ABSTRACT
A member of a new subfamily of G protein-coupled receptors, protease-activated receptor 2 (PAR2), is highly expressed on endothelial cells and plays an important role in inflammation. The purpose of this study was to determine the molecular mechanism used by PAR2 to induce IL-8 production and thereby mediate cell adhesion. We observed that PAR2-activating peptide (PAR2-AP) significantly increase peripheral blood mononuclear cells adhere to endothelial cells. Both PAR2-AP and the endogenous PAR2 activator trypsin caused concentration- and time-dependent increase in endothelial IL-8 production, and this effect was concentration dependently and selectively attenuated by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Western blotting analysis showed that PAR2-AP induced phosphorylation of p38 MAPK and its upstream protein kinase MAPK kinase 3/6 (MKK3/6) in a time-dependent manner. Using reverse-transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, PAR2-AP was found to cause an increase in IL-8 mRNA expression and its transcription factor activating transcription factor 2, respectively,. As expected, these signals were suppressed by SB203580 in a concentration-dependent manner. Furthermore, introduction of dominant-negative vectors targeting p38 MAPK, MKK3, and MKK6 abolished PAR2-AP-mediated IL-8 production and cell adhesion function. In conclusion, PAR2 via p38 MAPK signaling regulates IL-8 production and thereby mediates cell adhesion.
Subject(s)
Cell Adhesion/drug effects , Interleukin-8/metabolism , Leukocytes/cytology , Receptor, PAR-2/physiology , Signal Transduction/physiology , p38 Mitogen-Activated Protein Kinases/metabolism , Activating Transcription Factors , Blotting, Western , Cells, Cultured , DNA, Complementary , Flavonoids/pharmacology , Flow Cytometry , Humans , Imidazoles/pharmacology , Indoles/pharmacology , Integrin alpha4beta1/metabolism , Intercellular Adhesion Molecule-1/metabolism , Leukocytes/drug effects , Lymphocyte Function-Associated Antigen-1/metabolism , Macrophage-1 Antigen/metabolism , Pyridines/pharmacology , Receptor, PAR-2/agonists , Receptor, PAR-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Trypsin/pharmacology , Vascular Cell Adhesion Molecule-1/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
OBJECTIVE: Nitric oxide signaling pathways are of central importance in both the maintenance of vascular homeostasis and the progression of vascular disease. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, the authors investigated whether YC-1, a soluble guanylyl cyclase (sGC) activator, regulates apoptosis in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: Sodium nitroprusside (SNP) (1 mM) induced cGMP (guanosine 3':5'-cyclic monophosphate)-independent apoptosis in rat vascular smooth muscle cells using MTT assay and TUNEL-reaction techniques. Furthermore, sodium nitroprusside induced apoptosis via Bcl-2 down-regulation, cytochrome c release reaction, and caspase-3 activation by Western blotting analysis and enzymatic assay methods. YC-1 abolished these apoptotic signaling cascades and prevented apoptosis through a cGMP-involved pathway, and phosphatidylinositol (PI) 3-kinase behaved a downstream event in this pathway. CONCLUSIONS: These results suggest that YC-1 inhibits sodium nitroprusside-induced vascular smooth muscle cells apoptosis via a cGMP- and phosphatidylinositol 3-kinase-involved inhibition on Bcl-2 down-regulation/cytochrome c release/caspase-3 activation cascades. The ability of YC-1 to prevent smooth muscle cell apoptosis may play an important role in blocking lesion formation at sites of vascular injury.
Subject(s)
Guanylate Cyclase/metabolism , Indazoles/pharmacology , Muscle, Smooth, Vascular/metabolism , Nitroprusside/pharmacology , Receptors, Atrial Natriuretic Factor/metabolism , Vasodilator Agents/pharmacology , Animals , Apoptosis/drug effects , Caspase 3 , Caspases/metabolism , Cells, Cultured , Cytochromes c/metabolism , Depression, Chemical , Enzyme Activation/drug effects , Gene Expression/drug effects , Genes, bcl-2 , Muscle, Smooth, Vascular/drug effects , Rats , Rats, Wistar , Reactive Oxygen Species/analysisABSTRACT
An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population.
