title: Role of CL-100, a dual specificity phosphatase, in thrombin-induced endothelial cell activation.
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."
authors.affiliation: Department of Cell Biology, Cleveland Clinic Foundation and Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, Ohio 44195, USA.
authors.forename: Unni M
authors.initials: UM
authors.lastname: Chandrasekharan
authors.forename: Lin
authors.initials: L
authors.lastname: Yang
authors.forename: Alicia
authors.initials: A
authors.lastname: Walters
authors.forename: Philip
authors.initials: P
authors.lastname: Howe
authors.forename: Paul E
authors.initials: PE
authors.lastname: DiCorleto
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000096:biosynthesis*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D042783:Endothelial Cells / Q000201:enzymology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology*; D005786:Gene Expression Regulation; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis*; D047468:Immunoprecipitation; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis*; D054645:Protein Phosphatase 1; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D017874:Immediate-Early Proteins; D016376:Oligonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D003609:Dactinomycin; D019061:src-Family Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D013917:Thrombin