Cyclooxygenase-2 表現在脂臺口酸刺激巨噬細胞一氧化氮生成之路 徑探討
Role of Cyclooxygenase-2 Expression on Lipoteichoic Acid-Mediated Nitric Oxide Formation in RAW 264.7 Macrophages
中文摘要
本論文主要探討lipoteichoic acid (LTA) 刺激 RAW 264.7 巨噬細胞
cyclooxygenase-2 (COX-2) 表現對 inducible nitric oxide synthase (iNOS) 表現及 NO 釋放的調控。當以 LTA 處理不同時間，發現 LTA 以時間相關的方式刺激 COX-2 表現及 prostaglandin E2 (PGE2) 釋放。LTA 所引發之 iNOS 表現及 NO 生 成可被COX-2 抑制劑 indomethacin 及 NS-398 所抑制。且 adenylyl cyclase 抑制劑 dideoxyadenosine 及 protein kinase A 抑制劑 KT-5720 皆可抑制 LTA 所引發之 iNOS 表現及 NO 釋放。當以 PGE2 或 dibutyryl-cAMP (cAMP 類似物) 處理 RAW 264.7 細胞，發現 PGE2 或 dibutyryl-cAMP 可以刺激 iNOS 表現。由
electrophoretic mobility shift assay (EMSA) 的結果發現 LTA 可以時間相關的方式 增強NF-kB 與 DNA 結合的能力，當加入 indomethacin、NS-398、KT-5720 等抑制
劑前處理細胞，皆有抑制作用。這表示LTA 刺激 NF-kB 活性的增加可能受到
COX-2 及 protein kinase A 的調控。另外 LTA 也以時間相關的方式活化 p44/42 及 p38 mitogen-activated protein kinase (MAPK)。Indomethacin 及 NS-398 可抑制 LTA 所引發p44/42 或 p38 MAPK 之活化，而 PGE2 及 dibutyryl-cAMP 可以時間相關 的方式刺激p44/42 及 p38 MAPK 活化；且 dideoxyadenosine 及 KT-5720 可抑制 PGE2 所引發 p38 MAPK 之活化，但 p44/42 MAPK 則否。由此證明 LTA 經由刺 激COX-2 表現、PGE2 生成可引發 p44/42 及 p38 MAPK 活化，其中 p38 MAPK 的活化經由PKA-cAMP 的活化而來，但 p44/42 MAPK 則否。綜合以上的結果得 知，在RAW 264.7 巨噬細胞中，LTA 可刺激 COX-2 表現而導致 PGE2 釋放，
PGE2 釋放後再進一步刺激 iNOS 表現及 NO 合成，此 PGE2 可能以回饋作用的 方式引發adenylyl cyclase-cAMP-protein kinase A 路徑及 NF-kB 活化而調控 iNOS 表現及NO 釋放。另外在 LTA 刺激下，PGE2 的釋放也會刺激 p44/42 及 p38 MAPK 之活化，而此 p38 MAPK 之活化是經由 adneylylyl cyclase-cAMP-protein kinase A 路徑來調控，但 p44/42 則否。
英文摘要
In this study, we investigated the role of cyclooxygenase-2 (COX-2) expression on lipoteichoic acid (LTA)-mediated nitric oxide synthase (iNOS) expression and NO release in murine RAW 264.7 macrophages. LTA caused concentration- and time- dependent increases in COX-2 expression and PGE2 release. LTA also induced iNOS expression and NO release; these events were inhibited by COX-2 inhibitors
(indomethacin and NS-398), adenylyl cyclase (AC) inhibitor (DDA) or protein kinase
A (PKA) inhibitor (KT-5720). Furthermore, PGE2 and cAMP analog (dibutyryl- cAMP) induced iNOS expression in dose- and time-dependent manners. Moreover, LTA caused time-dependent increases in NF-kB activation; this stimulatory effect was inhibited by indomethacin, NS-398 or KT-5720. These results suggest that LTA induced COX-2 expression and PGE2 release which in turn result in iNOS expression and NO release via the pathway of adenylyl cyclase, PKA and NF-kB. In addition to NF-kB activation, we further investigated the role of PGE2/PKA on the p44/42 and p38 mitogen-activated protein kinase (MAPK) activation caused by LTA. LTA induced p44/42 and p38 MAPK activation. Indomethacin and NS-398 reduced LTA- induced p44/42 MAPK and p38 MAPK activation. PGE2 and dibutyryl-cAMP also resulted in activations of p44/42 and p38 MAPK. However, dideoxyadenosine and KT-5720 reduced the PGE2-induced p38 MAPK activation, but not p44/42 MAPK.
We demonstrate that LTA-induced COX-2-dependent PGE2 release and PKA
activation contribute to p38 MAPK activation, but not p44/42 MAPK. Taken together, Lta might induce COX-2 expression and PGE2 release which in turn upregulates NF- kB activation, and finally induced iNOS expression and NO release. PGE2 might induce NF-kB activation or p38 MAPK activation via cAMP-PKA, which also resulted in p44/42 MAPK activation without cAMP-PKA pathway.
