RESUMO
The serine protease thrombin is formed at sites of coagulation and inflammation and has been shown to have important proinflammatory cellular effects relevant to the pathogenesis of periodontal disease. Thrombin acts via specific cell surface receptors termed protease-activated receptor-1 (PAR-1) and PAR-3, which have a distinctive method of activation. Proteolytic cleavage of the extracellular domain by thrombin reveals a hidden amino terminus which then acts as a "tethered ligand". A short synthetic peptide (SFLLRN) can also mimic the tethered ligand and activate PAR-1 but not PAR-3. Also, a trypsin-sensitive receptor termed PAR-2 has been described which is activated by the PAR-1 activating peptide SFLLRN. Here we show conclusively by flow cytometric and Northern blot analysis that human gingival fibroblasts (HGF) express PAR-1 but not PAR-2. In functional studies we also show that thrombin and SFLLRN stimulated increased expression of mRNA encoding nuclear transcription factor NF-IL-6 and IL-6 in vitro. At optimal concentrations, thrombin (10(-7) M) induced 7.6 +/- 0.01 ng/ml immunoactive IL-6 and PAR-1 activating peptide (5 x 10(-5) M) induced 2.2 +/- 0.2 ng/ml (mean +/- standard error of mean). A proteolytically inactive recombinant thrombin (serine 195 to alanine) was without activity. These data show that HGF express PAR-1 and suggest that PAR-1 activation stimulates increased NF-IL-6 and IL-6 gene expression and IL-6 secretion by HGF in vitro. Whether HGF express PAR-3 is unknown, but the fact that SFLLRN was not a complete replacement for thrombin raises the possibility that HGF may express additional thrombin receptors. These findings add weight to the importance of the cytokine-like role played by thrombin and raise the possibility that protease-activated receptors may play a role in the pathogenesis of inflammatory periodontal disease.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fibroblastos/citologia , Gengiva/citologia , Interleucina-6/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Trombina/fisiologia , Fatores de Transcrição/fisiologia , Coagulação Sanguínea/fisiologia , Northern Blotting , Proteínas Estimuladoras de Ligação a CCAAT , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Gengiva/metabolismo , Humanos , Mediadores da Inflamação/fisiologia , Interleucina-6/genética , Ligantes , Proteínas Nucleares/genética , Fragmentos de Peptídeos/fisiologia , Doenças Periodontais/etiologia , Periodontite/etiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptor PAR-1 , Receptor PAR-2 , Receptores de Superfície Celular/fisiologia , Proteínas Recombinantes , Trombina/fisiologia , Fatores de Transcrição/genéticaRESUMO
P2X3 is one of seven cloned ATP-gated non-selective cation channels. We have isolated a full-length mouse P2X3 gene from a phage lambda-129/Sv genomic library. The gene consists of 12 exons spanning a locus of approximately 40 kb. No significant similarities have been found between the genomic organisation of the mouse P2X3 gene and genes encoding other ion channels. The encoded mouse P2X3 protein consists of 397 amino acids and shows 99% identity with rat P2X3. Using RNase protection and primer extension assays, multiple transcription initiation sites have been mapped in the mouse P2X3 promoter to a region 162-168 bp upstream of the translation initiation codon. The P2X3 gene has been mapped to mouse chromosome 2p by fluorescence in situ hybridisation. The RAG locus-associated gene T160 is located 1.8 kb upstream of the transcription start site of mouse P2X3 gene. The promoter region of the mouse P2X3 gene lacks a conventional TATA and CCAAT consensus sites, and initiator elements. P2X3 is the first member of the P2X gene family to be completely characterised.
