Critical Role of Flanking Residues in NGR-to-isoDGR Transition and CD13/Integrin Receptor Switching

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of αvβ3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the α-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind αvβ3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind αvβ3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (αvβ5, αvβ6, αvβ8, and α5β1), although with 10–100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas α-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Expression of Integrin ��v��6 and TGF-�� in Scarless vs Scar-forming Wound Healing

Oral mucosal wounds heal with reduced scar formation compared with skin. The epithelial integrin αvβ6 is induced during wound healing, and it can activate fibrogenic transforming growth factor β1 (TGF-β1) and anti-fibrogenic TGF-β3 that play key roles in scar formation. In this study, expression of β6 integrin and members of the TGF-β pathway were studied in experimental wounds of human gingiva and both gingiva and skin of red Duroc pigs using real-time PCR, gene microarrays, and immunostaining. Similar to human wounds, the expression of β6 integrin was induced in the pig wounds 7 days after wounding and remained upregulated >49 days. The αvβ6 integrin was colocalized with both TGF-β isoforms in the wound epithelium. Significantly higher expression levels of β6 integrin and TGF-β1 were observed in the pig gingival wounds compared with skin. Early gingival wounds also expressed higher levels of TGF-β3 compared with skin. The spatio-temporal colocalization of αvβ6 integrin with TGF-β1 and TGF-β3 in the wound epithelium suggests that αvβ6 integrin may activate both isoforms during wound healing. Prolonged expression of αvβ6 integrin along with TGF-β3 in the gingival wound epithelium may be important in protection of gingiva from scar formation. (J Histochem Cytochem 57:543–557, 2009)     

Matrix metalloproteinase-2 Promotes αvβ3 Integrin-Mediated Adhesion and Migration of Human Melanoma Cells by Cleaving Fibronectin

Background

Matrix metalloproteinase-2 (MMP-2) is a key regulator in the migration of tumor cells. αvβ3 integrin has been reported to play a critical role in cell adhesion and regulate the migration of tumor cells by promoting MMP-2 activation. However, little is known about the effects of MMP-2 on αvβ3 integrin activity and αvβ3 integrin-mediated adhesion and migration of tumor cells.

Methodology/Principal Findings

Human melanoma cells were seeded using an agarose drop model and/or subjected to in vitro analysis using immunofluorescence, adhesion, migration and invasion assays to investigate the relationship between active MMP-2 and αvβ3 integrin during the adhesion and migration of the tumor cells. We found that MMP-2 was localized at the leading edge of spreading cells before αvβ3 integrin. αvβ3 integrin-mediated adhesion and migration of the tumor cells were inhibited by a MMP-2 inhibitor. MMP-2 cleaved fibronectin into small fragments, which promoted the adhesion and migration of the tumor cells.

Conclusion/Significance

MMP-2 cleaves fibronectin into small fragments to enhance the adhesion and migration of human melanoma cells mediated by αvβ3 integrin. These results indicate that MMP-2 may guide the direction of the tumor cell migration.     

The Direct Binding of Insulin-like Growth Factor-1 (IGF-1) to Integrin ��v��3 Is Involved in IGF-1 Signaling

It has been proposed that ligand occupancy of integrin αvβ3 with extracellular matrix ligands (e.g. vitronectin) plays a critical role in insulin-like growth factor-1 (IGF-1) signaling. We found that expression of αvβ3 enhanced IGF-1-induced proliferation of Chinese hamster ovary cells in serum-free conditions (in the absence of vitronectin). We hypothesized that the direct integrin binding to IGF-1 may play a role in IGF-1 signaling. We demonstrated that αvβ3 specifically and directly bound to IGF-1 in cell adhesion, enzyme-linked immunosorbent assay-type binding, and surface plasmon resonance studies. We localized the amino acid residues of IGF-1 that are critical for integrin binding by docking simulation and mutagenesis. We found that mutating two Arg residues at positions 36 and 37 in the C-domain of IGF-1 to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, although the mutant still bound to IGF1R, it was defective in inducing IGF1R phosphorylation, AKT and ERK1/2 activation, and cell proliferation. Furthermore wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 to integrin αvβ3 plays a role in IGF-1 signaling through ternary complex formation (αvβ3-IGF-IGF1R), and integrin-IGF-1 interaction is a novel target for drug discovery.Integrins are a family of cell adhesion receptors that mediate cell-extracellular matrix (ECM)³ interaction and cell-cell interaction (1). It has been proposed that signaling from inside the cells regulates the ligand binding affinity of integrins (inside-out signaling) (2). Each integrin is a heterodimer containing α and β subunits. At present 18 α and 8 β subunits have been identified that combine to form 24 integrins (3).It has been reported that integrin αvβ3 plays a role in cancer proliferation and invasiveness. High levels of integrin αvβ3 correlate with growth and/or progression of melanoma (4, 5), neuroblastoma (6), breast cancer (7, 8), colon cancer (9), ovarian cancer (10), and cervical cancer (11). Moreover, individuals homozygous for the β3L33P polymorphism that enhances the ligand binding affinity of β3 integrins have an increased risk to develop breast cancer, ovarian cancer, and melanoma (12). However, it remains unclear whether and how increased levels of αvβ3 on tumor cells contribute to cancer development.Insulin-like growth factor-1 (IGF-1) is a polypeptide hormone (75 kDa) that has a high degree of structural similarity to human proinsulin. IGF-1 acts through binding to the type I IGF receptor (IGF1R), a receptor tyrosine kinase. The IGF1R is a heterotetramer that consists of two α-subunits that contain the ligand-binding domains and two β-subunits that contain the tyrosine kinase activity. After ligand binding, the receptor undergoes a conformational change resulting in the activation of the tyrosine kinase, which results in transphosphorylation of the opposite β-subunit on specific tyrosine residues. These phosphotyrosines then bind to adapter molecules such as Shc and IRS-1. Phosphorylation of these proteins leads to activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) signaling pathways (reviewed in Ref. 13).IGF-1 has been implicated in cancer progression (14). One of the major actions of IGF-1 is to inhibit apoptosis. IGF-1 confers resistance to chemotherapy and radiation therapy. IGF-1 expression levels are increased in breast, lung, prostate, and many other cancers. Several strategies to target IGF-1 signaling have been extensively studied, including small interfering RNA and monoclonal antibodies for IGF1R and kinase inhibitors to inhibit the enzymatic activity of the receptor. The IGF-1 system is a therapeutic target for cancer, and elucidation of the IGF-1 signaling pathway should have a major impact in designing new therapeutic strategies.It has been proposed that ligand occupancy of αvβ3 with ECM ligands such as vitronectin plays a critical role in enhancing IGF-1 signaling (14). It has been reported that inhibiting αvβ3-ECM interaction (“ligand occupancy”) of αvβ3 inhibited IGF-1 actions selectively in cell types that express αvβ3 (14). Inhibiting ligand occupancy of αvβ3 blocked IGF-1-induced cell migration (15), DNA synthesis, IRS-1 phosphorylation, and IGF1R-linked downstream signaling events, such as activation of phosphatidylinositol 3-kinase and ERK1/2 (16).In the present study, we demonstrated that expression of αvβ3 enhanced proliferation of ovarian cancer cells in the presence of fetal bovine serum (FBS) and in serum-free conditions if IGF-1 was present. This suggests that IGF-1 is involved in enhanced proliferation of αvβ3-expressing cells. We demonstrated that αvβ3 bound to IGF-1 in several different binding assays. We found that two Arg residues at positions 36 and 37 in the C-domain of IGF-1 are critical for integrin binding by docking simulation and mutagenesis. Mutation of these Arg residues to Glu (the R36E/R37E mutation) effectively reduced integrin binding. Interestingly, the R36E/R37E mutant was defective in inducing cell proliferation and IGF-1 intracellular signaling, although it still bound to IGF1R. We demonstrated that wild type IGF-1 mediated co-precipitation of αvβ3 and IGF1R, whereas the R36E/R37E mutant did not, suggesting that IGF-1 mediates the interaction between αvβ3 and IGF1R. These results suggest that the direct binding to IGF-1 plays a role in IGF-1 signaling.     

αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion

The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin–RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvβ8 integrin–RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion.     

Molecular Interplay between Endostatin, Integrins, and Heparan Sulfate

Endostatin is an endogenous inhibitor of angiogenesis. Although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanism of action is not fully elucidated. We used surface plasmon resonance assays to characterize interactions between endostatin, integrins, and heparin/heparan sulfate. α5β1 and αvβ3 integrins form stable complexes with immobilized endostatin (K_D = ∼1.8 × 10⁻⁸ m, two-state model). Two arginine residues (Arg²⁷ and Arg¹³⁹) are crucial for the binding of endostatin to integrins and to heparin/heparan sulfate, suggesting that endostatin would not bind simultaneously to integrins and to heparan sulfate. Experimental data and molecular modeling support endostatin binding to the headpiece of the αvβ3 integrin at the interface between the β-propeller domain of the αv subunit and the βA domain of the β3 subunit. In addition, we report that α5β1 and αvβ3 integrins bind to heparin/heparan sulfate. The ectodomain of the α5β1 integrin binds to haparin with high affinity (K_D = 15.5 nm). The direct binding between integrins and heparin/heparan sulfate might explain why both heparan sulfate and α5β1 integrin are required for the localization of endostatin in endothelial cell lipid rafts.Endostatin is an endogenous inhibitor of angiogenesis that inhibits proliferation and migration of endothelial cells (1–3). This C-fragment of collagen XVIII has also been shown to inhibit 65 different tumor types and appears to down-regulate pathological angiogenesis without side effects (2). Endostatin regulates angiogenesis by complex mechanisms. It modulates embryonic vascular development by enhancing proliferation, migration, and apoptosis (4). It also has a biphasic effect on the inhibition of endothelial cell migration in vitro, and endostatin therapy reveals a U-shaped curve for antitumor activity (5, 6). Short term exposure of endothelial cells to endostatin may be proangiogenic, unlike long term exposure, which is anti-angiogenic (7). The effect of endostatin depends on its concentration and on the type of endothelial cells (8). It exerts the opposite effects on human umbilical vein endothelial cells and on endothelial cells derived from differentiated embryonic stem cells. Furthermore, two different mechanisms (heparin-dependent and heparin-independent) may exist for the anti-proliferative activity of endostatin depending on the growth factor used to induce cell proliferation (fibroblast growth factor 2 or vascular endothelial growth factor). Its anti-proliferative effect on endothelial cells stimulated by fibroblast growth factor 2 is mediated by the binding of endostatin to heparan sulfate (9), whereas endostatin inhibits vascular endothelial growth factor-induced angiogenesis independently of its ability to bind heparin and heparan sulfate (9, 10). The broad range of molecular targets of endostatin suggests that multiple signaling systems are involved in mediating its anti-angiogenic action (11), and although several endothelial cell surface molecules have been reported to interact with endostatin, its molecular mechanisms of action are not as fully elucidated as they are for other endogenous angiogenesis inhibitors (11).Endostatin binds with relatively low affinity to several membrane proteins including α5β1 and αvβ3 integrins (12), heparan sulfate proteoglycans (glypican-1 and -4) (13), and KDR/Flk1/vascular endothelial growth factor receptor 2 (14), but no high affinity receptor(s) has been identified so far. The identification of molecular interactions established by endostatin at the cell surface is a first step toward the understanding of the mechanisms by which endostatin regulates angiogenesis. We have previously characterized the binding of endostatin to heparan sulfate chains (9). In the present study we have focused on characterizing the interactions between endostatin, α5β1, αvβ3, and αvβ5 integrins and heparan sulfate. Although interactions between several integrins and endostatin have been studied previously in solid phase assays (12) and in cell models (12, 15, 16), no molecular data are available on the binding site of endostatin to the integrins. We found that two arginine residues of endostatin (Arg²⁷ and Arg¹³⁹) participate in binding to integrins and to heparan sulfate, suggesting that endostatin is not able to bind simultaneously to these molecules displayed at the cell surface. Furthermore, we have demonstrated that α5β1, αvβ3, and αvβ5 integrins bind to heparan sulfate. This may explain why both heparan sulfate and α5β1 integrins are required for the localization of endostatin in lipid rafts, in support of the model proposed by Wickström et al. (15).     

Plasminogen Activator Inhibitor-1 Regulates Integrin ��v��3 Expression and Autocrine Transforming Growth Factor �� Signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.Fibrotic disorders can result from environmental toxins, persistent infection, autoimmune disease, or mechanical injury, leading to the hardening and scarring of tissues. In fibrotic diseases, such as liver cirrhosis, renal fibrosis, and idiopathic lung fibrosis, or in pathological wound healing, such as hypertrophic scarring, scleroderma, and Dupuytren disease, the persistence of myofibroblasts contributes to disease progression by overproduction of extracellular matrix (ECM)² and by excessive contraction (1–3). A shift in the balance of growth factors and cytokines that promote ECM deposition and proteases that degrade matrix often contributes to fibrotic disease (4, 5). Plasmin, a broad spectrum protease that is generated from plasminogen by uPA, is one of the proteases that degrades matrix and activates growth factors and other proteases (6). Since uPA activity is inhibited by PAI-1, the overexpression of PAI-1 results in matrix accumulation. For this reason, PAI-1 is a key prognostic marker for fibrotic disease. PAI-1 exerts its inhibitory activity on uPA by stimulating the endocytosis of the cell surface uPA·uPAR complex through the low density lipoprotein receptor-related protein (7). Integrin αvβ3 is also internalized with the uPA·uPAR·low density lipoprotein receptor-related protein complex (8). After endocytosis, uPAR and integrins are recycled back to the cell surface for another round of binding (8, 9). uPAR and αvβ3 promote cellular attachment and spreading, since they are receptors for the extracellular matrix molecule, vitronectin (10). Thus, cycling of the complex is thought to stimulate the attachment and detachment that is necessary for cell migration (8). Consequently, a shift in the expression of any of these components (PAI-1/uPA/uPAR/αvβ3) can result in either aggressive migration, as seen in cancer invasion, or a persistent increase in cell adhesion and cell tension, as seen in myofibroblasts in fibrotic tissue.The family of TGFβ growth factors has been intensively studied for their role in fibrotic wound healing. Up-regulation of TGFβ results in amplified and persistent overproduction of molecules, such as integrins and PAI-1 and other protease inhibitors (e.g. TIMPs) (2, 3). Up-regulated integrins continue the cycle of TGFβ signaling by participating in the sustained activation of TGFβ from its latent form. To date, studies have found that various αv integrins participate in the activation of TGFβ (αvβ3, αvβ5, αvβ6, and αvβ8), but the mechanism differs (11–15). Integrins can serve as docking proteins to localize proteases that cleave and activate latent TGFβ in the ECM, or they can directly activate latent TGFβ in a protease-independent manner. Recently, it was discovered that latent TGFβ is also activated by mechanical stress generated from an integrin-mediated interaction between myofibroblasts and the ECM, primarily involving αvβ5. The mechanical stress promotes a conformational change that activates the latent TGFβ complex (15). αv integrins also modulate TGFβ signaling through the binding of αvβ3 to TGFβ receptor II (TGFβRII) in the presence of TGFβ. This interaction was shown to promote a dramatic increase in the proliferation of lung fibroblasts and induce invasion of epithelial breast cancer cells (16, 17).Our data establish a role for the PAI-1-mediated control of αvβ3 expression and support a significant role for αvβ3 in TGFβ signaling. Using PAI-1 KO cells, we tested the hypothesis that the absence of PAI-1 would result in the accumulation of αvβ3 on the cell surface, since PAI-1 promotes the endocytosis of uPA·uPAR·αvβ3. PAI-1-mediated endocytosis of β3 was significantly reduced in the PAI-1 KO cells. Correspondingly, we report that β3 accumulated at the cell surface in enlarged β3-containing focal adhesions. Thus, we explored whether the accumulation of αvβ3 on the cell surface had fibrogenic effects even in the absence of profibrotic PAI-1. Our results demonstrate dramatically increased TGFβ activity and an increase in collagen expression in PAI-1 KO cells. Together, these findings suggest that PAI-1 modulates β3 expression and localization and, in turn, TGFβ signaling. Our data reveal that maintaining precise levels of PAI-1 is a key to preventing fibrosis. Understanding the consequence of regulating PAI-1 activity is critical in light of the many clinical therapies currently under development that target PAI-1 (18, 19).     

Application of a Novel Method of Double APAAP Staining With Subsequent Quantitative Image Analysis to the Examination of Integrin Expression in Undifferentiated-type Gastric Carcinomas

In undifferentiated-type gastric carcinoma (UGC), recognition of cancer cells is not easy, which has hampered its precise phenotypic analysis. To examine alterations of the integrin phenotype during the progression of UGC, we used double alkaline phosphatase anti-alkaline phosphatase staining and computer-aided image analyses for the expression of α1, α2, α3, α5, α6, αV, β1, and β4 integrin subunits and αVβ3, αVβ5, and αVβ6 integrins in cytokeratin-positive cells in the mucosal, the submucosal, and the deeper parts of 10 early and 17 advanced UGCs, their non-neoplastic counterparts, and 9 lymph node (LN) metastases. We revealed declining expression of epithelial integrin subunits (α2, α3, α6, β4) and increasing expression of mesenchymal integrin subunits (α1, α5) as the tumor invaded deeper, reflecting gradual epithelial-to-mesenchymal transition of the integrin phenotype during tumor invasion. Enhanced expression of the αV integrin subunit and αVβ3 and αVβ5 integrins correlated with tumor invasion, and that of αVβ6 integrins with LN metastasis. Our results have demonstrated that the method we introduced is suitable for analysis of dynamic alterations of the integrin repertoire in UGC progression. (J Histochem Cytochem 57:1183–1193, 2009)     

Generation of universal and hypoimmunogenic human pluripotent stem cells

There is a need to store very large numbers of conventional human pluripotent stem cell (hPSC) lines for their off‐the‐shelf usage in stem cell therapy. Therefore, it is valuable to generate “universal” or “hypoimmunogenic” hPSCs with gene‐editing technology by knocking out or in immune‐related genes. A few universal or hypoimmunogenic hPSC lines should be enough to store for their off‐the‐shelf usage. Here, we overview and discuss how to prepare universal or hypoimmunogenic hPSCs and their disadvantages. β2‐Microglobulin‐knockout hPSCs did not harbour human leukocyte antigen (HLA)‐expressing class I cells but rather activated natural killer (NK) cells. To avoid NK cell and macrophage activities, homozygous hPSCs expressing a single allele of an HLA class I molecule, such as HLA‐C, were developed. Major HLA class I molecules were knocked out, and PD‐L1, HLA‐G and CD47 were knocked in hPSCs using CRISPR/Cas9 gene editing. These cells escaped activation of not only T cells but also NK cells and macrophages, generating universal hPSCs.     

��1 Integrin Deficiency Results in Multiple Abnormalities of the Knee Joint

The lack of β1 integrins on chondrocytes leads to severe chondrodysplasia associated with high mortality rate around birth. To assess the impact of β1 integrin-mediated cell-matrix interactions on the function of adult knee joints, we conditionally deleted the β1 integrin gene in early limb mesenchyme using the Prx1-cre transgene. Mutant mice developed short limbed dwarfism and had joint defects due to β1 integrin deficiency in articular regions. The articular cartilage (AC) was structurally disorganized, accompanied by accelerated terminal differentiation, altered shape, and disrupted actin cytoskeleton of the chondrocytes. Defects in chondrocyte proliferation, cytokinesis, and survival resulted in hypocellularity. However, no significant differences in cartilage erosion, in the expression of matrix-degrading proteases, or in the exposure of aggrecan and collagen II cleavage neoepitopes were observed between control and mutant AC. We found no evidence for disturbed activation of MAPKs (ERK1/2, p38, and JNK) in vivo. Furthermore, fibronectin fragment-stimulated ERK activation and MMP-13 expression were indistinguishable in control and mutant femoral head explants. The mutant synovium was hyperplastic and frequently underwent chondrogenic differentiation. β1-null synoviocytes showed increased proliferation and phospho-focal adhesion kinase expression. Taken together, deletion of β1 integrins in the limb bud results in multiple abnormalities of the knee joints; however, it does not accelerate AC destruction, perturb cartilage metabolism, or influence intracellular MAPK signaling pathways.Chondrocytes of the articular cartilage (AC)² secrete a unique set of extracellular matrix (ECM) molecules that assemble into interactive associates composed of collagens, proteoglycans (PGs), and non-collagenous glycoproteins (1). The fibrillar collagen meshwork supplies cartilage with its tensile strength, whereas the hydrated glycosaminoglycan (GAG) chains of PGs (mainly aggrecan) generate an osmotic swelling pressure that resists compressive forces. In diarthrodial joints, the molecular composition and the physical properties of the cartilage are principal determinants for the shock-absorbing function of articular surfaces upon mechanical loading. During the development of osteoarthritis (OA), an imbalance between anabolic and catabolic processes increases the proteolysis of PGs and collagens (2, 3), which eventually leads to the mechanical weakening of the AC and culminates in its progressive destruction. Physiological and pathological remodeling of the AC ECM is primarily attributed to the activities of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin-like repeat (ADAMTS) proteases (4, 5) and is controlled by the communication between the cells and their environment.An increasing amount of evidence suggests that interactions between chondrocytes and the ECM through the integrin family of heterodimeric (αβ) transmembrane receptors play a central role in cartilage function (6). Integrins connect the pericellular matrix to cytoskeletal and intracellular signaling complexes and modulate various cellular functions, including survival, proliferation, differentiation, and matrix assembly and metabolism (7, 8). Chondrocytes express several integrin receptors for cartilage matrix ligands, such as α1β1, α2β1, and α10β1 (for collagen II); α5β1, αvβ3, and αvβ5 (for fibronectin); and α6β1 (for laminin) (6, 9). We have previously demonstrated that β1^fl/fl-Col2a1cre⁺ mice, in which the floxed β1 integrin gene (β1^fl/fl) was deleted using the chondrocyte-specific Col2a1cre transgene, display severe chondrodysplasia and a high mortality rate at birth (10). Homozygous mutant mice exhibit multiple growth plate abnormalities during endochondral bone formation, characterized by defects in chondrocyte adhesion, shape, proliferation, cytokinesis, and actin organization. In addition, the cartilage matrix shows a sparse, distorted collagen network. Similar, but milder abnormalities were found in mice lacking the collagen-binding integrin α10β1 or integrin-linked kinase in cartilage (11, 12).Although these works have identified β1 integrins as essential regulators of growth plate development, the role of integrins in joint morphogenesis, adult joint function, and pathology is incompletely understood. In the embryonic mouse limb culture system, administration of β1 and α5 blocking antibodies or RGD peptides induced ectopic joint formation between proliferating and hypertrophic chondrocytes of the growth plate, suggesting that α5β1 integrin controls the decision between cartilage differentiation and joint formation during development (13). In adult joints, increased immunostaining of β1 integrin was reported in osteoarthritic monkey cartilage compared with normal cartilage (14) and in human OA samples at minimally damaged locations compared with areas with more severe lesions (15). In another study, the neoexpression of α2, α4, and β2 integrins was observed in osteoarthritic human femoral head cartilage (16). In vitro experiments have suggested that signaling through the fibronectin (FN) receptor α5β1 integrin is pivotal to prevent cell death of normal and osteoarthritic human articular chondrocytes (17). FN fragments (FN-fs) present in synovial fluid and cartilage of OA patients have been implicated in cartilage breakdown (18–21). Human AC chondrocytes treated with the central, 110–120-kDa cell-binding FN-f but not with intact FN were shown to increase MMP-13 synthesis through the stimulation of α5β1 integrin and the subsequent activation of the proline-rich tyrosine kinase-2 and mitogen-activated protein kinases (MAPKs) ERK-1/2, JNK, and p38 (22, 23). Similarly, an adhesion-blocking antibody against α2β1 integrin induced the phosphorylation of MAPKs in human AC chondrocytes (22). Treatment of cultured rabbit synovial fibroblasts with central FN-fs or activating antibodies against α5β1 integrin elevated MMP-1 and MMP-3 expression (24). Although these experiments suggest that blocking integrin signaling through α2β1/α5β1 in response to degradation fragments may attenuate OA, mice lacking α1β1 integrin are prone to osteoarthritis (25). Knee joints of α1-null mice display precocious PG loss, cartilage erosion associated with increased MMP-2 and MMP-3 expression, and synovial hyperplasia.To further explore the role of β1 integrins in joint biology, here we report the deletion of the floxed β1 integrin gene in embryonic limb bud mesenchymal cells using the Prx1cre transgene (26). β1^fl/fl-Prx1cre⁺ mice were born alive with short limbs due to the lack of β1 integrin heterodimers on chondrocytes. We found that β1 integrin deficiency in knee joints leads to multiple abnormalities of the AC and the synovium, but it is not associated with accelerated AC destruction, perturbed AC metabolism, and MAPK signaling. Our data suggest that β1 integrins are required for the proper structural organization of the AC by anchoring chondrocytes to the ECM, but signaling through β1 integrins is less important for normal cartilage homeostasis.     

Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine

Background

NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far.

Methodology/Principal Findings

We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (K_D = 148±9.54 nM).

Conclusion/Significance

Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.     

Polymeric Osteopontin Employs Integrin ��9��1 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.Acidic phosphorylated secreted glycoprotein osteopontin (OPN),⁴ known as a cytokine, has multiple functions, including roles in tissue remodeling, fibrosis, mineralization, immunomodulation, inflammation, and tumor metastasis (1–3). OPN is also an integrin ligand. At least nine integrins can function as OPN receptors. α5β1, α8β1, αvβ1, αvβ3, αvβ5 (1), and αvβ6 (4) recognize the linear tripeptide RGD, and α9β1, α4β1, and α4β7 recognize the sequence, SVVYGLR (5), adjacent to RGD but only after OPN has been cleaved by the protease, thrombin (Fig. 1).Open in a separate windowFIGURE 1.Schematic diagram of OPN. Two integrin-binding sites (boxed), a thrombin cleavage site (arrow), and a putative transglutamination site (circled) are shown. The term thrombin-cleaved nOPN is defined as in the figure.The overlap of receptors for OPN does not necessarily mean that these integrins play redundant roles in cellular responses to OPN because the patterns of integrin expression and utilization vary widely among cell types. In addition, interactions of different integrins with a single ligand can exert distinct effects on cell behavior in a single cell type. For example, we have previously reported that signals by ligation of αvβ3, αvβ6, or α9β1 to a single ligand, tenascin-C, differently affected cell adhesion, spreading, and proliferation of the colon cancer cell line, SW480 (6). Furthermore, intact OPN or thrombin- or matrix metalloproteinase-cleaved OPN interact with distinct subsets of integrins and exhibit distinct effects on cell behavior (4, 7, 8). Collectively, some of the functional diversity of OPN could be attributed to this multiplicity of receptors and responses. We have recently shown that polymerization of OPN results in enhanced biological activity (9). We thus set out to determine whether polymerized OPN exerts its effects through unique interactions with integrins.OPN is polymerized by transglutaminase 2 (TG2, EC 2.3.2.13) (10) that catalyzes formation of isopeptide cross-links between glutamine and lysine residues in substrate proteins (11) including OPN. Polymeric OPN has been identified in vivo in bone (12) and calcified aorta (13). We have previously reported that upon polymerization, OPN displays increased integrin binding accompanied by enhanced cell adhesion, spreading, migration, and focal contact formation (9). However, very little is known about how polymeric OPN induces its biological effects.Integrin α9β1, highly expressed on neutrophils (14), does not act as a receptor for intact OPN but does bind to an N-terminal fragment of OPN (nOPN) that is generated by thrombin cleavage (15) through the new C-terminal sequence, SVVYGLR. Protein polymerization can expose otherwise cryptic domains (16), so we hypothesized that the SVVYGLR site might be exposed upon polymerization and serve as a binding site for α9β1. In the present study, we demonstrate that α9β1 is indeed a receptor for polymeric OPN and that neutrophil migration induced by polymeric OPN is largely mediated by this interaction. However, mutational analysis and antibody studies demonstrate that this interaction does not involve the SVVYGLR site, suggesting the presence of de novo binding site in polymeric OPN.     

�� Integrin Tyrosine Phosphorylation Is a Conserved Mechanism for Regulating Talin-induced Integrin Activation

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the β integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated ¹⁵N-labeled β3, β1A, and β7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of β3, β1A, and β7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of β integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.     

CCL5 and CCR5 interaction promotes cell motility in human osteosarcoma

Background

Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown.

Methodology/Principal Findings

Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells.

Conclusions/Significance

CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells.     

Targeted inactivation of β1 integrin induces β3 integrin switching,which drives breast cancer metastasis by TGF-β

Mammary tumorigenesis and epithelial–mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-β (TGF-β) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated β1 and β3 integrins with TGF-β stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-β remain unknown. We demonstrate that inactivation of β1 integrin impairs TGF-β from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of β3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory β3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-β. Of importance, compensatory expression of β3 integrin rescues the growth and pulmonary metastasis of β1 integrin–deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of β3 integrin. Collectively our findings demonstrate that inactivation of β1 integrin elicits metastatic progression via a β3 integrin–specific mechanism, indicating that dual β1 and β3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.     

Interactions of Soluble Recombinant Integrin αvβ5 with Human Adenoviruses

αv integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin αvβ5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin αvβ5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with αvβ5. Incubation of Ad particles with soluble αvβ5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble αv integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble αv integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.     

Crystal structure of α5β1 integrin ectodomain: atomic details of the fibronectin receptor

Integrin α5β1 is a major cellular receptor for the extracellular matrix protein fibronectin and plays a fundamental role during mammalian development. A crystal structure of the α5β1 integrin headpiece fragment bound by an allosteric inhibitory antibody was determined at a 2.9-Å resolution both in the absence and presence of a ligand peptide containing the Arg-Gly-Asp (RGD) sequence. The antibody-bound β1 chain accommodated the RGD ligand with very limited structural changes, which may represent the initial step of cell adhesion mediated by nonactivated integrins. Furthermore, a molecular dynamics simulation pointed to an important role for Ca²⁺ in the conformational coupling between the ligand-binding site and the rest of the molecule. The RGD-binding pocket is situated at the center of a trenchlike exposed surface on the top face of α5β1 devoid of glycosylation sites. The structure also enabled the precise prediction of the acceptor residue for the auxiliary synergy site of fibronectin on the α5 subunit, which was experimentally confirmed by mutagenesis and kinetic binding assays.