Search

Atherosclerotic lesions form at distinct sites in the arterial tree, suggesting that hemodynamic forces influence the initiation of atherogenesis. If NF-[Kappa]B plays a role in atherogenesis, then the activation of this signal transduction pathway in arterial endothelium should show topographic variation. The expression of NF-[Kappa]B/I[Kappa]B components and NF-[Kappa]B activation was evaluated by specific antibody staining, en face confocal microscopy, and image analysis of endothelium in regions of mouse proximal aorta with high and low probability (HP and LP) for atherosclerotic lesion development. In control C57BL/6 mice, expression levels of p65, I[Kappa]B[Alpha], and I[Kappa]B[Beta] were 5- to 18-fold higher in the HP region, yet NF-[Kappa]B was activated in a minority of endothelial cells. This suggested that NF-[Kappa]B signal transduction was primed for activation in HP regions on encountering an activation stimulus. Lipopolysaccharide treatment or feeding low-density lipoprotein receptor knockout mice an atherogenic diet resulted in NF-[Kappa]B activation and up-regulated expression of NF-[Kappa]B-inducible genes predominantly in HP region endothelium. Preferential regional activation of endothelial NF-[Kappa]B by systemic stimuli, including hypercholesterolemia, may contribute to the localization of atherosclerotic lesions at sites with high steadystate expression levels of NF-[Kappa]B/I[Kappa]B components. atherosclerosis | p65 | I[Kappa]B[Alpha] | VCAM-1 | E-selectin

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α4β1 integrin affinity and consequent adhesive events. Affinity upregulation of α4β1 integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α4β1 expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α4β1 and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α4β1 after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α4β1-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement. [ABSTRACT FROM AUTHOR]

The α chain of α4β1 integrins is unique in that it can bind directly to paxillin. Paxillin associates only with high affinity α4β1 that have a dephosphorylated serine-988 residue in the α4 cytoplasmic tail, which suggests that paxillin regulates leukocyte arrest and migration. On resting U937 cells, α4β1 is predominantly in a low affinity state. Inside-out signaling through GPCRs induces Ca2+-calmodulin activation, which is required for up-regulation of α4β1 affinity. Calmodulin activates calcineurin; hence we investigated whether calcineurin is involved in regulating paxillin association with high affinity α4β1. Using α4 immunoprecipitations, we observed that inhibition of calcineurin with cyclosporin A (CsA) or FK506 (tacrolimus), blocked formyl peptide (FP)-induced paxillin association in a dose-dependent manner. However, these inhibitors did not affect FP-induced α4β1 affinity up-regulation, determined by soluble ligand binding or FP- or SDF-1α-triggered U937 cell arrest and accumulation on VCAM-1-coated surfaces. Collectively, these data demonstrate that activation of calcineurin is required for the association of paxillin with high affinity, α4β1 integrins, but not for affinity modulation or leukocyte arrest. Future studies will focus on the role of paxillin in subsequent steps of emigration, such as leukocyte spreading and transendothelial migration. [ABSTRACT FROM AUTHOR]

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.

Monocyte recruitment or emigration to tissues is an essential component of host defense in both acute and chronic inflammatory responses. Sequential molecular interactions mediate a cascade of tethering, rolling, arrest, stable adhesion, and intravascular crawling that culminates in monocyte diapedesis across the vascular endothelium and migration through the basement membrane of postcapillary venules. Integrins are complex adhesion and signaling molecules. Dynamic alterations in their conformation and distribution on the monocyte cell surface are required for many steps of monocyte emigration. Intracellular signaling initiated by chemokine receptors induces conformational changes in integrins that upregulate their affinity for ligands, and this is essential for monocyte arrest. This review focuses on the activation of monocyte alpha4beta1 integrins by endothelial chemokines, which is required for the arrest of monocytes rolling on vascular cell adhesion molecule 1 under shear flow. Using soluble ligand-binding assays and adhesion assays in parallel-plate flow chambers, critical signaling mediators in chemokine-induced alpha4beta1 integrin affinity upregulation and monocyte arrest have been identified, including phospholipase C, calcium, and calmodulin.

During inflammation, monocytes roll on activated endothelium and arrest after stimulation by proteoglycan-bound chemokines and other chemoattractants. We investigated signaling pathways downstream of G protein-coupled receptors (GPCRs) that are relevant to alpha4beta1 integrin affinity up-regulation using formyl peptide receptor-transfected U937 cells stimulated with fMLP or stromal-derived factor-1alpha and human peripheral blood monocytes stimulated with multiple chemokines or chemoattractants. The up-regulation of soluble LDV peptide or vascular cell adhesion molecule-1 (VCAM-1) binding by these stimuli was critically dependent on activation of phospholipase C (PLC), inositol 1,4,5-triphosphate receptors, increased intracellular calcium, influx of extracellular calcium, and calmodulin, suggesting that this signaling pathway is required for alpha4 integrins to assume a high-affinity conformation. In fact, a rise in intracellular calcium following treatment with thapsigargin or ionomycin was sufficient to induce binding of ligand. Blockade of p44/42 and p38 mitogen-activated protein (MAP) kinases, phosphoinositide 3-kinase, or protein kinase C (PKC) signaling did not inhibit chemoattractant-induced LDV or VCAM-1 binding. However, activation of PKC by phorbol ester up-regulated alpha4beta1 affinity with kinetics distinct from those of GPCR signaling. A critical role for PLC and calmodulin was also established for leukocyte arrest and adhesion strengthening.

Dynamic regulation of beta(2) integrin-dependent adhesion is critical for a wide array of T cell functions. We previously showed that binding of high-affinity alpha(4)beta(1) integrins to VCAM-1 strengthens alpha(L)beta(2) integrin-mediated adhesion to ICAM-1. In this study, we compared beta(2) integrin-mediated adhesion of T cells to ICAM-1 under two different functional contexts: alpha(4) integrin signaling during emigration from blood into tissues and CD3 signaling during adhesion to APCs and target cells. Cross-linking either alpha(4) integrin or CD3 on Jurkat T cells induced adhesion to ICAM-1 of comparable strength. Adhesion was dependent on phosphatidylinositol (PI) 3-kinase but not p44/42 mitogen-activated protein kinase (extracellular regulated kinase 1/2), because it was inhibited by wortmannin and LY294002 but not U0126. These data suggest that PI 3-kinase is a ubiquitous regulator of beta(2) integrin-mediated adhesion. A distinct morphological change consisting of Jurkat cell spreading and extension of filopodia was induced by alpha(4) integrin signaling. In contrast, CD3 induced radial rings of cortical actin polymerization. Inhibitors of PI 3-kinase and extracellular regulated kinase 1/2 did not affect alpha(4) integrin-induced rearrangement of the actin cytoskeleton, but treatment with ionomycin, a Ca(2+) ionophore, modulated cell morphology by reducing filopodia and promoting lamellipodia formation. Qualitatively similar morphological and adhesive changes to those observed with Jurkat cells were observed following alpha(4) integrin or CD3 stimulation of human peripheral blood T cells.