埃及斑蚊TAK1參與調控基質金屬蛋白酶之功能性研究

Functional roles of TAK1 in the regulation of Aedes aegypti matrix metalloproteinase

王俊淵(Jiun-Yuan Wang)

指導教授 ： 蕭信宏

國立臺灣大學/醫學院/微生物學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10213

摘要

蚊子會造成許多不同傳染病之傳播，其主要為瘧疾、登革熱、黃熱病、日本腦炎與絲蟲病等疾病傳播之媒介。由於一直以來針對這些熱帶疾病所使用之疫苗或治療藥物相當缺乏，並且亦會有抗藥性的產生，因此發展有效控制病媒蚊疾病傳播之替代方法則為現今相當重要的課題。過去研究已知，在果蠅(Drosophila)系統中之Toll pathway與Immune deficiency (IMD) pathway主要負責調控果蠅抗菌胜肽的產生，其中Toll pathway在果蠅受到革蘭氏陽性菌或真菌感染時會啟動，而當果蠅受到革蘭氏陰性菌感染時則會啟動IMD pathway。除了調控免疫反應之外，屬於IMD pathway 下游且受到transforming growth factor-β-activated kinase1 (TAK1)調控之JNK pathway，其主要功能為控制果蠅發育過程中tissue remodeling作用。此外，過去研究發現MMP1 (Matrix Metalloprotienase 1) 亦會參與調控果蠅胚胎之tissue remodeling過程。本實驗室之前研究發現，埃及斑蚊之TAK1 (AaTAK1) 基因亦會調控埃及斑蚊受到革蘭氏陽性菌刺激時抗菌胜肽Cecropin A的表現，此外亦發現埃及斑蚊MMP1(AaMMP1)會影響埃及斑蚊吸血之後卵黃生成 (Vitellogenesis) 作用。我們利用RNA干擾方式抑制埃及斑蚊TAK1之表達，發現MMP1之轉譯層次的表達會受到抑制。因此本研究主要想要探討在埃及斑蚊中IMD pathway調控MMP1基因表現的分子機制為何。首先我們利用即時定量PCR (quantitative real-time PCR) 之方式探討AaTAK1轉錄層次之表現情形 (expression pattern)，我們觀察到AaTAK1之mRNA在埃及斑蚊吸血之後12小時到72小時會有大量表現的情形，且主要表現在埃及斑蚊之卵巢與中腸; 接著我我們利用抑制AaTAK1之表現的方式進一步研究AaTAK1之功能，透過利用顯微注射的方式將我們針對AaTAK1部分片段設計的雙股RNA (double-stranded RNA) 注射進入母蚊體內，再進一步進行功能分析。結果發現，當利用RNA干擾方式抑制AaTAK1表現時，埃及斑蚊之母蚊產卵量 (egg production) 會有下降的趨勢 ; 接著我們利用in vitro fat body culture的方式進行實驗，我們發現若利用RNA interference的方式抑制AaTAK1表現或利用AaJNK抑制劑 (SP600125) 抑制AaJNK表現的狀況下，皆會使Vitellogenin (Vg)表現受到抑制，此外，當AaJNK表現受到RNA 干擾方式抑制時，母蚊之產卵量亦會下降。當透過直接注射雙股RNA進入母蚊方式抑制AaTAK1與AaJNK表現時，則可造成母蚊之脂肪體中TOR pathway下游之S6K磷酸化反應受到抑制，並且更進一步發現受到TOR pathway 調控之Vitellogenin (Vg)與AaMMP1表現量亦會減少。總而言之，我們證明AaTAK1與AaJNK在埃及斑蚊之卵黃發育的過程中扮演相當重要的角色，並且其可能是透過調控TOR pathway之活化，再進一步影響AaMMP1之表現與卵黃生成之能力。

關鍵字

埃及斑蚊 ； 卵黃生成 ； MMP1 ； TAK1 ； JNK

並列摘要

Mosquitoes transmit several devastating infectious disease such as malaria, dengue fever, yellow fever, Japanese encephalitis and filariasis. Due to the lack of effective vaccine and the increasing drug and insecticide resistance, alternative approaches for these vector-borne disease are urgently required. It has been demonstrated that the Toll and the Immune Deficiency (IMD) signaling pathways play crucial roles in the production of antimicrobial peptides in the Drosophila. Toll pathway was primarily activated by the stimulation of Gram-positive bacteria or fungi, whereas IMD pathway was responsive to the challenge of Gram-negative bacteria. On the other hand, Matrix metalloproteinase 1 (MMP1) was demonstrated to be essential for the embryonic development in the Drosophila and JNK pathway was shown to be activated by transforming growth factor-β-activated kinase 1 (TAK1), the component of IMD pathway, and to play an important role in tissue modeling in Drosophila. Our previous study showed that Aedes aegypti TAK1 (AaTAK1) is responsible for the production of Cecropin A. We also showed the novel role of Aedes aegypti Matrix metalloproteinase 1 (AaMMP1) in the regulation of vitellogenesis. Our preliminary data revealed that silence of AaTAK1 by RNA interference approach resulted in the inhibition of AaMMP1 in the translational level. In this study, we will elucidate the molecular mechanism in the regulation of AaMMP1 by IMD pathway. We showed that the transcriptional pattern of AaTAK1 is highly expressed from 12 to 72 hours after a blood meal and particularly in the ovary and midgut. By RNAi-mediated silencing of AaTAK1, we showed that the egg production was reduced in the absence of AaTAK1. Interestingly, the expression of Vitellogenin (Vg) was inhibited in the absence of AaTAK1 or with the application of JNK inhibitor (SP600125) in the in vitro fat body culture system. In addition, by RNAi mediated silencing of AaJNK, the egg production was also reduced. Furthermore, we showed that silencing of AaTAK1 or AaJNK inhibit the phosphorylation of S6K, a key component of TOR pathway, and also inhibit the expression of Vitellogenin (Vg) and AaMMP1 in the fat body. Taken together, our data suggest a novel function of AaTAK1 and AaJNK in the regulation of vitellogenesis and AaMMP1 through TOR signaling pathway.

並列關鍵字

Aedes aegypti ； Vitellogenesis ； MMP1 ； TAK1 ； JNK

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