Signaling mechanisms involved in protease-activated receptor-1-mediated interleukin-6 production by human gingival fibroblasts.
Human gingival fibroblasts (HGFs) express protease-activated receptor-1 (PAR-1) at high levels. In cultured HGFs, we studied the signaling pathway of thrombin-induced interleukin-6 (IL-6) production. The PAR-1 agonist peptide SFLLRN mimicked the thrombin-induced IL-6 production in the presence of amastatin, an aminopeptidase inhibitor. Thrombin or a combination of SFLLRN and amastatin also strikingly induced the expression of IL-6 mRNA. Although continuous exposure of HGFs to thrombin rapidly desensitized Ca(2+) signaling, the cells did not lose their ability to produce IL-6 in response to thrombin. Similarly, although treatment of HGFs with BAPTA-AM [1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester], an intracellular Ca(2+) chelator, markedly attenuated the thrombin-induced increase in intracellular Ca(2+) concentration, the same treatment did not suppress the thrombin-induced IL-6 production. However, thrombin-induced IL-6 production was strongly inhibited by the p38 mitogen-activated protein (MAP) kinase and tyrosine kinase inhibitors, and Western blotting analyses showed that thrombin stimulates p38 MAP kinase phosphorylation. Specific inhibitors that inhibit extracellular signal-regulated kinase 1/2 kinase, phosphatidylinositol 3-kinase, and RhoA kinase also partially suppressed the thrombin-induced IL-6 production, but the effects were smaller than those of the p38 MAP and tyrosine kinase inhibitors. Thus, thrombin induces HGFs to produce IL-6 by activating PAR-1, and the tyrosine kinase- and p38 MAP kinase-dependent pathways, rather than the Ca(2+) signaling pathway, may play a crucial role in the IL-6 production.
Tanaka
Nobuhisa
N
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. tojyo@hoku-iryo-u.ac.jp
Morita
Takao
T
Nezu
Akihiro
A
Tanimura
Akihiko
A
Mizoguchi
Itaru
I
Tojyo
Yosuke
Y
D020013:Calcium Signaling; D002478:Cells, Cultured; D004347:Drug Interactions; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013917:Thrombin / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D044463:Receptor, PAR-1; C015482:protein kinase modulator; C082835:thrombin receptor peptide (42-47); C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D011505:Protein-Tyrosine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D013917:Thrombin