埃及斑蚊MMP1調節卵黃生成作用以及發育過程之特殊功能

Abstract

Matrix metalloproteinases (MMPs)是一酵素蛋白質家族(protein family)的統稱，MMP在哺乳動物中，主要參與了組織間重組(tissue remodeling)的過程，例如：胚胎生長、發育、生殖以及傷口修復等；然而，當個體受外來致病物感染時，免疫系統會活化而降低這些蛋白酶之表現。截至今日，在人類中已發現了超過二十種的MMPs，但和果蠅(Drosophila melanogaster)之MMPs具有同源性(homolorus)的只有兩種。雖然MMP的功能在哺乳動物以及果蠅已有相當多的研究，但是對於病媒蚊MMPs家族之研究幾乎付之闕如。我們在登革熱病媒蚊-埃及斑蚊(Aedes aegypti)體內，發現了共有六種不同的MMPs。而這些MMPs皆有一段序列具有高度保留的活性區以及在N端有一段訊號序列(signal peptide)。對於埃及斑蚊的MMP1(AaMMP1)的研究方面，首先我們分別利用即時定量PCR (quantitative Real-Time PCR)以及西方吸漬法(Western blot)來探討AaMMP1轉錄及轉譯之表現曲線(expression pattern)，我們觀察到AaMMP1的mRNA在蛹期早期以及羽化後三天有高量表現，而蛋白質則是只表現在吸血後六小時及十二小時，且主要是在fatbody及中腸(midgut)表現；接著我們利用抑制AaMMP1表現之方式來探討AaMMP1可能具有之功能，利用顯微注射方式，我們將針對AaMMP1設計的雙股RNA(double-stranded RNA)注射進入蚊子體內再進行功能分析。我們發現，若是AaMMP1的表現受到抑制會降低其產卵能力(oviposition rate)。而特別的是，我們發現若是抑制了Wnt signaling pathway中的其中一個蛋白質-Frizzled之表現，Aa MMP1之表現在吸血後則會被完全抑制。我們利用in vitro fat body culture的方式，也發現在AaTOR以及AaFz表現被抑制的情況下，AaMMP1的表現是完全被抑制的。此外我們也發現IMD pathway當中的TAK1會調控AaMMP1的表現。總而言之，我們證明了AaMMP1在埃及斑蚊卵黃發育所扮演之重要角色，以及其牽涉之多個不同信號傳遞路徑調控之機制。

Matrix Metalloproteinases (MMPs) are a family of enzymes that play major roles in tissue remodeling, such as embryonic growth, development, reproductive process and wound healing in mammals. In addition, deregulation production of these endopeptidases is associated with a number of pathological conditions resulting in the activation of innate immune responses. To date, more than 20 human MMPs have been identified and characterized and only two homologues are identified in the fruit fly Drosophila melanogaster. In spite of the intensive investigation in mammals and fruit fly, functional roles of MMP family in the mosquito are largely unknown. Recently, we identified six MMPs in the mosquito Aedes aegypti, the major vector of dengue virus. All of these MMPs contain a highly conserved catalytic domain and show signal peptide at the N-terminal part. The transcriptional and translational expression pattern of Ae. aegypti MMP1 (AaMMP1) were examined by quantitative Real-Time PCR and Western blot analysis, respectively. AaMMP1 mRNA is highly expressed at white pupal stage and 3 days post eclosion, whereas AaMMP1 protein is only expressed at 6 and 12 hours after a blood meal. In addition, AaMMP1 is highly expressed at fatbody and midgut after a blood meal. Next, we applied the well established reverse genetic approach by silencing specific MMPs in the mosquitoes to characterize the functions of MMPs. Silence of AaMMP1 by intrathoracically introduced the double-stranded RNA targeted to AaMMP1 revealed that oviposition rate is reduced in the absence of AaMMP1. Surprisingly, silence of Frizzled (Fz), a component of Wnt signaling, prior to blood meal resulted in the complete inhibition of AaMMP1. In addition, we showed that silence of AaTOR and AaFz completely inhibit the expression of AaMMP1 in the in vitro fat body culture. RNAi-mediated silence of the key component in the IMD pathway resulted in the depletion of AaMMP1 in vivo. Taken together, our results demonstrated the crucial role of AaMMP1 in the mosquito vitellogenesis and development, and the multiple regulatory machinery by different signal transduction pathways.