Abstract
BackgroundIntegrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule, which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α4β1-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic progenitors, stem cells, hematopoietic cancer cells, and others. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gαi-coupled GPCRs and down-regulated by Gαs-coupled GPCRs. However, other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. The goal of the current report was to study the effect of nitric oxide/cGMP signaling pathway on VLA-4 conformational regulation.ResultsUsing fluorescent ligand binding to evaluate the integrin activation state on live cells in real-time, we show that several small molecules, which specifically modulate nitric oxide/cGMP signaling pathway, as well as a cell permeable cGMP analog, can rapidly down-modulate binding of a VLA-4 specific ligand on cells pre-activated through three Gαi-coupled receptors: wild type CXCR4, CXCR2 (IL-8RB), and a non-desensitizing mutant of formyl peptide receptor (FPR ΔST). Upon signaling, we detected rapid changes in the ligand dissociation rate. The dissociation rate after inside-out integrin de-activation was similar to the rate for resting cells. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by nitric oxide had a statistically significant effect on real-time cell aggregation.ConclusionsWe conclude that nitric oxide/cGMP signaling pathway can rapidly down-modulate the affinity state of the VLA-4 binding pocket, especially under the condition of sustained Gαi-coupled GPCR signaling, generated by a non-desensitizing receptor mutant. This suggests a fundamental role of this pathway in de-activation of integrin-dependent cell adhesion.
Highlights
Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells
We found that the addition of a nitric oxide donor can rapidly induce dissociation of the (very late antigen 4 (VLA-4) specific ligand after cellular activation by any of three (guanine nucleotide binding protein coupled receptor) (GPCR) (CXCR4, CXCR2, and (formyl peptide receptor 1) (FPR))
It is composed of soluble guanylyl cyclase (GC) that serves as an intracellular receptor for nitric oxide (Figure 1)
Summary
Integrin activation in response to inside-out signaling serves as the basis for rapid leukocyte arrest on endothelium, migration, and mobilization of immune cells. VLA-4 conformation is rapidly up-regulated by inside-out signaling through Gai-coupled GPCRs and down-regulated by Gas-coupled GPCRs. other signaling pathways, which include nitric oxide-dependent signaling, have been implicated in the regulation of cell adhesion. Integrins are ubiquitous cell adhesion molecules that play an essential role in the regulation of leukocyte traffic, stem cell mobilization and homing, immune responses, development, hemostasis, and cancer [1,2,3]. On the cell surface at rest, a variety of integrin exhibit a non-adhesive inactive state and multiple signaling cascades are capable of rapidly and reversibly regulating integrin-dependent cell adhesion. This regulation is achieved without altering the integrin expression level. Interfering with integrin activation by targeting “the final steps of activation process” is envisioned as a novel approach for therapeutic intervention in integrin-related pathologies [13]
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