紅血球生成素影響 P19 分化神經元之神經纖維生長及生長相關基因
表現研究
The Effect of Erythropoietin on Growth and Survival-Related Gene Expressions in P19-Derived Neurons
中文摘要
紅血球生成素(EPO)是一種具有神經保護性的細胞激素，其與紅血球生成素受體 (EPO receptor)結合後，藉由活化 JAK/STAT 訊息傳遞路徑，調節抵抗細胞凋亡的 下游基因表現，以促進細胞存活。然而，在神經細胞的發育過程中，EPO 引發 的訊息路徑，與其他細胞存活或細胞生長相關的訊息路徑之間的相互關連研究尚 不明確。在本論文的研究中，我們利用畸胎瘤細胞株(P19 EC cells)分化而來的神 經細胞，進行 EPO 對 P19 分化神經元細胞存活與生長相關基因的影響。在 P19 EC cells 分化的過程中，除了原本使用維生素 A 酸(retinoic acid)誘導神經分化之外，
另加入了纖維母細胞生長因子(FGF8)，使 P19 可以 monolayer 的細胞培養形式直 接分化為神經細胞。螢光素酶活性分析(Luciferase activity assay)的結果可以得 知，於 P19 分化神經元外加 EPO，可促使生長相關蛋白(Growth-associated protein；GAP-43)的 P2 promotor 活性增加。除了以外加 EPO 進行實驗外，本論 文研究中也利用了含有 CMV promoter 驅動 epo 基因表現的 plasmid，轉染 (transfect)進入 P19 分化神經元，使 EPO 基因過度表現。研究結果顯示，無論是 外加的 EPO，抑或是利用 transfection 使 EPO 基因過度表現，皆有助於 P19 神經 纖維生長。GAP-43 為神經纖維生長的 marker；Bcl-xL 功能為抵抗細胞計畫性死 亡，此兩種基因皆受到 EPO 的調控，EPO 可以促進 GAP-43 及 Bcl-xL 的基因表 現。JAK 抑制劑-AG490、PI3K 抑制劑-LY294002、MAPK 抑制劑-PD98059 皆會 阻斷 EPO 對 P19 分化神經元於神經纖維生長及維持細胞存活的促進現象，此結 果意味了 EPO 的神經保護功能涉及了這三個訊息傳遞路徑。而 EPO 的神經保護 性不會被 NGF 受體抑制劑-AG879 所抑制，並且可以 AG879 抑制 P19 神經元細 胞存活的情形被緩解。實驗結果進一步發現 AG879 會抑制 P19 神經元 Bcl-xL 的 表現，但並不顯著抑制 GAP-43 的表現。epo 基因在 P19 神經元的過度表現，可 以回升被 AG879 抑制的 Bcl-xL 表現，同時也回升了 GAP-43 的表現量。因此，
本論文研究結果，除了證明了 EPO 會透過 JAK、PI3K、MAPK 等訊息傳遞路徑 促進神經纖維生長，並且發現 EPO 基因的過度表現，可以回升 NGF/TrkA 訊息 傳遞受到抑制時所降低的 Bcl-xL 表現量，也促使 GAP-43 的基因表現, 可能藉由 此一作用使得 EPO 可緩解因 NGF/TrkA 受到抑制所降低之神經存活。
英文摘要
Erythropoetin (EPO) is a neuroprotective cytokine that is known to mediate
anti-apoptotic gene expression via EPO receptor and the JAK/STAT signaling pathway. However, the interplay of EPO signaling with other survival or
growth-related signaling during neuronal development remains unclear. In the present study, we used pluripotent embryonal carcinoma (EC) P19 cell line-derived neurons as the experimental model to examine the effect of EPO on the survival and
growth-related gene expressions. We modified the neural induction protocol of the P19 cells with addition of fibroblast growth factor 8 (FGF-8) in the monolayer culture.
Luciferase activity assay shows that exogenous application of EPO could increase GAP-43 P2 promoter activity in P19-derived neurons. For the EPO effect, we applied exogenous EPO as well as established an epo gene overexpression system by
transfection of CMV promoter-driven epo gene plasmid into differentiated P19 neurons. Our results show that both EPO gene overexpression and exogenous EPO administration enhanced neurite outgrowth. The growth-associated protein-43 (GAP-43), a marker for outgrowth and nerve regeneration, and an anti-apoptotic protein Bcl-xL, an EPO-mediated gene expression product, were both enhanced by the EPO treatment. The EPO enhancement of neurite outgrowth and neuronal survival was blocked by the JAK inhibitor AG490, PI3 kinase inhibitor LY294002, and MAP kinase inhibitor PD98059, three of the EPO receptor downstream signaling pathway inhibitors, suggesting that these pathways are involved in the EPO neuroprotection.
However, the EPO neuroprotection was not blocked by the NGF receptor TrkA inhibitor AG879, and seems to reverse the AG879 impairment of neuronal survival.
Furthermore, we found that AG879 significantly reduced Bcl-xL but not GAP-43 gene expression, and overexpression of EPO could reverse the AG879
downregulation of Bcl-xL as well as further up-regulate GAP-43 expression. Together, our results suggest that EPO-mediated neurite outgrowth depends on the JAK, PI3 kinase, and MAP kinase signaling pathways, and EPO overexpression seems capable of attenuating the NGF/TrkA hypofunction-reduced neuronal survival by upregulating Bcl-xL and GAP-43 gene expression.
