Eosinophil Adhesion and Maturation is Modulated by Laminin

Eosinophils (Eo) participate in the inflammatory response to parasites, allergins, toxins, and epitopes recognized by autoimmune antibodies. Nonetheless, little attention has heretofore been paid to the interactions of Eo with extracellular matrix (ECM) proteins during their migration through the subendothelial basement membrane and into the surrounding tissue. Therefore, we have studied the adhesion of Eo to specific ECM proteins and the effect of this adhesion on Eo viability and maturation. Control Eo (from normal donors) adhere no better to substrates coated with laminin (LM), fibronectin (FN), cytotactin (CT), or collagen types I or IV (Col IV) than they do to human serum albumin coated substrates. In contrast, Eo activated in vitro with IL-5 or in vivo in patients with eosinophilia bind well to LM, FN and Col IV. LM is by far the most avid ligand among these molecules. For example, 43% of input cells bind to a substrate bearing 200 fmol/cm² of LM; a similar level of adhesion to FN requires 30 times as much adsorbed protein. Antibody inhibition experiments suggest that the αβ1 integrin heterodimer is the predominant LM receptor on these cells. Flow cytometry showed similar levels of these subunits on control and activated Eo, suggesting that Eo adhesion to LM is not regulated simply by cell surface integrin concentration. The effects of ECM proteins on Eo behavior were also examined. A LM-coated substrate (with no added cytokine) was found to be almost as effective as IL-5 in maintaining Eo viability while an equally adhesive FN-coated substrate had much less effect. Normally, even in the presence of 10% serum, no Eo survive a 5-day incubation in vitro unless IL-3, IL-5, or GM-CSF is added to the medium. Conditions that inhibit adhesion to LM (anti-integrin antibodies in the medium or CT on the substrate) and certain anti-cy-tokine antibodies inhibited the promotion of Eo viability by LM. During incubation on LM, Eo become hypodense, as they do in the presence of IL-5, indicating that they have become activated. These observations suggest that the interactions of Eo and ECM proteins may be important both for their potential to direct Eo migration and for their ability to regulate Eo viability, cytokine production, and maturation.     

Calmodulin-Binding Proteins in Human Y-79 Retinoblastoma and HTB-14 Glioma Cell Lines

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.     

The Tetraspanin CD9 Influences the Adhesion, Spreading, and Pericellular Fibronectin Matrix Assembly of Chinese Hamster Ovary Cells on Human Plasma Fibronectin

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and na?ve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.     

Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin

Background

Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules ⁷F3-¹⁰F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules ⁷F3-¹⁰F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly.

Methodology/Principal Findings

To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to ⁷F3-¹⁰F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules ²F3-¹⁴F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module ¹F3 or the C-terminal modules to modules ²F3-¹⁴F3 resulted in some activity, and addition of both ¹F3 and the C-terminal modules resulted in a construct, ¹F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs ¹F3-C V0, ¹F3-C V64, and ¹F3-C Δ(V¹⁵F3¹⁰F1) were all able to support fibronectin assembly, suggesting that ¹F3 through ¹¹F1 and/or ¹²F1 were important for activity. Coatings in which the active parts of ¹F3-C were present in different proteins were much less active than intact ¹F3-C.

Conclusions

These results suggest that ¹F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells.     

Laminin extraction and disorganization of collagen fibrils in snail muscles by EDTA treatment

Summary— Snail muscles were extracted by a solution of EDTA and electron microscopy showed that the extract contained dispersed, depolymerized collagen fibrils and cross-shaped laminin-like structures. The extracts were purified by ultracentrifugation followed by two different procedures which enriched the content of laminin-like structures. The laminin-related molecules displayed unique properties when analyzed by biochemical, immunological and morphological methods. Electrophoretic patterns of the molecular form purified primarily by ion exchange chromatography, resembled EHS-tumor laminin and displayed a cruciform shape when viewed by electron microscopy. Immunohistology, using antiserum obtained against the agarose gel-purified protein, showed that this laminin was primarily located in the extracellular matrix surrounding muscle fibers. Western blots using anti-EHS laminin antibody showed reaction of a 300 kDa subunit of this snail laminin. The protein obtained by another procedure, initially using gel filtration, followed by ion exchange chromatography, also appeared to be a laminin. It had a collapsed cruciform appearance when viewed by electron microscopy. It contained several different subunits, one of which, ca 300 kDa, reacted with anti-EHS-laminin antibody and with anti-snail laminin antibody. In contrast, EHS laminin did not react with the anti-snail laminin antibody. The composite results suggest that at least two different forms of laminin are extractable from snail muscle and that they share molecular properties and immune determinants with mouse tumor laminin.     

Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists

In vascular smooth muscle cells (VSMCs) integrin-mediated adhesion to extracellular matrix (ECM) proteins play important roles in sustaining vascular tone and resistance. The main goal of this study was to determine whether VSMCs adhesion to type I collagen (COL-I) was altered in parallel with the changes in the VSMCs contractile state induced by vasoconstrictors and vasodilators. VSMCs were isolated from rat cremaster skeletal muscle arterioles and maintained in primary culture without passage. Cell adhesion and cell E-modulus were assessed using atomic force microscopy (AFM) by repetitive nano-indentation of the AFM probe on the cell surface at 0.1 Hz sampling frequency and 3200 nm Z-piezo travelling distance (approach and retraction). AFM probes were tipped with a 5 μm diameter microbead functionalized with COL-I (1mg\ml). Results showed that the vasoconstrictor angiotensin II (ANG-II; 10⁻⁶) significantly increased (p<0.05) VSMC E-modulus and adhesion probability to COL-I by approximately 35% and 33%, respectively. In contrast, the vasodilator adenosine (ADO; 10⁻⁴) significantly decreased (p<0.05) VSMC E-modulus and adhesion probability by approximately −33% and −17%, respectively. Similarly, the NO donor (PANOate, 10⁻⁶ M), a potent vasodilator, also significantly decreased (p<0.05) the VSMC E-modulus and COL-I adhesion probability by −38% and −35%, respectively. These observations support the hypothesis that integrin-mediated VSMC adhesion to the ECM protein COL-I is dynamically regulated in parallel with VSMC contractile activation. These data suggest that the signal transduction pathways modulating VSMC contractile activation and relaxation, in addition to ECM adhesion, interact during regulation of contractile state.     

Roles for Laminin in Embryogenesis: Exencephaly,Syndactyly, and Placentopathy in Mice Lacking the Laminin α5 Chain

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin''s roles in embryonic development. Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are α5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin α chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.     

Adhesion,spreading, and proliferation of cells on protein carpets: Effects of stability of a carpet

Summary In the present report we have investigated the role that the physical properties of substrata play in modulating the effects which components of extracellular matrix (ECM) exert on adhesion, spreading, and growth of retinal pigmented epithelial cells. By simple modifications of conditions for protein adsorption on glass we obtained a set of substrata all coated with proteins of ECM (protein carpets) but with different physical properties. Using these protein carpets we have shown that their stability (desorption rate) in tissue culture conditions varies according to the technique with which they were prepared. Both semiremovable and immobilized carpets are stable, whereas removable protein carpets desorb readily. Therefore, the protein concentration or composition or both may change with time in tissue culture depending on the technique used to prepare the carpet. In addition, efficacy of cell attachment to given protein may vary depending on whether a technique used to prepare the protein carpet involves denaturation of the protein. Adherent cells quickly remove (clear) weakly adsorbed protein carpets and it seems that the carpet removal is a mechanical process. During the carpet removal cells are rounded, which indicates that a spread cell phenotype normally associated with stress fibers and focal contacts occurs when the substratum is rigid enough to sustain cell traction. In addition, substrata lacking the rigidity to support the spread phenotype do not support cell proliferation either.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Identification of Proteins Sensitive to Thermal Stress in Human Neuroblastoma and Glioma Cell Lines

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.     

细丝蛋白A在细胞黏附和迁移中的作用

细胞迁移在发育、伤口愈合、炎症反应和肿瘤转移等多种病理生理过程中发挥重要作用。细丝蛋白A（filamin A,FlnA）是一种在各组织细胞中广泛表达的微丝结合蛋白,其表达异常导致细胞迁移功能障碍。该文回顾了相关的文献,首先介绍生理情况下细丝蛋白A的功能,接着介绍细丝蛋白A基因突变和表达异常导致的多种遗传性疾病及其与肿瘤转移的关系,突出细丝蛋白A对迁移的影响在这些疾病发病中的作用,最后深入探讨了细丝蛋白A影响细胞迁移和黏附的可能机制。     

Underlying Mechanism of Quercetin-induced Cell Death in Human Glioma Cells

There has been considerable interest in recent years in the anti-tumor activities of flavonoids. Quercetin, a ubiquitous bioactive flavonoid, can inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the precise molecular mechanism by which quercetin induces apoptosis in cancer cells is poorly understood. The present study was undertaken to examine the effect of quercetin on cell viability and to determine its underlying mechanism in human glioma cells. Quercetin resulted in loss of cell viability in a dose- and time-dependent manner and the decrease in cell viability was mainly attributed to cell death. Quercetin did not increase reactive oxygen species (ROS) generation and the quercetin-induced cell death was also not affected by antioxidants, suggesting that ROS generation is not involved in loss of cell viability. Western blot analysis showed that quercetin treatment caused rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Transient transfection with constitutively active forms of MEK and Akt protected against the quercetin-induced loss of cell viability. Quercetin-induced depolarization of mitochondrial membrane potential. Caspase activity was stimulated by quercetin and caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin resulted in a decrease in expression of survivin, antiapoptotic proteins. Taken together, these findings suggest that quercetin results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of ERK, Akt, and survivin.     

Effects of substrata and method of tissue dissociation on adhesion,cytoskeleton, and growth of chick retinal pigmented epithelium in vitro

Summary In this report we compare attachment, morphology, and growth of retinal pigmented epithelial (RPE) cells isolated by either EDTA or dispase digestion and plated onto either uncoated substrata (plastic or glass) or substrata derivatized by covalent conjugation of proteins of reconstituted basement membrane gel. We show that the derivatized substrata promote better initial attachment and subsequent cell growth than the uncoated substrata. These effects are independent of the method of dissociation of cells from the tissue. Cell morphology, however, is strongly affected by the method used for tissue dispersion. The dispase-dissociated cells are very flat, display a circumferential arrangement of microfilaments and elaborate extensive arrays of vinculin-containing cell-to-cell junctions. In contrast, EDTA-dissociated cells are much less spread, display straight microfilament bundles criss-crossing the cytoplasm and have less extensive cell-to-cell junctions. The protein-derivatized substrata also promote maintenance of differentiated traits such as pigmentation, by the RPE cells. Supported by Medical Research Council grant MA-9713 and by a grant from the R P Eye Research Foundation.     

Effects of Interferon and PKC Modulators on Human Glioma Protein Kinase C,Cell Proliferation,and Cell Cycle

The in-vitro effects of human interferon -2b (HuIFN -2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN -2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC₅₀ 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN -2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN -2b is similar to TPA (potent PKC activation and S phase arrest).     

牛蛙皮胶原蛋白酸提取条件优化及其对细胞生长和粘附性的影响

应用羟脯氨酸法测定了牛蛙皮胶原蛋白含量,通过正交实验对牛蛙皮胶原蛋白酸法提取条件进行了优化,并结合MTT法测定了胶原蛋白对细胞生长和粘附性的影响。结果显示,牛蛙皮胶原蛋白的含量约为45．1％;温度条件对提取率影响最大,其次依次为酸种、时间和浓度,最优组合为乙酸、1．5mol／L、37℃、24h;在低浓度时,牛蛙皮胶原蛋白对正常人类肝细胞的生长和粘附性无显著影响,当浓度升高到一定值时,对细胞生长和粘附性有显著促进作用。     

Establishment and Partial Characterization of a Continuous Human Malignant Glioma Cell Line: NG97

1. A human glioma cell line, NG97, was established from tissue obtained from a patient diagnosed with a grade III astrocytoma.2. The NG97 cell line has been subcultured for more than 100 passages in standard culture media without feeder layer or collagen coatings.3. NG97 cells grow in vitro as two subpopulations with distinct morphological appearance: stellate cells with pleomorphic nuclei, and small round cells with few processes. The cells have a doubling time of about 72 h and a plating efficiency of 1%. The injection of NG97 cells into congenitally athymic mice induced the formation of solid tumor masses that could be retransplanted every 4 weeks. The cells obtained from tumor mass when cultivated in vitro had a morphology comparable to those of the initial culture.4. This cell line may prove useful for cellular and molecular studies as well as in studies of gliomas treatment.     

Binding of CD157 Protein to Fibronectin Regulates Cell Adhesion and Spreading

CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.