雌激素在人體內是重要的固醇類荷爾蒙，其中17β-estradiol (E2)為人體內表現量最多的一種雌激素，大部分研究亦以此種雌激素為探討目標；已知雌激素在多數細胞會經由雌激素受體調控細胞增生及抑制細胞凋亡，然而另一方面，多數研究亦同樣顯示雌激素會造成乳癌細胞的凋亡；雌激素主要藉由雌激素受體α和β作為媒介調控不同的訊息傳遞路徑，並可進一步細分成estrogen response element (ERE)依賴性機制和ERE非依賴性機制，在ERE非依賴性機制中的調控路徑種類包括有：雌激素受體受刺激後與其協同因子一同進行基因調控，或者由激酶瀑布性效應而活化轉錄因子的基因調控。已知KLF10為雌激素、TGFβs (TGFβ1-3)、表皮生長因子 (EGF)和骨型態發生蛋白(BMP-2)所引發表現的一種轉錄因子，TGFβ1調控KLF10的機制已廣泛被研究，已知KLF10在細胞內部的表現增加會產生類似TGFβ1加入的效應，其會抑制表皮細胞的增生且增加細胞凋亡，由以上可知KLF10在TGFβ1所引導的訊息傳遞路徑中扮演重要角色，但雌激素與KLF10之間的關係則尚未明瞭。
本研究使用小鼠睪丸組織細胞株作為實驗探討的材料，分別為具有雌激素受體α和β的萊迪吉氏細胞株-TM3(+/+)及只具有雌激素受體β的賽透力氏細胞株-TM4(-/+)，運用此兩種細胞分析KLF10是否在高劑量雌激素所造成細胞凋亡過程中扮演重要角色。藉由存活率分析(MTT assay)測試不同劑量的雌激素對於兩種細胞株的影響，發現使用高劑量雌激素處理時(＞10 μM)，兩種細胞的存活率皆顯著下降，且在免疫螢光染色實驗中發現兩者皆有Cytochrome C自粒線體釋放的情形，因此兩者細胞在高雌激素存在下皆有發生細胞凋亡的現象；然而，進一步以細胞流式儀(Flow cytometry)分析兩者細胞週期時，卻發現高劑量雌激素使TM3細胞株的細胞週期偏向停留於G2/M時期，而TM4細胞株卻會直接趨使細胞凋亡；試驗以西方墨點轉漬法(Western blot)觀察KLF10蛋白質的表現，發現在TM4細胞株中KLF10蛋白質的表現量隨著雌激素劑量增加而有提升的現象，而TM3細胞株則無此現象，將TM4細胞株僅過量表現KLF10蛋白質時，即會促使類似高劑量雌激素所引起的細胞凋亡，而以shRNA抑制Klf10基因表現時，則可避免被高劑量雌激素所誘導之細胞凋亡；在探討KLF10調控機制時，以即時定量聚合酶連鎖反應(Real-time PCR)測定TM4細胞Klf10 mRNA表現，當加入高劑量雌激素30分鐘後表現量即上升，且於Klf10啟動子活化分析實驗(Luciferase assay)中亦發現高劑量雌激素刺激之下可活化Klf10，因此推論Klf10可經由高劑量雌激素刺激而提高轉錄表現量；已知雌激素除了核內基因體路徑(genomic pathway)調控之外，也會透過非核內基因體路徑(non-genomic pathway)引發訊息傳遞路徑，因此利用轉譯抑制藥物(Cycloheximide)以抑制蛋白新生成，且分析KLF10蛋白質於雌激素加入下的表現，由結果可知高劑量雌激素可能會經由核內非基因體路徑調控KLF10蛋白質的增加，其可能與MAPK磷酸化KLF10上特殊位點以增加其穩定性有關，但相關的機制需再進一步探討。
本研究發現當TM4細胞株受高劑量雌激素的刺激時，會經由雌激素受體β增加KLF10蛋白質的表現，進而促使TM4細胞株走向凋亡，此結果不僅發現另一種高劑量雌激素造成細胞凋亡的訊息傳遞路徑，對於高劑量雌激素造成男性生殖系統上的影響也可略為了解。

Estrogens are important steroid hormones in human. Among them, 17β-estradiol (E2) is the most abundant estrogen in the physiological metabolism of human, and this type estrogen is the major topic for molecular study. Estrogen has been demonstrated to stimulate growth and inhibit apoptosis through estrogen receptor-mediated mechanisms in many cell types. However, there is strong evidence shows another dimension for estrogen action of which estrogen induces apoptosis in breast cancer cells. Estrogen has previously known that regulates various signaling transduction pathways mainly through estrogen receptor α and β. To further group the mechanism of estrogen, it can be distributed to two mechanisms which are estrogen response element (ERE)-dependent and ERE-independent. One of them, ERE-independent mechanism involves the binding of nuclear ER to co-activator and activation of transcriptional factor by the kinase cascade through the ER on the plasma membrane within the indirectly regulating of target gene. KLF10 has been shown that it is particularly induced by estrogen, TGF-1, 2, 3, EGF and BMP-2. In compare with other cytokines, regulation of Klf10 transcription by TGF-1 has been demonstrated more. These observations indicated that the increased intracellular levels of KLF10 mimic the anti-proliferative and apoptotic effects of TGF-β1 on epithelial cell growth, suggesting that KLF10 is an important factor for mediating TGF-β1 signaling, but within estrogen signaling is still unknown.
In order to find out the role of KLF10 in estrogen signaling, the mouse testicular cell lines were used which are the Leydig cell line-TM3 (ERα/β, +/+) and Sertoli cell line-TM4 (ERα/β, -/+) with different estrogen receptor component. Using these two cell lines to observe whether the KLF10 plays an important role in high-dose estrogen induced apoptosis through a serial assays including MTT, immunochemistry and flow cytometry etc. The MTT assay showed that both cell lines survival progressively decreased when treatment of different doses of estrogen. Moreover, addition of high-dose estrogen also resulted in a release of cytochrome C from mitochondria in both cell lines. However, to further analyze the cell cycle phase profile by flow cytometry, it presented that high-dose estrogen arrested the TM3 cells in the G2/M phase, but induced apoptosis merely in TM4 cells. In order to address the role of KLF10 in estrogen induced apoptosis, the expression levels of KLF10 were observed when different doses estrogen were added. The results showed that estrogen significantly induced the KLF10 expression in a dose-dependent manner in TM4, but not in TM3. When overexpressing the KLF10 protein in TM4, it induced apoptotic effect similar to high-dose estrogen dose; however, using shRNA to knock-down the expression of Klf10 rescued the high-dose estrogen-induced apoptosis. To further understand the regulation mechanism of KLF10, we used real-time PCR to analyze the expression of Klf10 mRNA in TM4. It revealed that Klf10 mRNA was rapidly increased within 30 mins of high-dose estrogen treatment. This transcriptional increase was further be verified by luciferase assay in TM4. Besides the well-documented genomic pathways, biological effects of estrogen can also be mediated by a non-genomic pathway control. Base on this possibility, this study also applied a translational inhibitor (Cycloheximide) to block protein synthesis, and then detected the level of KLF10 after estrogen treatment. The results showed that both genomic and non-genomic effects of estrogen exhibited a significantly increased or stabilized expression of KLF10. The non-genomic effect of estrogen may affect the KLF10 level through phosphorylating KLF10 on a specific site by a MAPK-dependent pathway. However, more experiments are needed to do for this hypothesis.
The results not only show that induction of KLF10 gene expression is one of the high-dose estrogen-induced apoptosis signaling pathways but also showed the important impacts of male reproductive system triggered by high-dose estrogen.

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