PRSS23必需透過TCF12調控Snail相關的內皮到間葉細胞型態之轉化

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研究生：	謝宜珊 Hsieh, Yi-Shan
論文名稱：	PRSS23必需透過TCF12調控Snail相關的內皮到間葉細胞型態之轉化 PRSS23 is essential for Snail-dependent endothelial to mesenchymal transition via TCF12
指導教授：	莊永仁 Chuang, Yung-Jen
口試委員:	王學孝張壯榮莊永仁
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	41
中文關鍵詞：	內皮到間葉細胞型態之轉化、人類動脈內皮層細胞、絲氨酸蛋白酶23 、轉錄因子Snail 、乙型轉化生長因子
外文關鍵詞：	Endothelial to mesenchymal transition, Human aortic endothelial cell, PRSS23, Snail transcription factor, Transforming growth factor β
相關次數：	點閱：3 下載：0
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內皮細胞自原型態轉化到間葉細胞型態的改變，是心臟瓣膜發育過程中重要的步驟。此一細胞型態轉化的過程，與表皮到間葉細胞型態轉化的過程非常類似，其調控機制也極為相近。先前的研究指出，內皮到間葉細胞型態的轉化可以由乙型轉化生長因子所引發，再經由下游轉錄因子Snail調控許多與轉化相關基因的表現。然而此調控機制複雜多變，仍有許多細節尚待釐清。
PRSS23是我們實驗室早先於內皮細胞中發現並已研究多年的新穎絲氨酸蛋白酶。在先前的斑馬魚研究上顯示，我們利用反義寡核苷酸抑制PRSS23的蛋白質表現後，會導致斑馬魚心臟瓣膜發育異常。我們進一步利用組織免疫螢光切片染色發現，在PRSS23表現受抑制時，位於瓣膜處的內皮細胞並未進行內皮到間葉細胞型態的轉化。於是為了更進一步研究PRSS23在內皮到間葉細胞型態轉化中扮演的角色，本篇研究主要使用短夾干擾型核醣核酸在人類動脈內皮層細胞上抑制PRSS23的表現，進行細胞層次的實驗。
我們的結果指出，抑制PRSS23的表現會使得人類動脈內皮層細胞不進行乙型轉化生長因子所誘發的細胞型態轉化。進一步研究指出，PRSS23不會影響轉化生長因子路徑中SMAD的磷酸化，而是進入胞核影響SMAD下游轉錄因子Snail的轉錄活性。另外根據Snail啟動子的序列分析，我們在各物種間較為保留的片段中，找到E-box的序列 (CANNTG)。在前人研究中，已知TCF12轉錄因子會與E-box結合，進而調控與心臟發育相關下游基因的表現。利用免疫共沈澱法，我們確定了PRSS23會與TCF12結合。這個分析指出，PRSS23和TCF12的複合物可能會藉由調控Snail的啟動子，影響內皮到間葉細胞型態的轉化。本篇研究闡述PRSS23是Snail相關的內皮到間葉細胞型態轉化過程中，不可或缺的因子，並推測其調控心臟瓣膜發育的反應機制。

Endothelial to Mesenchymal Transition (EnMT) is an important process during cardiac valve formation. It is believed the general mechanism of EnMT is similar to that of epithelial to mesenchymal transition (EMT). Previous studies indicate that EnMT can be induced by transforming growth factor β (TGF-β). TGF-β first triggers the downstream signal transduction, such as SMAD pathway, and then activates transcription factor like Snail. The activated Snail represses the expression of endothelial markers while promotes the expression of mesenchymal markers, to drive the EnMT process. Nevertheless, the EnMT has a complex molecular mechanism with many details still remains unclear. PRSS23 is a novel endothelial serine protease identified in our previous studies. In previous in vivo assays, we have used morpholino to knockdown PRSS23 expression in zebrafish embryo, and found the loss of PRSS23 could cause cardiac valve malformation in developing heart. Immunohistochemcal data further showed the cardiac endothelial cells fail to initiate EnMT during valve formation. To characterize the role of PRSS23 in EnMT, human aortic endothelial cells (HAECs) and shRNA knockdown were used as key experimental systems in this study. The results indicated that knockdown of PRSS23 inhibited TGF-β2-induced EnMT in HAECs. Further studies revealed knockdown of PRSS23 did not affect the phosphorylation of SMAD, but repressed the transcription of Snail in TGF-β2-treated HAECs. By co-immunoprecipitation (co-IP), we also validated the interaction of PRSS23 and TCF12. TCF12 is a transcription factor involved in heart development, and is known to bind E-box (CANNTG) in promoter. Interestingly, we found E-box is conserved in the Snail promoter of various vertebrates. Therefore, PRSS23/TCF12 complex may bind the promoter of Snail to regulate EnMT. Our results suggest that PRSS23 is essential for Snail-dependent EnMT, and provide clue to reveal the possible mechanism of PRSS23 in EnMT.

List of Contents
中文摘要    I
Abstract    II
List of Supplemental Figures    IV
List of Tables    IV
Abbreviations    V
1.Introduction    1
1.1PRSS23 and endothelial to mesenchymal transition    1
1.2TCF12    3
1.3The objective in this study    4
2.Materials and Methods    5
2.1Cell culture of HAEC, HEK293T and TLA-HEK293TTM    5
2.2Lentivirus preparation and infection    5
2.3TGF-β2 induced EnMT assay    6
2.4RNA preparation and cDNA synthesis    6
2.5Real-time quantitative polymerase chain reaction (RT-qPCR)    7
2.6Western blot    7
2.7Immunofluorescent staining    8
2.8Construction of protein expression plasmids    8
2.9HEK293T cells transfection    9
2.10Co-immunoprecipitation (Co-IP)    9
2.11Promoter analysis    10
3.Results    11
3.1PRSS23 expression was knockdown by lentiviral shRNA approach    11
3.2Knockdown of PRSS23 inhibited TGF-β2-induced EnMT in HAECs.    11
3.3PRSS23 knockdown repressed Snail transcription.    13
3.4Expression and localization of PRSS23 is independent of TGF-β2 signaling.    14
3.5PRSS23 co-localized and interacted with TCF12 in the HAEC nucleus.    14
3.6Snail promoter analysis indicated possible regulation role of PRSS23/TCF12 complex on EnMT.    16
4.Discussion    18
4.1PRSS23 and TGF-β-induced EnMT    18
4.2PRSS23 and SMAD-independent pathway    19
4.3Nuclear PRSS23    19
4.4PRSS23 and cell proliferation    20
4.5The upstream regulator of PRSS23/TCF12    20
4.6Comparison the mechanism of EMT and EMT    21
4.7Future study    21
5.References    22

List of Figures
Figure 1. PRSS23 expression were reduced at both mRNA and protein level by lentiviral shRNA system.    26
Figure 2. Knockdown of PRSS23 caused HAECs fail to undergo TGF-β2-induced EnMT.    27
Figure 3. Knockdown of PRSS23 did not affect the Smad2 phosphorylation in TGF-β2 treated HAECs.    28
Figure 4. PRSS23 knockdown represses Snail transcription after TGF-β2 treatment.    29
Figure 5. TGF-β2 signaling did not change the expression level and distribution of PRSS23 in HAECs.    30
Figure 6. PRSS23 co-localized with TCF12 at nucleus in HAECs.    31
Figure 7. Treating TGF-β2 in HAECs did not affect the expression pattern of PRSS23 and TCF12 in HAECs.    32
Figure 8. PRSS23 interacts with TCF12.    33
Figure 9. TCF12 DNA binding site, E-box, is found in the conserved region of Snail promoter in various vertebrates.    34
Figure 10. Model of PRSS23 regulated Snail-dependent EnMT via TCF12.    35


List of Supplemental Figures
Figure S1. zPRSS23 knockdown resulted in failed to initiate EnMT during valve formation at 72 hours post fertilization (hpf).    36
Figure S2. Knockdown of PRSS23 inhibited TGF-β1-induced EnMT in HAECs.    37
Figure S3. Knockdown of PRSS23 inhibited Snail transcription during TGF-β1-induced EnMT in HAECs.    38


List of Tables
Table 1. RT-qPCR primers list    39
Table 2. Antibodies list    40
Table 3. Comparison the mechanism of EMT and EMT    41

                                

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