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Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion - PubMed

  • ️Sat Jan 01 2011

Nitric oxide/cGMP pathway signaling actively down-regulates α4β1-integrin affinity: an unexpected mechanism for inducing cell de-adhesion

Alexandre Chigaev et al. BMC Immunol. 2011.

Abstract

Background: Integrin 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.

Results: Using 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.

Conclusions: We 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.

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Figures

Figure 1
Figure 1

NO/cGMP signaling cascade and small molecules that modulate this pathway, used in the current study. Nitric oxide, generated by nitric oxide synthase, diffuses across the plasma membrane and through the cytoplasm. In leukocytes NO reacts with the active site of guanylyl cyclase (guanylate GC), and stimulates the production of the intracellular mediator cyclic GMP (cGMP). Next, cGMP interacts with the cGMP-dependent protein kinase (PKG), which phosphorylates multiple substrates, and participates in signal propagation. Cyclic nucleotide phosphodiesterases (PDEs, not shown) can rapidly hydrolyze cGMP and terminate signal propagation. The NO/cGMP signaling pathway can be specifically targeted using small molecules. The nitric oxide donor provides an exogenous source of NO. The activator of soluble guanylyl cyclase binds to GC, and induces enzyme activation in the absence of NO. The cell permeable analog of cGMP diffuses across the plasma membrane, and thus, activates cGMP-dependent signaling.

Figure 2
Figure 2

Effect of nitric oxide addition on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gαi-coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of DEA-NONOate (nitric oxide donor). Dashed line indicates the non-specific binding of the LDV-FITC probe determined using an excess of unlabelled LDV competitor (as shown in Fig. 2D,E). B, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). Rapid and reversible binding of the probe reflects the VLA-4 affinity change [14]. C, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). D, The experiment involved sequential addition of the DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM). Excess unlabelled competitor LDV (1 μM) is added at the end of the experiment to determine the non-specific binding of the probe (panels D, and E). E, The experiment involved sequential addition of DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM) (arrows). According to the unpaired t test, the means are significantly different (p<0.05) at the peak of activation (marked on panels D and E as “*”), and at the steady state (marked on panels B-E as “**”). Experiments shown in the different panels were performed using different instruments, and therefore MCF values are not identical.

Figure 3
Figure 3

Effect of guanylyl cyclase activator on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gαi-coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of the Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of BAY 41-2272 (guanylyl cyclase activator). B, The experiment involved sequential addition of the BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL12/SDF-1 (12 nM). C, The experiment involved sequential addition of BAY 41-2272 (100 μM, guanylyl cyclase activator) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), and CXCL8/IL-8 (20 nM). The means are significantly different (p<0.05) at the peak of activation (marked on panels B and C as “*”), and at the steady state (marked in panels B and C as “**”). D, Kinetic analysis of binding and dissociation of the LDV-FITC probe. Cells were sequentially treated with the LDV-FITC probe (25 nM, near saturation), the Gαi-coupled receptor ligand (fMLFF, 100 nM), BAY 41-2272 (guanylyl cyclase activator, 50 μM). At time points indicated by arrows, cells were treated with excess unlabeled LDV containing small molecule (2 μM), and the dissociation of the fluorescent molecule was followed. Dissociation rate constants (koff) were obtained by fitting dissociation curves to a single exponential decay equation. E, Dissociation rate values, obtained in experiments analogous to panel D, summarized as a bar graph showing mean and SEM (n=4). Colors of the dissociation curves in panel D and bars on panel E are matching. The difference between koffs for “resting” and “fMLFF activated”, and between “fMLFF activated” and “fMLFF activated and treated with BAY 41-2272” is statistically significant (P < 0.05) as calculated by one-way ANOVA.

Figure 4
Figure 4

Effect of the cell permeable analog of cGMP on binding and dissociation of the LDV-FITC probe on U937 cells stably transfected with the non-desensitizing mutant of FPR. LDV-FITC probe binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant of FPR plotted as mean channel fluorescence (MCF) versus time. The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of the Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of dibutyrylguanosine 3',5'-cyclic monophosphate (cell permeable cGMP analog) (arrows). Control cells were treated with vehicle. The MCF value corresponding to cell autofluorescence is indicated by the horizontal arrow. Dashed line indicates the non-specific binding of the LDV-FITC probe determined using excess unlabelled LDV competitor (as shown on Figure 3B). Rapid and reversible binding of the probe reflects the VLA-4 affinity change [14]. Curves are means out of two independent determinations calculated on a point-by-point basis (n = 2).

Figure 5
Figure 5

Changes in cell adhesion between U937 FPR (ΔST) and VCAM-1-transfected B78H1 cells in the resting state and in response to receptor stimulation. Real-time aggregation experiments were conducted as described under "Methods". U937/ΔST FPR stably transfected cells, which constitutively express VLA-4, were labeled with red fluorescent dye, and B78H1/VCAM-1 transfectants were stained with green fluorescent dye. Labeled cells were preincubated for 10 min at 37°C with fMLFF only (100 nM, activated control), DMSO (vehicle, resting cells control), or with fMLFF and DEA-NONOate (250 μM, nitric oxide donor) in a manner analogous to the experiment showed in Figure 2A. Next, cells were mixed and real-time cell aggregation (red and green double positive events) was followed. To determine the level of VLA-4 dependent cell aggregation, 6 min after cell mixing, excess unlabelled VLA-4 specific ligand was added (arrow, LDV block, 2 μM). This induced rapid cellular disaggregation to the level of non-specific binding. A representative experiment out of three experiments is shown.

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