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  • 學位論文

Protein Kinase C及NF-?B在Lipoteichoic Acid引發巨噬細胞誘導型一氧化氮合成酶表現之訊息傳遞角色探討

The Signaling Role of Protein Kinase C and NF-?B on Lipoteichoic Acid-Mediated Inducible Nitric Oxide Synthase Expression in RAW 264.7 Macrophages

指導教授 : 林建煌
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摘要


本論文主要在探討lipoteichoic acid (LTA) 刺激RAW 264.7巨噬細胞inducible nitric oxide synthase (iNOS) 表現和NO釋放之訊號傳遞路徑。當以LTA處理不同的濃度及不同的時間,發現LTA以濃度相關及時間相關的方式刺激iNOS的表現及NO的釋放。蛋白轉錄抑制劑actinomycin D和蛋白轉譯抑制劑cycloheximide可抑制LTA所引發之iNOS表現及NO增加,然而內毒素抑制劑polymycin B則不影響LTA所引發之反應。Tyrosine kinase抑制劑genistein及tyrphostin AG126可抑制LTA所引發之iNOS表現及NO增加。PC-PLC抑制劑D-609、PI-PLC抑制劑U-73122及phosphatidate phosphohydrolase抑制劑propranolol皆可抑制LTA所引發之iNOS表現及NO增加。PKC抑制劑Ro 31-8220、Go 6976及以長時間PMA處理造成PKC down-regulation皆可顯著抑制LTA所引發之iNOS表現及NO增加。由Western blotting之分析發現RAW 264.7細胞存在有PKC-?, -?I, ?II, -?, -?, -?, -?, -?,和-?等九種isoforms。以1 ?M的PMA處理RAW 264.7 細胞24小時後,發現在九種isoforms中只有PKC-?, -?I, -?II, -? 四種isoforms會有down-regulation的現象。表示LTA所引發的iNOS表現及NO增加的路徑中可能包含PKC-?, -?I, -?II或 -? 的活化。由PKC activity analysis發現在LTA刺激下RAW 264.7細胞質部份的PKC activity有下降趨勢,而細胞膜部份的PKC activity則有上升的趨勢。表示LTA確實會造成PKC活化並且進行轉位。加入tyrphostin AG126、D-609、U-73122和propranolol可抑制LTA所造成的PKC的轉位,表示LTA刺激PKC活性增加可能受到上游tyrosine kinase、PC-PLC、PI-PLC及PC-PLD的調控。轉錄因子NF-?B抑制劑PDTC及I?B protease抑制劑TPCK、calpain inhibitor I皆可抑制LTA所引發的iNOS表現及NO增加。以LTA刺激RAW 264.7細胞30分鐘可使p65 NF-?B及p50 NF-?B由細胞質轉位至細胞核,亦會造成細胞質中I?B-?分解及磷酸化I?B-?的增加。Electrophoretic mobility shift assay (EMSA) 的結果也發現LTA可使NF-?B與DNA的結合的活性隨時間而增加,在30分鐘時達最大反應,但加入PDTC、TPCK、calpain inhibitor I、tyrphostin AG126、genistein、D-609、U-73122、propranolol、Go 6976、Ro 31-8220及以長時間PMA處理皆可抑制LTA所刺激的NF-?B活化,表示LTA刺激NF-?B活性的增加可受到上游tyrosine kinase、PC-PLC、PI-PLC、PC-PLD及PKC的調控。綜合以上的結果得知,在RAW 264.7 macrophage中,LTA作用於細胞膜上的受體後活化tyrosine kinase,使得PC-PLC、PI-PLC、PC-PLD活化而產生DAG,DAG再促使PKC-?, -?I, -?II及-?活化,之後造成NF-?B的活化並轉位到細胞核中,作用在iNOS的promoter上使得iNOS表現,最後造成NO的釋放。

並列摘要


The signaling pathway of lipoteichoic acid (LTA)-mediated inducible nitric oxide synthase (iNOS) expression was studied in murine RAW 264.7 macrophage. LTA caused concentration- and time-dependent increases in iNOS expression and NO release. Actinomycin D and cycloheximide blocked the LTA-induced iNOS expression and NO release, while polymyxin B, an agent that binds and inactivates endotoxin, had no the effect. The tyrosine kinase inhibitors (genistein and tyrphostin AG126) attenuated the LTA-induced iNOS expression and NO release. The phosphatidylcholine-phospholipase C inhibitor (D-609), phosphatidylinositol-phospholipase C inhibitor (U-73122) and phosphatidate phosphohydrolase inhibitor (propranolol) prevented the LTA-mediated iNOS expression and NO increase. The PKC inhibitors (Ro 31-8220 and Go 6976) or long term (24h) PMA (1?M) treatment also resulted in inhibition of LTA-stimulated iNOS expression and NO release. Western blot analysis using PKC isoenzyme specific antibodies indicated that RAW cells expressed PKC-?, -?I, ?II, -?, -?, -?, -?, -?, and -?. Furthermore, the down-regulation of PKC-?, -?I, -?II, and -?, but not -?, -?, -?, -?, and -?, was seen after long term PMA treatment, indicating the possible involvement of PKC-?, -?I, -?II, and -?, but not -?, -?, -?, -?, and -?, in LTA-mediated effects. PKC activity was decreased in the cytosol and increased in the membrane after treatment with LTA for 30 min in RAW cells. This phenomenon indicates that LTA stimulates PKC activation and translocation. The LTA-induced increase in membrane PKC activity was inhibited by pretreatment with tyrphostin AG126, D-609, U-73122 or propranolol. Thus, LTA may act through the tyrosine kinase, PI-PLC, PC-PLC and PC-PLD pathway to induce PKC activation in RAW 264.7 cells. The NF-?B inhibitor (PDTC), I?B protease inhibitors (TPCK and calpain inhibitor I) attenuated the LTA-induced iNOS expression and NO release. Treatment of RAW 264.7 cells with LTA resulted in a translocation of p65 NF-?B and p50 NF-?B from the cytosol to the nucleus. LTA also induced transient decrease of I?B-???and increase of phosphorylated I?B-??in the cytosol of RAW 274.7 cells. Stimulation of RAW 264.7 cells with LTA for 30 min caused activation of NF-?B in the nucleus by detection of NF-?B-specific DNA-protein binding; this effect was inhibited by PDTC, TPCK, calpain inhibitor I, tyrphostin AG126, genistein, D-609, U-73122, propranolol, Go 6976, Ro 31-8220 or long term PMA treatment. PKC activation signal pathway was found to be involved in the regulation of LTA-induced NF-?B activation. These data suggest that LTA activates PI-PLC, PC-PLC and PC-PLD via an upstream tyrosine kinase to induce PKC activation, which in turn initiates stimulation of NF-?B activation, and finally induces of iNOS expression and NO release. Furthermore, the PKC isoforms, -?、-?I、-?II and -??? present in RAW 264.7 macrophages may involved in the regulation of LTA-induced responses.

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