Α4 Β1 Research Articles

Silencing of spontaneous activity at α4β1/3δ GABAA receptors in hippocampal granule cells reveals different ligand pharmacology

The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy. Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed. Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1 -/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells. Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

DS-70, a novel and potent α4 integrin antagonist, is an effective treatment for experimental allergic conjunctivitis in guinea pigs.

Allergic conjunctivitis is an eye inflammation that involves the infiltration of immune cells into the conjunctiva via cell surface-adhesion receptors, such as integrin α4 β1 . These receptors interact with adhesion molecules expressed on the conjunctival endothelium and may be a target to treat this disease. We synthesized DS-70, a novel α/β-peptidomimetic α4 integrin antagonist, to prevent the conjunctival infiltration of immune cells and clinical symptoms in a model of allergic conjunctivitis. In vitro, DS-70 was pharmacologically characterized using a scintillation proximity procedure to measure its affinity for α4 β1 integrin, and its effect on cell adhesion mediated by different integrins was also evaluated. The effects of DS-70 on vascular cell adhesion molecule-1 (VCAM-1)-mediated degranulation of a human mast cell line and an eosinophilic cell line, which both express α4 β1 , and on VCAM-1-mediated phosphorylation of ERK 1/2 in Jurkat E6.1 cells were investigated. Effects of DS-70 administered in the conjunctival fornix of ovalbumin-sensitized guinea pigs were evaluated. DS-70 bound to integrin α4 β1 with nanomolar affinity, prevented the adhesion of α4 integrin-expressing cells, antagonized VCAM-1-mediated degranulation of mast cells and eosinophils and ERK 1/2 phosphorylation. Only 20% was degraded after an 8h incubation with serum. DS-70 dose-dependently reduced the clinical symptoms of allergic conjunctivitis, conjunctival α4 integrin expression and conjunctival levels of chemokines and cytokines in ovalbumin-sensitized guinea pigs. These findings highlight the role of α4 integrin in allergic conjunctivitis and suggest that DS-70 is a potential treatment for this condition.

α4 β1 integrin promotes accumulation of tissue-resident memory CD8+ T cells in salivary glands.

The salivary glands (SGs) of virus-immune mice contain substantial numbers of tissue-resident memory CD8+ T cells (TRM cells) that can provide immunity to local infections. Integrins regulate entry of activated T cells into nonlymphoid tissues but the molecules that mediate migration of virus-specific CD8+ T cells to the SGs have not yet been defined. Here, we found that polyinosinic-polycytidylic acid (poly(I:C)) strongly promoted the accumulation of P14 TCR-transgenic CD8+ TRM cells in SGs in an α4 β1 integrin-dependent manner. After infection with lymphocytic choriomeningitis virus, accumulation of P14 TRM cells in SGs and intestine but not in kidney was also α4 integrin dependent. Blockade of α4 β7 by monoclonal antibodies (mAbs) inhibited lymphocytic choriomeningitis virus-induced accumulation of P14 TRM cells in the intestine but not in SGs. In conclusion, our data reveal that α4 β1 integrin mediates CD8+ TRM accumulation in SGs and that poly(I:C) can be used to direct activated CD8+ T cells to this organ.

Peptide REDV-modified polysaccharide hydrogel with endothelial cell selectivity for the promotion of angiogenesis.

Rapid and controlled vascularization of engineered tissues remains one of the key limitations in thick tissue engineering. Although many studies have focused on improving the rapid vascularization through the immobilization of bioactive molecules, the competition in growth between endothelial cells (ECs) and other cell types is to some extent neglected. In this study, we developed a peptide GREDV-modified scaffold for selective adhesion of human umbilical vein endothelial cells (HUVECs) through the specific recognition of the REDV peptide and integrin α4 β1 . In vitro studies showed that GREDV-conjugated alginate (ALG-GREDV) improved HUVEC adhesion, migration and proliferation when compared with a non-modified group. Furthermore, ALG-GREDV exhibited a superior capability for promoting the proliferation and selective adhesion of HUVEC over that of other peptide (RGD and YIGSR) modified groups (ALG-Pep). In vivo angiogenic assays demonstrated that the ALG-GREDV scaffold induced an angiogenic potential by stimulating new vessel formation and showed the highest blood vessel density among all samples after 21 days of implanting (83.7 vessels/mm(2) ). More importantly, the blood vessel density in cambium fibrous tissue of ALG-GREDV was about 1.5 times greater than other ALG-Pep groups, indicating facilitation of ALG-GREDV on selective angiogenesis in vivo. These results demonstrated that REDV-conjugated alginate could be a useful scaffold for stimulating and inducing angiogenesis in tissue-engineered applications.

Pharmacology of AMG 181, a human anti‐α4β7 antibody that specifically alters trafficking of gut‐homing T cells

AMG 181 is a human anti-α4 β7 antibody currently in phase 1 and 2 trials in subjects with inflammatory bowel diseases. AMG 181 specifically targets the α4 β7 integrin heterodimer, blocking its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand that mediates α4 β7 T cell gut-homing. We studied the in vitro pharmacology of AMG 181, and the pharmacokinetics and pharmacodynamics of AMG 181 after single or weekly i.v. or s.c. administration in cynomolgus monkeys for up to 13 weeks. AMG 181 bound to α4 β7 , but not α4 β1 or αE β7 , and potently inhibited α4 β7 binding to MAdCAM-1 (but not vascular cell adhesion molecule-1) and thus inhibited T cell adhesion. Following single i.v. administration, AMG 181 Cmax was dose proportional from 0.01 to 80 mg·kg(-1) , while AUC increased more than dose proportionally. Following s.c. administration, dose-proportional exposure was observed with single dose ranging from 5 to 80 mg·kg(-1) and after 13 weekly doses at levels between 20 and 80 mg·kg(-1) . AMG 181 accumulated two- to threefold after 13 weekly 80 mg·kg(-1) i.v. or s.c. doses. AMG 181 had an s.c. bioavailability of 80%. The linear elimination half-life was 12 days, with a volume of distribution close to the intravascular plasma space. The mean trend for the magnitude and duration of AMG 181 exposure, immunogenicity, α4 β7 receptor occupancy and elevation in gut-homing CD4+ central memory T cell count displayed apparent correlations. AMG 181 has in vitro pharmacology, and pharmacokinetic/pharmacodynamic and safety characteristics in cynomolgus monkeys that are suitable for further investigation in humans.

Interaction of (α4 β1) integrin/VCAM‐1 induces Myosin Light Chain phosphorylation in monocytes and endothelial cells

We undertook this study to investigate the role of actin cytoskeleton and Myosin Light Chain (MLC) phosphorylation in transendothelial migration (TEM) of monocytes. Our studies demonstrated that exposure of THP‐1 to integrin‐activating antibody induced both Extracellular Signal‐Regulated Kinase (ERK) and MLC phosphorylation. These inductions were more pronounced in the presence of α4β1 ligand, CS‐1fibronectin. Exposure of THP‐1 cells to chemokines (MCP‐1, SDF‐1) induced MLC‐phosphorylation by activation of MLC kinase and inactivation of MLC phosphatase. The chemokine‐induced MLC phosphorylation was accompanied by ERK‐phosphorylation and was inhibited by pre‐exposure of the cells to the inhibitors of MLC kinase, protein kinase C and MEK/ERK. The integrin‐mediated clustering of VCAM‐1 on the endothelial cells (ECs) is mimicked by the crosslinking of antibodies to VCAM‐1. Our studies demonstrated that crosslinking of VCAM‐1 on ECs induces ERK and MLC phosphorylation. The induction of phosphorylation of MLC was attenuated when the cells were pre‐exposed to the inhibitors of MLC kinase, protein kinase C and MEK/ERK. Experiments in the transwell system showed that TEM of monocytes were reduced and enhanced after inhibition and induction of MLC phosphorylation in THP‐1 cells. Our studies demonstrated that interaction of α4β1/VCAM‐1 leads to a critical reaction in TEM of monocytes, phosphorylation of MLC.

RUNX3 regulates the activity of the CD11a and CD49d integrin gene promoters

The leukocyte integrins CD11a/CD18 (LFA-1, αL β2) and CD49d (VLA-4, α4 β1, α4 β7) mediate leukocyte transendothelial migration during immune and inflammatory responses and provides co-stimulatory signals for the activation of T lymphocytes. Our previous studies demonstrate that the CD11a gene promoter directs CD11a/CD18 integrin expression, and it depends on two overlapping sequences within the MS7 element, RUNX-110 and CEBP-100, which are recognized by RUNX and C/EBP transcription factor families, respectively. Recognition of MS7 differs in lymphoid (RUNX) and myeloid (C/EBP and RUNX) cells and its in vivo occupancy is regulated in a competitive and differentiation-dependent manner. The functional relevance of these elements are illustrated by the fact that RUNX3 overexpression leads to enhanced CD11a/CD18 levels, whereas RUNX1-ETO-expressing cells exhibit a weak/absent CD11a/CD18 integrin cell surface expression. We now provide evidence that RUNX3 also transactivates the CD49d gene promoter, and that the increased expression of CD49d mRNA and CD49d integrins on mature monocyte-derived dendritic cells correlates with an up-regulation of RUNX3 mRNA. The regulation of CD49d and CD11a integrins by RUNX3 could potentially contribute to the enhancement of transendothelial migration, antigen presentation and T cell stimulatory capabilities of mature dendritic cells.

A 3D Structure Model of Integrin α4 β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4 β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4 β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4 β1, we began constructing a structure model for the integrin α4 β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg 2+ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4 β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/ β7 ligand binding selectivity.

Differences in phosphatase modulation of α4 β1 and α5 β1 integrin-mediated adhesion and migration of B16F1 cells

It is well established that a biphasic relationship exists between the adhesive strength of beta1 integrins and their ability to mediate cell movement. Thus, cell movement increases progressively with adhesive strength, but beyond a certain point of optimal interaction, cell movement is reduced with further increases in adhesive function. The interplay between the various kinase and phosphatase activities provides the balance in beta1 integrin-mediated cell adhesion and migration. In the present study, the significance of protein tyrosine phosphatases (PTP) and ser/thr protein phosphatases (PP) in alpha4beta1 and alpha5beta1 integrin-mediated mouse melanoma B16F1 cell anchorage and migration on fibronectin was characterized using phosphatase inhibitors. At low fibronectin concentration, alpha5beta1 functioned as the predominant receptor for cell movement; a role for alpha4beta1 in B16F1 cell migration increased progressively with fibronectin concentration. Treatment of B16F1 cells with PTP inhibitors, sodium orthovanadate (Na3VO4) and phenylarsine oxide (PAO), or PP-1/2A inhibitor, okadaic acid (OA), abolished cell movement. Inhibition of cell movement by PAO and OA was associated by a reduction in the adhesive strength of alpha4beta1 and alpha5beta1. In contrast, treatment of B16F1 cells with Na3VO4 resulted in selective stimulation of the adhesive function of alpha5beta1, but not alpha4beta1. Therefore, our results demonstrate that (i) both PTP and PP-1/2A have roles in cell movement, (ii) modulation of cell movement by PTP and PP-1/2A may involve either a stimulation or reduction of beta1 integrin adhesive strength, and (iii) distinct phosphatase-mediated signaling pathways for differential regulation of the various beta1 integrins exist.

Differences in phosphatase modulation of α4 β1 and α5 β1 integrin-mediated adhesion and migration of B16F1 cells

Differences in phosphatase modulation of α4 β1 and α5 β1 integrin-mediated adhesion and migration of B16F1 cells

Cell Adhesion via Murine α4 Human β1 Integrin Chimera on Transfected K562 Cells to Endothelial Cells

To evaluate the property of binding activity of α4β1 integrin in cell–cell interaction, we newly established a cell line, α4mK562, by transfecting cDNA of murine integrin α4 subunit into human erythroleukemic K562 cells. α4mK562 transfectant expressed both murine α4 and human β1 subunit, which generated a functional heterodimer. α4mK562 cells more efficiently bound to murine endothelial cell lines and recombinant human TNFα (rhTNF)-treated human umbilical vein endothelial cells (HUVEC) than the parental K562 cells. These adhesion resulted from the interaction between α4β1 and VCAM-1. Interestingly, treatment with mAb against human β1 (4B4 clone), which has been known as inhibitory mAb, enhanced binding of α4mK562 cells to rhTNF-treated HUVEC but not to murine endothelial cells. This increase in binding induced by 4B4 mAb was completely inhibited by another mAb against human β1 (mAb13), but only partially by anti-α4 mAb (PS/2). The increase in binding induced by 4B4 mAb was also abolished by metabolic inhibitors, indicating that the increased binding is energy dependent. These observations suggest that the binding of 4B4 mAb to the chimeric α4β1 induces an unique outside-in signaling and enhances the specific binding of α4mK562 cells to rhTNF-treated HUVEC.