title: Modulation of endotoxin-induced endothelial function by calcium/calmodulin-dependent protein kinase.
abstract: Endothelial cells facilitate sepsis-induced neutrophil adherence through the production of adhesion molecules and proinflammatory cytokines. The production of these factors requires coordinated intracellular inflammatory signaling. Recently, patients prone to sepsis-induced complications have been shown to have derangements in intracellular calcium and potentially calcium/calmodulin-dependent protein kinase (CaMK) activity, but the impact of these impairments is unknown. Human umbilical vein endothelial vein endothelial cells (HUVECs) were exposed to lipopolysaccharide (LPS) for various periods of time. Select HUVECs were pretreated with an inhibitor of CaMK II, KN62. Total cellular and nuclear proteins were extracted and analyzed for various components of the Toll-mediated signal cascade. Neutrophil adhesion was assayed fluorometrically using calcein-labeled neutrophils on treated HUVECs. LPS stimulation led to mitogen-activated protein kinase activation and translocation of activator protein-1 (AP-1) and nuclear factor (NF)-kappaB. CaMK blockade inhibited LPS induced ERK 1/2 and JNK but enhanced p38 activity. This selective MAPK inhibition was associated with a reduction in AP-1 activity, with no affect on NF-kappaB activity. Associated with this altered cell signaling was increased ICAM-1 production and enhanced neutrophil adhesion. Altered CaMK activity resulted in dysregulated mitogen-activated protein kinase signaling, demonstrated by reduced ERK 1/2 and JNK activity but enhanced p38 activity. This altered signaling is associated with reduced AP-1 activation and unaffected NF-kappaB activation. Neutrophil adhesion, however, is enhanced presumably through increased ICAM-1 production. Therefore, CaMK inhibition of endothelial cells, characteristic of sustained increases in intracellular calcium, appears to result in a dysregulated proadhesive phenotype.
authors.affiliation: Department of Surgery, University of Cincinnati, Cincinnati, Ohio 45267, USA. cuschij@ucmail.uc.edu
authors.forename: Joseph
authors.initials: J
authors.lastname: Cuschieri
authors.forename: David
authors.initials: D
authors.lastname: Gourlay
authors.forename: Iris
authors.initials: I
authors.lastname: Garcia
authors.forename: Sandra
authors.initials: S
authors.lastname: Jelacic
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Maier
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002448:Cell Adhesion; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D018805:Sepsis; D013997:Time Factors; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D004731:Endotoxins; D005452:Fluoresceins; D008070:Lipopolysaccharides; D018808:Transcription Factor AP-1; D018799:Intercellular Adhesion Molecule-1; D004247:DNA; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D002118:Calcium; C007740:fluorexon