Integrin Alpha 4 (Itga 4)
Integrins are heterodimeric glycoproteins composed of non-covalently associated α and β subunits, and constitute one of the largest families of adhesion molecules. Integrins mediate cell-to-cell and cell-to-extracellular matrix interactions and are involved in a wide range of physiological processes such as development, immune regulation, and hemostasis. Mammalian genomes contain 18 α subunit and 8 β subunit genes which generate 24 different αβ heterodimer combinations.
The α4 integrin subunit is expressed on the cell surface associated with β1 or β7 integrin chains (Hemler et al. 1987; Sanchez-Madrid et al. 1986). These α4 integrins (α4β1, also known as VLA-4, and α4β7) are involved in important cell differentiation processes such as neural and muscle cell differentiation and hematopoiesis. The importance of α4 integrins in development is demonstrated by the embryonic lethality of disruption of the α4 integrin gene in mice; a fraction of these embryos die due to a defect in the development of the placenta, while the rest die after hemorrhage in the heart region. The generation of chimeric mice revealed that α4 is essential for hematopoiesis and also for epicardial development (Arroyo et al. 1996). α4 integrins also play a key role in the adhesion interaction between stem/progenitor cells and bone marrow stromal cells and their matrix (Miyake et al. 1991). The most well-documented role of α4 integrins is in leukocyte adhesion, motility, and extravasation during the inflammatory response. All of these functions are mediated through the binding of α4 integrins to their ligands, which include fibronectin, and the Ig Superfamily members VCAM-1, JAM-B, and MadCAM.
α4 Integrin Expression
The α4 integrin subunit is expressed as an integral membrane protein on the surface of several cell types, mostly restricted to the hematopoietic lineage in adults (Hemler et al. 1987; Sanchez-Madrid et al. 1986). Bone marrow progenitor cells and several mature cells, including mononuclear leukocytes and eosinophils, express α4, mainly as α4β1. Myocytes, thymocytes, osteoclasts, cardiac cells, placental cells, erythrocytes, neutrophils, and fibroblasts also express α4, but expression on these cells depends on developmental stage and stimulation. Melanoma and neural-crest-derived tumor cell lines express α4β1. α4β7 is expressed at high levels by a small subset of circulating memory T cells that preferentially localize to gut-associated lymphoid tissue. This homing is mediated by the interaction of this integrin with MAdCAM-1, an addressin molecule selectively expressed on intestinal endothelial cells (Holzmann and Weissman 1989).
α4 Integrin Ligands
The most important α4β1 integrin ligands include the extracellular matrix molecules fibronectin (FN; (Wayner et al. 1989)), osteopontin and thrombospondin, the immunoglobulin superfamily member VCAM-1 (Elices et al. 1990), which is expressed by activated endothelium, and the junctional adhesion molecule JAM-B (also known as JAM-2) (Cunningham et al. 2002), which localizes at endothelial cell–cell junctions. α4β1, like other β1 integrins, also interacts with the bacterial coat protein invasin (Isberg and Leong 1990), mediating bacterial entry into mammalian cells. α4β7 integrin binds to fibronectin, VCAM-1, and the gut addressin MAdCAM-1 (Berlin et al. 1995).
α4 Integrin Function
α4 integrins are important for the motility of many types of leukocytes, including hematopoietic stem cells, eosinophils, mast cells, and T and B lymphocytes. During inflammatory responses leukocytes interact with the activated endothelium in order to migrate from the bloodstream to peripheral tissue. This extravasation process consists of the steps of tethering and rolling, firm adhesion, and diapedesis. α4 integrins, by interacting first with VCAM-1 (vascular cell adhesion molecule-1) and then with JAM-B (junctional adhesion molecule-B), participate in all of these steps (Berlin et al. 1995; Elices et al. 1990). α4 integrins are involved in the recruitment of bone marrow-derived endothelial progenitors to the neovasculature and mediate their adhesion to VCAM-1 on endothelial and mural cells in neovessels (Garmy-Susini et al. 2005). Integrin α4β1 is enriched at the immune synapse during activation of T lymphocytes and acts as a co-stimulatory molecule driving T helper (TH)1/TH2 cell differentiation (Mittelbrunn et al. 2004). Integrin α4β7, through its interaction with MAdCAM-1 expressed on intestinal endothelial cells (Holzmann and Weissman 1989), acts as a homing receptor for a subset of memory T cells that preferentially migrate to gut-associated lymphoid tissue.
Regulation of α4 Integrin Function
The capacity of α4 integrins to bind their ligands is regulated both by intracellular signaling mechanisms and by extracellular stimuli. Intracellular signals induce conformational changes (integrin activation) that result in increased ligand-binding affinity, while extracellular stimuli modify the expression and activation status as well as the level of receptor aggregation/clustering on the plasma membrane. The conformational change of integrin activation involves the cytoplasmic domains of the α and β subunits. Under resting conditions, these domains associate with each other. This association is thought to function as a “clasp” that keeps integrins in a low ligand-affinity bent conformation. When the cells are activated, the cytoplasmic domains dissociate and the extracellular domains form an open conformation that allows interaction with ligands. The association of the α and β cytoplasmic domains is regulated by the GFFKR sequence, a well-preserved motif in the α4 cytoplasmic domain.
The signaling pathway that underlies the inside-out activation mechanism of α4β1 upon stimulation of the B-cell receptor has been described, and involves the consecutive activation of Lyn, Syk, phosphatidylinositol-3′ hydroxy kinase, Bruton’s tyrosine kinase (Btk), phospholipase C (PLC)γ2, Ins(1,4,5)P3-receptor-mediated Ca2+ release, and protein kinase C (Spaargaren et al. 2003). The affinity of α4 integrins is also regulated by divalent cations; Mn2+ and Mg2+ modulate the strength of binding to VCAM-1 and MAdCAM-1, while Ca2+ facilitates the binding of α4β1 to VCAM-1. The small GTPase Rap 1 and its effector RAPL play a role in the activation of β1 and β2 integrins induced by the T-cell antigen receptor (TCR), CD31, and cytokines (Katagiri et al. 2004). Clustering of integrins on the cell membrane also increases their adhesiveness as a result of cooperative binding (Hogg et al. 2003). An additional factor that might contribute to α4 integrin function is their lateral association with CD44 and the tetraspanins CD53, CD63, CD81, and CD82, which might facilitate integrin receptor clustering.
The cytoplasmic domain of the α4 subunit can bind directly to paxillin, a signaling adaptor molecule that has been reported to regulate the function of α4 integrins in immune cells (Liu et al. 1999). Paxillin binds to the α4 cytoplasmic domain upon dephosphorylation of Ser988. The paxillin–α4 interaction inhibits the formation of focal adhesions, stress fibers, and lamellipodia by triggering the activation of various tyrosine kinases, such as focal adhesion kinase (FAK), Pyk2, Src, and Abl. The paxillin–α4 complex inhibits the formation of stable lamellipodia by sequestering ADP-ribosylation factor (Arf)-GTPase-activating protein, thereby decreasing Arf activity and inhibiting Rac. It has recently been reported that dissociation of Vav1 from talin generates alpha4beta1-talin complexes, resulting in high-affinity alpha4beta1 conformations and efficient integrin activation (Garcia-Bernal et al. 2009). Moreover, recent evidence suggests that Kindlins, a group of 3 structurally related adaptors, cooperate with talin in activating integrins through binding to the integrin Î2 subunits (Montanez et al. 2008).
It is well recognized that inflammatory responses form part of the pathogenesis of many disorders, including autoimmune diseases. The integrins α4β1 and α4β7 are fundamental for the adhesion and migration of leukocytes to different inflammatory scenarios and have therefore been investigated as potential therapeutic targets. Administration of anti-α4 mAb or various synthetic blockers has shown beneficial effects in several animal models, including experimental allergic encephalomyelitis, adjuvant-induced arthritis, diabetes mellitus of NOD mice, experimental graft-versus-host disease, allograft rejection, and immediate hypersensitivity reactions (Yusuf-Makagiansar et al. 2002).
In a clinical setting, the humanized anti-α4 mAb natalizumab (Tysabri) has been approved for treatment of the relapsing forms of multiple sclerosis and Crohn’s disease (Gonzalez-Amaro et al. 2005). Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system that is presumably caused by activated T cells specific for myelin antigens. Crohn’s disease is an inflammatory bowel disease (IBD), a group of chronic systemic diseases involving inflammation of the gastrointestinal tract. Patients with multiple sclerosis or Crohn’s disease show significant improvements with natalizumab (Gonzalez-Amaro et al. 2005).
Another recently suggested application for α4 blocking agents is the treatment and prevention of epilepsy. Recent findings indicate that seizure activity is associated with leukocytic inflammatory changes in the central nervous system vasculature. α4-specific antibodies decrease leukocyte adhesion to the brain vessel wall and have anti-epileptogenic properties, reducing and preventing subsequent seizures (Fabene et al. 2008).
It has been assumed that the therapeutic effect of this type of mAb is a consequence of the inhibition of the interaction of α4 integrins with VCAM-1 and MAdCAM-1, thus inhibiting leukocyte extravasation to inflammatory foci. However, the complex role of α4 in immune cells, including its involvement in leukocyte activation and TH1/TH2 polarization (Mittelbrunn et al. 2004), might lead to undesired side effects with anti-α4 mAbs. This possibility should be considered, especially in relation to the treatment of autoimmune and inflammatory diseases associated with TH1/TH2 imbalance. Further studies are needed to minimize risks and optimize benefits of long-term maintenance therapy with these antibodies.
Integrins are one of the largest families of adhesion molecules that mediate cell–cell and cell–matrix interactions. α4 integrins are expressed on many types of hematopoietic cells, including stem/progenitor cells, and are critical regulators of hematopoiesis and leukocyte trafficking.
Inhibition of leukocyte trafficking by antibody blockade of α4-integrins is a validated therapeutic approach for the treatment of multiple sclerosis and Crohn’s disease. It remains unclear, however, why anti-α4-based therapies are effective in some chronic autoimmune and inflammatory conditions but not in others, and this is an area that deserves further research. The onset of demyelinating diseases such as progressive multifocal leukoencephalopathy (PML) in some antibody-treated patients suggests that the mechanism of action of these drugs is not fully understood and forces us to carefully consider the potential risks associated with long-term administration of anti- α4-integrin antibodies. These risks might include alterations to T helper cell differentiation, thereby distorting the population profile of T helper cells and subverting the type of immune response. Further studies are therefore needed to define the regulatory mechanisms that mediate α4 function. The development of a new generation of modulators of α4 integrin function, small molecules that block interaction with ligands or intracellular-associated proteins, is likely to lead to novel therapies not only for multiple sclerosis and Crohn’s disease but also for others inflammatory/autoimmune diseases.
- Mittelbrunn M, Molina A, Escribese MM, Yanez-Mo M, Escudero E, Ursa A, Tejedor R, Mampaso F, Sanchez-Madrid F. VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses. Proc Natl Acad Sci USA. 2004;101:11058–63.PubMedCrossRefPubMedCentralGoogle Scholar