Tanaka Nobuhisa, Morita Takao, Nezu Akihiro, Tanimura Akihiko, Mizoguchi Itaru, Tojyo Yosuke
Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
J Pharmacol Exp Ther. 2004 Nov;311(2):778-86. doi: 10.1124/jpet.104.068569. Epub 2004 Jun 21.
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.
人牙龈成纤维细胞（HGFs）高水平表达蛋白酶激活受体-1（PAR-1）。在培养的HGFs中，我们研究了凝血酶诱导白细胞介素-6（IL-6）产生的信号通路。PAR-1激动剂肽SFLLRN在氨肽酶抑制剂抑肽酶存在的情况下模拟了凝血酶诱导的IL-6产生。凝血酶或SFLLRN与抑肽酶的组合也显著诱导了IL-6 mRNA的表达。尽管HGFs持续暴露于凝血酶会迅速使Ca(2+)信号脱敏，但细胞对凝血酶产生IL-6的能力并未丧失。同样，尽管用细胞内Ca(2+)螯合剂BAPTA-AM [1,2-双（O-氨基苯氧基）乙烷-N,N,N',N'-四乙酸-乙酰氧甲酯]处理HGFs显著减弱了凝血酶诱导的细胞内Ca(2+)浓度升高，但相同处理并未抑制凝血酶诱导的IL-6产生。然而，凝血酶诱导的IL-6产生受到p38丝裂原活化蛋白（MAP）激酶和酪氨酸激酶抑制剂的强烈抑制，蛋白质印迹分析表明凝血酶刺激p38 MAP激酶磷酸化。抑制细胞外信号调节激酶1/2激酶、磷脂酰肌醇3-激酶和RhoA激酶的特异性抑制剂也部分抑制了凝血酶诱导的IL-6产生，但作用小于p38 MAP和酪氨酸激酶抑制剂。因此，凝血酶通过激活PAR-1诱导HGFs产生IL-6，酪氨酸激酶和p38 MAP激酶依赖性途径而非Ca(2+)信号通路可能在IL-6产生中起关键作用。
