Functions of PXR and bHLH-PAS Genes in Fish Gastrointestinal Tract Developmnet (III)

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Project title

Code/計畫編號

NSC96-2313-B019-004

Translated Name/計畫中文名

PXR與bHLH-PAS基因對於魚類消化器官發育的功能(III)

Project Coordinator/計畫主持人

Chin-Hwa Hu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=1415734

Year

2007

Start date/計畫起

01-08-2007

Expected Completion/計畫迄

01-07-2008

Bugetid/研究經費

1528千元

ResearchField/研究領域

漁業
生物技術(農)

在我們先前的研究中發現斑馬魚ARNT2 基因受到抑制後會造成肝臟與胰臟外分泌系統發育停滯的現象，顯示bHLH-PAS 基因及其相關的細胞訊息傳遞系統參與了肝臟及胰臟外分泌系統的細胞分化工作。在另外一方面，我們先前亦從斑馬魚胚胎中選殖出一個與人類最重要的藥物代謝基因CYP3A4 相似的基因CYP3A65，並且此基因對抗生素rifampicin 以及類皮質醣dexamethasone 有類似人類CYP3A4 的敏感反應增加基因表現的能力，顯示斑馬魚的CYP3A65 基因的調控機制可能與人類及哺乳動物的CYP3A 基因相似，接受細胞內的PXR::RXR 的訊息傳遞系統控制。但是CYP3A65 除了對PXR 的受質有敏感反應外，在發育時期對於戴奧辛（TCDD）亦有相當敏感的反應而增加表現的能力，而將其訊息傳遞的接受器AHR2 基因抑制後則CYP3A65 的表現消失，顯示CYP3A65 亦同時受到 AHR::ARNT 訊息傳遞系統的調控。目前對於PXR::RXR 以及AHR::ARNT 兩個細胞傳遞系統在YP3A65 基因調控的機制中相互的關係尚待釐清。在本計畫中，針對上述兩項重要的研究結果探討其分子機制。我們發現將PXR或AHR2基因弱化（knockdown）後均會造成 CYP3A65基因的表現受到抑制，顯示兩者均直接調控CYP3A65基因的表現。另外我們分析 CYP3A65基因的啟動子，發現在其上游5800 bp區域共有5個AHR::ARNT的辨識序列（XRE）以及21個腸道專一性基因轉錄子Cdx2的辨識序列。為了進一步分析CYP3A65基因的調控機制以及消化道發育的機制，我們利用Tol2系統，建立CYP3A65-GFP轉殖基因品系斑馬魚，發現從2 dpf時期起可以在消化到出現明顯的GFP螢光的表現，表現部位與時間均與原生性 CYP3A65基因的活性相同。當我們以morpholino弱化AHR2後會抑制GFP的表現，但是並不會抑制另一消化道的標示基因IFABP的表現，顯示AHR2確實參與了CYP3A65的調控，但是並不影響消化道的發育。另外我們將AHR2基因弱化後，發現並不會對PXR的表現產生顯著的影響，顯示AHR與PXR兩者間並無相互的關連性。在肝臟及胰臟發育方面，我們將 bHLH-PAS 基因（包括ARNT2與HIF-1/2/3 alpha）以morpholino 進行基因弱化之後，發現會抑制肝臟與胰臟的發育。其中在HIF-1/2/3 alpha的基因弱化後，會引起細胞的凋亡現象，此外對於腹部Shh基因的表現也會顯著受到抑制，我們判斷這可能是造成消化道發育受阻的主要原因。綜合以上的實驗結果，我們在本計劃中已完成下列的幾項研究工作，包括建立 CYP3A65 -GFP螢光蛋白品系斑馬魚，探討AHR2與PXR在CYP3A65基因調控的功能，以及探討HIF-1/2/3 alpha在消化道發育中所扮演的功能。這些成果提供未來進一步活體觀察消化道發育的一項重要工具，並且可開發成為藥物篩選的重要工具。Previously we have shown that the ARNT2 knockdown in zebrafish embryo caused severe defects in liver and pancreatic exocrine development. It suggests that the bHLH-PAS proteins and their related signaling systems have important roles in fish gastrointestinal development. In addition, we have cloned a CYP3A-like gene, CYP3A65, from zebrafish embryo. Like human and mammalian CYP3A genes, the CYP3A65 transcription was sensitive to rifampicin and dexamethasone. It suggests that the PXR::RXR signaling pathway may have important role in CYP3A65 regulation as what does in human CYP3A4 transcription. Unlike mammalian CYP3A gene, zebrafish CYP3A65 transcription could be enhanced by TCDD during larva stage. Knockdown AHR2 caused CYP3A65 inhibition. It suggests that the CYP3A65 is also controlled by AHR::ARNT signaling pathway. In this research project, we have investigated the following issues: 1. the functions of bHLH-PAS genes in liver and pancreas development. 2. regulatory mechanism of PXR::RXR and AHR::ARNT signaling pathways in CYP3A65 transcription. 3. the function of PXR::RXR in fish development. We have established a transgenic CYP3A65-EGFP fish line which display a similar expression pattern as the endogenous CYP3A65 gene. The EGFP expression was greatly enhanced by TCDD and it was inhibited by AHR2 knockdown. The endogenous CYP3A65 transcription was also repressed by PXR knockdown. It confirmed that CYP3A65 gene is regulated by both of AHR and PXR-related pathways. We have also found that blocking HIF-1/2/3 alpha translation inhibited liver and the pancreas exocrine cells differentiation, but had little or no effect on gastrointestinal development.

Keyword(s)

bHLH-PAS 蛋白
PXR::RXR
CYP3A65基因

DSpace CRIS

Functions of PXR and bHLH-PAS Genes in Fish Gastrointestinal Tract Developmnet (III)

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