Identification of osteopontin as a novel ligand for the integrin alpha8 beta1 and potential roles for this integrin-ligand interaction in kidney morphogenesis.
ABSTRACT: Epithelio-mesenchymal interactions during kidney organogenesis are disrupted in integrin alpha8 beta1-deficient mice. However, the known ligands for integrin alpha8 beta1-fibronectin, vitronectin, and tenascin-C-are not appropriately localized to mediate all alpha8 beta1 functions in the kidney. Using a method of general utility for determining the distribution of unknown integrin ligands in situ and biochemical characterization of these ligands, we identified osteopontin (OPN) as a ligand for alpha8 beta1. We have coexpressed the extracellular domains of the mouse alpha8 and beta1 integrin subunits as a soluble heterodimer with one subunit fused to alkaline phosphatase (AP) and have used the alpha8 beta1-AP chimera as a histochemical reagent on sections of mouse embryos. Ligand localization with alpha8 beta1-AP in developing bone and kidney was observed to be overlapping with the distribution of OPN. In "far Western" blots of mouse embryonic protein extracts, bands were detected with sizes corresponding to fibronectin, vitronectin, and unknown proteins, one of which was identical to the size of OPN. In a solid-phase binding assay we demonstrated that purified OPN binds specifically to alpha8 beta1-AP. Cell adhesion assays using K562 cells expressing alpha8 beta1 were used to confirm this result. Together with a recent report that anti-OPN antibodies disrupt kidney morphogenesis, our results suggest that interactions between OPN and integrin alpha8 beta1 may help regulate kidney development and other morphogenetic processes.
Project description:The integrin alpha 8 beta 1 has been reported to bind to fibronectin, vitronectin, and tenascin-C in cell adhesion or neurite outgrowth assays. Here, we describe cDNA cloning of the murine alpha 8 subunit, purification of a recombinant soluble heterodimer consisting of the extracellular domains of the murine alpha 8 and beta1 subunits, and development of a sensitive binding assay using a modified form of this heterodimer fused to alkaline phosphatase (AP). In binding assays, the purified alpha 8 beta 1-AP chimera exhibited the same divalent ion requirements for activation and binding specificity as cell surface alpha 8 beta 1: in the presence of Mn2+ it bound to fibronectin and vitronectin in an RGDS-peptide inhibitable manner. Contrary to previous reports, we found no evidence that alpha 8 beta 1, expressed on K562 cells or as an AP chimera, interacts strongly with native tenascin-C. In binding, adhesion, and spreading assays, significant interactions were observed only to short fragments of tenascin-C containing the third fibronectin type III repeat which contains an RGD sequence. Full length tenascin-C and longer fragments containing this repeat did not appear to serve as ligands, implying that the RGD site in native tenascin-C is a cryptic binding site for this integrin, exposed by removal of adjacent domains. Soluble integrin-AP chimeras should be generally useful for identifying and characterizing integrin interactions with ligands.
Project description:N-glycosylation of the I-like domain of beta1 integrin plays an essential role in integrin structure and function, and the altered sialylation of beta1 integrin regulates beta1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the beta1 I-like domain on beta1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated beta1 I-like domain-FN-III(9-10) complex caused by altered sialylation of the beta1 I-like domain. Binding free energy analyses showed that desialylation of beta1 I-like domain increased beta1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the beta1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the beta1 I-like domain caused significant conformational changes in key functional sites of both the beta1 I-like domain and fibronectin. In addition, altered sialylation of the beta1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect beta1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of beta1 integrin in regulating beta1 integrin binding to fibronectin and it's induced cellular activities.
Project description:The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a beta1 integrin, rather than alphav beta3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to alpha2 and beta1 integrin subunits and to IAP. A combination of antialpha2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antialphav beta3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between alpha2 beta1 and IAP was detected by the coimmunoprecipitation of both alpha2 and beta1 integrin subunits with IAP. These data suggest that IAP can associate with alpha2 beta1 integrin and modulate its function.
Project description:beta1 integrins are major cell surface receptors for fibronectin. Some integrins, including beta1 integrins, are known to undergo constitutive endocytosis and recycling. Integrin endocytosis/recycling has been implicated in the regulation of cell migration. However, the mechanisms by which integrin endocytosis/recycling regulates cell migration, and other biological consequences of integrin trafficking are not completely understood. We previously showed that turnover of extracellular matrix (ECM) fibronectin occurs via receptor-mediated endocytosis. Here, we investigate the biological relevance of beta1 integrin endocytosis to fibronectin matrix turnover. First, we demonstrate that beta1 integrins, including alpha5beta1 play an important role in endocytosis and turnover of matrix fibronectin. Second, we show that caveolin-1 constitutively regulates endocytosis of alpha5beta1 integrins, and that alpha5beta1 integrin endocytosis can occur in the absence of fibronectin and fibronectin matrix. We also show that downregulation of caveolin-1 expression by siRNA results in marked reduction of beta1 integrin and fibronectin endocytosis. Hence, caveolin-1-dependent beta1 integrin and fibronectin endocytosis plays a critical role in fibronectin matrix turnover, and may contribute to abnormal ECM remodeling that occurs in fibrotic disorders.
Project description:Prenatal inflammation prevents normal lung morphogenesis and leads to bronchopulmonary dysplasia (BPD), a common complication of preterm birth. We previously demonstrated in a bacterial endotoxin mouse model of BPD that disrupting fibronectin localization in the fetal lung mesenchyme causes arrested saccular airway branching. In this study we show that expression of the fibronectin receptor, integrin alpha8beta1 is decreased in the lung mesenchyme in the same inflammation model suggesting it is required for normal lung development. We verified a role for integrin alpha8beta1 in lung development using integrin alpha8-null mice, which develop fusion of the medial and caudal lobes as well as abnormalities in airway division. We further show in vivo and in vitro that alpha8-null fetal lung mesenchymal cells fail to form stable adhesions and have increased migration. Thus we propose that integrin alpha8beta1 plays a critical role in lung morphogenesis by regulating mesenchymal cell adhesion and migration. Furthermore, our data suggest that disruption of the interactions between extracellular matrix and integrin alpha8beta1 may contribute to the pathogenesis of BPD.
Project description:Mutation of the functionally redundant Hoxa 11/Hoxd 11 genes gives absent or rudimentary kidneys resulting from a dramatic reduction of the growth and branching of the ureteric bud. To understand better the molecular mechanisms of Hoxa 11/Hoxd 11 function in kidney development, it is necessary to identify the downstream target genes regulated by their encoded transcription factors. To this end, we conducted a screen for Hoxa 11-responsive genes in two kidney cell lines. HEK293 cells, which usually do not express Hoxa 11, were modified to allow inducible Hoxa 11 expression. The mK10 cells, derived specifically for this study from Hoxa 11/Hoxd 11 double-mutant mice, were also modified to give cell populations with and without Hoxa 11 expression. Differential display, Gene Discovery Arrays, and Affymetrix genechip probe arrays were used to screen for genes up- or down-regulated by Hoxa 11. Nine genes, PDGF A, Cathepsin L, annexin A1, Mm.112139, Est2 repressor factor, NrCAM, ZNF192, integrin-associated protein, and GCM1, showed reproducible 3-fold or smaller changes in gene expression in response to Hoxa 11. One gene, the Integrin alpha8, was up-regulated approximately 20-fold after Hoxa 11 expression. The Integrin alpha8 gene is expressed together with Hoxa 11 in metanephric mesenchyme cells, and mutation of Integrin alpha8 gives a bud-branching morphogenesis defect very similar to that observed in Hoxa 11/Hoxd 11 mutant mice. In situ hybridizations showed a dramatic regional reduction in Integrin alpha8 expression in the developing kidneys of Hoxa 11/Hoxd 11 mutant mice. This work suggests that the Integrin alpha8 gene may be a major effector of Hoxa 11/Hoxd 11 function in the developing kidney.
Project description:Interactions between specific cell-surface molecules, which include the urokinase receptor (uPAR) and integrins, are crucial to processes of tumor invasion and metastasis. Here we demonstrate that uPAR and beta1-integrins may cluster at distinct sites at the cell surface of metastatic MDA-MB-231 breast cancer cells and form functional complexes. Attachment assays performed in the presence of a synthetic peptide (p25), which interferes with the formation of uPAR-integrin complexes, reveal that uPAR is able to regulate the adhesive function of integrins in breast cancer cells. On dissociation of the uPAR-integrin complexes by p25, tumor cell attachment to the extracellular matrix was either decreased (vitronectin) or increased (fibronectin). Moreover, the tumor cells display remarkable morphological changes when cultured on fibronectin in the continuous presence of p25, leading to increased cell spreading and attachment. In marked contrast to control conditions, increased cellular adhesion to fibronectin after p25 treatment was entirely beta1-integrin-mediated. The role of uPAR-integrin complexes in tumor progression was studied in an in vivo bone xenograft model. Stably transfected MDA-MB-231 cells that overexpress p25 showed a significant reduction in tumor progression in bone (P < or = 0.0001 versus mock-control). In line with these observations, continuous administration of p25 (25 microg/mouse/day, osmotic minipumps) for 28 days resulted in significantly reduced tumor progression of MDA-MB-231 cells in bone (P < or = 0.005) when compared to scrambled control peptide. In conclusion, our data demonstrate that uPAR can act as an adhesion receptor in breast cancer and is capable of regulating integrin function. Our findings strongly suggest that adhesive and proteolytic events are tightly associated in metastatic breast cancer cells and that functional integrin-uPAR complexes are involved in tumor progression in vivo.
Project description:Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR. Two of these antibodies compete against urokinase plasminogen activator (uPA) for uPAR binding, whereas a third competes with beta1 integrins for uPAR binding. These competitive antibodies inhibit uPAR-dependent cell signaling and invasion in the non-small cell lung cancer cell line, H1299. Additionally, the integrin-blocking antibody abrogates uPAR/beta1 integrin-mediated H1299 cell adhesion to fibronectin and vitronectin. This antibody and one of the uPAR/uPA antagonist antibodies shows a significant combined effect in inhibiting cell invasion through Matrigel/Collagen I or Collagen I matrices. Our results indicate that these antagonistic antibodies have potential for the detection and treatment of uPAR-expressing tumors.
Project description:To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM.Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2.HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it.Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.
Project description:The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of ??-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of ??-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.