S. Kent Dickeson

β1 Integrin Mediates Internalization of Mammalian Reovirus

ABSTRACT Reovirus infection is initiated by interactions between the attachment protein σ1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein λ2, which serves as the structural base for σ1, suggests that integrins mediate reovirus endocytosis. A β1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a β1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in β1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing β1. However, reovirus bound equivalently to cells that differed in levels of β1 expression, suggesting that β1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into β1-deficient cells was substantially diminished in comparison to viral uptake into β1-expressing cells. These data provide evidence that β1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.

Collagen Receptor Control of Epithelial Morphogenesis and Cell Cycle Progression

To define the unique contributions of the alpha subunit cytoplasmic tails of the alpha(1)beta(1) and alpha(2)beta(1) integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking alpha(1)beta(1) and alpha(2)beta(1) integrin expression was stably transfected with the full-length alpha(2) integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the alpha(2) subunit and the cytoplasmic domain of the alpha(1) subunit (X2C1), or alpha(2) cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three-dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the alpha(2) integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact alpha(1) or alpha(2) integrin subunit cytoplasmic domain can promote cell cycle progression.

Negative regulation of activated α2 integrins during thrombopoiesis

Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, alpha2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface alpha2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, alpha2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated alpha2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated alpha2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.

A comparison of the effects of factor XII deficiency and prekallikrein deficiency on thrombus formation

Studies with animal models implicate the plasma proteases factor XIIa (FXIIa) and α-kallikrein in arterial and venous thrombosis. As congenital deficiencies of factor XII (FXII) or prekallikrein (PK), the zymogens of FXIIa and α-kallikrein respectively, do not cause bleeding disorders, inhibition of these enzymes may have therapeutic benefit without compromising hemostasis. The relative contributions of FXIIa and α-kallikrein to thrombosis in animal models are not clear. We compared mice lacking FXII or PK to wild type mice in established models of arterial thrombosis. Wild type mice developed carotid artery occlusion when the vessel was exposed to a 3.5% solution of ferric chloride (FeCl3). FXII-deficient mice were resistant to occlusion at 5% FeCl3 and partially resistant at 10% FeCl3. PK-deficient mice were resistant at 3.5% FeCl3 and partially resistant at 5% FeCl3. Mice lacking high molecular weight kininogen, a cofactor for PK activation and activity, were also partially resistant to thrombosis at 5% FeCl3. Induction of carotid artery thrombosis with Rose Bengal was delayed in FXII-deficient mice compared to wild type or PK-deficient animals. In human plasma supplemented with silica, DNA or collagen to induce contact activation, an antibody to the FXIIa active site was more effective at preventing thrombin generation than an antibody to the α-kallikrein active site. Similarly, the FXIIa antibody was more effective at reducing fibrin formation in human blood flowing through collagen coated-tubes. The findings suggest that inhibitors of FXIIa will have more potent anti-thrombotic effects than inhibitors of α-kallikrein.

BINDING OF THE ALPHA 2 INTEGRIN I DOMAIN TO EXTRACELLULAR MATRIX LIGANDS :STRUCTURAL AND MECHANISTIC DIFFERENCES BETWEEN COLLAGEN AND LAMININ BINDING

The alpha 2 beta 1 integrin functions as a cell surface receptor for collagen on some cells and as both a collagen and laminin receptor on a more restricted subset of cell types including endothelial and epithelial cells. The alpha 2 integrin subunit I domain binds collagen in a divalent cation-dependent manner. In contrast, I domain binding to laminin occurs via both divalent cation-dependent and -independent mechanisms. Saturable binding was observed in the presence of either Mn2+ or EDTA, although the extent of binding in Mn2+ was twice that observed in EDTA. Half-maximal binding occurred at about 22 nM I domain in either case. Whereas laminin binding was significantly enhanced by Mn2+, with half-maximal binding occurring at 1.9 mM Mn2+, Mg2+ was much less effective. Deletion of the N-terminal 35 residues of the I domain, including the DXSXS portion of the MIDAS motif, caused a significant diminution of laminin binding activity. Laminin binding by the I domain was significantly inhibited by the alpha 2 beta 1 function-blocking antibody 6F1 in the presence of either EDTA or Mn2+. The non-function-blocking antibody 12F1 had no effect. In contrast to the binding of the alpha 2 integrin I domain to collagen, the laminin binding activity of the I domain was not enhanced by the addition of the first EF hand motif of the integrin.

Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the α2β1 integrin (α2β1) and the glycoprotein VI (GPVI)/FcRγ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (α2-CRP) containing the α2β1-specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to α2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was α2β1-dependent and GPVI/FcRγ-independent as revealed in experiments with α2β1- or FcRγ-deficient mouse platelets. We further show that suboptimal activation of other platelet Gq-linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to α2-CRP. The enhanced α2β1-mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of αIIbβ3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet Gq-linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced α2β1 binding to collagens containing GFOGER sites.

Perlecan domain V inhibits α2 integrin-mediated amyloid-β neurotoxicity

Amyloid-β (Aβ) peptide is a key component of amyloid plaques, one of the pathological features of Alzheimers disease. Another feature is pronounced cell loss in the brain leading to an enlargement of the ventricular area and a decrease in brain weight and volume. Aβ plaque deposition and neuronal toxicity can be modeled by treating human cortical neuronal cultures with Aβ and showing robust Aβ deposition and neurotoxicity that is mediated by α2β1 and αvβ1 integrins. The current study expands on these findings by showing that the domain V of perlecan, a known α2 integrin ligand, inhibits Aβ neurotoxicity in an α2 integrin-dependent manner. Additionally, Aβ binds more efficiently to cells expressing activated α2 integrin. Finally the inhibition of Aβ neurotoxicity with domain V is synergistic with inhibitors of αv integrin and β1 integrin. We propose that domain V and potentially other α2 integrin ligands could be a new therapeutic approach for inhibiting the Aβ plaque deposition and neurotoxicity observed in Alzheimers disease.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

Mitigation of oxygen-induced retinopathy in α2β1 integrin-deficient mice.

PURPOSE The α2β1 integrin plays an important but complex role in angiogenesis and vasculopathies. Published GWAS studies established a correlation between genetic polymorphisms of the α2β1 integrin gene and incidence of diabetic retinopathy. Recent studies indicated that α2-null mice demonstrate superior vascularization in both the wound and diabetic microenvironments. The goal of this study was to determine whether the vasculoprotective effects of α2-integrin deficiency extended to the retina, using the oxygen-induced retinopathy (OIR) model for retinopathy of prematurity (ROP). METHODS In the OIR model, wild-type (WT) and α2-null mice were exposed to 75% oxygen for 5 days (postnatal day [P] 7 to P12) and subsequently returned to room air for 6 days (P12-P18). Retinas were collected at postnatal day 7, day 13, and day 18 and examined via hematoxylin and eosin and Lectin staining. Retinas were analyzed for retinal vascular area, neovascularization, VEGF expression, and Müller cell activation. Primary Müller cell cultures from WT and α2-null mice were isolated and analyzed for hypoxia-induced VEGF-A expression. RESULTS In the retina, the α2β1 integrin was minimally expressed in endothelial cells and strongly expressed in activated Müller cells. Isolated α2-null primary Müller cells demonstrated decreased hypoxia-induced VEGF-A expression. In the OIR model, α2-null mice displayed reduced hyperoxia-induced vaso-attenuation, reduced pathological retinal neovascularization, and decreased VEGF expression as compared to WT counterparts. CONCLUSIONS Our data suggest that the α2β1 integrin contributes to the pathogenesis of retinopathy. We describe a newly identified role for α2β1 integrin in mediating hypoxia-induced Müller cell VEGF-A production.

Perlecan Domain V Inhibits Amyloid-β Induced Activation of the α2β1 Integrin-Mediated Neurotoxic Signaling Cascade

Alzheimers disease (AD) is characterized by neuronal death, neurofibrillary tangles, and senile plaques. Amyloid-beta (Aβ) is the major component of plaques and consists of two prominent isoforms, Aβ40 and Aβ42. As many risk factors for AD are vascular in origin and blood vessel defects in clearing Aβ from the brain are a potential key component of AD pathology, we have focused on the neuron-blood vessel interface, and in particular, the vascular basement membrane, which coats blood vessels and physically separates them from neurons. A prominent component of the vascular basement membrane is the extracellular matrix proteoglycan perlecan. Domain V (DV) is the C-terminal domain and is generated by perlecan proteolysis. DV interacts with the α2 integrin and Aβ is a ligand for both α2β1 and αvβ1. Due to the known interaction of DV with α2β1 and α2β1s requirement for Aβ deposition and neurotoxicity, we hypothesized that DV and/or its C-terminal domain, LG3, might alter neurotoxic signaling pathways by directly blocking or otherwise interfering with α2β1 binding by Aβ. Our study suggests that α2β1 mediates Aβ-induced activation of c-Jun and caspase-3, key components of the neurotoxic pathway, in primary cortical and hippocampal neurons. We further demonstrate that DV and/or LG3 may therapeutically modulate these α2β1 mediated neurotoxic effects suggesting that they or other α2β1 integrin modulators could represent a novel approach to treat AD. Finally, our results suggest different neurotoxicity susceptibilities between cortical and hippocampal neurons to Aβ40 and Aβ42 as further underscored by differing neuroprotective potencies of LG3 in each cell type.