Integrin α1/β1 and α2/β1 as a receptor for IgA1 in human glomerular mesangial cells in IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of IgA1 and galactose-deficient IgA1 is expected to play a pathogenic role. However, the identity of the receptor for IgA1 is still controversial. Hence, the aim of this study was to explore the receptor for galactose-deficient IgA1. Human monoclonal IgA1 was treated with exoglycosidase and FITC-conjugated control, asialo- and agalactosyl-IgA1 was used as a probe to detect the receptor in cultured human mesangial cells. Tumor necrosis factor-α or transforming growth factor-β1 treatment accelerated IgA1-binding on mesangial cells, and these effects were diminished by the addition of dexamethasone, whereas these changes were not dependent on galactose-deficiency of IgA1. According to comprehensive gene expression analysis, we focused on integrin β1. Pre-treatment by Mn(2+), which activates integrin by changing its structure, enhanced the binding of IgA1 in cultured mesangial cells. Furthermore, pre-incubation with collagens specifically enhanced binding of IgA1 in the cultured human mesangial cells without activation by Mn(2+). Collagen type IV distributed in the mesangial region of the glomeruli as well as Bowman's capsule and tubular basal membrane in IgAN patients, and the IgA1 with collagen type IV induced proliferative signals on mesangial cells by phosphorylating extracellular signal-regulated kinase more effectively than the IgA1 alone. Immunoprecipitation assay revealed the binding of IgA1 and integrin α1/β1 and α2/β1 heterodimer and down-regulation of integrin α1, α2 and β1 expression in human mesangial cells induced by each specific small interfering RNA diminished the ability to bind IgA1 probe. Integrin α1/β1 and α2/β1 would be a candidate receptor for IgA1.     

Rotavirus-neutralizing antibodies inhibit virus binding to integrins α2β1 and α4β1

Summary Rotavirus outer capsid proteins VP5^*, VP8^* and VP7 elicit neutralizing, protective antibodies. The α2β1 integrin is a cellular receptor for rotavirus that is bound by VP5^*. Some rotaviruses also recognize the α4β1 integrin. In this study, the effects of antibodies to rotavirus on virus binding to recombinant α2β1 and α4β1 expressed on K562 cells were determined. All neutralizing monoclonal antibodies to VP5^* tested (YO-2C2, 2G4, 1A10) and two to VP7 (RV-3:2, RV-4:2) inhibited rotavirus binding to α2β1. Rotavirus binding to α4β1 was reduced by 2G4 and neutralizing antibody F45:2, directed to VP7. However, a neutralizing antibody to VP8^* (RV-5:2) and one to VP7 (RV-3:1) did not affect rotavirus binding to these integrins. Virus-cell binding was unaffected by non-neutralizing antibody RVA to the rotavirus inner capsid protein VP6. The attachment of human rotavirus strain Wa to these integrins was inhibited by infection sera with neutralizing activity collected from two children hospitalised with severe rotavirus gastroenteritis. A negative reference serum did not affect rotavirus-cell attachment. As the binding of rotaviruses to α2β1 and α4β1 is inhibited by neutralizing antibodies to VP5^* and VP7, and serum from children with rotavirus disease, rotavirus recognition of these integrins may be important for host infection.     

Fibroblast expression of α-smooth muscle actin, α2β1 integrin and αvβ3 integrin: influence of surface rigidity

Open wound contraction necessitates cell and connective tissue interactions, that produce tension. Investigating fibroblast responses to tension utilizes collagen coated polyacrylamide gels with differences in stiffness. Human foreskin fibroblasts were plated on native type I collagen-coated polyacrylamide gel cover slips with different rigidities, which were controlled by bis-acrylamide concentrations. Changes in alpha smooth muscle actin (αSMA), α2β1 integrin (CD49B) and αvβ3 integrin (CD-51) were documented by immuno-histology and Western blot analysis. Cells plated on rigid gels were longer, and expressed αvβ3 integrin and αSMA within cytoplasmic stress fibers. In contrast, cells on flexible gels were shorter, expressed α2β1 integrin and had fine cytoskeletal microfilaments without αSMA. Increased tension changed the actin makeup of the cytoskeleton and the integrin expressed on the cell's surface. These in vitro findings are in agreement with the tension buildup as an open wound closes by wound contraction. It supports the notion that cells under minimal tension in early granulation tissue express α2β1 integrin, required for organizing fine collagen fibrils into thick collagen fibers. Thicker fibers create a rigid matrix, generating more tension. With increased tension cytoskeletal stress fibers develop that contain αSMA and αvβ3 integrin that replaces α2β1 integrin, consistent with cell switching from collagen to non-collagen proteins interactions.     

NLS-RARα通过与importin α1/β1(KPNA2/KPNB1)结合入核并抑制U937细胞分化

目的探究核定位信号-维甲酸受体α(NLS-RARα)异常的核定位对细胞分化的抑制作用以及其核转运机制。方法过表达带有血凝素(HA)标签的NLS-RARα慢病毒和空载体慢病毒转染HEK293T细胞和U937细胞,设为NLS-RARα过表达组(NR组)和阴性对照组(NC组)。提取HEK293T细胞与U937细胞NC组和NR组细胞核和细胞质蛋白,Western blot法检测NLS-RARα的定位。同时采用免疫荧光细胞化学染色检测NLS-RARα的定位情况。1α,25-二羟维生素D3(1,25D3)诱导U937细胞分化,实时定量PCR,Western blot法检测细胞CD11b、 CCAAT/增强子结合蛋白β(CEBPβ)在mRNA和蛋白水平,采用质谱和免疫共沉淀技术(CoIP)筛选与NLS-RARα相作用的转运蛋白,并且通过小干扰RNA(siRNA)转染验证其对NLS-RARα的核定位作用。结果 NLS-RARα主要位于细胞核中。与对照组相比,过表达NLS-RARα组CD11b和CEBPβ的增加减少,说明NLS-RARα对1,25D3诱导的细胞分化有抑制作用。质谱和CoIP结果发现核转运蛋白α2/输入蛋白α1(KPNA2/importinα1)和核转运蛋白β1/输入蛋白β1(KPNB1/importinβ1)与NLS-RARα有相互作用,敲低KPNA2/KPNB1则抑制NLS-RARα入核。结论 NLS-RARα通过结合KPNA2/KPNB1入核,抑制U937细胞分化。     

T cell-associated α4β7 but not α4β1 integrin is required for the induction and perpetuation of chronic colitis

Anti-adhesion therapies that target α₄ integrins (e.g., natalizumab) are thought to work by blocking T-cell recruitment to the intestinal tissues in patients with Crohn's disease (CD); however, little direct evidence is available to confirm this contention. We wished to evaluate the importance of T cell-associated α₄ integrins in a chronic colitis model in mice and to determine the effect of natalizumab treatment on intestinal tissue T-cell accumulation in human CD. Adoptive transfer of T cells lacking α4 (α₄^−/−) but not β₁ integrin into immunodeficient mice produced significantly attenuated disease. This was correlated with reduced numbers of colon CD4 T cells compared with the control mice; however, tissue distribution of T helper type 1 (Th1) and T helper type 17 (Th17) cells and regulatory T cells (Tregs) was not affected by the lack of α₄. Furthermore, α4^−/− T cells demonstrated defective homing to the chronically inflamed small intestines and colons. Finally, patients treated with natalizumab showed significant reduction in mucosal CD4 T cells and no skewing in the foxp3⁺ Treg or T-bet⁺Th1 fractions thereof. These results demonstrate a direct role for T cell-associated α₄β₇ but not α₄β₁ integrins during initiation and perpetuation of chronic colitis. Moreover, our data demonstrated that natalizumab treatment reduced mucosal CD4 T-cell accumulation in CD patients.     

Expression of Integrin α6β1 Enhances Tumorigenesis in Glioma Cells

The integrin α6β1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the α6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the α6β1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of α6β1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to α6β1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of α6β1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that α6β1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of α6β1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin α6β1 in glioma progression.Malignant brain tumors have an increasing incidence in both children and adults. In adults, the most common type of primary brain tumor, malignant glioma, is considered as one of the deadliest of human cancers. Despite recent advances in both diagnostic modalities and therapeutic strategies, the 5-year survival rate of less than 3% in patients with glioblastoma is among the lowest for all cancers.¹ Patients with the most malignant histopathological subtype, glioblastoma, carry the worst prognosis, with median survival rate of less than 1 year, despite aggressive surgery associated with adjuvant radiotherapy and chemotherapy.¹ Glioblastoma are characterized by rapidly dividing cells, high degree of vascularity, invasion into normal brain tissue, and an intense resistance to death-inducing stimuli.^2,3 Since integrins, the major family of extracellular matrix (ECM) receptors, are involved in these events, they are one of the most promising molecules to consider for a targeted therapy.Integrins are cell surface transmembrane αβ heterodimers that recognize specific ECM ligands. The combination of α and β subunits, leading to the formation of at least 24 receptors, determines the ligand specificity.⁴ Glioblastoma commonly displays enhanced expression of several integrins along with their ECM ligands: αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin receptor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1 (laminins receptors).⁵ Numerous studies have focused on the αv integrin family. The integrins αvβ3 and αvβ5 are markers of glioblastoma malignancy⁶ and influence a variety of processes in glioblastoma progression in vivo, including proliferation, apoptosis, and angiogenesis.⁷ Furthermore, cilengitide, an αvβ3 and αvβ5 integrins antagonist, extends mouse survival by delaying the tumor growth^8,9 and is nowadays in clinical trial for recurrent malignant glioma. Two other integrins, α5β1 and α3β1, have been shown to be implicated in glioma cell adhesion and migration in vitro.^10,11 In addition, the use of α5β1 antagonists reduces glioma cell proliferation in vitro,¹⁰ while α3β1 antagonists inhibited glioma invasion in vivo.¹¹The α6 integrin subunit associates with β1 or β4 subunits to form functional heterodimers that selectively bind laminins. The α6β4 integrin is essential for the organization and maintenance of epithelial hemidesmosomes that link the intermediate filaments with the extracellular matrix.¹² The major ligand of α6β4 is the laminin-332, while α6β1 is a well-characterized laminin-111 receptor. Overexpression of α6β1 integrin has been associated with the progression of many epithelial tumors. In particular, induction of α6β1 expression is an early event in hepatocellular carcinogenesis.^13,14 In the same way, during prostate cancer progression α6β1 is continually expressed and found in micrometastases.¹⁵ Expression of α6β1 integrin has also been linked to metastatic potential of melanoma cells,¹⁶ and has been involved in the survival and metastatic potential of human breast carcinoma cells.^17,18 Moreover, in a recent study using the α6-blocking antibody GoH3, Lee et al¹⁹ inhibited angiogenesis and breast carcinoma growth in vivo.Several studies concerning gliomas and the α6β1 ligand laminin-111 have been reported in the literature. Using immunohistochemistry studies, Gingras et al²⁰ showed that α6 integrin was strongly expressed in glioblastoma tissue, whereas it was weakly expressed in normal brain. Previtali et al²¹ confirmed that the expression of α6 was increased in glioblastoma and in other central nervous system tumors, such as meningioma, astrocytoma, and neuroblastoma, when compared with the autologous normal tissue counterpart. In glioblastoma biopsies, laminin-111 is highly expressed on tumor blood vessels, but also within the brain tumor as punctuate deposits and at the tumor invasion front.²² In vitro, glioma cells can both secrete laminin-111 and induce its expression in normal brain tissue.^22,23,24 Moreover, laminin-111 is one of the most permissive substrates for adhesion and migration of glioma cells in vitro.^25,26,27 Additionally, over laminin-111, migrating glioma cells are protected from apoptosis.²⁸ For all these reasons, we hypothesized that laminin-111 and its main receptor α6β1 may contribute to glioblastoma progression.In the present study we investigated the role of integrin α6β1 in glioblastoma malignancy by using U87, a well-characterized glioblastoma cell line. We report that stable expression of α6β1 in this α6-negative cell line leads to enhanced tumor progression and tumor growth in vivo. We demonstrate that α6β1 is pro-angiogenic and acts on the balance between proliferation and apoptosis. Additionally, we show that α6β1 is involved in glioblastoma cell migration and invasion. Our results highlight for the first time the considerable role of integrin α6β1 in the malignant phenotype of glioblastoma cells and demonstrate that the α6β1-expressing cell is an appropriate model for the study of glioblastoma progression.     

Endothelial α3β1-Integrin Represses Pathological Angiogenesis and Sustains Endothelial-VEGF

Integrin α3β1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of α3β1 can affect angiogenesis either positively or negatively, either a direct in vivo role for α3β1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of α3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where α3 is deleted specifically in endothelial cells (ec-α3−/−). Here we show that ec-α3−/− mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that α3β1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial α3β1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.Angiogenesis, the formation of new blood vessels from pre-existing ones, is enhanced in various pathological conditions including rheumatoid arthritis, diabetic retinopathy, and cancer development. Angiogenic processes are regulated both by growth factors, such as vascular endothelial growth factor (VEGF), and adhesion molecules, such as integrins.^1,2,3 Inhibitors of VEGF signaling can extend progression-free survival in colorectal, lung, and breast cancers when used in combination with chemotherapy^4,5 and in renal cancer when used as a monotherapy.⁶ However, recent data suggest that complications with acquired resistance to these agents and how they affect metastasis may compromise their efficacy.^7,8 Thus, understanding the molecular mechanisms that underlie the regulation of angiogenesis, and especially VEGF function, is essential for the development of safe and effective antiangiogenic therapies.Endothelial integrins include the fibronectin receptor α5β1, collagen receptors α2β1 and α1β1, vitronectin receptors αvβ3 and αvβ5, and laminin receptors α6β1, α6β4, and α3β1. Although many integrins have been shown to be involved in tumor angiogenesis,¹ the role of endothelial-α3β1 in this process is unknown. In addition, integrin α3β1 is a binding partner for several other ligands, and together these have been implicated in either promoting or inhibiting angiogenesis. For example, deletion of CD151 or embryonic laminin α4-subunit, molecules that can both bind α3β1, results in impaired angiogenesis,^9,10,11 whereas adult laminin-8–deficient mice display increased tumor vascularization.¹² In addition, it has also been proposed that the antitumorigenic effects of recombinant α3(IV)NC1 can, in part, be attributed to its binding to αvβ3 and α3β1-integrin,^13,14 whereas the antiangiogenic functions of thrombospondin-1 are reversed by binding αvβ3 and α3β1.^15,16 And finally, the binding of VEGF to α3β1 in vitro is thought to enhance angiogenic responses,¹⁷ whereas the interaction of TIMP-2 with α3β1-integrin has been reported to inhibit VEGF receptor-2 function.¹⁸ Taken together, these contradictory results indicate that a precise and, more importantly, direct role of α3β1-integrin in pathological angiogenesis is not well understood. Accordingly, because α3β1-directed inhibitors are being designed to either block tumor cell growth or angiogenesis,^19,20 the requirement to test directly the role of α3β1 in pathological angiogenesis becomes a priority.Genetic ablation of the α3-integrin-subunit in mice results in a lethal phenotype where mice die within hours after birth,²¹ rendering them inappropriate for pathological angiogenesis studies. We therefore have generated mice where the α3-integrin-subunit is deleted in endothelial cells (ec-α3−/−). These mice are viable and fertile, and here we report that mice deficient in endothelial-α3β1-integrin display enhanced tumor growth and elevated tumor angiogenesis. In addition we show that the deletion of α3-integrin in endothelial cells results in enhanced VEGF-mediated angiogenic responses both ex vivo and in vivo. Moreover, we present evidence for a novel and unexpected mechanism by which deficiency in α3β1 regulates angiogenesis by the regulation of endothelial VEGF. Although autocrine signaling by endothelial VEGF has been shown to regulate endothelial cell survival,²² its role in angiogenesis or a mechanism by which it is regulated has not been demonstrated. We show that α3β1 reduces the expression of endothelial VEGF, which surprisingly, at low levels, elevates VEGFR2 expression and thus enhances VEGF-induced angiogenic responses.     

Interleukin-1β-induced brain injury and neurobehavioral dysfunctions in juvenile rats can be attenuated by α-phenyl-n-tert-butyl-nitrone

Our previous study showed that perinatal exposure to interleukin-1β (IL-1β), an inflammatory cytokine, induces acute injury to developing white matter in the neonatal rat brain, and α-phenyl-n-tert-butyl-nitrone (PBN), a free radical scavenger and antioxidant, protects against IL-1β-induced acute brain injury. The objective of the present study was to further examine whether perinatal exposure to IL-1β resulted in persistent brain damage and neurological disabilities, and whether PBN offers lasting protection. Intracerebral injection of IL-1β (1 μg/kg) was performed in postnatal day 5 (P5) Sprague-Dawley rat pups and PBN (100 mg/kg) or saline was administered intraperitoneally 5 min after IL-1β injection. Perinatal IL-1β exposure significantly affected neurobehavioral functions in juvenile rats. Although some neurobehavioral deficits such as performance in negative geotaxis, cliff avoidance, beam walking, and locomotion were spontaneously reversible, sustained deficits such as poor performance in the vibrissa-elicited forelimb-placing test, the pole test, the passive avoidance task, and the elevated plus-maze task were still observable at P21. Perinatal IL-1β exposure resulted in persistent brain damage including enlargement of ventricles, loss of mature oligodendrocytes, impaired myelination as indicated by the decrease in myelin basic protein immunostaining, axonal and dendritic injury, and loss of hippocampal CA1 neurons and tyrosine hydroxylase positive neurons in the substantia nigra and ventral tegmental areas of the rat brain. Treatments with PBN provided lasting protection against the IL-1β-induced brain injury and improved the associated neurological dysfunctions in juvenile rats, suggesting that prompt treatments for brain injury induced by perinatal infection/inflammation might have important long-term consequences.     

Comparing the expression of integrins αvβ3, αvβ5, αvβ6, αvβ8, fibronectin and fibrinogen in human brain metastases and their corresponding primary tumors

Aims: To evaluate the expression of αv-series integrins in brain metastases. Inhibitors targeting these integrins are being tested for their therapeutic potential. Material and Method: The extracellular regions of the αvβ3, αvβ5, αvβ6, αvβ8, the cytoplasmic domain of β3, the αv-chain, and the ECM molecules fibronectin and fibrinogen were studied immunohistochemically in a series of 122 carcinoma and 60 melanomas metastatic to the central nervous system. In addition, 38 matched primary and metastatic tumors to the brain were compared directly. Results: The αv-subunit was generally moderately to highly expressed in most tumors. αvβ3 and cytoplasmic β3 were weakly to moderately detectable in metastatic renal cell carcinomas and melanomas, αvβ5 was prominently expressed in metastatic renal and colorectal carcinomas, αvβ6 was most abundantly detectable in metastatic lung adenocarcinomas, but absent in melanomas. The tumor associated vessels in CNS metastases consistently expressed αvβ3, αvβ5, αv-, fibronectin and fibrinogen, however, mostly at low levels, while αvβ6, αvβ8 were lacking in vasculature. The comparative analysis of 38 matched primary tumors and brain metastases showed comparable levels of expression only for αvβ3 and αvβ8, while αvβ6 and αvβ5 were higher in primaries. Conclusion: We confirmed that integrin expression exhibits considerable heterogeneity according to tumor origin. αvβ5 is the most promising target for integrin targeted treatment in brain metastases.     

αvβ3, αvβ5 and αvβ6 integrins in brain metastases of lung cancer

Integrins are transmembranous adhesion molecules postulated to be involved in the brain metastatic cascade. We investigated the correlation of alpha v beta 3 (αvβ3), alpha v beta 5 (αvβ5) and alpha v beta 6 (αvβ6) integrin isoform expression with clinical characteristics including survival times in lung cancer patients with brain metastases (BM). All BM from lung cancer operated at our institution between 1990 and 2011, were identified; where available, primary tumors were retrieved as well. Immunohistochemical analysis for αvβ3, αvβ5 and αvβ6 integrin subunits was performed and correlated with Ki67 and hypoxia-inducible factor (HIF)-1α indexes. Clinical data including survival data were obtained by chart review. 191 BM specimens of 191 patients with histologically confirmed lung cancer (172 non-small cell lung cancer and 19 small cell lung cancer) were included. In 18 patients matched primary tumor samples were available. αvβ6 expression was commonly found on BM tumor cells (103/191; 53.9 %) and showed a significant association with low Ki67 proliferation indices (46 vs. 36 %, p = 0.001, Mann–Whitney U test) and favorable survival times (p = 0.020; log rank test) in patients with non-squamous NSCLC BM. αvβ5 expression was highly expressed on vascular structures (167/191; 87.4 %) and tumor stroma in BM (151/191; 79.1 %) and associated with high HIF-1α indices (60 vs. 90, p = 0.007, Mann–Whitney U test). αvβ3 expression was more frequently found on vascular structures in BM than in primary tumors (68.1 vs. 5.6 %; p = 0.645; Chi square test) and its expression in BM tumor cells correlated with low Ki67 indices (41 vs. 28 %; p = 0.046, Mann–Whitney U test). Expression of αv integrin subunits seem to be of pathobiological and clinical relevance in patients with NSCLC BM. Further investigations of their involvement in the brain metastatic cascade and their role as biomarkers are warranted.     

大鼠脑缺血区IL-1β和TNF α表达的实验研究

炎症和免疫因素与脑缺血的联系，近来倍受观注。本实验旨在探查脑缺血区ＩＬ１β和ＴＮＦα表达的结构和时程，进而探讨其表达与脑缺血性损伤之间的病理联系。采用局灶性脑缺血／再灌模型（缺血１小时／再灌流０小时、２小时、４小时、８小时和１６小时），原位杂交和免疫组织化学染色技术对４０只成年雄性ＳＤ（ＳｐｒａｇｕｅＤａｗｌｅｙ）大鼠进行了研究。结果显示，脑缺血区ＩＬ１βｍＲＮＡ阳性信号主要存在微血管内皮细胞，在脑缺血周围区的ＩＬ１βｍＲＮＡ信号比中央区更强烈。阳性信号出现于脑缺血１小时（与对照组比较…     

Integrin α5β1: a potent inhibitor of experimental lung metastasis

The integrin alpha5beta1 seems to be the most relevant receptor of tumor cells for binding to fibronectin. Although numerous studies suggest a role of tumor cell fibronectin interaction in tumor metastasis, differential integrin expression on tumor cells has, however, not been correlated with metastatic capabilities. We addressed this question by transfection of the integrin alpha5beta1 cDNA into HT-29 human colon carcinoma cells which led to de novo expression of functional integrin alpha5beta1. Similar to other reports, expression of the integrin alpha5beta1 in HT-29 tumor cells exerted an inhibitory action on cell proliferation as indicated in our study by formation of fewer colonies in soft agar. The tumor growth inhibitory property of the integrin alpha5beta1 was also shown by reduction of subcutaneous xenograft growth in nude mice to approximately 50% of that of control transfectants. For the first time, we found that several clones of integrin alpha5 subunit transfectants displayed dramatically reduced formation of lung colonies and cutaneous metastasis after intravenous injection into nude mice. While most animals inoculated with control transfectant cells formed macroscopically visible lung colonies ranging from 12.6 +/- 2.6 to 22.0 +/- 6.6 (mean colony number +/- SEM), mice inoculated with HT-29 cell clones expressing the integrin alpha5beta1 were almost completely free of lung colonies (ranging from 0.0 +/- 0 to 0.2 +/- 0.1). Our results imply that integrin alpha5beta1 expression inhibits circulating tumor cells in pursuing late steps of the metastatic process as represented by the artificial metastasis (lung colonisation) model.     

1例GNPTAB基因变异致黏脂贮积症Ⅲ α/β型患儿的临床和遗传学分析

目的 对1例表现为骨骼畸形，关节异常，特殊面容，运动障碍，智力障碍的患儿进行遗传学分析，明确其病因。方法 提取患儿及其父母、妹妹外周血DNA，应用全外显子组测序技术结合临床表型系统分析相关基因的致病变异，通过Sanger测序对先证者及父母、妹妹进行验证。结果 全外显子组测序结果显示患儿的GNPTAB基因第13外显子存在c.2715+1G>A和第9外显子存在c.1090C>T(p.Arg364*)复合杂合变异，分别遗传自父亲和母亲，且变异为已报道的致病性变异，患儿妹妹存在c.1090C>T(p.Arg364*)变异，遗传自母亲。结论 GNPTAB基因c.2715+1G>A和c.1090C>T复合杂合变异可能为患儿的致病原因。我们的结果为家系的遗传咨询提供了依据。     

Contribution of sialic acids to integrin α5β1 functioning in melanoma cells

PurposeTo establish the relationship between sialylation of integrin α5β1 and possible alteration in the function of α5β1 receptor in melanoma cells.Materials and methodsIntegrin α5β1 was isolated from primary WM115 (RGP/VGP-like phenotype) and metastatic WM266-4 (lymph node metastasis) cells via affinity chromatography. Integrin α5β1 sialylation and the shift in relative masses of the enzymatically desialylated subunits were confirmed by confocal microscopy and SDS-PAGE, respectively. The ELISA assay was performed to evaluate sialic acid (SA) influence on integrin α5β1 binding to fibronectin (FN). Cell invasion was investigated by the Transwell invasion assay. The effect of neuraminidases treatment on melanoma cells was assessed by flow cytometry using Maackia amurensis and Sambucus nigra lectins.ResultsBoth subunits of integrin α5β1 were found to be more abundantly sialylated in primary than in metastatic cells. The removal of SA had no effect on the purified integrin α5β1 binding to FN. Although metastatic cells underwent more pronounced desialylation than primary cells, invasion of primary WM115 cells was more dependent on the presence of α2-3 linked SA than it was in the case of metastatic WM266-4 cells. In both melanoma cell lines not only integrin α5β1 was involved in invasion, however simultaneous desialylation and usage of anti-integrin α5β1 antibodies resulted in lower invasion abilities of primary WM115 cells.ConclusionsOur data suggest that in primary melanoma cells integrin α5β1 action is more likely dependent on its glycosylation profile, i.e. the presence of SA residues, which influence (decreased) their invasion properties and may facilitate malignant melanoma progression.     

Integrin α2β1 targeted GdVO4:Eu ultrathin nanosheet for multimodal PET/MR imaging

Multifunctional nanoprobes open exciting possibilities for accurate diagnosis and therapy. In this research, we developed a ⁶⁴Cu-labeled GdVO₄:4%Eu two-dimension (2D) tetragonal ultrathin nanosheets (NSs) that simultaneously possess radioactivity, fluorescence, and paramagnetic properties for multimodal imaging. The carboxyl-functionalized Eu³⁺-doped GdVO₄ NSs were synthesized by a facile solvothermal reaction, followed by ligand exchange with polyacrylic acid (PAA). With ultrathin thickness of ∼5 nm and width of ∼150 nm, the carboxyl-functionalized NSs were further modified by DOTA chelator for ⁶⁴Cu labeling and Asp-Gly-Glu-Ala (DGEA) peptide for integrin α₂β₁ targeting. After initial evaluation of the cytotoxicity and targeting capability with PC-3 cells, the obtained multifunctional nanoprobes (⁶⁴Cu-DOTA-GdVO₄:4%Eu-DGEA) were further explored for targeted positron emission tomography (PET) and T₁-weighted magnetic resonance imaging (MRI) of PC-3 tumor (prostate cancer, high integrin α₂β₁ expression) in vivo. Based on the strong fluorescence of the NSs, the particle distribution in mouse tissues was also determined by fluorescent microscopy. In summary, GdVO₄:4%Eu NS is a potential multimodal multiscale nanoprobe that could not only be used for in vivo imaging, but also be tracked in cellular scale and ex vivo due to its fluorescent property.     

Expression and ligand binding of α2β1 integrin on breast carcinoma cells

We examined the expression and ligand specificity of the 21 integrin on human mammary epithelial cells (HMEC) and a panel of breast carcinoma cell lines in vitro. We found that the 21 integrin was universally, but quite variably expressed on these cells by FACS analysis. No significant correlation was observed between its expression and other known cellular phenotypes. Substrate attachment assays using blocking antibodies demonstrated that 21 integrin served as a receptor for collagen on HMEC and almost all breast carcinoma cells. However, its contribution to laminin binding of these cells appeared to be related to cellular differentiation as evaluated by sex steroid receptor status and by markers of epithelial-mesenchymal transition, i.e. loss of E-cadherin and expression of vimentin. Two different populations of non-malignant immortalized HMEC (184A1N4 and MCF-10A) contained cells capable of using 21 integrin as a laminin receptor. Breast cancer cell lines positive for estrogen receptor (ER) and E-cadherin (MCF-7, T47D, ZR75-1) could also use 21 integrin as a laminin receptor. Conversely, 21 integrin appeared to be incapable of binding to laminin or to be a very minor receptor for laminin on metastatic ER-negative breast carcinoma cells that expressed vimentin (MDA-MB 231, MDA-MB 435, and MDA-MB 436). These findings suggest that the ligand specificity of 21 integrin, i.e. its function as a laminin receptor, may be regulated during the malignant progression of breast carcinoma cells. A reduced contribution of 21 integrin to the cellular laminin binding appears to be associated with an increased malignant phenotype and with an epithelial-mesenchymal transition of breast carcinoma cells.     

Integrin α4β1/VCAM-1 pathway mediates primary adhesion of RAW117 lymphoma cells to hepatic sinusoidal endothelial cells under flow

Adhesion and stabilization of circulating tumor cells to endothelial cells in target blood vessels play an important role in the complex process of metastasis. We examined the cell surface receptors involved in the liver-metastatic adhesive interactions of murine RAW117 large-cell lymphoma cells to unstimulated hepatic sinusoidal endothelial cells (HSE) under physiological flow conditions. Flow cytometric analysis indicated that VCAM-1, ICAM-1 and PECAM-1 are constitutively expressed on the surfaces of both HSE and RAW117 cells. However, monoclonal antibody (mAb) blockade studies showed that ICAM-1 and PECAM-1 affected neither the attachment nor the stabilization step of the adhesion of RAW117 cells to HSE cell monolayers under flow. In contrast, RAW117 cells required a significantly lower shear stress to establish adhesion to HSE cells when VCAM-1 receptors on HSE cells were blocked with mAb. Furthermore, the presence of the anti-VCAM-1 mAb significantly decreased the extent of adhesion compared to that of the control, without affecting adherent cell stabilization times. Blocking the α₄integrin subunits present mainly on RAW117 cells produced similar results to those previously observed with anti-VCAM-1 mAb. Although constitutively present mainly on the surfaces of RAW117 cells, MAdCAM-1 and β₇ integrin subunit do not appear to play a role in either the arrest or stabilization of RAW117 cells on HSE cell monolayers. However, blocking the β₁integrin subunit on the RAW117-H10 cells reduced adhesion to the same extent as anti-α₄ and anti-VCAM-1 treatments. These observations suggest that an interaction of integrin α₄β₁on RAW117 cells with liver endothelial VCAM-1 occurs during the early stages of the adhesion process and may be important in liver metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

α4β1 and α5β1 Control Cell Migration on Fibronectin by Differentially Regulating Cell Speed and Motile Cell Phenotype

Computer-aided time lapse fluorescence videomicroscopy was used to study single cell migration behavior of human aortic endothelial cells on fibronectin coated substrates of varying protein surface density. The role of receptors _₅ _₁, __v _₃, and ₄₁ in mediating cell adhesion and migration on fibronectin was characterized using integrin specific monoclonal antibodies. Matrix density had a direct effect on controlling the proportion of migrating cells and the directional persistence of cell movement (p < 0.01). While there was relatively little influence of fibronectin surface density on absolute migration speed, the ability of endothelial cells to disperse over a surface, as measured by the dispersion coefficient, was biphasic with respect to the surface density of this matrix protein (p < 0.005). Both cell speed and the proportion of migrating cells was controlled by ₄₁ (p < 0.01). However, ₅₁ selectively regulated the transformation of stationary cells to those exhibiting motile behavior (p <0.05). Migratory responses on fibronectin were not influenced by blockade of the _v3 receptor. It is noteworthy that cell surface adhesive receptors which control commitment to a motile phenotype are not necessarily the same as those that control migration speed. © 1998 Biomedical Engineering Society. PAC98: 8722-q