粘着斑激酶活化对平滑肌细胞粘附和迁移的影响

目的:研究粘着斑激酶磷酸化在细胞外基质成份诱导平滑肌细胞粘附和迁移中的作用。方法:通过纤粘连蛋白(FN)诱导培养的平滑肌细胞粘附迁移,以免疫沉淀和Western blolt方法检测粘着斑激酶(FAK)及其磷酸化的表达量。将FAK反义寡核苷酸(ODNs)经脂质体转染细胞,观察对FAK磷酸化、细胞粘附铺展和迁移的影响。结果:FN在显著诱导平滑肌细胞粘附和迁移时,FAK也呈明显表达,20 mg/L FN可使其磷酸化处于较高表达量。脂质体可有效地介导ODNs转染,转染效率为(86.7±4.5)%,FAK磷酸化表达量明显减少,5-60 mg/L不同浓度FN组,细胞铺展率减少17.89%-27.67%,10、20、40和60 mg/L FN组迁移细胞数也分别显著少23.26%、21.63%、19.31%、17.88%(P＜0.05)。结论:活化的FAK是细胞外基质诱导SMCs粘附和迁移的重要信号分子,由其介导的信号转导促进了这一过程,反义FAK ODNs可有效地对此进行抑制。     

PI3K对IL-8／Rac1信号通路介导的内皮细胞迁移的影响

目的研究磷脂酰肌醇-3激酶（PI3K）对IL-8／Rac1信号通路所介导的内皮细胞迁移的影响。方法选择P13K抑制剂wortmannin及划痕创伤愈合修复分析,研究不同wortmannin的作用浓度和不同时间对IL-8／Rac1信号通路诱导的内皮细胞迁移的影响。结果不同wortmannin的作用浓度和不同时间预处理均可抑制IL-8／Rac1信号通路介导的内皮细胞迁移,其中wortmannin预处理浓度为100nmol／L,预处理时间为20min时IL-8诱导的内皮细胞迁移水平最低。结论P13K能够影响IL-8／Rac1信号通路介导的内皮细胞迁移;P13K抑制剂wortmannin的最佳作用浓度为100nmol／L,最佳作用时间为20min。     

OPN促进大鼠肝星状细胞黏附、迁移及黏着斑激酶活性

目的探讨细胞外间质(ECM)成分骨桥蛋白(OPN)和精-甘-天冬-丝氨酸(Arg-G ly-Asp-Ser,RGDS)四肽对大鼠肝星状细胞(HSCs)黏附、迁移与黏着斑激酶(FAK)活性的影响。方法应用体外细胞培养技术,MTT法检测HSCs增殖,甲苯胺蓝法检测HSCs黏附情况,并观察HSCs迁移;采用免疫沉淀和W estern b lot技术测定黏着斑激酶活性变化。结果①不同浓度OPN和RGDS四肽作用于HSCs 2 h后,8、16、32 mg/L OPN组细胞黏附促进率均高于对照组(P<0.05);25 mg/L~100 mg/L RGDS四肽不同浓度组细胞黏附率明显下降(P<0.05)。②不同浓度OPN干预细胞48 h后,促进HSCs增殖,而RGDS四肽组抑制HSCs增殖。③16 mg/L OPN作用12、24和36 h,细胞迁移距离显著高于对照组(P<0.05);而100 mg/L RGDS四肽组则显著性低于对照组(P<0.05)。④经OPN作用后各浓度组HSCs中磷酸化FAK均有显著表达;RGDS四肽干预后,磷酸化FAK表达量降低。结论①OPN促进HSCs增殖、黏附和迁移;RGDS四肽对HSCs增殖、黏附和迁移具有抑制作用。②OPN可以诱导FAK活化,RGDS四肽可抑制磷酸化FAK的表达。     

粘着斑激酶反义寡核苷酸对平滑肌细胞粘附迁移和凋亡的影响

为研究粘着斑激酶反义寡核苷酸对平滑肌细胞粘附迁移和凋亡的调控作用 ,采用纤粘连蛋白 (fibronectin ,FN)诱导平滑肌细胞 (smoothmusclecells,SMCs)粘附迁移并活化粘着斑激酶 (focaladhesionkinase ,FAK) ,将FAK反义寡核苷酸 (antisenseoligodeoxynucleotides,ODNs)经脂质体转染细胞 ,观察对FAK磷酸化、细胞粘附迁移以及凋亡的影响。结果表明 ,FN在有效地诱导SMCs粘附迁移并活化FAK的同时 ,凋亡细胞数显著低于对照 ,(8 96± 1 2 ) %和(2 3 45± 9 6 ) %。脂质体可有效地介导ODNs转染 ,转染效率为 (86 7± 4 5 ) % ,FAK磷酸化表达量明显减少 ,不同浓度FN组 5～ 6 0mg L ,细胞铺展率减少 17 89%～ 2 7 6 7% ,10、2 0、40和 6 0mg LFN组迁移细胞数也分别显著减少2 3 2 6 %、2 1 6 3%、19 31%、17 88% ,凋亡细胞数比对照高 33 5 7% (P <0 0 5 ) ,也显著高于FN组。据此可以认为FAK活化及其介导的信号转导通路是细胞外基质诱导SMC粘附迁移并抑制其凋亡的重要因素。反义FAKODNs可有效地对此进行调控     

FAK通路在RA发病机制中的作用

1 粘着斑激酶(FAK)的结构与功能 粘着斑激酶(Focal adhesion kinase,FAK)是一种广泛表达的胞浆内非受体蛋白酪氨酸激酶,定位于整联蛋白聚集处,具有多种功能,临床研究显示其在多种人类肿瘤中均有表达,与肿瘤细胞的侵袭与转移特性关系密切,并且能够促进细胞增殖、迁移和存活.     

1-磷酸鞘氨醇在高糖诱导血管内皮细胞功能损伤中的作用

 目的: 探讨1-磷酸鞘氨醇(S1P)在高糖诱导血管内皮细胞功能损伤中的作用及其机制。方法: 在高糖培养的人主动脉内皮细胞模型中,分别或同时给予S1P、鞘氨醇激酶1抑制剂和Akt抑制剂处理后,观察一氧化氮(NO)、粒细胞-内皮细胞黏附率、细胞间黏附分子1(ICAM-1)蛋白表达、内皮细胞迁移以及Akt/内皮型一氧化氮合酶(eNOS)信号途径的改变。结果: S1P明显降低高糖诱导的内皮细胞培养上清液中NO含量,促进粒细胞与内皮细胞黏附,显著增加内皮细胞ICAM-1蛋白表达,抑制内皮细胞迁移和Akt/eNOS信号通路激活。鞘氨醇激酶1抑制剂减少S1P生成后上述内皮细胞功能指标得以明显改善,Akt/eNOS信号通路恢复激活。结论: S1P促进高糖培养的内皮细胞功能障碍的形成,可能与其抑制Akt/eNOS信号通路激活有关。抑制S1P生成有望成为减轻内皮细胞功能损伤的治疗策略之一。     

骨桥蛋白对骨髓间充质干细胞核力学特性的影响及相关分子机制

目的研究骨桥蛋白(osteopontin,OPN)对骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)核力学特性的影响及相关分子机制。方法采用Transwell法分析BMSCs迁移能力。利用原子力显微镜(atomic force microscope,AFM)检测细胞核弹性模量,分析OPN作用下BMSCs细胞核硬度的变化。通过Western blot技术检测OPN作用对黏着斑激酶(focal adhesion kinase,FAK)和胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK 1/2)的影响,并利用FAK或ERK1/2抑制剂考察FAK-ERK1/2信号通路在OPN影响BMSCs核力学特性中的作用。通过RT-PCR和Western blot技术检测OPN作用下核纤层蛋白Lamin A/C的表达变化。结果 OPN处理组细胞核弹性模量与对照组相比明显降低。OPN作用显著上调FAK、ERK1/2磷酸化水平,加入FAK或ERK1/2抑制剂在一定程度上回救OPN降低的细胞核弹性模量,并显著抑制BMSCs迁移。OPN处理BMSCs后Lamin A/C mRNA和蛋白水平的表达出现下调,但FAK或ERK1/2抑制剂能够抑制OPN诱导的Lamin A/C表达下调。结论 OPN可能通过FAK-ERK1/2信号通路下调BMSCs核骨架蛋白Lamin A/C表达,降低细胞核硬度,促进BMSCs迁移。该研究结果为深入认识OPN调控BMSCs迁移行为的机制及其临床应用提供了实验依据。     

FAK基因表达对人结直肠癌细胞增殖及运动的影响

目的:探讨黏着斑激酶(FAK)表达水平对结直肠癌细胞增殖及运动的影响.方法:针对FAK基因不同靶点设计siRNA序列, 构建siRNA重组子, 转染Caco-2细胞, 以RT-PCR和免疫细胞化学方法检测FAK mRNA和蛋白表达变化及时间效应, 同时检测FAK基因敲低对Caco-2细胞的凋亡、增殖及迁移的影响.结果:FAK siRNA导入Caco-2细胞后, FAK mRNA和蛋白表达水平明显下调, 细胞增殖及迁移能力受抑, 呈时间依赖关系, FAK mRNA水平下调在转染后48 h达到最大.结论:FAK siRNA可有效抑制靶基因表达, FAK表达水平下调后Caco-2细胞的增殖及运动明显受抑制.     

血管内皮生长因子C促进HeLa宫颈癌细胞粘着斑激酶活性的研究

目的探讨粘着斑激酶（focal adhesion kinase,FAK）介导血管内皮细胞生长因子C（vascular endothelial growth factor C,VEGF—C）促宫颈癌恶性进展的作用。方法提取不同病理分期的宫颈癌组织标本蛋白．采用Western-blot检测VEGF．C及FAK蛋白的表达情况。在体外培养宫颈癌细胞株HeLa细胞,采用Western—blot测定VEGF—C对FAK蛋白的表达和磷酸化的调控作用。结果随着宫颈癌恶性程度的增高,VEGF-C、FAK蛋白及磷酸化FAK蛋白表达水平均随之增加。与正常宫颈组织比较,宫颈原位癌（CIN）组织中VEGF．C、FAK、磷酸化FAK蛋白表达分别增加了（48±10）％、（78±14）％、（83±15）％,P〈0．05;宫颈鳞癌Ⅰ期各蛋白增高幅度分别为（104±22）％、（121±28）％、（143±30）％（P〈0．01）;宫颈鳞癌Ⅱ期（未放疗、化疗）各蛋白表达增高更为显著,其幅度分别为（195±28）％、（186±22）％、（204±31）％,P〈0．001。在培养的宫颈癌细胞株HeLa细胞上,VEGF—C（100μg／L）处理24h后,可显著增高FAK蛋白、磷酸化FAK蛋白的表达,该作用可被VEGF—C单克隆抗体明显抑制。结论VEGF—C、FAK蛋白表达与宫颈癌恶性程度密切相关,VEGF—C可能通过上调FAK表扶殛苴磷酪化水平而但讲宫颈癌的熏忡讲犀     

阿霉素对K562细胞凋亡及FAK mRNA的影响

目的探讨阿霉素体外作用于K562细胞后,观察其对细胞增殖的影响,促进细胞凋亡情况,以及调节细胞周期和粘着斑激酶（FAK）mRNA基因,进一步探讨通过调控FAK表达,研究抗白血病的作用机制。方法应用细胞增殖实验（CCK8法）观察不同浓度阿霉素作用不同时间对K562细胞增殖的影响,应用流式细胞仪观察不同浓度阿霉素对K562细胞细胞凋亡,细胞周期的影响,应用RT-PCR和Western blot技术检测不同浓度阿霉素作用对K562细胞36h后FAK mRNA以及蛋白表达水平的变化。结果随着阿霉素浓度增加及作用时间延长,K562细胞的增殖抑制率逐渐升高,同一时间不同浓度之间比较,或者同一浓度不同时间组之间比较,差异均有统计学意义（P〈0.05）;阿霉素能引起K562细胞凋亡,且随着药物浓度增加,凋亡率也逐渐增加,差异均有统计学意义（P〈0.05）;阿霉素能引起K562细胞周期阻滞,多停留在S期;阿霉素能引起K562细胞FAK mRNA表达显著降低。阿霉素能引起K562细胞FAK蛋白表达水平的降低。结论阿霉素抑制分裂期细胞的增殖,诱导细胞凋亡增加,对细胞FAK基因和蛋白水平均显著下调,为进一步研究FAK基因表达的调控与肿瘤细胞凋亡的分子机制提供了实验基础。     

造影红细胞对剪切流中肿瘤细胞黏附的影响

目的研究造影红细胞对剪切流环境中白细胞介导肿瘤细胞在内皮细胞上黏附的影响。方法在平行平板流动腔中加入20%比容的造影红细胞,分析不同剪切率(62.5、100、200 s-1)下内皮细胞上黏附白细胞数目、肿瘤细胞与黏附白细胞的碰撞事件以及稳定黏附肿瘤细胞数目的变化。结果造影红细胞促进白细胞在内皮细胞上黏附,增加肿瘤细胞与黏附白细胞的碰撞频率,并最终促进肿瘤细胞在内皮细胞上的黏附,且这一现象在高剪切率(200 s-1)下更为明显;但造影红细胞对肿瘤细胞的黏附效率并无显著影响。结论剪切流中造影红细胞的存在对肿瘤细胞在内皮细胞上的黏附起到促进作用,研究结果为探索癌症治疗方法提供理论基础。     

SSeCKS对牛肺动脉内皮细胞黏附和迁移的影响

目的： 研究SSeCKS在细胞外基质成份诱导内皮细胞黏附和迁移中的作用。方法： 用纤黏连蛋白(FN)诱导内皮细胞，以Western blotting分析SSeCKS表达量的变化；用Ro31-8220、calphostin C（蛋白激酶C抑制剂）预处理内皮细胞，观察对细胞黏附和迁移的影响，以共聚焦显微镜观察其对SSeCKS、F-actin和vinculin在细胞定位的影响。结果： FN能够显著诱导内皮细胞黏附和迁移，SSeCKS的表达量也随之增加，具有时间和剂量依赖性；经Ro31-8220、calphostin C处理细胞后，SSeCKS表达量明显减少，细胞黏附率和迁移细胞数也较处理前显著减少，SSeCKS由细胞质散在分布向核周聚集，且SSeCKS与F-actin、vinculin在细胞边缘共定位比处理前减少。结论： SSeCKS参与细胞外基质诱导内皮细胞黏附和迁移的过程，由其介导的PKC信号转导促进了这一过程，蛋白激酶C抑制剂可有效抑制此过程。     

Potential roles for focal adhesion kinase in development

 Focal adhesion kinase (pp125^FAK or FAK) is a protein tyrosine kinase which is associated with intracellular signalling cascades which are initiated when the integrin family of cell adhesion molecules engage extracellular matrix molecules. In cultured cells, this molecule is physically associated with focal adhesions, which are well-defined regions of intimate cell-to-substratum adhesion. In this location, it interacts with other proteins of the focal adhesion to activate intracellular signalling events associated with cell adhesion. The in vitro expression of FAK and its level of phosphorylation appear to be related to several physiological phenomena, including cell spreading, cell differentiation, cell locomotion and cell death. Because these phenomena are all of critical importance during morphogenesis, and because FAK is expressed in embryonic cells, evidence has been accumulating to indicate that FAK may be an important modulator of developmental processes. In this review, this evidence is surveyed together with evidence from analogous situations, such as tumour cell migration and invasiveness. Although evidence suggesting a role for FAK in morphogenesis is accumulating, current uncertainties regarding its cytoplasmic location and its molecular interactions in vivo make it difficult to reach definitive conclusions regarding the significance of its contributions to developmental processes. Accepted: 14 September 1998     

Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy.     

Abnormal cleavage of APP impairs its functions in cell adhesion and migration

Amyloid precursor protein (APP) is expressed ubiquitously but its wrong cleavage only occurs in central nervous system. In this research, overexpression of wild type human APP695 was found to stimulate the adhesion and migration of N2a cells. In the cells co-transfected by familial Alzheimer’s disease (FAD)-linked Swedish mutant of APP695 gene plus E9 deleted presenilin1 gene (N2a/Swe.9), however, this stimulating function was impaired compared to that in the cells co-transfected by Swedish mutant of APP695 gene plus dominant negative mutant of presenilin1 D385A gene (N2a/Swe.385). Furthermore, it was also found that the phosphorylation of FAK Tyr-861 and GSK-3β Ser-9 was reduced in N2a/Swe.Δ9 cells, which can be possibly taken as a reasonable explanation for the underlying mechanism. Our results suggest that impaired cell adhesion and migration induced by abnormal cleavage of APP could contribute to the pathological effects in FAD brain.     

Keratinocyte growth factor-transfection-stimulated adhesion of colorectal cancer cells to extracellular matrices

The keratinocyte growth factor (KGF) regulates cell growth and behavior in an autocrine or paracrine manner. In colorectal cancer tissues, KGF is expressed in tumor cells and adjacent stromal fibroblasts. We have constructed a KGF-gene-transfected cell line (HCT15-KGF) from a colorectal cancer cell line, HCT-15, that expresses the KGF receptor, and studied the effects of KGF on cell behavior, particularly growth and adhesion to extracellular matrices (ECMs). The amount of KGF secreted from HCT15-KGF was significantly higher than that from a mock-transfected cell line (HCT15-MOCK). The modes of growth of these cell lines were similar. The degree of adhesion of HCT15-KGF to ECMs, including type-IV collagen and fibronectin was higher than that of HCT15-MOCK. The expressions of integrins in both cell lines were not significantly different. However, extracellular-regulated kinase-1 and -2 (ERK1/2) phosphorylation and focal adhesion kinase (FAK) expression that regulate the adhesive functions of integrin families were enhanced in HCT15-KGF. U0126, an inhibitor of the ERK upstream regulator MEK, attenuated the adhesion and spreading of HCT15-KGF cells to type-IV collagen. These results indicate that KGF enhances the adhesion of colorectal cancer cells to type-IV collagen through ERK and FAK signaling pathways.     

Mediation of the migration of endothelial cells and fibroblasts on polyurethane nanocomposites by the activation of integrin-focal adhesion kinase signaling

Model surfaces of polyurethane-gold nanocomposites (PU-Au) were used to examine cell behavior on nanophase-segregated materials. Previously we showed that endothelial cell (EC) migration on these materials was modulated by the PI3K/Akt/eNOS pathway. The present study, investigated the expressions of alpha5/beta3 (α5β3) integrin, focal adhesion kinase (FAK), and other downstream signal molecules such as the Rho family and matrix metalloproteinases 2 (MMP-2) induced by the materials in two different cells, that is bovine arterial endothelial cells (BAEC) and human skin fibroblasts (HSF). Both cells proliferated better on the more phase-separated PU-Au 43.5 ppm than on the less phase-separated controls (PU and PU-Au 174 ppm). On PU-Au 43.5 ppm, BAEC compared to HSF had denser actin fibers and were more extended. BAEC became rounded with Y-27632 treatment and shrunk with LY294002 treatment. Treatment by inhibitors only caused slight changes in HSF. The migration distance of BAEC on PU-Au 43.5 ppm was greater than that of HSF, and was significantly reduced by LY294002 or Y-27632 but not SU-1498. The expressions of p-FAK, p-RhoA, p-Rac/Cdc42, MMP2, and α5β3 integrin induced by PU-Au 43.5 ppm were more pronounced in BAEC versus HSF. Further enhancement in MMP2 and α5β3 integrin expressions by FAK-GFP transfection was more remarkable for cells on PU-Au 43.5 ppm. Our findings suggested that the integrin α5β3/FAK pathway may be induced by nanophase-separated materials in both ECs and fibroblasts to promote their proliferation/migration, while the crosstalk between the PI3K/Akt/eNOS pathway and FAK/Rho-GTPase activation may account for the greater effect in ECs than in fibroblasts.     

粘着斑激酶在血管内皮细胞迁移中的作用

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase (PTK) that can localize indirectly to sites of clustering integrin family of heterodimeric receptors. As an important structure and signaling molecule in the adhesive complexes, which are large and stable referred as ‘focal adhesions‘ or relatively small and transient within filopodia and lamellipodia named ‘focal complexes‘, FAK is closely related with cell death, proliferation and migration. In this review, we discuss the function of FAK in the regulation of endothelial cell migration based on current data.     

Focal adhesion kinase functions as a receptor-proximal signaling component required for directed cell migration

In performing host-defense functions, cells of the immune system become activated by soluble chemokine signals and must migrate through endothelial cell or solid tissue barriers to reach sites of inflammation or infection. Regulated adhesive interactions of immune cells with endothelium, extracellular matrix components, and cells of solid organs are critical control points of the overall immune response. Both the soluble chemokine and cell adhesion receptor-mediated migration signals must converge on common intracellular targets to engage the cell migration machinery. In this article, we focus on the role of focal adhesion kinase (FAK) and its homolog Pyk2 as cytoplasmic mediators of motility events in multiple cell types. We introduce the overall domain structure of the FAK and Pyk2 nonreceptor protein tyrosine kinases (PTKs), high-light some of the signals that activate these PTKs, and detail the molecules that functionally interact and signal transduction pathways that may mediate cell migration responses. Emphasis is placed on the knowledge gained from studies using FAK-null cells as a model system to decipher the role of this PTK in promoting cell motility.