Wntless蛋白促使抗細胞凋亡藉由活化NF-κB訊息傳遞路徑

Wntless increases anti-apoptotic activity by activating NF-κB signal pathway

劉奎延(Kwei-Yan Liu)

指導教授 ： 許世賢

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

在人類腫瘤的形成中，Wnt和NF-κB的訊息調節扮演著十分關鍵的角色，然而現今的文獻中卻很少提及兩者之間在腫瘤形成的關聯性，而近年有學者研究證明在果蠅中Wntless為一個Wnt路徑的新調控因子，但對於Wntless在哺乳類細胞中的功能還不是很清楚。本篇研究主要是探討Wntless 在正常和腫瘤細胞中所扮演的功能與特性。經由北方點墨法發現Wntless mRNA主要表現在老鼠的脾臟、肺臟、腎臟和胸腺中，而於老鼠腦部、睪丸和胃組織有少量的表現。但是使用西方點墨法和免疫組織染色法進行檢測時，發現Wntless蛋白僅於大腸組織之黏膜下層、肌肉細胞、神經節細胞和大腸中的神經細胞可以被檢測到。進一步地，利用免疫螢光染色法發現Wntless蛋白主要以點狀分布在細胞內，再者，利用細胞核、質、膜和細胞骨架分析法得知Wntless主要存在於細胞膜及胞器膜部分。此外，大量表現wntless可促使IκB磷酸化並啟動NF-κB/IκB複合物分離進而活化NF-κB下游的特定基因，例如c-Myc和Cyclin D1。進一步地分析，經由酵母菌雙雜交實驗、免疫沉澱法及免疫螢光染色則證明wntless會直接與NF-κB路徑中的NIK和IKKα結合，而非IKKβ和IKKγ。用TNFα刺激NF-κB路徑活化後，可以發現Wntless與IKKα的結合有明顯增加的情形。分析結果顯示在許多人類的腫瘤細胞株及神經膠質瘤、急性兒童白血病和乳癌病人檢體的免疫螢光染色中，發現Wntless和NF-κB(p65 和p52)有大量表現，並作用座落於同一個位置的現象發生。在本研究中亦使用shRNA的方式抑制腫瘤細胞中的Wntless表現，發現其具有增強TNF-α與cycloheximide誘發腫瘤細胞的細胞凋亡情形。當Wntless被抑制時降低腫瘤在soft agar的轉型和裸鼠上腫瘤形成。綜括上述，我們提出Wntless在活化NF-κB訊號路徑中，扮演一個十分重要的角色，並且調控腫瘤的形成。

關鍵字

Wntless ； NF-κB訊息路徑

並列摘要

Wnt and NF-κB signalings are two important pathways leading to human tumor formation. However, less evidence was available to link them in a common tumorigenic mechanism. In recent studies, Wntless is a new regulator of Wnt signals in Drosophila, but the cellular function in mammalian remains unclear. In this study, we characterized the cellular function of Wntless in tumor cells. Wntless mRNA was highly expressed in mouse spleen, lung, kidney, thymus and stomach, but less in brain and testis of mouse . However, Western blot and immunohistochemistry analysises showed that Wntless is specifically detected in submucosa, muscle cells, ganglion cells and nerve cells of the large intestine. Immunofluorescence staining showed that Wntless protein is expresssed predominantly in the plasma membrane in a dot-shape morphology, The subcellular fraction extraction analyses confirm Wntless expression in membrane and organelle. In addition, over-expression of Wntless led to phosphorylation of IκB that caused further activation of NF-κB/IκB complex and induced expression of downstream target genes, such as c-Myc and Cyclin D1. Further analysis indicated that Wntless can interact specifically with NIK and IKKα, but not IKKβ and IKKγ. The increased interaction of Wntless and IKKα was observed upon TNF α treatment.Furthermore, the expression of both Wntless and NF-κB (p65 and p52) was up-regulated and co-localized in human tumor cell, including glioma, acute childhood leukemia, and breast cancer. The knock-down Wntless expression by shRNA in tumor cells led to increase of apoptosis upon TNF-α/cycloheximide treatment,which resulted in the decrease of cancer cells. In conclusion, we suggest that Wntless plays a crucial role in activation of NF-κB signaling pathway and benefits tumorigenesis.

並列關鍵字

Wntless ； NF-κB signal pathway

參考文獻

1. Aggarwal, B. B. (2004). Nuclear factor-kappaB: the enemy within. Cancer Cell 6, 203-208.

2. Balkwill, F., and Mantovani, A. (2001). Inflammation and cancer: back to Virchow Lancet 357, 539-545.

3. Banziger, C., Soldini, D., Schutt, C., Zipperlen, P., Hausmann, G., and Basler, K. (2006). Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells. Cell 125, 509-522.

4. Beg, A. A., and Baldwin, A. S., Jr. (1994). Activation of multiple NF-kappa B/Rel DNA-binding complexes by tumor necrosis factor. Oncogene 9, 1487-1492.

5. Biliran, H., Jr., Wang, Y., Banerjee, S., Xu, H., Heng, H., Thakur, A., Bollig, A., Sarkar, F. H., and Liao, J. D. (2005). Overexpression of cyclin D1 promotes tumor cell growth and confers resistance to cisplatin-mediated apoptosis in an elastase-myc transgene-expressing pancreatic tumor cell line. Clin Cancer Res 11, 6075-6086.