity. Exp. Anim.2006, 46: 279-286.
6. The International Agency for Research on Cancer (IARC) 1995,62:217-362.
7. Weissbach L, Settleman J, Kalady MF, et al. Identification of a human rasGAP-related protein containing calmodulin-binding motifs. J. Biol.Chem. 1994,269:20517-20521.
8. Futaka M, Nakagawa M,Kaibuchi K. Roles of Rho-family GTPases in cell polarization and directional migration. Curr. Opin. Cell Biol. 2003, 15: 590-597.
9. Watanabe T, Wang S, Noritake J, et al. Interaction with IQGAP1 links APC to Racl,Cdc42 and actin filaments during cell polarization and migration. Dev. Cell 2004, 7:871-883.
10. Erickson JW, Cerione RA, Hart MJ. Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase. J. Biol. Chem. 1997, 272:24443-24447.
11. Roy M, Li Z, Sacks DB. IQGAP1 binds ERK2 and modulates its activity. J. Biol.Chem. 2004, 279: 17329-17337.
12. Roy M, Li Z, Sack DB. 1QGAP1 is a scaffold for mitogen-activated protein kinase signaling. Mol Cell Biol 2005, 25: 7940-7952.
13. Hart MJ, Callow MG, Souza B, et al.IQGAP1,a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs. EMBO J. 1996, 15:2997-3005.
14. Kuroda S, Fukata M, Nakagawa M, et al. Role of IQGAP1,a target of the small GTPases Cdc42 and Racl,in regulation of E-cadherin-mediated cell-cell adhesion.Science 1998, 281: 832-835.
15. Li Z, Kim SH, Higgins JM, et al. IQGAP1 and calmodulin modulate E-cadherin function. J. Biol. Chem.1999, 274: 37885-37892.
16. Fukata M, Watanabe T, Noritake J, et al. Racl and Cdc42 capture microtubules through IQGAP1 and CLIP-170.Cell 2002, 109: 873-885.
17. Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by P-catenin is modulated by calmodulin. J. Biol. Chem. 2002, 277:7453-7465.
18. Suqimoto N, Imoto I, Fukuda Y, et al. IQGAP1,a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A. J. Hum. Genet. 2001,46:21-25.
19. Sun W, Zhang K, Zhang X, et al. Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.Cancer Lett. 2004, 212: 83-93.
20. Nabeshima K, Shimao Y, Inoue T, et al. Immunohistochemical analysis of IQGAP1 expression in human colorectal carcinomas: its overexpression in carcinomas and association with invasion fronts. Cancer Lett. 2002, 176: 101-109.
21. Dong P, Nabeshima K, Nishimura N, et al. Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent prognostic parameters in ovarian carcinomas. Cancer Lett. 2006, 243: 120-127.
22. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000, 406: 532-535.
23.Miyoshi T,Shirakusa T,Ishikawa Y,et al.Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern.Pathol.Int.2005,55:419-424.
24.Jadeski L,Mataraza JM,Jeong HW,et al.IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells.J.Biol.Chem.2008,283:1008-1017.
25.Dong PX,Jia N,Xu ZJ,et al.Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro.J.Exp.Clin.Cancer Res.2008,27:77-85.
26.Wang Y,Wang M,Wang F,et al.IQGAP1 promotes cell proliferation and is involved in a phosphorylation-dependent manner in wound closure of bronchial epithelial cells.Int.J.Mol.Med.2008,22:79-87.
27.Wang Y,Wang A,Wang F,et al.IQGAP1 activates Tcf signal independent of Racl and Cdc42 in injury and repair of bronchial epithelial cells.Exp.Mol.Pathol.2008,85:122-128.
28.Hatsell S,Rowlands T,Hiremath M,et al.Beta-catenin and Tcfs in mammary development and cancer.J.Mammary Gland Biol.Neoplasia 2003,8:145-158.
29．许三林，吴人亮，陈春莲，等．上皮钙黏附素在吸烟小鼠呼吸道上皮损伤修复中表达的影响．中华结核和呼吸杂志1999，22：417-419．
30．陈芳，吴人亮，王曦，等．香烟烟雾提取物对培养猪气道上皮细胞β-连环素及酪氨酸磷酸化的影响．中华医学杂志2001，81：406-410．
31.Chen WS,Wu RL,Tian D,et al.Role of glycogen synthase kinase 3 in squamous differentiation of pig airway epithelial cells:A primary study.Acta Physiologica Sinica 2005,57:467-472.
32.Tian D,Zhu M,Chen WS,et al.Role of glycogen synthase kinase 3 in squamous differentiation induced by cigarette smoke in porcine tracheobronchial epithelial cells.Food Chem.Toxicol.2006,44:1590-1596.
33.Zhu Min,Tian Dan,Li Jiansha,et al.Glycogen synthase kinase 3beta and beta-catenin are involved in the injury and repair of bronchial epithelial cells induced by scratching.Exp.Mol.Pathol.2007,83:30-38.
34.Tian Dan,Zhu Min,Li Jiansha,et al.Cigarette smoke extract induces activation of β-catenin/TCF signaling through inhibiting GSK3β in human alveolar epithelial cell line.Toxicol.Lett.2009,187:58-62.
35．刘明阁，李娜萍，吴人亮，等．APC蛋白、GSK3β在吸烟小鼠气道上皮损伤修复中的动态变化．生理学报2006，58：255-261．
36．朱敏，李建莎，田丹，等．GSK3β和APC蛋白在气道上皮细胞机械损伤修复过程的时空分布．生理学报．2007，59：197-203．
37.Sena P,Saviano M,Monni S,et al.Subcellular localization of beta-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions.Cancer Lett.2006,241:203-212.
38.Neufeld KL and White RL.Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein.Proc.Natl.Acad.Sci.USA 1997,94:3034-3039.
39.Neufeld KL,Nix DA,Bogerd H,et al.Adenomatous polyposis coli protein contains two nuclear export signals and shuttles between the nucleus and cytoplasm.Proc.Natl.Acad.Sci.USA 2000,97:12085-12090.
40.Rosin-Arbesfeld R,Cliffe A,Brabletz T,et al.Nuclear export of the APC tumour suppressor controls β-catenin function in transcription.EMBO J.2003,22:1101-1113.
41.Yang YH,Xi ZG,Chao FH,et al.Effects of FA inhalation on lung of rats.Biomed.Environ.Sci.2005,18:164-168.
42.Fukata M,Nakagawa M,Itoh N,et al.Involvement of IQGAP1,an effector of Racl and Cdc42 GTPases,in cell-cell dissociation during cell scattering.Mol.Cell.Biol.2001,21:2165-2183.
43.Korinek V,Barker N,Morin P J,et al.Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science 1997,275:1784-1787.
44.Etienne-Manneville S and Hall A.Rho GTPases in cell biology.Nature 2002,420:629-635.
45. Roemer E, Anton HJ, Kindt R. Cell proliferation in the respiratory tract of the rat after acute inhalation of formaldehyde or acrolein. J. Appl.Toxicol. 1993, 13:103-107.
46. Hsu SC, Gakceran J, Grosschedl R. Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin. Mol. Cell Biol. 1998,18:4807-4818.
47. Cheon SS, Nadesan P, Poon R, et al. Growth factors regulate Beta-catenin-mediated TCF- dependent transcriptional activation in fibroblasts during the proliferative phase of wound healing. Exp. Cell Res. 2004, 293: 267-274.
48. Tetsu O and McCormick F. Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature 1999, 398: 422-426.
49. Roose J and Clevers H. TCF transcription factors: molecular switches in carcinogenesis. Biochim. Biophys. Acta. 1999, 1424: M23-M37.
50. Monticello TM, Morgan KT, Everitt JI,et al. Effects of formaldehyde gas on the respiratory tract of rhesus monkeys. Pathology and cell proliferation. Am. J. Pahtol.1989,134:515-527.
51. Sacco O, Silvestri M, Sabatini F, et al. Epithelial cells and fibroblasts: structural repair and remodeling in the airways. Paediatr. Respir. Rev. 2004, 5: S35-40.
52. Raftopoulou M and Hall A. Cell migration: Rho GTPases lead the way. Dev Biol 2004, 265: 23-32.
53. Eaton S, Auvinen P, Luo L, et al. Cdc42 and Racl control different actin-dependent processes in the Drosophila wing disc epithelium. J. Cell Biol. 1995, 131:151-164.
54. Wu X, Tu X, Joeng KS, et al. Racl activation controls nuclear localization of beta-catenin during canonical Wnt signaling. Cell 2008, 133: 340-353.
55. Willert K and Nusse R. Beta-catenin: a key mediator of Wnt signaling. Curr. Opin.Gene Dev.1998,8:95-102.
56. Natsume H, Sasaki S, Kitagawa M, et al.β-Catenin/Tcf-1-mediated transactivation of cyclin D1 promoter is negatively regulated by thyroid hormone. Biochem.Biophys. Res. Comm.2003, 309: 408-413.
1.Weissbach L,Settleman J,Kalady MF,et al.Identification of a human rasGAP-related protein containing calmodulin-binding motifs.J.Biol.Chem.1994,269:20517-20521.
2.Noritake J,Watanabe T,Sato K,et al.IQGAP1:a key regulator of adhesion and migration.J.Cell Sci.2005,118:2085-2092.
3.Brown MD and Sacks DB.IQGAP1 in cellular signaling:bridging the GAP.Trends Cell Biol.2006,16:242-249.
4.Suqimoto N,Imoto I,Fukuda Y,et al.IQGAP1,a negative regulator of cell-cell adhesion,is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A.J.Hum.Genet.2001,46:21-25.
5.Sun W,Zhang K,Zhang X,et al.Identification of differentially expressed genes in human lung squamous cell carcinoma using suppression subtractive hybridization.Cancer Lett.2004,212:83-93.
6.Nabeshima K,Shimao Y,Inoue T,et al.Immunohistochemical analysis of IQGAP1 expression in human colorectal carcinomas:its overexpression in carcinomas and association with invasion fronts.Cancer Lett.2002,176:101-109.
7.Dong P,Nabeshima K,Nishimura N,et al.Overexpression and diffuse expression pattern of IQGAP1 at invasion fronts are independent prognostic parameters in ovarian carcinomas.Cancer Lett. 2006,243:120-127.
8. Clark EA, Golub TR, Lander ES, et al. Genomic analysis of metastasis reveals an essential role for RhoC.Nature 2000,406: 532-535.
9. Miyoshi T, Shirakusa T, Ishikawa Y, et al. Possible mechanism of metastasis in lung adenocarcinomas with a micropapillary pattern. Pathol. Int. 2005, 55: 419-424.
10. McCallum SJ, Wu WJ, Cerione RA. Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAPl and a Cdc42Hs binding partner with similarity to IQGAP2. J. Biol. Chem. 1996, 271:21732-21737.
11. Briggs MW and Sacks DB. IQGAPl as signal integrator: Ca~(2+),calmodulin, Cdc42 and the cytoskeleton. FEBS Lett. 2003, 542: 7-11.
12. Fukata M,Kuroda S, Nakagawa M, et al. Cdc42 and Rac1 regulate the interaction of IQGAP1 with #####-catenin. J. Biol. Chem. 1999, 274: 26044-26050.
13. Kuroda S, Fukata M, Nakagawa M, et al. Role of IQGAP1, a target of the small GTPases Cdc42 and Rac1, in regulation of E-cadherin-mediated cell-cell adhesion.Science 1998,281: 832-835.
14. Briggs MW, Li Z, Sacks DB. IQGAP1-mediated stimulation of transcriptional co-activation by #######-catenin is modulated by calmodulin. J. Biol. Chem. 2002, 277:7453-7465.
15. Wang Y, Wang A, Wang F, et al. IQGAP1 activates Tcf signal independent of Rac1 and Cdc42 in injury and repair of bronchial epithelial cells. Exp. Mol. Pathol. 2008,85: 122-128.
16. Wang Y, Wang M, Wang F, et al. IQGAP1 promotes cell proliferation and is involved in a phosphorylation-dependent manner in wound closure of bronchial epithelial cells. Int. J. Mol. Med. 2008, 22: 79-87.
17. Akhmanova A and Hoogenroad CC. Microtubule plus-end-tracking proteins:mechanisms and function. Curr. Opin. Cell Biol. 2005, 17:47-54.
18. Fukata M, Watanabe T, Noritake J, et al. Racl and Cdc42 capture microtubules through IQGAP1 and CLIP-170.Cell 2002, 109: 873-885.
19. Watanabe T, Wang S, Noritake J, et al. Interaction with IQGAP1 links APC to Rac1,Cdc42 and actin filaments during cell polarization and migration. Dev. Cell 2004, 7:871-883.
20. Walch A, Seidl S, Hermannstadter C, et al. Combined analysis of Racl, IQGAP1,Tiam1 and E-cadherin expression in gastric cancer. Mod. Pathol. 2008, 21:544-552.
21. Takemoto H, Doki Y, Shiozaki H, et al. Localization of IQGAP1 is inversely correlated with intercellular adhesion mediated by E-cadherin in gastric cancers. Int.J.Cancer 2001, 91: 783-788.
22. Morris LE, Bloom GS, Frierson HF Jr, et al. Nucleotide variants within the IQGAP1 gene in diffuse-type gastric cancers. Genes Chromosomes Cancer 2005,42: 280-286.
23. Li S, Wang Q, Chakladar A, et al. Gastric hyperplasia in mice lacking the putative Cdc42 effector IQGAP1. Mol.Cell Biol.2000, 20: 697-701.
24. Schmidt VA, Chiariello CS, Capilla E, et al. Development of hepatocellular carcinoma in Iqgap2-deficient mice is IQGAP1 dependent. Mol. Cell Biol. 2008, 28:1489-1502.
25. Jin SH, Akiyama Y, Fukamachi H, et al. IQGAP2 inactivation through aberrant promoter methylation and promotion of invasion in gastric cancer cells. Int. J.Cancer 2008, 122:1040-1046.
26. Nakamura H, Fujita K, Nakagawa H, et al. Expression pattern of the scaffold protein IQGAP1 in lung cancer. Oncol. Rep. 2005,13: 427-431.
27. McDonald KL, O'Sullivan MG, Parkinson JF, et al. IQGAP1 and IGFBP2: valuable biomarkers for determining prognosis in glioma patients. J. Neuropathol. Exp.Neurol. 2007, 66:405-417.
28. Balenci L, Clarke ID, Dirks PB, et al. IQGAP1 protein specifies amplifying cancer cells in glioblastoma multiforme. Cancer Res. 2006, 66: 9074-9082.
29.Jadeski L,Mataraza JM,Jeong HW,et al.IQGAP1 stimulates proliferation and enhances tumorigenesis of human breast epithelial cells.J.Biol.Chem.2008,283:1008-1017.
30.Bourguignon LY,Gilad E,Rothman K,et al.Hyaluronan-CD44 interaction with IQGAP1 promotes Cdc42 and ERK signaling,leading to actin binding,Elk-1/estrogen receptor transcriptional activation,and ovarian cancer progression.J.Biol.Chem.2005,280:11961-11972.
31.Dong PX,Jia N,Xu Z J,et al.Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro.J.Exp.Clin.Cancer Res.2008,27:77.