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標題: | Interleukin-4經多條訊息傳遞路徑誘導HSD3B1表現於HT-29人類大腸癌細胞 HSD3B1 is up-regulated by interleukin-4 in HT-29 human colon cancer cells via multiple signaling pathways |
作者: | 陳昕梅 Hsin-Mei Chen |
指導教授: | 胡孟君 Meng-Chun Hu |
關鍵字: | 介白素-4,HSD3B1,大腸癌,訊息傳遞路徑, Interleukin-4,HSD3B1,colon cancer,signaling pathway, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 類固醇荷爾蒙主要由腎上腺、性腺或胎盤所分泌。近年發現,腸道也會表現類固醇生成基因,並局部性地合成糖類皮質素,調節腸道免疫恆定。類固醇荷爾蒙的生成需仰賴3beta-hydroxysteroid dehydrogenase/△5-△4 isomerase (3beta-HSD) 的作用,其為HSD3B基因的產物。人類有HSD3B1與HSD3B2兩種基因,前者主要於胎盤、周邊組織與腫瘤細胞中表現,後者則在腎上腺與性腺中表現。過去研究顯示,周邊組織中的HSD3B1在interlukin-4 (IL-4) 的刺激下會增加表現,而我們先前於人類大腸癌細胞HT-29發現,IL-4能誘導HSD3B1大量表現且此現象受PI3K/Akt/GSK3訊息傳遞路徑所調控。本論文則更深入探討其他可能參與調控的因子。結果顯示,STAT6抑制劑AS1517499能大量抑制IL-4誘導的HSD3B1表現,且IL-4會促進STAT6磷酸化,也能提升STAT6與HSD3B1啟動子結合的能力,說明IL-4會活化STAT6路徑來增加HSD3B1在HT-29細胞中表現。另外,我們亦發現,ERK抑制劑PD98059和p38 MAPK抑制劑SB202190能降低50 ~ 70% IL-4誘導的HSD3B1表現量,可是IL-4似乎不會影響細胞內ERK與p38 MAPK磷酸化的程度。此外,NF-κB抑制劑Bay117082約減少50% 的HSD3B1表現,且我們發現IL-4能增加NF-κB p65 Ser536的磷酸化,並促進NF-κB與HSD3B1啟動子的結合,說明IL-4會活化NF-κB來增加HSD3B1的表現。接著,IKK抑制劑 BMS345541與TPCA-1以及其激酶TAK-1抑制劑5Z-7-oxozeaenol皆能降低約90% 的HSD3B1蛋白質量,但IKK knockdown並不影響IL-4誘導的HSD3B1表現,說明IKK可能並非調控NF-κB活化的因子。BMS345541、TPCA-1以及5Z-7-oxozeaenol的作用機制尚需更多的研究。綜合所有結果,本論文發現IL-4能透過多條訊息傳遞路徑調控HSD3B1於HT-29細胞表現,包括:STAT6、NF-κB、ERK與p38 MAPK。 In humans, most of steroid hormones are produced by adrenal glands, gonads and placenta. Recently, intestine has also been found to express steroidogenic genes and has the ability to locally synthesize glucocorticoid (GC) for maintenance of intestinal immune homeostasis. The 3beta-hydroxysteroid dehydrogenase (3beta-HSD), which is encoded by HSD3B gene, is essential for the biosynthesis of steroid hormones. There are two HSD3B genes in human. HSD3B1 is predominantly expressed in the placenta, peripheral tissues and tumor cells, while HSD3B2 is expressed in the adrenals and gonads. Previous studies indicated that interleukin-4 (IL-4) induces the expression of HSD3B1 in several peripheral tissues and tumor cells. Our recent data demonstrated that IL-4 dramatically increased the protein and mRNA levels of HSD3B1 in HT-29 human colon cancer cells. In addition, PI3K/Akt/GSK3 signaling pathway is critical for IL-4-stimulated HSD3B1 expression. In this study, we further investigate whether additional signaling factors induced by IL-4 that mediate the HSD3B1 expression. Our data revealed that IL-4-induced HSD3B1 expression was markedly decreased by STAT6 inhibitor AS1517499. The results showed that IL-4 induced the phosphorylation of STAT6 and enhanced the binding of STAT6 to the proximal region of HSD3B1 promoter, suggesting that IL-4 increased HSD3B1 expression via STAT6 activation. Additionally, we found that MEK inhibitor PD98059 and p38 MAPK inhibitor SB202190 suppressed IL-4 induced HSD3B1 by 50~70%. However, IL-4 treatment had no effect on the phosphorylation level of ERK and p38 in HT-29 cells. Moreover, NF-κB inhibitor Bay117082 attenuated HSD3B1 protein by 50%. We found that IL-4 increased the level of NF-κB p65 Ser536 phosphorylation and enhanced the binding of NF-κB to the HSD3B1 promoter, demonstrating that NF-κB is involved in IL-4-induced HSD3B1 expression. Furthermore, treatment with IKK inhibitors (BMS345541 and TPCA-1) and TAK-1 (kinase for IKK) inhibitor 5Z-7-oxozeaenol resulted in ~90% decrease in IL-4-induced HSD3B1 protein in HT-29 cells. However, knockdown of IKK alpha and IKK beta had no effect on IL-4-mediated HSD3B1 expression, implying that IKKs may not be the upstream regulator of NF-κB. As for the inhibition mechanism of BMS345541, TPCA-1 and 5Z-7-oxozeaenol, more research is need. In summary, our finding demonstrated that IL-4 induces HSD3B1 expression in HT-29 cells via multiple signaling pathways, including STAT6, NF-κB, ERK and p38 MAPK. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78970 |
DOI: | 10.6342/NTU201803273 |
全文授權: | 未授權 |
電子全文公開日期: | 2028-08-16 |
顯示於系所單位: | 生理學科所 |
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