Osteopontin mediates glioblastoma-associated macrophage infiltration and is a potential therapeutic target.

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

The Interaction of CD154 with the α5ß1 Integrin Inhibits Fas-Induced T Cell Death.

CD154, a critical regulator of the immune response, is usually associated with chronic inflammatory, autoimmune diseases as well as malignant disorders. In addition to its classical receptor CD40, CD154 is capable of binding other receptors, members of the integrin family, the αIIbß3, αMß2 and α5ß1. Given the role attributed to integrins and particularly the ß1 integrins in inhibiting apoptotic events in normal as well as malignant T cells, we were highly interested in investigating the role of the CD154/α5ß1 interaction in promoting survival of malignant T cells contributing as such to tumor development and/or propagation. To support our hypothesis, we first show that soluble CD154 binds to the T-cell acute lymphoblastic leukemia cell line, Jurkat E6.1 in a α5ß1-dependent manner. Binding of soluble CD154 to α5ß1 integrin of Jurkat cells leads to the activation of key survival proteins, including the p38 and ERK1/2 mitogen-activated protein kinases (MAPKs), phosphoinositide 3 kinase (PI-3K), and Akt. Interestingly, soluble CD154 significantly inhibits Fas-mediated apoptosis in T cell leukemia-lymphoma cell lines, Jurkat E6.1 and HUT78 cells, an important hallmark of T cell survival during malignancy progression. These anti-apoptotic effects were mainly mediated by the activation of the PI-3K/Akt pathway but also involved the p38 and the ERK1/2 MAPKs cascades. Our data also demonstrated that the CD154-triggered inhibition of the Fas-mediated cell death response was dependent on a suppression of caspase-8 cleavage, but independent of de novo protein synthesis or alterations in Fas expression on cell surface. Together, our results highlight the impact of the CD154/α5ß1 interaction in T cell function/survival and identify novel targets for the treatment of malignant disorders, particularly of T cell origin.

Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis.

The extracellular matrix (ECM) has been demonstrated to facilitate angiogenesis. In particular, fibronectin has been documented to activate endothelial cells, resulting in their transition from a quiescent state to an active state in which the cells exhibit enhanced migration and proliferation. The goal of this study is to examine the role of polymerized fibronectin during vascular tubulogenesis using a 3 dimensional (3D) cell-derived de-cellularized matrix. A fibronectin-rich 3D de-cellularized ECM was used as a scaffold to study vascular morphogenesis of endothelial cells (ECs). Confocal analyses of several matrix proteins reveal high intra- and extra-cellular deposition of fibronectin in formed vascular structures. Using a small peptide inhibitor of fibronectin polymerization, we demonstrate that inhibition of fibronectin fibrillogenesis in ECs cultured atop de-cellularized ECM resulted in decreased vascular morphogenesis. Further, immunofluorescence and ultrastructural analyses reveal decreased expression of stromal matrix proteins in the absence of polymerized fibronectin with high co-localization of matrix proteins found in association with polymerized fibronectin. Evaluating vascular kinetics, live cell imaging showed that migration, migration velocity, and mean square displacement, are disrupted in structures grown in the absence of polymerized fibronectin. Additionally, vascular organization failed to occur in the absence of a polymerized fibronectin matrix. Consistent with these observations, we tested vascular morphogenesis following the disruption of EC adhesion to polymerized fibronectin, demonstrating that block of integrins α5ß1 and αvß3, abrogated vascular morphogenesis. Overall, fibronectin deposition in a 3D cell-derived de-cellularized ECM appears to be imperative for matrix assembly and vascular morphogenesis.

Identification of novel chicken CD4⁺ CD3⁻ blood population with NK cell like features.

Chicken NK cells have been defined in embryonic spleen and intestinal epithelium as CD8(+) lymphoid cells that lack BCR and TCR, whereas blood NK cells have not been phenotypically defined. Here we employed the mab, 8D12 directed against CHIR-AB1, a chicken Fc receptor, to define a previously uncharacterized lymphoid cell population in the blood. Although CHIR-AB1 expression was found on several cell populations, cells with extraordinary high CHIR-AB1 levels ranged between 0.4 and 2.8% in five different chicken lines. The widespread applicability of the CHIR-AB1 mab was unexpected, since CHIR-AB1-like genes form a polygenic and polymorphic subfamily. Surprisingly the CHIR-AB1 high cells coexpressed low MHCII, low CD4 and CD5, while other T cell markers CD3 and CD8, the B cell marker Bu1, the macrophage marker KUL01 were absent. Moreover, they stained with the mab 28-4, 20E5 and 1G7, which define chicken NK cells and they also expressed CD25, CD57, CD244 and the vitronectin receptor (αVß3 integrin). In functional assays, PMA stimulation led to high levels of IFNγ release, while spontaneous cytotoxicity was not detectable. The expression of typical NK cell markers in the absence of characteristic B- or T-cell markers, and their IFNγ release is suggestive of a yet unidentified NK like population.

Anti-transforming growth factor ß-induced protein antibody ameliorates vascular barrier dysfunction and improves survival in sepsis.

AIM: Sepsis is a systemic inflammatory response syndrome resulting from a microbial infection. Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein expressed by human endothelial cells and platelets that induces sepsis through interaction with integrin αvß5. The aim of this study was to investigate the role of TGFBIp in vascular permeability and the underlying mechanisms using TGFBIp-neutralizing antibody. METHODS: Mice were subjected to caecal ligation and puncture (CLP) with or without neutralizing anti-TGFBIp antibody (300 µg kg(-1), intravenously). Wild-type or integrin ß5-null mice received TGFBIp (0.1 mg kg(-1), intravenously) or were subjected to CLP. Human umbilical vein endothelial cells were exposed to lipopolysaccharide (100 ng mL(-1)) with or without neutralizing anti-TGFBIp antibody (50 µg mL(-1)). RESULTS: Administration of neutralizing anti-TGFBIp antibody in mice attenuated CLP-induced secretion of TGFBIp, leucocyte migration and vascular permeability and reduced septic mortality. Injected TGFBIp did not enhance vascular barrier permeability or leucocyte migration in ß5-null mice. Finally, neutralizing anti-TGFBIp antibody inhibited the specific interactions between TGFBIp and its receptor, integrin αvß5. CONCLUSION: Our findings demonstrate that treatment with a TGFBIp-neutralizing antibody can ameliorate the deleterious effects of sepsis.

Osteoactivin promotes osteoblast adhesion through HSPG and αvß1 integrin.

Osteoactivin (OA), also known as glycoprotein nmb (gpnmb) plays an important role in the regulation of osteoblast differentiation and function. OA induced osteoblast differentiation and function in vitro by stimulating alkaline phosphatase (ALP) activity, osteocalcin production, nodule formation, and matrix mineralization. Recent studies reported a role for OA in cell adhesion and integrin binding. In this study, we demonstrate that recombinant osteoactivin (rOA) as a matricellular protein stimulated adhesion, spreading and differentiation of MC3T3-E1 osteoblast-like cells through binding to αv ß1 integrin and heparan sulfated proteoglycans (HSPGs). MC3T3-E1 cell adhesion to rOA was blocked by neutralizing anti-OA or anti-αv and ß1 integrin antibodies. rOA stimulated-osteoblast adhesion was also inhibited by soluble heparin and sodium chlorate. Interestingly, rOA stimulated-osteoblast adhesion promoted an increase in FAK and ERK activation, resulting in the formation of focal adhesions, cell spreading and enhanced actin cytoskeleton organization. In addition, differentiation of primary osteoblasts was augmented on rOA coated-wells marked by increased alkaline phosphatase staining and activity. Taken together, these data implicate OA as a matricellular protein that stimulates osteoblast adhesion through binding to αv ß1 integrin and cell surface HSPGs, resulting in increased cell spreading, actin reorganization, and osteoblast differentiation with emphasis on the positive role of OA in osteogenesis.

CCN4 induces IL-6 production through αvß5 receptor, PI3K, Akt, and NF-κB singling pathway in human synovial fibroblasts.

INTRODUCTION: Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells. METHODS: CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65. RESULTS: Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvß5 but not α5ß1 and αvß3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor (LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation. CONCLUSIONS: Our results suggest that CCN4 activates αvß5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.

αV ß5 and CD44 are oxygen-regulated human embryonic stem cell attachment factors.

Human embryonic stem cells (hESCs) have great potential for clinical therapeutic use. However, relatively little is known of the mechanisms which dictate their specificity of adhesion to substrates through adhesion proteins including integrins. Previous observations demonstrated enhanced clonogenicity in reduced oxygen culture systems. Here, we demonstrated via antibody blocking experiments that αV ß5 and α 6 significantly promoted hESC attachment in 2% O2 only, whereas blockage of CD44 inhibited cell attachment in 21% O2 alone. Immunofluorescence confirmed expression of αV ß5 and CD44 in both 2% O2 and 21% O2 cultured hESCs while flow cytometry revealed significantly higher αV ß5 expression in 2% O2 versus 21% O2 cultured hESCs and higher CD44 expression in 21% O2 versus 2% O2 cultured hESCs. Adhered hESCs following blockage of αV ß5 in 2% O2 displayed a reduction in nuclear colocalisation of Oct-4 and Nanog with little effect observed in 21% O2. Blockage of CD44 had the converse effect with dramatic reductions in nuclear colocalisation of Oct-4 and Nanog in 21% O2 cultured hESC which retained adherence, but not in 2% O2 cultured cells. Identification of oxygen-dependent substrate attachment mechanisms in hESCs has the potential to play a role in the development of novel substrates to improve hESC attachment and culture.

Expression, purification, and characterization of RGD-mda-7, a His-tagged mda-7/IL-24 mutant protein.

RGD peptide (Arg-Gly-Asp tripeptide) binds to integrin αVß(3) and αVß(5), which is selectively expressed in tumor neovasculature and on the surface of some tumor cells. Some studies showed that coupling the RGD peptides to anticancer drugs yielded compounds with increased efficiency against tumors and lowered toxicity to normal tissues. The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, we inserted a glycine residue into the wild type (mda-7/IL-24) between (164)Arg and (165)Asp to form a RGD peptide, named RGD-mda-7, then expressed RGD-mda-7 in Escherichia coli. Herein, we describe the expression and purification of RGD-mda-7. We detected the characterizations of immunostimulatory activity, tumor targeting, potent cytopathic effect, and apoptosis inducing exploited by RGD-mda-7 in tumor cells, and also compared these characterizations with wtmda-7/IL-24. The data showed that RGD-mda-7 had more potent tumor targeting and apoptosis-inducing effects than wtmda-7/IL-24.

Thrombin stimulates integrin ß1-dependent adhesion of human pancreatic cancer cells to vitronectin through protease-activated receptor (PAR)-1.

BACKGROUND/AIMS: The aim of this study was to investigate the effect of thrombin and the thrombin receptor protease-activated receptor (PAR)-1 on adhesion of human pancreatic cancer cell lines to extracellular matrices (ECMs) and to identify related integrins with these effects. METHODOLOGY: Human pancreatic cancer cell lines SUIT-2 and its four sublines, and Panc- 1, AsPC-1 and MiaPaCa-2 were treated with thrombin, PAR-1 agonist TRAP-6, PAR-1 antagonist SCH79797, or anti-integrin ±vß3, ±vß5 and ß1 monoclonal antibodies. Cells were incubated for 45 minutes on micro titer plates that were pre-coated with ECMs (fibronectin, laminin, vitronectin, type IV collagen). The number of adherent cells was measured by the MTT method. RESULTS: Eight human pancreatic cancer cell lines expressed PAR-1. Thrombin significantly enhanced adhesion of SUIT-2 and its sublines and MiaPaCa-2 to vitronectin, especially in the SUIT-2 subline S2-007. We obtained similar results on S2-007 cells through treatment with TRAP-6. However, SCH79797 inhibited the effect of thrombin. Furthermore, anti-integrin ß1 antibody conspicuously inhibited 1U/mL thrombin-induced enhancement of adhesion to vitronectin. CONCLUSIONS: Thrombin significantly enhanced adhesion of pancreatic cancer cells to vitronectin through PAR- 1 depending on the presence of integrin ß1. Suppression of thrombin action by anti-integrin ß1 antibody will become a useful therapy against pancreatic cancer.

Degradation of internalized αvß5 integrin is controlled by uPAR bound uPA: effect on ß1 integrin activity and α-SMA stress fiber assembly.

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvß5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFß-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin ß5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin ß5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin ß5 total and cell-surface levels were increased (2-4 fold). Integrin ß5 accumulation resulted from a significant decrease in ß5 ubiquitination leading to a decrease in the degradation rate of internalized ß5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin ß1 binding to the collagen matrix, with reduced activation of ß1. Elevated cell-surface integrin ß5 was necessary for these changes after uPA-silencing since blocking αvß5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvß5 cell-surface protein levels that regulate the activity of ß1 integrins, promoting characteristics of the persistent Mf.

Cyr61 increases matrix metalloproteinase-3 expression and cell motility in human oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) has a striking tendency to migrate and metastasize. Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effects of Cyr61 on human OSCC cells are largely unknown. In this study, we found that Cyr61 increased the migration and the expression of matrix metalloproteinases-3 (MMP)-3 in human OSCC cells. αvß5 or α6ß1 monoclonal antibody (mAb), focal adhesion kinase (FAK) inhibitor, and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) inhibited the Cyr61-induced increase of the migration and MMP-3 up-regulation of OSCC cells. Cyr61 stimulation increased the phosphorylation of FAK, MEK, and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors suppressed the cell migration and MMP-3 expression enhanced by Cyr61. Moreover, Cyr61 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-3 promoter. Taken together, our results indicate that Cyr61 enhances the migration of OSCC cells by increasing MMP-3 expression through the αvß3 or α6ß1 integrin receptor, FAK, MEK, ERK, and NF-κB signal transduction pathway.

Integrin αvß5-mediated TGF-ß activation by airway smooth muscle cells in asthma.

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-ß is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-ß is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-ß is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-ß activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-ß activation by HASM cells via the integrin αvß5. Our findings highlight the importance of the ß5 cytoplasmic domain because a polymorphism in the ß5 subunit rendered the integrin unable to activate TGF-ß. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-ß. These data demonstrate that murine airway smooth muscle cells express αvß5 integrins and activate TGF-ß. Finally, these data show that inhibition, or genetic loss, of αvß5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-ß activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-ß activation.

CTGF inhibits cell motility and COX-2 expression in oral cancer cells.

Oral squamous cell carcinoma (SCC) has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity and COX-2 expression in human oral cells is mostly unknown. Here we found that CTGF reduced the migration and expression of COX-2 in human oral cancer cells. αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin) and Akt inhibitor reversed the CTGF-inhibited the migration and COX-2 down-regulation of oral cancer cells. CTGF stimulation decreased the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt. In addition, c-Jun siRNA also antagonized the CTGF-inhibited migration and COX-2 expression. Moreover, CTGF decreased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Taken together, our results indicated that CTGF inhibits the migration of oral cancer cells by decreasing COX-2 expression through the αvß5 integrin receptor, FAK, PI3K, Akt, c-Jun and AP-1 signal transduction pathways.

CCN3 increases cell motility and MMP-13 expression in human chondrosarcoma through integrin-dependent pathway.

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. CCN3, also called nephroblastoma overexpressed gene (NOV), regulates proliferation and differentiation of cancer cells. However, the effect of CCN3 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). αvß3 or αvß5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase (PI3K) inhibitors (Ly294002 and wortmannin) and Akt inhibitor inhibited the CCN3-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. CCN3 stimulation increased the phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. In addition, NF-κB inhibitors also suppressed the cell migration and MMP-13 expression enhanced by CCN3. Moreover, CCN3 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-13 promoter. Taken together, our results indicate that CCN3 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the αvß3/αvß5 integrin receptor, FAK, PI3K, Akt, p65, and NF-κB signal transduction pathway.

Preclinical evaluation of monoclonal antibody 14C5 for targeting pancreatic cancer.

The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.

Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin.

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived, patient-specific pluripotent cells for use in cell-based regenerative therapies. However, current methods of cell culture are tedious and expensive, and the mechanisms underlying cell proliferation are not understood. In this study, we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion, survival, and proliferation. We show that iPSC lines generated using Oct-3/4, Sox-2, Nanog, and Lin-28 express a repertoire of integrins similar to that of hESCs, with prominent expression of subunits alpha5, alpha6, alphav, beta1, and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin, in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel, as shown by immunological blockade experiments. On vitronectin, the integrin alphavbeta5 was required for initial attachment, but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore, iPSCs cultured on vitronectin for 9 passages retained normal karyotype, pluripotency marker expression, and capacity to differentiate in vitro. These studies suggest that vitronectin, or derivatives thereof, might substitute for Matrigel in a more defined system for iPSC culture.

Integrin alphavbeta1 promotes infection by human metapneumovirus.

Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes lower respiratory infections in children and adults worldwide. The hMPV fusion (F) protein is a membrane-anchored glycoprotein and major protective antigen. All hMPV F protein sequences determined to date contain an Arg-Gly-Asp (RGD) sequence, suggesting that F engages RGD-binding integrins to mediate cell entry. The divalent cation chelator EDTA, which disrupts heterodimeric integrin interactions, inhibits infectivity of hMPV but not the closely related respiratory syncytial virus (RSV), which lacks an RGD motif. Function-blocking antibodies specific for alphavbeta1 integrin inhibit infectivity of hMPV but not RSV. Transfection of nonpermissive cells with alphav or beta1 cDNAs confers hMPV infectivity, whereas reduction of alphav and beta1 integrin expression by siRNA inhibits hMPV infection. Recombinant hMPV F protein binds to cells, whereas Arg-Gly-Glu (RGE)-mutant F protein does not. These data suggest that alphavbeta1 integrin is a functional receptor for hMPV.

Kaposi's sarcoma-associated herpesvirus forms a multimolecular complex of integrins (alphaVbeta5, alphaVbeta3, and alpha3beta1) and CD98-xCT during infection of human dermal microvascular endothelial cells, and CD98-xCT is essential for the postentry stage of infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and alpha3beta1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with alpha3beta1 and alphaVbeta3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-alphaV and -beta1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against alphaVbeta3 and alphaVbeta5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble alpha3beta1, alphaVbeta3, and alphaVbeta5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that alphaVbeta3 and alphaVbeta5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin alphaVbeta5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas alpha3beta1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and alphaVbeta3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of alphaVbeta5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with alpha3beta1 and KSHV. Preincubation of KSHV with soluble heparin and alpha3beta1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (alphaVbeta3, alpha3beta1, and alphaVbeta5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.

[Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules].

OBJECTIVE: To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro. METHODS: The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture. RESULTS: The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5. CONCLUSION: alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.