Yasuyuki Yokosaki

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin.

The integrin α9β1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH2-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α9β1. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α9β1 recognition sequence in osteopontin. α9-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH2-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α9 monoclonal antibody Y9A2, indicating the presence of a non-RGD α9β1recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α9β1-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr165, blocked α9β1-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β1 subunit on α9-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α9β1 and is responsible for α9β1-mediated cell adhesion to the NH2-terminal fragment of osteopontin.

Differential Effects of the Integrins α9β1, αvβ3, and αvβ6 on Cell Proliferative Responses to Tenascin ROLES OF THE β SUBUNIT EXTRACELLULAR AND CYTOPLASMIC DOMAINS

Members of the integrin family manifest considerable overlap in ligand specificity, and many cells have the capacity to express multiple integrin receptors for the same ligand. For example, at least 5 different integrins recognize tenascin as a ligand, and 4 of these bind to the same region of the protein, the third fibronectin type III repeat (TNfn3). We utilized colon carcinoma cells (SW480) that do not normally attach to TNfn3 to examine the possibility that ligation of different integrin receptors for this ligand would induce different effects on cell behavior and intracellular signaling. Heterologous expression of the tenascin receptors αvβ3 and α9β1 produced comparable effects on cell adhesion and spreading on TNfn3, but αvβ3-transfectants proliferated considerably better on each concentration examined. αvβ6-transfectants attached (although less avidly), but completely failed to spread or proliferate. Expression of a chimeric β subunit composed of the β3 extracellular domain fused to the β6 transmembrane and cytoplasmic domains resulted in adhesion and spreading similar to that seen with β3-transfectants, but considerably less proliferation. When the same cell lines were plated on fibronectin, αvβ6-transfectants spread and proliferated as well as cells transfected with the chimeric β3/β6 subunit, but, again, neither cell line proliferated as well as cells expressing αvβ3. Cell proliferation was always associated with spreading and with phosphorylation of the focal adhesion kinase, paxillin, and the mitogen-activated kinase, Erk2, but cell attachment in the absence of spreading or proliferation was not associated with phosphorylation of any of these proteins. These data suggest that different integrin receptors for a single ligand can produce markedly different effects on cell proliferation, and that both the extracellular and cytoplasmic domains of integrin β subunits contribute to these differences.

Significance of α9β1 and αvβ6 integrin expression in breast carcinoma

BackgroundBoth α9β1 and αvβ6 integrins have been newly identified from the tracheal epithelium of guinea pig. It has been pointed out that α9β1 functions as a receptor for tenascin-C and osteopontin. As for the ligands of αvβ6, fibronectin and tenascin-C have been identified. It has not been ascertained whether α9β1 and αvβ6 are expressed in normal breast tissue, benign breast lesion or breast carcinoma.MethodsImmunohistochemical staining for α9β1 and αvβ6 was performed in benign breast lesion and breast carcinoma specimens. Western blotting was carried out on 11 breast carcinoma cases.Resultsα9β1 was expressed in the cytoplasm of carcinoma cells in 23 of 90 cases (26%) and αvβ6 in the membrane of carcinoma cells in 16 of 90 cases (18%). However, these findings of α9β1 and αvβ6 did not correlate with any clinicopathological factors including the patients’ age, tumor size, histological type of carcinoma, location of carcinoma cells and hormone receptor status. With regard to the histological grade of carcinoma, αvβ6 and α9β1 expression did not statistically correlate, although no expression of αvβ6 was observed in 14 cases of Grade I. On Western-blott analysis strong and weak bands consistent with αvβ6 were noted in the membrane fraction extracted from breast carcinoma cells. On the other hand weak bands consistent with α9 subunit were noted in the whole cell lysates of breast carcinoma cells and very weak or no bands consistent with α9 subunit were noted in the membrane fraction extracted from the breast carcinoma cells.ConclusionsSignificance of α9β1 and αvβ6 integrins expression in breast carcinoma was still unknown on clinicopathological examination. The findings of Western blot analysis may indicate that the transportation system of glycoproteins such as integrins to the cell membrane of carcinoma cells is disturbed, although these integrins can be produced.

The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma

The status of Fas and Fas ligand (Fas L) expression was investigated in this study for 103 hepatocellular carcinomas (HCC). We studied the expression of the following three factors, Fas and Fas L expression in carcinoma cells and Fas L expression in stromal mononuclear cells (defined as stromal Fas L index). Fas expression in HCC cells was significantly decreased in cases with poor differentiation (P< 0.0001) and of larger size (P = 0.0058). Fas L expression in carcinoma cells was observed exclusively in moderately or poorly differentiated cases. Furthermore, each factor had prognostic significance for disease-free survival (DFS) (P< 0.0001, P = 0.0222 and 0.0027 respectively). We then scored the results of each factor and defined the total score as ‘Fas-Fas L risk score’. The P -value of the score for DFS was even lower than that of the clinical stage by multivariate analysis. These results suggest that the evaluation of Fas and Fas ligand expression potentially has a significant prognostic value for DFS of HCC patients, in addition to the clinical stage, and can be regarded as a new prognostic marker.

Tissue Transglutaminase, Coagulation Factor XIII, and the Pro-polypeptide of von Willebrand Factor Are All Ligands for the Integrins α9β1 and α4β1

We previously reported that MOLT-3 human lymphocyte-like leukemia cells adhere to tissue-type transglutaminase (tTG) through the integrin α4β1. We now report that G-361 human melanoma cells also adhere to tTG, although they do not express α4β1. G-361 cells utilize two additional integrins, α9β1 and α5β1 to adhere to tTG. Furthermore, blood coagulation factor XIII (FXIII), another member of the transglutaminase family that is highly homologous to tTG, and propolypeptide of von Willebrand factor (pp-vWF) also promoted cell adhesion through α9β1 or α4β1 in G-361 or MOLT-3 cells, respectively. In the case of pp-vWF, α9β1 and α4β1both bind to the same site, comprised of 15 amino acid residues and designated T2–15. Moreover, SW480 human colon cancer cells stably transfected to express α9β1, but not mock transfectants, adhered to tTG, FXIII, pp-vWF, and T2–15/bovine serum albumin conjugate. These data identify tTG, FXIII, and pp-vWF as shared ligands for the integrins α9β1 and α4β1. This report is the first to unambiguously show that these two integrins share the same cell adhesion site within one protein and provides strong support for classifying α9β1- and α4-integrins as functionally related members of an integrin subfamily.

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPNs functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence‐specific reagents, along with the previously described anti‐OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD‐dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit α9 integrin‐dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell‐binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration. J. Cell. Biochem. 84: 420–432, 2002.

Inhibitory Effects of MLDG-containing Heterodimeric Disintegrins Reveal Distinct Structural Requirements for Interaction of the Integrin α9β1 with VCAM-1, Tenascin-C, and Osteopontin

The integrin α9β1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The α9 subunit is structurally similar to the integrin α4 subunit, and α9β1 and α4β1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of α4 integrins to ligands, would also be a functional inhibitor of α9β1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of α9β1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited α9β1- and α4β1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of α9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other α9β1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of α9β1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the α9β1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.

Enhanced biological activity of polymeric osteopontin

Osteopontin is a multifunctional glycoprotein with roles in immunomodulation, inflammatory response, tissue mineralization, and tissue remodeling, which are mediated primarily through integrins. Transglutaminase 2 selectively cross‐links proteins by isopeptide bonding. Osteopontin is one of the substrates of this enzyme and undergoes polymerization; however, the biological meaning of this polymerization remains unknown. Using recombinant osteopontin polymerized with purified transglutaminase 2, we examined cell adhesion, spreading, focal contact formation, and migration of SW480 or HUVE cells. All of these cellular behaviors were dramatically enhanced with polymeric osteopontin. These enhancements of cellular functions imply that polymerization might modulate physiological and pathological functions of osteopontin.

Polymeric Osteopontin Employs Integrin α9β1 as a Receptor and Attracts Neutrophils by Presenting a de Novo Binding Site

Osteopontin (OPN) is a cytokine and ligand for multiple members of the integrin family. OPN undergoes the in vivo polymerization catalyzed by cross-linking enzyme transglutaminase 2, which consequently increases the bioactivity through enhanced interaction with integrins. The integrin α9β1, highly expressed on neutrophils, binds to the sequence SVVYGLR only after intact OPN is cleaved by thrombin. The SVVYGLR sequence appears to be cryptic in intact OPN because α9β1 does not recognize intact OPN. Because transglutaminase 2-catalyzed polymers change their physical and chemical properties, we hypothesized that the SVVYGLR site might also be exposed on polymeric OPN. As expected, α9β1 turned into a receptor for polymeric OPN, a result obtained by cell adhesion and migration assays with α9-transfected cells and by detection of direct binding of recombinant soluble α9β1 with colorimetry and surface plasmon resonance analysis. Because the N-terminal fragment of thrombin-cleaved OPN, a ligand for α9β1, has been reported to attract neutrophils, we next examined migration of neutrophils to polymeric OPN using time-lapse microscopy. Polymeric OPN showed potent neutrophil chemotactic activity, which was clearly inhibited by anti-α9β1 antibody. Unexpectedly, mutagenesis studies showed that α9β1 bound to polymeric OPN independently of the SVVYGLR sequence, and further, SVVYGLR sequence of polymeric OPN was cryptic because SVVYGLR-specific antibody did not recognize polymeric OPN. These results demonstrate that polymerization of OPN generates a novel α9β1-binding site and that the interaction of this site with the α9β1 integrin is critical to the neutrophil chemotaxis induced by polymeric OPN.

Osteopontin Undergoes Polymerization in Vivo and Gains Chemotactic Activity for Neutrophils Mediated by Integrin α9β1

Osteopontin (OPN) is an integrin-binding inflammatory cytokine that undergoes polymerization catalyzed by transglutaminase 2. We have previously reported that polymeric OPN (polyOPN), but not unpolymerized OPN (OPN*), attracts neutrophils in vitro by presenting an acquired binding site for integrin α9β1. Among many in vitro substrates for transglutaminase 2, only a few have evidence for in vivo polymerization and concomitant function. Although polyOPN has been identified in bone and aorta, the in vivo functional significance of polyOPN is unknown. To determine whether OPN polymerization contributes to neutrophil recruitment in vivo, we injected OPN* into the peritoneal space of mice. Polymeric OPN was detected by immunoblotting in the peritoneal wash of mice injected with OPN*, and both intraperitoneal and plasma OPN* levels were higher in mice injected with a polymerization-incompetent mutant, confirming that OPN* polymerizes in vivo. OPN* injection induced neutrophil accumulation, which was significantly less following injection of a mutant OPN that was incapable of polymerization. The importance of in vivo polymerization was further confirmed with cystamine, a transglutaminase inhibitor, which blocked the polymerization and attenuated OPN*-mediated neutrophil recruitment. The thrombin-cleaved N-terminal fragment of OPN, another ligand for α9β1, was not responsible for neutrophil accumulation because a thrombin cleavage-incompetent mutant recruited similar numbers of neutrophils as wild type OPN*. Neutrophil accumulation in response to both wild type and thrombin cleavage-incompetent OPN* was reduced in mice lacking the integrin α9 subunit in leukocytes, indicating that α9β1 is required for polymerization-induced recruitment. We have illustrated a physiological role of molecular polymerization by demonstrating acquired chemotactic properties for OPN.