Human erythroleukemia (HEL) 細胞經PMA處理後會分化成類單核球/巨噬細胞 (monocyte/ macrophage like cells) 之貼壁細胞。然而HEL細胞經phorbol ester (PMA) 處理一天後再投予EP2或EP4致效劑，則這些細胞會進一步分化成類棘狀細胞 (dendritic cells; DC)。在棘狀細胞的鑑定中，EP2及EP4致效劑會促使棘狀細胞專一性標記CD83表現增加，其表現約為單獨投予PMA之2倍，然而在HLA-DR 的表現上，只有EP2致效劑會促使其表現增加約為單獨投予PMA之1.6倍。在功能性分析上，EP2及EP4致效劑所誘導出的棘狀細胞其細胞吞噬能力較低，顯示所誘導出的細胞為成熟之棘狀細胞。而在細胞訊息傳遞之研究中發現，投予MEK抑制劑U0126、PD98059皆能抑制EP2/EP4致效劑誘導CD83表現，並且反轉EP2/EP4致效劑誘導細胞吞噬能力的降低，以及抑制EP2致效劑誘導ERK1/2磷酸化，因此可知EP2/EP4誘導棘狀細胞的生成具MEK 依賴性。已知EP2及EP4受體主要與Gs蛋白質作用而促使細胞內cAMP 的作用，然而在投予PKA抑制劑H89後發現，H89並不能抑制EP2誘導CD83表現增加或ERK1/2磷酸化，顯示EP2誘導棘狀細胞形成無PKA依賴性。而投予PI3-Kinase(PI3K)抑制劑LY294002、Wortmannin亦得相同結果。而在投予PKC抑制劑Ro318425以及Gö6976中發現，Ro318425能明顯抑制EP2致效劑所誘導CD83表現增加及ERK1/2磷酸化，而classical PKC抑制劑Gö6976卻無此作用，已知HEL 細胞在PMA長時間刺激下會促使classical PKC的down-regulated，因此排除classical PKC的參與。在PKC磷酸化分析中發現EP2致效劑能夠誘導PKC-δ Ser-643及Thr-505 位置磷酸化，且此磷酸化現象可被Ro318425 所抑制，因此可以證實PKC-δ在其中扮演著重要的角色。

　　綜合上述實驗結果可以得知，EP2/EP4致效劑主要經由具PKC-δ依賴性的MEK 路徑而誘導棘狀細胞形成。

　　Human erythroleukemia (HEL) cells differentiate into monocytic/ macrophage-like cells after phorbol ester (PMA) treatment. In our finding, EP2 and EP4 agonists promoted dendrite outgrowth in PMA-pretreated HEL cells.EP2 and EP4 agonists induced a 2 fold increase in the expression of DC specific surface maker CD83 in these differentiated cells. However, only EP2 agonist could induce a 1.6 fold increase in the expression of HLA-DR. In functional analysis, EP2 and EP4 agonists reduced the phagocytic activity on PMA treated HEL cells, which is characteristic of maturated dendritic cells. In the study of signal transduction involved in EP2 receptor mediated dendrite formation, we found that MEK inhibitors, U0126 and PD98059, reduced the expression of CD83 and ERK1/2 phosphorylation induced by EP2 agonist. Moreover, they reversed EP2 agonist reduced phagocytotic activity. It’s well known that EP2/EP4 receptors were coupled to Gs protein and induce intracellular cAMP formation. However, PKA inhibitor H89 has no effect on EP2 agonist induced increase in CD83 expression and ERK1/2 phosphorylation, while PI3 Kinase inhibitors Wortmannin and LY294002 faled also. We suggested that EP2 agonist promoted DC formation was PKA- and PI3K- independent. A genernal PKC inhibitor,
Ro318425 was found to inhibit EP2 agonist induced CD83 expression and ERK1/2 phosphorylation, while classical PKC inhibitor Gö6976 failed. It suggested that classical PKCs were not involved in EP2 receptor signaling in our system. Furthermore, EP2 agonist promotes PKC-δ Ser-643 and Thr-505 phosphorylation, which can be abolished by Ro318425. PKC-δ may plays an important role in EP2 agonist induced DC formation.
　
　　In conclusion, EP2/EP4 agonists induced DC differentiation in PMA-pretreated HEL cells via a PKC-δ dependent MEK activation signaling pathway.

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