Thrombin modulates the expression of a set of genes including thrombospondin-1 in human microvascular endothelial cells.
Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.
McLaughlin
Joseph N
JN
Department of Pharmacology, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 444 Robinson Research Building, 23rd Avenue South at Pierce, Nashville, TN 37232 , USA. joseph.mclaughlin@vanderbilt.edu
Mazzoni
Maria R
MR
Cleator
John H
JH
Earls
Laurie
L
Perdigoto
Ana Luisa
AL
Brooks
Joshua D
JD
Muldowney
James A S
JA
Vaughan
Douglas E
DE
Hamm
Heidi E
HE
D000244:Adenosine Diphosphate / Q000737:chemistry; D000465:Algorithms; D000577:Amides / Q000494:pharmacology; D017209:Apoptosis; D002478:Cells, Cultured; D016000:Cluster Analysis; D003470:Culture Media; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008833:Microcirculation / Q000378:metabolism*; D008954:Models, Biological; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D010455:Peptides / Q000737:chemistry; D037342:Pertussis Toxin / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D019700:Thrombospondin 1 / Q000096:biosynthesis*; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation
D000577:Amides; D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D007211:Indoles; D008301:Maleimides; D010455:Peptides; D011725:Pyridines; D044463:Receptor, PAR-1; D019700:Thrombospondin 1; C108830:Y 27632; D000244:Adenosine Diphosphate; D012313:RNA; D037342:Pertussis Toxin; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C070515:bisindolylmaleimide I