| 摘要: | 摘要
本論文主要在探討mitogen-activated protein kinases (MAPKs) 路徑在lipoteichoic acid (LTA) 刺激RAW264.7巨噬細胞iNOS表現及NO釋放所扮演的角色。先前的實驗已證實LTA以時間及劑量相關的方式刺激iNOS表現及NO釋放。而我們發現，phosphatidylinositol 3-kinase (PI3K) 抑制劑wortamanin及LY294002以劑量相關方式抑制LTA所引發iNOS表現及NO釋放。而MEK抑制劑PD 98059及p38抑制劑SB 203580也以劑量相關的方式抑制LTA所引發之iNOS表現及NO釋放；然而Ras活化的抑制劑FPT inhibitor 對LTA引發之反應則沒有任何影響。LTA以時間及劑量相關的方式引發p44/42 MAPK之活化，當加入tyrosine kinase抑制劑 (tyrphostin AG126、genistein)、PKC抑制劑 (Go 6976、Ro 31-8220)、wortamanin、PD 98059、SB 203580及phorbol 12-myristate 13-acetate (PMA) 長時間 (24 h) 處理，發現tyrphostin AG126、genistein及PD 98059會抑制LTA引發p44/42 MAPK之活化，而wortamanin、Go 6976、Ro 31-8220、SB 203580及PMA長時間處理則不影響 LTA 的作用，表示LTA所引發之p44/42 MAPK的活化可受到上游 tyrosine kinase之調控，但並不會受到PI3K、PKC及p38 MAPK的調控。LTA也可以時間相關的方式引發 p38 MAPK活性的增加，當加入 wortamanin、LY294002、tyrphostin AG126、genistein、Go 6976、Ro 31-8220 或 SB 203580 等抑制劑及PMA長時間處理，這些抑制劑及PMA長時間處理皆有抑制作用，表示LTA刺激p38 MAPK的活性增加可受到上游 PI3K、tyrosine kinase及 PKC之調控。NF-κB 抑制劑 pyrrolidine dithiocarbamate (PDTC) 與IκB protease抑制劑 L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) 皆可抑制LTA所引發iNOS表現及NO釋放。由Electrophoretic mobility shift assay (EMSA) 的結果發現LTA可使NF-κB 的與DNA 結合的作用增加，於30分鐘時達最大反應，但120分鐘後反應明顯地減少，當加入wortamanin、LY294002、tyrphostin AG126、genistein、PD 98059、SB 203580、PDTC與TPCK，發現這些抑制劑皆可抑制LTA的作用。綜合以上的結果得知，在RAW264.7巨噬細胞中，LTA可經由p44/42及p38 MAPK二條訊號傳遞路徑活化NF-κB，而進一步調控iNOS表現及NO釋放。而 p38 MAPK可受到上游 PI3K、tyrosine kinase及 PKC之調控。而 p44/42 MAPK之活化僅受到上游 tyrosine kinase之調控，但並不會受到 PI3K及 PKC之調控。
Abstract
Our previous study has shown that lipoteichoic acid (LTA) caused time- and concentration-dependent increases of inducible nitric oxide synthase (iNOS) expression and NO release. In this study, we investigated the role of mitogen-activated protein kinases (MAPKs) on LTA-mediated iNOS expression and NO release in RAW264.7 macrophages. The phosphatidylinositol 3-kinase (PI3K) inhibitors (wortamanin and LY294002) inhibited the LTA-induced iNOS expression and NO release in a concentration-dependent manner. The LTA-induced increases in iNOS expression and NO release were also attenuated by the MEK inhibitor (PD 98059) and p38 MAPK inhibitor (SB 203580), but not by FPT inhibitor II. Treatment of RAW 264.7 macrophages with LTA caused time- and concentration-dependent activations of p44/42 MAPK. Moreover, the LTA-induced p44/42 MAPK activation was inhibited by the tyrosine kinase inhibitors (tyrphostin AG126 and genistein) and PD 98059, but not wortamanin, the PKC inhibitors (Go 6976 and Ro 31-8220), long term PMA (24 h) treatment, or SB 203580.These results suggested that LTA-mediated activation of p44/42 MAPK was regulated by upstream tyrosine kinase, but not by PI3K, PKC and p38 MAPK. Stimulation of RAW 264.7 macrophages with LTA also induced p38 MAPK activation; this effect was inhibited by wortamanin, LY294002, tyrphostin AG126, geinstein, Go 6976, Ro 31-8220 and long term PMA treatment, indicating that LTA might act through the pathways of PI3K, tyrosine kinase and PKC to induce p38 MAPK activation. The NF-B inhibitor (PDTC) and IB protease inhibitor (TPCK) reduced concentration-dependently the LTA-induced NO release and iNOS expression. Treatment of RAW 264.7 macrophages with LTA stimulated NF-B specific DNA-protein complex formation in nuclear extracts; this effect was inhibited by wortamanin, LY294002, genistein, tyrphostin AG126, PD 98059, SB 203580, PDTC and TPCK. Taken together, these results indicated that in RAW264.7 macrophages, LTA might activate p44/42 and p38 MAPK, which in turn resulting in stimulation NF-B DNA-protein binding, and finally initiated iNOS expression and NO release. Both events required the activation of an upstream protein tyrosine kinase. The activation of p38 MAPK was downstream signal of LTA-mediated activation of PI3K and PKC, whereas that of p44/42 MAPK was PI3K- and PKC-independent. |
