Home About us Contact
|
Home About us Contact | ||
|
|
||
Integrin Subunits (integrin + subunit)
Selected AbstractsThe microanatomy of the distal arrector pili: possible role for ,1,1 and ,5,1 integrins in mediating cell-cell adhesion and anchorage to the extracellular matrixJOURNAL OF CUTANEOUS PATHOLOGY, Issue 2 2000Jeri Kersten Mendelson The arrector pili (AP) muscle is a small band of smooth muscle that attaches proximally to the bulge area of the pilosebaceous apparatus in the reticular dermis and extends up toward the epidermis. The distal anatomy of the AP and the anchorage mechanism allowing hair erection have not been previously described. Integrins are likely candidates mediating this attachment. Immunohistochemical techniques were used to determine the distribution of the following integrins: ,1, ,2, ,3, ,4, ,5, ,6 and ,1 as well as fibronectin. Frozen human scalp tissue was sectioned in traditional planes, obliquely and horizontally to visualize microanatomy in three dimensions. Histological examination revealed that the distal portions of smooth muscle fibers splay extensively between collagen bundles of the upper dermis. Integrin subunits ,1, ,5 and ,1 were expressed by the AP muscle. Analysis of the relative density of immunoreactivity in digitized sections revealed increased ,5 subunit expression at the extracellular matrix (ECM)-muscle interface. These data suggest that anchorage of the AP muscle to the ECM is via ,5,1 integrin and ,1,1 integrin functions in muscle cell-cell adhesion. Extensive splaying of smooth muscle fibers may allow increased surface area contact between the ECM and smooth muscle cells expressing peripherally situated ,5 integrin. [source] ,6 integrin subunit mediates laminin enhancement of cisplatin-induced apoptosis in testicular tumor germ cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Maoulana Andjilani Abstract Our study demonstrates that laminin potentiates cisplatin-induced apoptosis in NCCIT, a testicular tumor germ cell line. When cultured on laminin, NCCIT cells displayed a significantly higher susceptibility to cisplatin-induced apoptosis than on plastic or on other ECM components including fibronectin, Type IV collagen and vitronectin. This high cisplatin sensitivity observed on NCCIT cell cultured on laminin was mediated by the ,6-integrin signaling. The knockdown of the ,6-integrin subunit by small interfering RNAs suppressed the higher cisplatin-sensitivity supporting the existence of a crosstalk between laminin-,6-integrin signaling and cisplatin-induced apoptosis. Our findings indicate that in cisplatin-treated NCCIT cells, the laminin-,6-integrin signaling induces the activation of executioner procaspase-3 and -6 as well as apoptosis-inducing factor (AIF) transcription and expression. The ability of integrin-mediated specific stroma,tumor cell interactions to modulate the chemosensitive phenotype of a tumor cell might provide new insights to overcome cisplatin resistance of tumor cells. © 2005 Wiley-Liss, Inc. [source] Integrin ,3,1 interacts with I1PP2A/lanp and phosphatase PP1JOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Diana Mutz Abstract Integrin ,3,1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin ,3,1, we looked for proteins that interact with the cytoplasmic part of the ,3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the ,3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a ,3A/cyto-binding protein. PP1 and integrin ,3,1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the , integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin ,3,1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the ,3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the ,3A integrin subunit and might possibly regulate the phosphorylation status of integrin ,3,1 and/or integrin downstream targets. © 2006 Wiley-Liss, Inc. [source] The cytoplasmic tail of the ,3 integrin subunit promotes neurite outgrowth in PC12 cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005Nadja Mechai Abstract Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein,protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the ,3 integrin subunit of ,3,1 integrin is required for ,3,1 integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) , subunit and the cytoplasmic sequence of the ,3 integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic ,3 tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic ,3 sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-,3 can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase ,. © 2005 Wiley-Liss, Inc. [source] Expression and Cytoskeletal Association of Integrin Subunits Is Selectively Increased in Rat Perivenous Hepatocytes After Chronic Ethanol AdministrationALCOHOLISM, Issue 12 2001Courtney S. Schaffert Background: For normal function and survival, hepatocytes require proper cell,extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the ,1 integrin subunit, the common , subunit for two major hepatocyte-ECM receptors, ,1,1 and ,5,1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of ,1, ,5, and ,1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. Methods: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. Results: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. Conclusions: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration. [source] Temporal and spatial regulation of ,6 integrin expression during the development of the cochlear-vestibular ganglionTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2007Dawn Davies Abstract The neurons of the cochlear-vestibular ganglion (CVG) that innervate the sensory hair cells of the inner ear are derived from the otic epithelium early in development. Neuroblasts detach from neighboring cells, migrate into the mesenchyme where they coalesce to form the ganglion complex, then send processes back into the epithelium. Cell migration and neuronal process formation involve changes in cellular interactions with other cells and proteins in the extracellular matrix that are orchestrated by cell surface-expressed adhesion molecules, including the integrins. I studied the expression pattern of the ,6 integrin subunit during the early development of the CVG using immunohistochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR) in murine tissue sections, otocyst, and ganglion explants. At embryonic day (E)10.5 ,6 integrin was expressed in the otic epithelium but not in migrating neuroblasts. Importantly, the loss of ,6 was associated with exit from the epithelium, not neuronal determination, revealing differentiation cues acutely associated with the cellular environment. Markers of glial and neuronal phenotype showed that ,6-expressing cells present in the CVG at this stage were glia of neural crest origin. By E12.5 ,6 expression in the ganglion increased alongside the elaboration of neuronal processes. Immunohistochemistry applied to otocyst cultures in the absence of glia revealed that neuronal processes remained ,6-negative at this developmental stage and confirmed that ,6 was expressed by closely apposed glia. The spatiotemporal modulation of ,6 expression suggests changing roles for this integrin during the early development of inner ear innervation. J. Comp. Neurol. 502:673,682, 2007. © 2007 Wiley-Liss, Inc. [source] Expression of Integrin ,v,3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor VasculatureBRAIN PATHOLOGY, Issue 3 2008Oliver Schnell MD Abstract In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of ,v,3 integrin caused by malignancy. The aim of our study was to assess the extent and pattern of ,v,3 integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of ,v,3 integrin and quantified by an imaging software. The expression of ,v,3 was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of ,v,3 integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the ,3 integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of ,v,3 integrin in gliomas and are of relevance for the inhibition of ,v,3 integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth. [source] Activation and deactivation of periventricular white matter phagocytes during postnatal mouse developmentGLIA, Issue 1 2010Mariya Hristova Abstract Brain microglia are related to peripheral macrophages but undergo a highly specific process of regional maturation and differentiation inside the brain. Here, we examined this deactivation and morphological differentiation in cerebral cortex and periventricular subcortical white matter, the main "fountain of microglia" site, during postnatal mouse development, 0,28 days after birth (P0,P28). Only macrophages in subcortical white matter but not cortical microglia exhibited strong expression of typical activation markers alpha5, alpha6, alphaM, alphaX, and beta2 integrin subunits and B7.2 at any postnatal time point studied. White matter phagocyte activation was maximal at P0, decreased linearly over P3 and P7 and disappeared at P10. P7 white matter phagocytes also expressed high levels of IGF1 and MCSF, but not TNFalpha mRNA; this expression disappeared at P14. This process of deactivation followed the presence of ingested phagocytic material but correlated only moderately with ramification, and not with the extent of TUNEL+ death in neighboring cells, their ingestion or microglial proliferation. Intravenous fluosphere labeling revealed postnatal recruitment and transformation of circulating leukocytes into meningeal and perivascular macrophages as well as into ramified cortical microglia, but bypassing the white matter areas. In conclusion, this study describes strong and selective activation of postnatally resident phagocytes in the P0,P7 subcortical white matter, roughly equivalent to mid 3rd trimester human fetal development. This presence of highly active and IGF1- and MCSF-expressing phagocytes in the neighborhood of vulnerable white matter could play an important role in the genesis of or protection against axonal damage in the fetus and premature neonate. © 2009 Wiley-Liss, Inc. [source] Expression of FGFR3 with the G380R Achondroplasia Mutation Inhibits Proliferation and Maturation of CFK2 Chondrocytic CellsJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Janet E. Henderson Abstract A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression. [source] ,1,3 fucosyltransferase-VII up-regulates the mRNA of ,5 integrin and its biological functionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Qiu-yan Wang Abstract After transfection of ,1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of ,5, but not ,1 integrin was significantly up-regulated. This was evidenced by the increase of ,5 integrin on cell surface as well as the increase of ,5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLex, the product of ,1,3FucT-VII) on both ,5 and ,1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin ,5,1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of ,1,3FucT-VII in different ,1,3FucT-VII transfected H7721 cell lines. In addition, after ,1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by ,5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both ,5 integrin and SLex. These findings suggest that the increased surface ,5 integrin caused by the up-regulation of ,5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of ,5,1 integrin. The up-regulation of surface SLex originated from the over expression of ,1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that ,1,3FucT-VII can regulate the mRNA expression of integrin. J. Cell. Biochem. 104: 2078,2090, 2008. © 2008 Wiley-Liss, Inc. [source] Expression of RNAs encoding for , and , integrin subunits in periodontitis and in cyclosporin A gingival overgrowthJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 11 2003A.-L. Bolcato-Bellemin Abstract Background: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling. Aim: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth. Methods: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls. Results: The RNA encoding for ,1, ,2 and ,5 integrin subunits were reduced in periodontitis gingiva. The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for ,1 subunit was increased. The RNA encoding for ,6 integrin was only reduced in cyclosporin A-treated gingiva. Immunohistochemistry showed that i) integrin ,2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of ,6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) ,1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva. Conclusion: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth. Zusammenfassung Die Variation der Integrin Expression bei gesunder und erkrankter Gingiva zeigt eine potentielle biologische Rolle für diese Zellmatrixrezeptoren während der gingivalen Erneuerung. Wir bestimmten hier die Level von RNA und die Gewebelokalisation von unterschiedlichen Integrin Untereinheiten bei Parodontitis und Cyclosporin A induzierter gingivaler Wucherung. Die Level der Expression wurden mit der reversen Transscriptase Polymerase Kettenreaktion bei 12 Parodontitis-Patienten, 4 Patienten mit schwerer Cyclosporin A induzierter gingivaler Wucherung und sieben gesunden Kontrollpatienten bestimmt. Die kodierende RNA für ,1, ,2 und ,5 Integrin Untereinheiten waren in der Gingiva mit Parodontitis reduziert. Die beobachtete Reduktion war stärker bei den mit Cyclosporin A behandelten Patienten verglichen mit den gesunden Kontrollen, während kodierende RNA für ,1 Untereinheiten erhöht war. Die kodierende RNA für ,6 Integrin war nur bei der Cyclosporin A behandelten Gingiva reduziert. Die Immunhistochemie zeigte (i) die Integrin ,2 Expression ist auf das gingivale Epithel von Cyclosporin A behandelten Patienten beschränkt, (ii) die Reduktion von ,6 Integrin Expression bei Cyclosporin A behandelter Gingiva ist die Folge von fokalen Expressionverlusten und (iii) ,1 Integrin ist gleichmäßig verteilt in den drei Populationen mit einer Intensitätsabnahme bei Parodontitis und Cyclosporin A behandelter Gingiva. Zusammenfassend zeigen die Ergebnisse eine Rolle für die Integrin Rezeptoren bei den parodontalen Erkrankungen und Cyclosporin A induzierter gingivalen Wucherung. Résumé La variation d'expression des intégrines dans les tissus gingivaux sain et pathologique a démontré le rôle biologique potentiel de ces récepteurs de la matrice extracellulaire au cours du remodelage tissulaire gingival. La quantité d'ARN et la localisation tissulaire de certaines sous-unités d'intégrines dans la parodontite et l'hyperplasie gingivale induite par la ciclosporine A ont été déterminées. Le niveau d'expression a étéévalué par transcription inverse des ARN et réaction de polymérisation en chaine chez douze patients atteints de parodontite, quatre patients présentant une hyperplasie gingivale sévère induite par la ciclosporine A et sept patients sains ayant servi de témoins. L'expression des ARN codant pour les sous-unités ,1, ,2 et ,5était diminuée dans le tissu gingival atteint de parodontite. La diminution observée était plus importante chez les patients traités par la ciclosporine A, comparée aux témoins sains alors que l'expression de l'ARN codant pour la sous-unité,1était augmentée. L'expression de l'ARN codant pour la sous-unité,6était diminuée uniquement dans le tissu gingival traité par la ciclosporine A. L'immohistochimie a montré que (1) l'expression de la sous-unité,2 est limitée à l'épithélium gingival des patients traités par la ciclosporine A, (2) la diminution de l'expression de la sous-unité,6 dans le tissu gingival traité par la ciclosporine A est due à une perte des contacts focaux et (3) la sous-unité,1 est répartie de manière uniforme dans les trois groupes avec une diminution de l'intensité dans les cas de parodontite et d'hyperplasie gingivale induite par la ciclosporine A. Ces résultats montrent un rôle des récepteurs de type intégrine dans la pathologie parodontale et l'hyperplasie gingivale induite par la ciclosporine A. [source] Functional integrin subunits regulating cell,matrix interactions in the intervertebral discJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2007Christopher L. Gilchrist Abstract Cellular interactions with the extracellular matrix are key factors regulating cell survival, differentiation, and response to environmental stimuli in cartilagenous tissues. Much is known about the extracellular matrix proteins in the intervertebral disc (IVD) and their variations with region, age, or degenerative state of the tissue. In contrast, little is known of the integrin cell surface receptors that directly bind to and interact with these matrix proteins in the IVD. In almost all tissues, these integrin-mediated cell,matrix interactions are important for transducing environmental cues arising from mechanical stimuli, matrix degradation fragments, and cytokines into intracellular signals. In this study, cells from the nucleus pulposus and anulus fibrosus regions of porcine IVDs were analyzed via flow cytometry to quantify integrin expression levels upon isolation and after monolayer culture. Assays of cell attachment to collagens, fibronectin, and laminin were performed after functional blocking of select integrin subunits to evaluate the role of specific integrins in cell attachment. In situ distribution and co-localization of integrins and laminin were also characterized. Results identify integrin receptors critical for IVD cell interactions with collagens (,1,1) and fibronectin (,5,1). Additionally, dramatic differences in cell,laminin interactions were observed between cells of the nucleus and anulus regions, including differences in ,6 integrin expression, cell adhesion to laminin, and in situ pericellular environments. These findings suggest laminin,cell interactions may be important and unique to the nucleus pulposus region of the IVD. The results of this study provide new information on functional cell,matrix interactions in tissues of the IVD. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25: 829,840, 2007 [source] Expression and Cytoskeletal Association of Integrin Subunits Is Selectively Increased in Rat Perivenous Hepatocytes After Chronic Ethanol AdministrationALCOHOLISM, Issue 12 2001Courtney S. Schaffert Background: For normal function and survival, hepatocytes require proper cell,extracellular matrix (ECM) contacts mediated by integrin receptors and focal adhesions. Previous studies have shown that chronic ethanol consumption selectively impairs perivenous (PV) hepatocyte attachment and spreading on various ECM substrates but increases expression of the ,1 integrin subunit, the common , subunit for two major hepatocyte-ECM receptors, ,1,1 and ,5,1 integrins. This study examined the effects of ethanol treatment on the expression and cytoskeletal distribution of ,1, ,5, and ,1 integrin subunits, the epidermal growth factor receptor (EGF-R), and the cytoskeletal proteins focal adhesion kinase, paxillin, vinculin, and actin in periportal and PV hepatocytes. Methods: Periportal and PV hepatocytes were isolated from control and ethanol-fed rats. For expression analysis, lysates were examined by SDS-PAGE and immunoblotting procedures. For cytoskeletal distribution studies, Triton-soluble and -insoluble (cytoskeletal) fractions from hepatocytes cultured on collagen IV were analyzed by SDS-PAGE and immunoblotting. Results: Chronic ethanol administration caused PV-specific increases in expression and cytoskeletal association of the integrin subunits. Although ethanol treatment did not affect expression of the EGF-R in either cell type, it did increase the association of the EGF-R with the cytoskeleton selectively in PV hepatocytes. Ethanol treatment had no significant effect on either the expression or the cytoskeletal distribution of focal adhesion kinase, paxillin, vinculin, or actin in either cell type. Conclusions: The increases in integrin expression and cytoskeletal association observed after chronic ethanol administration suggest that a process downstream of integrin-ECM interactions is impaired selectively in PV hepatocytes, possibly involving altered focal adhesion assembly or turnover, processes essential for efficient cell-ECM adhesion. Alterations in these processes could contribute to the impaired hepatocyte function and structure observed after chronic ethanol administration. [source] Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin functionJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2006M. GAUR Summary.,Background:,The platelet fibrinogen receptor, a heterodimer consisting of integrin subunits ,IIb and ,3, is required for platelet aggregation, spreading, and hemostasis. Platelet agonists such as thrombin and adenosine diphosphate (ADP) lead to the activation of ,IIb,3, thereby enhancing its affinity and avidity for binding fibrinogen (inside-out signaling). Furthermore, fibrinogen binding to ,IIb,3 triggers cytoskeletal changes and granule release (outside-in signaling).Aim:,Genetic approaches to characterize the molecular pathways involved in ,IIb,3 signaling are not possible with anucleate blood platelets. Therefore, we have established an OP9 stromal cell co-culture system to generate megakaryocytes from human embryonic stem cells (hESCs).Results:,,IIb,3 activation, measured by soluble fibrinogen binding to hESC-derived megakaryocytes, /GPIb,+ cells, is readily detectable following stimulation with known platelet agonists. Dose,response curves for peptide agonists specific for the two platelet thrombin receptors, protease-activated receptor 1 (PAR1) and PAR4, show a relative responsiveness that mirrors that of human platelets, and sub-maximal ADP responses are augmented by epinephrine. Moreover, hESC-derived megakaryocytes undergo lamellipodia formation, actin filament assembly, and vinculin localization at focal adhesions when plated on a fibrinogen-coated surface, characteristic of ,IIb,3 outside-in signaling. Undifferentiated hESCs genetically modified by lentiviral infection can be cloned and maintained in an undifferentiated state and then differentiated into megakaryocytes capable of ,IIb,3 activation.Conclusion:,Using hESCs, we have developed a renewable source of human megakaryocytes, and a genetically tractable system for studying megakaryocytopoiesis and ,IIb,3 signaling in the native cellular environment. [source] The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin ,2,1JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 8 2004J. M. Stevens Summary.,Background: Activation of the platelet integrin ,2,1 is closely regulated due to the high thrombogenicity of its ligand. As a ,1 interacting kinase, ILK represents a candidate intracellular regulator of ,2,1 in human platelets. Objectives We investigated the regulation of ILK in human platelets and the role of ILK in regulating ,2,1 activation in HEL cells, a megakaryocytic cell line. Methods: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with ,1 -integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating ,2,1 function assessed by overexpression studies in HEL cells. Results: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the ,1 -integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced ,2,1 -mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of ,2,1. Conclusions: Our findings that ILK regulates ,2,1 in HEL cells, is activated in platelets and associates with ,1 -integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets. [source] Effect of phenytoin on collagen accumulation by human gingival fibroblasts exposed to TNF- ,in vitroORAL DISEASES, Issue 2 2006T Kato Objective:, Tumor necrosis factor (TNF)- , is associated with chronic gingival inflammation and reported to induce gingival overgrowth (GO), while phenytoin (PHT) is also known to be a causative agent of GO. We examined the synergistic effect of PHT and TNF- , on collagen metabolism in human gingival fibroblasts (HGFs). Materials and methods:, HGFs were cultured with TNF- , and PHT. Quantitative real-time RT-PCR was employed to determine the mRNA levels for collagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and integrin subunits. Cellular collagen endocytosis was determined using a flow-cytometry. Results:, The proliferation of HGFs was not affected by TNF- , or PHT individually, whereas both synergistically increased collagen accumulation in HGFs. Further, collagen mRNA expression was not increased by TNF- , or PHT, although together they markedly prevented cellular collagen endocytosis, associated with the suppression of ,2,1-integrin mRNA expression. The mRNA expression of MMP-1 and-2 was suppressed by PHT, while TIMP-1 mRNA expression was enhanced by both TNF- , and PHT. Conclusion:, Our results suggest that TNF- , and PHT together cause impaired collagen metabolism by suppression of enzymatic degradation with MMPs/TIMP-1 and integrin-mediated endocytosis. These synergistic effects may also be involved in TNF- , - and PHT-induced collagen accumulation, leading to GO. [source] Glucocorticoids increase ,5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasionARTHRITIS & RHEUMATISM, Issue 12 2009Torsten Lowin Objective In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. Methods We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. Results In RA, expression of integrin subunits ,5, ,v, and ,1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17,-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the ,5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to ,5 integrin increased SF migration, indicating that up-regulated ,5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. Conclusion Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of ,5 integrin. [source] Expression of Integrin ,v,3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor VasculatureBRAIN PATHOLOGY, Issue 3 2008Oliver Schnell MD Abstract In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of ,v,3 integrin caused by malignancy. The aim of our study was to assess the extent and pattern of ,v,3 integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of ,v,3 integrin and quantified by an imaging software. The expression of ,v,3 was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of ,v,3 integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the ,3 integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of ,v,3 integrin in gliomas and are of relevance for the inhibition of ,v,3 integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth. [source] | |||||||||||||