Concerted Actions of Multiple Transcription Elements Confer Differential Transactivation of HSP90 Isoforms in Geldanamycin-Treated 9L Rat Gliosarcoma Cells

膠達那黴素所誘發之9L大鼠腦瘤細胞逆境反應與熱休克蛋白90表達之調控

趙致忠(Chih-Chung Chao)

指導教授 ： 黎耀基

國立清華大學/生命科學院/生命科學系/博士(2008年)

https://doi.org/10.6843/NTHU.2008.00146

摘要

本論文以觀察膠達那黴素 (geldanamycin, GA) 引發大鼠腦瘤細胞 (RBT 9L cells) 的逆境反應為始，研究其對熱休克蛋白HSP90同源異構體 (isoforms) 表達的調控機制。膠達那黴素為袢霉素 (ansamycin) 所衍生之苯二酮 (benzoquinone) 化合物，原本由天然抗真菌活性的產物中分離而得到。其經由專一性競爭作用抑制熱休克蛋白HSP90之腺三磷酸合成脢 (ATPase) 活性，進而造成受HSP90保護之客戶蛋白質 (client proteins) 失活、不穩定而分解；這類蛋白質包括調節細胞生長、分化等為數眾多的訊息傳導蛋白，如蛋白質激酶，及固醇類荷爾蒙受體等。
近年來，許多報導已指出GA會引發熱休克反應，且以RBT 9L細胞為研究題材之文獻中，已知至少可引發HSP70、GRP78、GRP94等多種蛋白表現。在本研究中，我們觀察到低GA劑量誘發HSP90，僅一般處理劑量的十分之一。而HSP90在哺乳動物細胞中，皆存在兩種同源異構體：即HSP90α 與HSP90β，且不同層級的物種間都有極高的相似度。當改變電泳條件可成功分離HSP90α與β，並發現GA處理下之不同誘導規模。由分析蛋白質與RNA層次的誘導表現量，得知HSP90α誘導量大於HSP90β均有其一致性，故可推測GA誘導HSP90之不同表現量調控在基因層次。
為探討HSP90α 與HSP90β 基因層次的調控機制，我們依據 hsp90α 與 hsp90β 之啟動子基因序列，由其上分析得到多個可能的轉錄蛋白結合區域，進一步利用電泳凝膠遷移檢測法(Electrophoresis Mobility Shift Assay)，比較GA處理RBT 9L細胞之核蛋白與轉錄元(transcriptional elements)的結合情形。據此結果可以得知HSP90的誘導與熱休克因子HSF1對於HSE的結合有極大相關，且HSP90α 與 HSP90β序列上分布不同的HSE的結合情形也有所不同，並據此由兩組HSE之間找出各有一個相對重要的HSE；同時核蛋白在 hsp90α-HSE2 與hsp90β-HSE1的不同結合力也顯示了兩者誘導量不同的可能原因。另外，針對其他重要的基本轉錄元分析，則又發現核蛋白中對於GC-box (SP-1)的結合力也有所不同，可能與HSP90α的誘發規模比HSP90β高，以及HSP90β基礎表現量有關；而TATA-box上的結合力則可能顯示HSP90α誘導量會與TATA-box上結合的轉錄蛋白相關；另一方面，對於CRE的結合力則可能說明CRE上的轉錄蛋白與GA處理下對HSP90β的誘導作用有關。
綜言之，本研究發現GA的作用能透過HSF1對啟動子上特定HSE的結合來誘發HSP90，並對於兩種HSP90同源異構體在轉錄層次同樣具有重要的調控作用，這可能決定了它們的誘導量由基因層次乃至於蛋白質層次的顯著差異。

關鍵字

膠那達黴素 ； 熱休克蛋白90 ； 熱休克元素 ； 熱休克因子1 ； 差異性基因活化 ； 及時定量聚合脢連鎖反應 ； 基因表現

並列摘要

This thesis started by using geldanamycin (GA) to treat rat brain tumor (RBT) 9L cells. By observing cell stress response, we initiated the study of the differentially inductive mechanisms on heat shock protein 90 isoforms. Geldanamycin (GA) is an ansamycin-derivative benzoquinone compound, which was originally isolated as a natural product with anti-fungal activity. GA could inhibit the essential ATPase activity of HSP90 and results in inactivation, destabilization, and degradation of HSP90 client proteins, including a wide variety of signal-transducing proteins that regulate cell growth and differentiation, such as protein kinases and steroid hormone receptors.
But reports had shown that GA treatment could also induce heat-shock response; especially including HSP70, GRP78 and GRP94 in RBT 9L cells. In this study, we found only a 1/10 dose of GA (i.e., 0.5 贡M) could induce HSP90, compared to 5 贡M GA to induce those heat shock proteins like HSP70, GRP78 and GRP94. Furthermore, HSP90 exists two highly consistent isoforms, HSP90α and HSP90β, in mammalian cells. We further separated HSP90 isoforms by decrease pH (to 8.0) and percentage (9%) of PAGE gel; the results showed apparently differential induction of HSP90 isoforms through GA treatment (HSP90α > HSP90β). On the other hand, the consistencies were confirmed from RNA to protein level by real time qPCR analysis, Western Blotting and de novo synthetic analysis. Our results demonstrated that gene level regulation controlled the differential induction of HSP90 isoforms.
According to the promoter sequences of hsp90α and hsp90β, we evaluated the different importance of some transcriptional elements by Electrophoresis Mobility Shift Assay (EMSA). Interestingly, differential induction of HSP90α and β is related to the differential binding activities to HSEs in hsp90α and hsp90β promoters. In addition, we also observed the binding strengths on HSEs might imply how HSP90α is more inducible isoforms than HSP90β. On the other hand, the results of binding activities on basic transcription elements showed that GC-box (sp-1 site) involved in inductive level of HSP90α汹and HSP90β the binding on TATA-box and CRE were respectively related to the induction of HSP90α and HSP90β.
In conclusion, treatment with GA facilitate HSF1 binding to the distinct HSE sites on the promoters of hsp90α and hsp90β and further induce HSP90s. It agreed with the observations of differential induction on mRNA and protein level in 9L cells under treatment with GA.

並列關鍵字

Geldanamycin ； Heat shock protein 90 ； Heat shock element ； Heat shock factor 1 ； Differential transactivation ； real-time qPCR ； gene expression

參考文獻

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