Ligand recognition by the I domain-containing integrins

Seven of the integrin α subunits described to date, α ₁ , α ₂ , α _L , α _X , α _d , α _M and α _E , contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant α _M and α _L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.     

The role of VEGF receptors in angiogenesis; complex partnerships

Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development and homeostasis but also have profound effects on neural cells. VEGFs are predominantly produced by endothelial, hematopoietic and stromal cells in response to hypoxia and upon stimulation with growth factors such as transforming growth factors, interleukins or platelet-derived growth factor. VEGFs bind to three variants of type III receptor tyrosine kinases, VEGF receptor 1, 2 and 3. Each VEGF isoform binds to a particular subset of these receptors giving rise to the formation of receptor homo- and heterodimers that activate discrete signaling pathways. Signal specificity of VEGF receptors is further modulated upon recruitment of coreceptors, such as neuropilins, heparan sulfate, integrins or cadherins. Here we summarize the knowledge accumulated since the discovery of these proteins more than 20 years ago with the emphasis on the signaling pathways activated by VEGF receptors in endothelial cells during cell migration, growth and differentiation. Received 15 September 2005; received after revision 11 November; accepted 24 November 2005     

Interactions of the cell adhesion molecule nectin with transmembrane and peripheral membrane proteins for pleiotropic functions

Cell adhesion molecules (CAMs) have been implicated in the control of a wide variety of cellular processes, such as cell adhesion, polarization, survival, movement, and proliferation. Nectins have emerged as immunoglobulin-like CAMs that participate in calcium-independent cell-cell adhesion by homophilic and heterophilic trans-interactions with nectins and nectin-like molecules. Nectin-based cell-cell adhesion exerts its function independently or in cooperation with other CAMs including cadherins and is essential for the formation of intercellular junctions, including adherens junctions, tight junctions, and puncta adherentia junctions. Nectins cis-interact with integrin α_vβ₃ and platelet-derived growth factor receptor and facilitate their signals to regulate the formation and integrity of intercellular junctions and cell survival. Nectins intracellularly associate with peripheral membrane proteins, including afadin and Par-3. This review focuses on recent progress in understanding the interactions of nectins with other transmembrane and peripheral membrane proteins to exert pleiotropic functions. Received 27 June 2007; received after revision 14 August 2007; accepted 12 September 2007     

Cathepsin X-mediated β2 integrin activation results in nanotube outgrowth

Membrane nanotubes were recently described as a new principle of cell–cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; α_Lβ₂) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 26 December 2008; received after revision 26 January 2009; accepted 27 January 2009 N. Obermajer, Z. Jevnikar: These authors contributed equally to the present work.     

七鳃鳗口腔腺重组蛋白rLj-RGD3对人肝癌HepG2细胞活性的影响

七鳃鳗口腔腺含有RGD功能基因,为了确认其表达蛋白在抑制肿瘤方面的功能,在前期获得带有组氨酸标签的分子量为14．5 kD 的基因重组蛋白 rLj‐RGD3基础上,研究了不同浓度rLj‐RGD3对HepG2细胞的增殖、凋亡、黏附、迁移、侵润的影响．结果显示：rLj‐RGD3能显著抑制HepG2细胞的增殖,IC50为3．8μmol／L ,诱导HepG2细胞发生凋亡,有效抑制 HepG2细胞黏附玻蛋白（VN）,抑制 HepG2细胞的迁移,且抑制率达58％,明显抑制以 bFGF 为趋化剂的 HepG2细胞穿透 Matrigel 的侵润行为．以上研究表明,rLj‐RGD3具有典型的RGD毒素蛋白抑制肿瘤细胞的功能,能够应用于抗肿瘤基因工程药物开发,具有重要的应用意义．     

Molecular imaging of tumor angiogenesis using RGD-labeled iron oxide nanoparticles

Integrin is often significantly up­regulated in activated endothelial cells during tumor angiogenesis. The arginine-glycine-aspartic acid (RGD) peptide sequence is a specific recognition motif to α_νβ₃ integrin. In this study, a RGD labeled, Poly lactic acid (PLA) coated ultrasmall paramagnetic iron oxide (USPIO) (referred to as RGD-PLA-USPIO) were developed and the ability to detect tumor angiogenesis was investigated in vitro and in vivo. Increased uptake of RGD-PLA-USPIO by human umbilical vein endothelial cells (HUVECs) was detected by Prussian blue stain and transmission electronic microscopy (TEM). Pronounced signal decrease in T₂^*-weighted magnetic resonance image (MRI) and heterogeneous arrangement of neovasculature of tumor tissue were clearly identified in Vx-2 tumor model. The MR signal of contralateral muscle only could be seen a slight background change after either RGD-PLA-USPIO or PLA-USPIO injection. These studies demonstrate the efficiency of RGD-PLA-USPIO to visualize α_νβ₃ integrin in activated tumor endothelial cells and its potential for detecting and monitoring tumor vasculature change after therapy.     

聚乙烯亚胺-整合素配体复合物介导bcl-2反义核酸对结肠癌细胞的作用

目的:观察聚乙烯亚胺(PEI) -整合素蛋白的配体(Arg -Gly -Asp ,RGD)介导的bcl - 2反义核酸(antisenseoligodeoxynucleotide ,ASODN)对结肠癌细胞caco - 2的作用效果。方法:将阳离子复合物jetPEI-RGD与bcl- 2反义核酸混合形成ASODN -PEI-RGD三聚体复合物,转染caco - 2细胞。用台盼蓝拒染法计数活细胞,观察ASODN -PEI-RGD对caco - 2细胞的生长抑制作用,用流式细胞仪检测细胞的亚二倍体百分率,Heochst332 5 8染色观察细胞凋亡。结果:与ASODN对照组和jetPEI-RGD对照组相比,ASODN -PEI -RGD能够明显抑制caco - 2细胞增殖和诱导细胞凋亡(P<0 0 5 ) ,呈剂量和时间-效应关系。ASODN -PEI-RGD作用4 8h ,荧光染色可见大量的caco - 2细胞核固缩、核碎裂。结论:jetPEI-RGD介导的bcl- 2反义核酸能抑制caco - 2细胞增殖和诱导细胞凋亡。     

汉滩病毒受体与细胞因子TNFRⅠ真核表达载体的构建及稳定转染细胞系的建立

摘要 目的：构建带有荧光标签的汉滩病毒受体整合素β3与细胞因子TNFRⅠ的稳定转染细胞系。方法：构建目的基因的真核表达载体pIRES2-EGFP-β3和pIRES2-DsRed-TNFRⅠ(EC),并转染CHO细胞,直接荧光观察报告基因和荧光定量RT-PCR检测目的基因mRNA表达。结果：转染重组质粒pIRES2-EGFP-β3的细胞可检测到荧光报告基因和目的基因β3 mRNA的表达,转染pIRES2-DsRed-TNFRⅠ(EC)的细胞可检测到荧光报告基因和目的基因TNFRⅠmRNA的表达。结论：成功构建真核表达整合素β3与TNFRⅠ的稳定细胞系。     

Role of αVβ3 integrin in embryo implantation in the mouse

Integrin, a heterodimeric adhesive molecule composed of α and β subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the “implantation window” stage, α Vβ 3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that α Vβ 3 integrin was clearly expressed in the mouse blastocyst. Injection of α Vβ 3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, α Vβ 3 integrin antisera at 1︰100 and 1︰200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of α Vβ 3 integrin antiserum was dosage/dilution-dependent. Thus, α Vβ 3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at “implantation” stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.     

Interaction between integrin αIIbβ3 and synthesized cyclic hexapeptide containing RGD

The RGD sequence generally exists in the extracellular matrix proteins and can be recognized by many integrin proteins. The binding ability of immobilized biotinylated cyclic hexapeptide [cyclo(-Arg-Gly-Asp-D-Phe-Lys-Gly-)] containing RGD to integrin α_αbβ₃ was tested by the methods of ELISA and SPR. Results showed that a spacer of 1.48–2.2 nm between the peptide and the biotin residue was long enough to send the RGD sequence into the binding center embccedded within α_IIbβ₃, and the equilibrium dissociation constant was 1.1 μm. The work provides an ideal model system for the research of cell adhesion on solid surfaces.