Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation

J Biol Chem. 2004 Nov 5;279(45):46678-85. doi: 10.1074/jbc.M406441200. Epub 2004 Aug 31.

Abstract

Using a cDNA microarray screening approach, we have identified seven novel thrombin-responsive genes in human umbilical vein endothelial cells that were verifiable by Northern blot analysis. Among them CL-100, a dual-specificity phosphatase also known as MAP kinase phosphatase-1 (MKP-1), showed greatest induction by thrombin. Steady-state levels of CL-100 mRNA induction by thrombin peaked at 1 h and declined rapidly (t1/2 approximately 45 min). Induction by thrombin was protease-activated receptor-1-mediated, protein synthesis-independent, and transcriptionally regulated. Metabolic labeling followed by immunoprecipitation verified that the thrombin-induced CL-100 mRNA was translated into protein. We found that both Src-kinase and p42/p44 ERK activity are critical for thrombin-induced CL-100 expression, whereas phosphatidylinositol 3-kinase and protein kinase C activity were not required. Antisense-mediated inhibition of CL-100 was shown to prolong thrombin-induced ERK activity in endothelial cells, concomitant with an inhibition in thrombin-induced PDGF-A (platelet-derived growth factor A) and PDGF-B gene expression and an up-regulation in thrombin-induced VCAM-1 and E-selectin gene expression. Inhibition of ERK activation by PD98059 in endothelial cells was shown to potentiate thrombin-induced expression of PDGF-B (approximately 3-fold) while inhibiting thrombin-induced VCAM-1 and E-selectin gene expression by 60 and 70%, respectively. These results suggested that induced expression of the CL-100 phosphatase and its subsequent regulation of ERK activity play a key regulatory role in the thrombin signaling pathway and in the transcriptional regulation of pathologically important "endothelial cell activation genes."

Publication types

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

MeSH terms

  • Blotting, Northern
  • Blotting, Western
  • Cell Cycle Proteins / biosynthesis*
  • Cell Nucleus / metabolism
  • Cells, Cultured
  • DNA, Complementary / metabolism
  • Dactinomycin / pharmacology
  • Dose-Response Relationship, Drug
  • Dual Specificity Phosphatase 1
  • Endothelial Cells / enzymology*
  • Endothelial Cells / metabolism
  • Endothelium, Vascular / cytology*
  • Gene Expression Regulation
  • Humans
  • Immediate-Early Proteins / biosynthesis*
  • Immunoprecipitation
  • Kinetics
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Oligonucleotides, Antisense / pharmacology
  • Phosphoprotein Phosphatases / biosynthesis*
  • Protein Phosphatase 1
  • Protein Synthesis Inhibitors / pharmacology
  • Protein Tyrosine Phosphatases / biosynthesis*
  • RNA, Messenger / metabolism
  • Receptor, PAR-1 / metabolism
  • Signal Transduction
  • Thrombin / metabolism*
  • Time Factors
  • Transcription, Genetic
  • Transcriptional Activation
  • Umbilical Veins / cytology
  • Umbilical Veins / metabolism
  • src-Family Kinases / metabolism

Substances

  • Cell Cycle Proteins
  • DNA, Complementary
  • Immediate-Early Proteins
  • Oligonucleotides, Antisense
  • Protein Synthesis Inhibitors
  • RNA, Messenger
  • Receptor, PAR-1
  • Dactinomycin
  • src-Family Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1
  • DUSP1 protein, human
  • Dual Specificity Phosphatase 1
  • Protein Tyrosine Phosphatases
  • Thrombin