p44/42 MAPK及NF-κB 在Phorbol-12-Myristate-13-Acetate引發人類肺臟上皮細胞環氧酵素-2表現的訊息傳遞路徑之角色

The Role of p44/42 MAPK and NF-κB on Phorbol-12-Myristate-13-Acetate-Induced Cyclooxygenase-2 Expression in Human Pulmonary Epithelial Cells (A549)

簡銘賢(Ming-Shyan Chang)

指導教授 ： 林建煌

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2000年)

摘要

本論文主要探討phorbol-12-myristate-13-acetate (PMA) 引發人類肺臟上皮細胞（A549）之PGE2釋放、COX活性增加及COX-2表現之訊息傳遞路徑以及PKC同功酵素 (isoforms) 在此路徑所扮演的角色。PMA以劑量及時間相關的方式刺激PGE2釋放、COX活性增加及COX-2表現。而蛋白轉錄抑制劑actinomycin D和蛋白轉譯抑制劑cycloheximide可抑制PMA引發PGE2釋放、COX活性增加及COX-2表現。PKC抑制劑（Ro 31-8220, Go 6976）或以PMA (1 μM) 長時間 (24 hr) 處理也會減少PMA所引發之PGE2釋放、COX活性增加及COX-2表現，然而Ras活化的抑制劑FPT inhibitor II則沒有任何影響。先前的研究報告已指出，在A549細胞中存在有PKC-α,-γ,-ι,-λ,-μ及 -ζ六種isoforms。以PMA (1 μM) 刺激A549細胞後，發現只有 PKC-α 及PKC-γ兩種isoforms會從細胞質轉位到細胞核中，其它isoforms則否。而以PMA (1 μM) 長時間 (24 hr) 處理A549細胞會造成 PKC-α 及-γ的down-regulation，意味著PKC-α 及-γ包含於PMA引發COX-2表現的訊號傳遞路徑當中。 MEK抑制劑PD 98059可抑制PMA所引發之PGE2釋放、COX活性增加及COX-2表現，然而p38MAPK抑制劑SB 203580對PMA所引發的反應則沒有任何影響。以PMA刺激A549細胞可導致p44/42 MAPK的活化，當加入抑制劑Ro 31-8220、PD 98059、genistein、SB 203580或長時間 (24 hr) 之PMA處理，發現Ro 31-8220、PD 98059及長時間PMA的處理皆會抑制p44/42 MAPK的活化，genistein及SB 203580則沒有抑制作用。 NF-κB 抑制劑pyrrolidine dithiocarbamate (PDTC) 及Iκ-Bαprotease 抑制劑TPCK皆可抑制PMA所引發之PGE2釋放、COX活性增加及COX-2表現。以PMA刺激A549細胞可引發p65 NF-κB 從細胞質轉位到細胞核，以及細胞質中Iκ-Bα的分解。當預先以Ro 31-8220、PD 98059、 SB 203580及PDTC處理A549細胞，發現Ro 31-8220、PD 98059 及PDTC皆可抑制p65 NF-κB的轉位，只有SB 203580沒有抑制作用。由以上結果說明，在A549細胞中，PMA是藉由先活化PKC之後再使p44/42 MAPK活化，接著導致NF-κB 的活化，最後才導致COX-2表現及PGE2釋放。而存在於A549細胞中的PKC isoforms，只有PKC-α 及 PKC-γ是包含在PMA引發COX-2表現及PGE2釋放的反應當中。

關鍵字

環氧酵素-2 ； 訊息傳遞

並列摘要

We examined the role of PKC isoforms and signaling pathway involved in phorbol-12-myristate-13 acetate (PMA)-induced prostaglandin E2 (PGE2) release, the increase of cyclooxygenase (COX) activity and COX-2 expression in human pulmonary epithelial cell line (A549). PMA caused a concentration- and time-dependent increase in PGE2 formation, the increase of COX activity and COX-2 expression. Actinomycin D and cycloheximide inhibited PMA-induced PGE2 release, the increase of COX activity, and COX-2 expression. The PKC inhibitors (Go 6976 and Ro 31-8220) or long-term (24 hr) PMA treatment attenuated PMA-induced PGE2 release, the increase of COX activity and COX-2 expression, while FPT inhibitor II (Ras farnesyl protein transferase inhibitor) had no effect on PMA-induced responses. The tyrosine kinase inhibitor, genistein, attenuated PMA-induced PGE2 release, but not PMA-induced the increase of COX activity and COX-2 expression. In our previously studies have demonstrated that PKC-α, -γ, -ι, -λ, -μand -ζwere detected in A549 cells. Treatment of A549 cells with PMA (1 μM) caused the translocation of PKC-α and -γbut not other isoforms from cytosol to the membrane fraction. Long-term treatment of PMA (1μM) resulted in complete down-regulation of PKC-α and -γ, indicating the activation of PKC-α and -γis involved in PMA-mediated responses. The MEK inhibitor (PD 98059) attenuated PMA-induced PGE2 release, the increase of COX activity and COX-2 expression, while p38 mitogen-activated protein kinase inhibitor, SB203580, had no effect. Treatmemt of A549 cells with PMA caused p44/42 MAPK activation ; the activation was inhibited by Ro 31-8220, PD 98059 or long-term PMA treatment, but not genistein or SB 203580. The NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) and the Iκ-B protease inhibitor, l-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), prevented PMA-induced PGE2 release, the increase of COX activity, and COX-2 expression. PMA also caused the translocation of p65 NF-κB from cytosol to the nucleus as well as the degradation of Iκ-Bαin cytosol. Furthermore, the PMA-induced p65 NF-κB activation was inhibited by Ro 31-8220, PD 98059 or PDTC, but not SB 203580.These results indicate that PMA might activate PKC to elicite p44/42 MAPK activation, which in turn initiates NF-κB activation, and finally induces COX-2 expression and PGE2 release in A549 cells. Of the PKC isoforms present in A549 cells, only PKC-α and -γactivation are involved in regulating the PMA-induced responses.

並列關鍵字

cyclooxygenase-2 ； signal transduction ； phorbol-12-myristate-13-acetate

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