Phospholipase C, calcium, and calmodulin are critical for alpha4beta1 integrin affinity up-regulation and monocyte arrest triggered by chemoattractants.
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.
Hyduk
Sharon J
SJ
Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada.
Chan
Jason R
JR
Duffy
Stewart T
ST
Chen
Mian
M
Peterson
Mark D
MD
Waddell
Thomas K
TK
Digby
Genevieve C
GC
Szaszi
Katalin
K
Kapus
Andras
A
Cybulsky
Myron I
MI
D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002147:Calmodulin / Q000502:physiology*; D002460:Cell Line; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000494:pharmacology; D002630:Chemotactic Factors / Q000494:pharmacology*; D002633:Chemotaxis / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000502:physiology; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000187:drug effects*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D010671:Phenylurea Compounds / Q000378:metabolism* / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D044042:Receptors, Formyl Peptide / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology*; D020298:U937 Cells / Q000166:cytology / Q000187:drug effects; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*
C517048:4-((N'-2-methylphenyl)ureido)phenylalanyl-leucyl-alpha-aspartyl-valyl-prolyl-alanyl-alanyl-lysine; C515255:CXCL12 protein, human; D002147:Calmodulin; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D039041:Integrin alpha4beta1; D009842:Oligopeptides; D010671:Phenylurea Compounds; D044042:Receptors, Formyl Peptide; D043562:Receptors, G-Protein-Coupled; D019010:Vascular Cell Adhesion Molecule-1; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019284:Thapsigargin; D019869:Phosphatidylinositol 3-Kinases; D010738:Type C Phospholipases; D002118:Calcium