整合素相关激酶（ILK）研究进展

细胞和细胞外基质以及细胞－细胞之间的相互作用往往是通过整合素和生长因子体介导的信号所引发。细胞内的效应因子将整合素和生长因子受体同下游组分相联系从而传递着相关的信息。整合素相关激酶（inte-grin-linked kinase,ILK),已被证实了参与了许多信号传导通路,包括整合素,生长因子及Wnt信号传导通路。     

ILK—整合素信号传导通路中的关键激酶

胸外基质与细胞的相互作用主要由整合素介导。由整合素介导的机械和生化信号调控胞浆激酶、生长因子受体、离子通道的活性并掏胞内肌动蛋白细胞骨架的组装。众多由整合素介导的信号传导通路最后可归于对细胞周期的调节、决定细胞存活或死亡、增殖或者退出细胞周期和分化。近年的研究发现,整合素连接激酶（inte-grin-linked kinase,ILK）在这些信号传导通路中具有关键作用。     

整合素连接激酶与肾小球疾病

目前肾小球疾病系病因及发病机理尚不明确,许多细胞因子均参与了肾小球疾病的发生与发展.其中整合素连接激酶(integrin-linkedkinase,ILK)在肾脏疾病的发生发展过程起了一定的作用.它是一种丝氨酸/苏氨酸蛋白激酶,参与多种信号传导通路,在调节细胞凋亡、胚胎发生、肿瘤发生等过程中起重要作用,近期研究发现它与肾小球疾病密切相关.本文就ILK在肾小球疾病中的研究新进展作一综述.     

整合素及其信号传导通路

整合素是位于细胞表面的重要黏附分子,通过其双向信号传导通路,介导细胞与细胞外基质及细胞与细胞间的黏附.整合素由胞外域、跨膜域和胞内域3部分组成.胞内域与细胞内信号分子结合,启动胞内一胞外信号传导激活整合素,提高与相应配体亲合力.而胞外域与相应配体结合后,通过胞外-胞内信号传导,调节细胞生存、增殖、黏附、分化功能.近年研究显示,整合素结构功能及信号传导通路异常与多种疾病有关.     

Notch信号传导通路相关疾病的研究进展

Notch信号传导通路是影响细胞命运决定的重要通路之一,相邻细胞间通过Notch受体传递信号可以调节包括干细胞在内的多种细胞的分化、增殖和凋亡,影响器官形成和形态发生.Notch信号传导通路中某些分子的基因突变与多种疾病的发生发展有关.在深入研究Notch信号传导通路的基础上,以其作为靶点设计药物,对于治疗包括肿瘤、CADASIL等遗传性疾病在内的相关疾病,或发展干细胞医疗技术治疗阿尔茨海默症(Alzheimer!sdisease,AD)、帕金森病、糖尿病等细胞组织功能减退或受损性疾病具有重要的科学意义和应用价值.     

骨唾液酸蛋白通过整合素αvβ3调控ILK信号通路

旨在探究骨唾液酸蛋白(BSP)是否通过整合素αvβ3对整合素连接激酶(ILK)信号通路进行调控。BSP基因沉默乳腺癌MDA-MB-231细胞,流式细胞仪在细胞水平检测BSP不同水平的细胞株中整合素αvβ3的表达量。Western blotting检测磷酸化ILK水平的变化,MTT法检测细胞增殖能力。与对照组231BO-Scrambled细胞相比,BSP基因沉默组231BO-BSP27细胞中整合素αvβ3的表达水平明显下调(61.32±1.94)%(P<0.01)。整合素αvβ3鼠抗单克隆抗体(LM609)处理前的BSP基因沉默组231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(39.38±1.38)%(P<0.01);LM609处理后的231BO-BSP27细胞与21BO-Scrambled细胞相比,ILK磷酸化水平下调明显(33.78±1.51)%(P<0.01)。向乳腺癌细胞231BO-scrambled和231BO-BSP27中添加LM609,MTT试验结果显示两株乳腺癌细胞的增殖能力均有降低(P<0.05)。BSP通过整合素αvβ3对乳腺癌MDA-MB-231细胞ILK信号通路进行调控,并影响细胞增殖。     

肿瘤坏死因子信号传导的分子机理

肿瘤坏死因子(tumor necrosis factor,TNF)是一种具有多种生物学效应的细胞因子,其生物学效应包括促进细胞生长、分化、凋亡及炎症诱发等.TNF的生物学效应都是通过细胞表面的两种TNF受体引发的.TNF的信号传导通路主要包括细胞凋亡及转录因子NF-kB和JNK蛋白激酶的激活.这3条信号传导通路之间及各通路内部含有各种调节机制,使TNF的各种生物学功能协调发挥出来.从1994年到现在,对肿瘤坏死因子信号传导通路的分子机理研究取得了一系列突破性进展,在细胞信号传导研究领域中树立了成功的典范.     

整合素连接激酶生物学功能研究进展

整合素连接激酶(integrin-linked kinase,ILK)是一种联接整合素和细胞骨架的关键蛋白因子,具有丝氨酸和苏氨酸蛋白激酶活性,主要有343和473两个丝氨酸磷酸化位点。ILK具有C端的激酶催化结构域、磷脂酰肌醇结合结构域和锚蛋白重复序列三个功能结构域。ILK能与MAPK、PKB、GSK3、NF-κB等多条信号通路和细胞骨架蛋白相互作用。研究表明,ILK在细胞生物学活动中发挥了重要作用,如参与调控细胞周期G1/S/G2期,促进肿瘤细胞迁移,抑制细胞凋亡,此外ILK还通过TGF-β1等参与了上皮细胞-间充质细胞转换(epithelial-mesenchymal transition,EMT)。研究表明,ILK在血管新生中也发挥了重要作用;不但促进血管生成,而且能逆转高压导致的血管内皮紊乱,减少氧化应激产物e NOS的产生,降低血管动脉粥样硬化的发病率和严重程度。     

Src介导骨桥蛋白诱导的血管平滑肌细胞黏附和迁移过程

为阐明酪氨酸激酶Src在整合素被骨桥蛋白(OPN)激活所触发的细胞黏附和迁移信号途径中所起的作用,应用Src特异性抑制剂PP2阻断Src,观察OPN诱导的血管平滑肌细胞(VSMC)黏附和迁移活性的改变,并利用免疫沉淀检查PP2对整合素下游信号分子黏着斑激酶(FAK)和整合素偶联激酶(ILK)磷酸化及其相互作用的影响。结果显示,PP2可明显抑制OPN诱导的VSMC黏附和伤口愈合(黏附和迁移活性分别为对照组的76.6%和33.8%);OPN可显著诱导FAK磷酸化(磷酸化水平达对照组的1.9倍),促进ILK去磷酸化,并使FAK与ILK的结合减少(降至对照组的46.4%)。10μmol/LPP2可明显抑制OPN诱导的FAK磷酸化、拮抗OPN诱导对ILK的去磷酸化作用、促进FAK与ILK之间的结合。研究结果表明,Src作为OPN-整合素-FAK信号途径中的信号分子,通过影响FAK和ILK的磷酸化以及两者之间的相互作用来调节VSMC的黏附和迁移活性。     

胞内接头蛋白TRAFs家族

肿瘤坏死因子（tumor necrosis factor,TNF）受体相关因子（receptor-associated factor,TRAF）家族是一类胞内接头蛋白,能介导TNF受体和Toll-like／IL-1受体超家族成员与多种下游信号通路包括NF—κB（nuclear factor κB）和JNK（Jun N-terminal kinase）的信号传导。TRAF蛋白家族参与调控细胞增殖、分化乃至凋亡等生理过程。由于其在信号通路中的关键作用,TRAFS蛋白的失调与多种疾病的发生相关。本文结合最新的研究进展对TRAFs蛋白家族在信号传导通路中的地位进行介绍,并探讨了TRAFs及其相关蛋白在临床上可能的应用前景。     

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.     

Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway

A beta1 integrin phosphatidylinositol 3-kinase/Akt pathway regulates fibroblast survival in collagen matrices. When fibroblasts attach to collagen, Akt becomes phosphorylated, providing a survival signal. In contrast, in response to mechanical forces generated during collagen contraction, Akt is dephosphorylated and fibroblasts undergo apoptosis. The kinase(s) responsible for regulating Akt phosphorylation in response to matrix-derived mechanical signals are unclear. Integrin-linked kinase (ILK) is associated with the beta1 integrin in the focal adhesion complex and as such is a candidate kinase that may regulate Akt phosphorylation and fibroblast viability. Nevertheless, there is no direct evidence that matrix-derived mechanical forces regulate cell viability by modulating ILK activity. Here, we show that ILK activity decreased in response to collagen matrix contraction, which correlated with Akt dephosphorylation and induction of fibroblast apoptosis. In contrast, enforced activation of beta1 integrin by activating antibody preserved ILK and Akt activity during collagen matrix contraction, and this is associated with protection from collagen contraction-induced apoptosis. Knock-down of ILK by small, interfering RNA (siRNA) attenuated Akt phosphorylation in response to ligation of beta1 integrin by collagen or activating antibody and enhanced fibroblast apoptosis in response to collagen contraction. Kinase dead ILK attenuated Akt phosphorylation and enhanced fibroblast apoptosis, whereas hyperactive and wild type ILK augmented Akt phosphorylation and protected fibroblasts from apoptosis. Constitutively active Akt preserved Akt activity and rescued ILK siRNA-treated fibroblasts from collagen contraction-induced apoptosis. These data establish that matrix-derived mechanical forces sensed by beta1 integrin are capable of modulating ILK activity which regulates fibroblast viability via an Akt-dependent mechanism.     

Integrin-linked kinase controls neurite outgrowth in N1E-115 neuroblastoma cells

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against beta(1) integrin inhibits neurite outgrowth. Thus, beta(1) integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of beta(1) integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whereas that in the DN-ILK-transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinase kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK-expressed cells. The time course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells.     

Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling.

Interaction of cells with the extracellular matrix (ECM) results in the regulation of cell growth, differentiation and migration by coordinated signal transduction through integrins and growth-factor receptors. Integrins achieve signalling by interacting with intracellular effectors that couple integrins and growth-factor receptors to downstream components. One well-studied effector is focal-adhesion kinase (FAK), but recently another protein kinase, integrin-linked kinase (ILK), has been identified as a receptor-proximal effector of integrin and growth-factor signalling. ILK appears to interact with and be influenced by a number of different signalling pathways, and this provides new routes for integrin-mediated signalling. This article discusses ILK structure and function and recent genetic and biochemical evidence about the role of ILK in signal transduction.     

The integrin-linked kinase regulates cell morphology and motility in a rho-associated kinase-dependent manner

The integrin-linked kinase (ILK) is a multidomain focal adhesion protein implicated in signal transmission from integrin and growth factor receptors. We have determined that ILK regulates U2OS osteosarcoma cell spreading and motility in a manner requiring both kinase activity and localization. Overexpression of wild-type (WT) ILK resulted in suppression of cell spreading, polarization, and motility to fibronectin. Cell lines overexpressing kinase-dead (S343A) or paxillin binding site mutant ILK proteins display inhibited haptotaxis to fibronectin. Conversely, spreading and motility was potentiated in cells expressing the "dominant negative," non-targeting, kinase-deficient E359K ILK protein. Suppression of cell spreading and motility of WT ILK U2OS cells could be rescued by treatment with the Rho-associated kinase (ROCK) inhibitor Y-27632 or introduction of dominant negative ROCK or RhoA, suggesting these cells have increased RhoA signaling. Activation of focal adhesion kinase (FAK), a negative regulator of RhoA, was reduced in WT ILK cells, whereas overexpression of FAK rescued the observed defects in spreading and cell polarity. Thus, ILK-dependent effects on ROCK and/or RhoA signaling may be mediated through FAK.     

Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1)

Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.     

Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction

The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked by substrate adhesion induces affixin-alpha-actinin interaction. The overexpression of a peptide corresponding to the alpha-actinin-binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK-affixin complex serves as an integrin-anchoring site for alpha-actinin and thereby mediates integrin signaling to alpha-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.     

C. elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes

BACKGROUND: Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain. ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes. RESULTS: We identified the C. elegans pat-4 gene in previous genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments. Here, we report that pat-4 encodes the sole C. elegans homolog of ILK. In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked. Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments. Transgenically expressed "kinase-dead" ILK fully rescues pat-4 loss-of-function mutants. We also identify UNC-112 as a new binding partner for ILK. CONCLUSIONS: Our data strengthens the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner.     

Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase

Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. Integrins regulate cell-ECM (extracellular matrix) adhesion and must associate with phosphorylating proteins such as ILK (integrin-linked kinase). Dysregulation of ILK expression is associated with anchorage-independent growth, cell survival and inhibition of apoptosis. Glucocorticoids influence differentiation and adhesion of osteoblasts and can affect bone protein synthesis. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts, together with its influence on the expression of ILK and β1 integrin. For this, primary cultures of human osteoblasts were exposed to DEX at 10-9 M (physiological dose) and 10-6 M (pharmacological dose) for 24 and 48 h. Cell viability, apoptosis and cell adhesion were analysed, as well as protein expression of β1 integrin and ILK. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of β1 integrin was significantly lower. Both proteins under study were co-localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased β1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti-apoptotic role.     

Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane

Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.