Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Calcium / physiology*
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Calcium Signaling / drug effects
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Calcium Signaling / physiology
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Calmodulin / physiology*
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Cell Line
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Chemokine CXCL12
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Chemokines, CXC / pharmacology
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Chemotactic Factors / pharmacology*
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Chemotaxis / drug effects
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Endothelial Cells / cytology
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Endothelium, Vascular / cytology
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Humans
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Hydrogen-Ion Concentration
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Inositol 1,4,5-Trisphosphate Receptors / physiology
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Integrin alpha4beta1 / metabolism*
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Monocytes / cytology
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Monocytes / drug effects*
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N-Formylmethionine Leucyl-Phenylalanine / pharmacology
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Oligopeptides / metabolism*
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Oligopeptides / pharmacology
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Phenylurea Compounds / metabolism*
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Phenylurea Compounds / pharmacology
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Phosphatidylinositol 3-Kinases / physiology
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Receptors, Formyl Peptide / drug effects
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Receptors, Formyl Peptide / genetics
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Receptors, Formyl Peptide / physiology
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Receptors, G-Protein-Coupled / physiology*
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Thapsigargin / pharmacology
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Type C Phospholipases / physiology*
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U937 Cells / cytology
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U937 Cells / drug effects
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Vascular Cell Adhesion Molecule-1 / metabolism*
Substances
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4-((N'-2-methylphenyl)ureido)phenylalanyl-leucyl-alpha-aspartyl-valyl-prolyl-alanyl-alanyl-lysine
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CXCL12 protein, human
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Calmodulin
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Chemokine CXCL12
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Chemokines, CXC
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Chemotactic Factors
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Inositol 1,4,5-Trisphosphate Receptors
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Integrin alpha4beta1
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Oligopeptides
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Phenylurea Compounds
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Receptors, Formyl Peptide
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Receptors, G-Protein-Coupled
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Vascular Cell Adhesion Molecule-1
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N-Formylmethionine Leucyl-Phenylalanine
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Thapsigargin
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Phosphatidylinositol 3-Kinases
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Type C Phospholipases
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Calcium