Cobalt chloride-induced signaling in endothelium leading to the augmented adherence of sickle red blood cells and transendothelial migration of monocyte-like HL-60 cells is blocked by PAF-receptor antagonist

J Cell Physiol. 1999 Apr;179(1):67-78. doi: 10.1002/(SICI)1097-4652(199904)179:1<67::AID-JCP9>3.0.CO;2-0.

Abstract

In response to hypoxia, sickle red blood cells (SS RBC) and leukocytes exhibit increased adherence to the vascular endothelium, while diapedesis of leukocytes through the blood vessel increases. However, the cellular signaling pathway(s) caused by hypoxia is poorly understood. We utilized CoCl2 as a mimetic molecule for hypoxia to study cellular signaling pathways. We found that in human umbilical vein endothelial cells (HUVEC), CoCl2 at 2 mM concentration induced the surface expression of a subset of CAMs (VCAM-1) and activation of transcription factor NF-kappaB in the nuclear extracts of HUVEC. Furthermore, CoCl2 also caused time-dependent tyrosine phosphorylation of mitogen-activated protein (MAP) kinase isoform ERK2 without significantly affecting ERK1, indicating ERK2 is the preferred substrate for upstream kinase of the MAPK pathway. Inhibitors of MAP kinase (PD98059) or platelet-activating factor (PAF)- receptor antagonist (CV3988) inhibited the CoCl2-induced NF-kappaB activation and VCAM-1 expression. Augmented expression of VCAM-1 led to increased SS RBC adhesion, inhibitable by a VCAM-1 antibody. Additionally, CoCl2 caused a two- to threefold increase in the rate of transendothelial migration of monocyte-like HL-60 cells and a twentyfold increase in phosphorylation of platelet endothelial cell adhesion molecules (PECAM-1). The transendothelial migration of monocytes was inhibited by an antibody to PECAM-1. Both phosphorylation of PECAM-1 and transendothelial migration of monocytes in response to CoCl2 were inhibited by protein kinase inhibitor (GF109203X) and augmented by protein phosphatase inhibitor (Calyculin A). Our data suggests that CoCl2-induced cellular signals directing increased expression of VCAM-1 in HUVEC involve downstream activation of MAP kinase and NF-kappaB, while the phosphorylation of PECAM-1 occurs as a result of activation of PKC. We conclude that PAF-receptor antagonist inhibits the CoCl2- or hypoxia-induced increase in the adhesion of SS RBC, PECAM-1 phosphorylation, and the concomitant transendothelial migration of monocytes.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Anemia, Sickle Cell / pathology*
  • Base Sequence
  • Calcium-Calmodulin-Dependent Protein Kinases / antagonists & inhibitors
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinases / physiology
  • Cell Adhesion / drug effects
  • Cell Movement / drug effects
  • Cells, Cultured
  • Cobalt / pharmacology*
  • E-Selectin / biosynthesis
  • E-Selectin / genetics
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / pathology
  • Enzyme Inhibitors / pharmacology
  • Erythrocytes, Abnormal / drug effects*
  • Erythrocytes, Abnormal / pathology
  • Flavonoids / pharmacology
  • HL-60 Cells / drug effects*
  • Humans
  • Hypoxia / metabolism
  • Indoles / pharmacology
  • Intercellular Adhesion Molecule-1 / biosynthesis
  • Intercellular Adhesion Molecule-1 / genetics
  • Maleimides / pharmacology
  • Marine Toxins
  • Mitogen-Activated Protein Kinase 1
  • Molecular Sequence Data
  • NF-kappa B / metabolism
  • Oxazoles / pharmacology
  • Phospholipid Ethers / pharmacology*
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphorylation / drug effects
  • Platelet Endothelial Cell Adhesion Molecule-1 / metabolism
  • Platelet Membrane Glycoproteins / antagonists & inhibitors*
  • Protein Processing, Post-Translational / drug effects
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • Receptors, Cell Surface*
  • Receptors, G-Protein-Coupled*
  • Signal Transduction / drug effects*
  • Transcription Factor AP-1 / metabolism
  • Umbilical Veins
  • Vascular Cell Adhesion Molecule-1 / biosynthesis
  • Vascular Cell Adhesion Molecule-1 / genetics

Substances

  • E-Selectin
  • Enzyme Inhibitors
  • Flavonoids
  • Indoles
  • Maleimides
  • Marine Toxins
  • NF-kappa B
  • Oxazoles
  • Phospholipid Ethers
  • Platelet Endothelial Cell Adhesion Molecule-1
  • Platelet Membrane Glycoproteins
  • RNA, Messenger
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Transcription Factor AP-1
  • Vascular Cell Adhesion Molecule-1
  • platelet activating factor receptor
  • Intercellular Adhesion Molecule-1
  • Cobalt
  • calyculin A
  • CV 3988
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 1
  • Phosphoprotein Phosphatases
  • cobaltous chloride
  • bisindolylmaleimide I
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one