Mitogen-Activated Protein Kinases
在 Lipoteichoic Acid 引發巨噬
細胞一氧化氮合成酵素表現之訊號
本論文主要在探討 mitogen-activated protein kinases (MAPKs) 路徑在 lipoteichoic acid (LTA) 刺激 RA
W264.7 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 wortamani
n 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 p4
4/42 MAPK 之活化，當加入 tyrosine kinase 抑制劑 (tyrphostin AG126 、 genistein) 、 PKC 抑制劑 (G
o 6976 、 Ro 31-8220) 、 wortamanin 、 PD 98059 、 SB 203580 及 phorbol 12-myristate 13-acetate (PM
A) 長時間 (24 h) 處理，發現 tyrphostin AG126 、 genistein 及 PD 98059 會抑制 LTA 引發 p44/42 MA
PK 之活化，而 wortamanin 、 Go 6976 、 Ro 31-8220 、 SB 203580 及 PMA 長時間處理則不影響 LT
A 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 pyrr
olidine dithiocarbamate (PDTC) 與 IκB protease 抑制劑 L-1-tosylamido-2-phenylethyl chloromethyl keton
e (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 RAW2
64.7 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 i
NOS 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。
Studies on the Role of Mitogen-Activated Protein Kinases in Lipoteichoi
c Acid-Mediated Nitric Oxide Synthase Expression in RAW 264.7 Macr
ophages
Our previous study has shown that lipoteichoic acid (LTA) caused time- and concentration-dependent incr
eases of inducible nitric oxide synthase (iNOS) expression and NO release. In this study, we investigated t
he 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 LY2940
02) 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 macr
ophages 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 ter
m PMA (24 h) treatment, or SB 203580.These results suggested that LTA-mediated activation of p44/42 M
APK was regulated by upstream tyrosine kinase, but not by PI3K, PKC and p38 MAPK. Stimulation of RA
W 264.7 macrophages with LTA also induced p38 MAPK activation; this effect was inhibited by wortama
nin, LY294002, tyrphostin AG126, geinstein, Go 6976, Ro 31-8220 and long term PMA treatment, indicati
ng that LTA might act through the pathways of PI3K, tyrosine kinase and PKC to induce p38 MAPK activ
ation. The NF- B inhibitor (PDTC) and I B protease inhibitor (TPCK) reduced concentration-dependently
the LTA-induced NO release and iNOS expression. Treatment of RAW 264.7 macrophages with LTA stim
ulated NF- B specific DNA-protein complex formation in nuclear extracts; this effect was inhibited by wor
tamanin, LY294002, genistein, tyrphostin AG126, PD 98059, SB 203580, PDTC and TPCK. Taken togeth
er, these results indicated that in RAW264.7 macrophages, LTA might activate p44/42 and p38 MAPK, wh
ich 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 p
38 MAPK was downstream signal of LTA-mediated activation of PI3K and PKC, whereas that of p44/42
MAPK was PI3K- and PKC-independent.
