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標題: | 斑馬魚Dickkopf-3-related gene (Dkk3r) 藉由膜蛋白受器Integrin alpha 6b影響肌肉調控蛋白myf5基因啟動子的活性 Zebrafish Dickkopf-3-related gene (Dkk3r) regulates the promoter activity of myogenic regulatory factor myf5 gene through interaction with membrane receptor of Integrin alpha 6b |
作者: | Ying-Fang Su 蘇盈方 |
指導教授: | 蔡懷楨 |
關鍵字: | Dkk3r,Integrin alpha 6b,myf5,肌肉發育,斑馬魚, Dkk3r,Integrin alpha 6b,myf5,myogenesis,zebrafish, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | Myf5是肌肉發育調控因子MRFs (myogenic regulatory factors) 的成員,對於肌肉發育的特化和分化上具有重要功能。斑馬魚myf5 intron I中含有miR-3906,會抑制其目標基因dickkopf-3-related gene (dkk3r) 的mRNA轉譯。Dkk3r為外泌型蛋白質,會調控細胞質訊息傳遞路徑,使得下游p38a磷酸化比例增加,進而增加Smad4蛋白質穩定性,進一步促使Smad2/Smad3a/Smad4複合體形成,進入細胞核以活化myf5啟動子,影響體節肌肉的生成發育。然而外泌型的Dkk3r是藉由與何種膜蛋白受器結合來調控胚胎發育仍然為未知。Fu (unpublished data) 以昆蟲細胞大量表現斑馬魚Dkk3r-Flag蛋白質,於斑馬魚總蛋白質中藉由蛋白質免疫沉澱實驗和蛋白質質譜分析篩選出Integrin alpha6b (Itga6b) 為可能結合的膜蛋白受器基因之一。為了進一步確認這個假設,本實驗中首先以whole-mount in situ hybridization (WISH) 觀察dkk3r mRNA與itga6b mRNA表現位置,發現於肌肉發育時期 (16 hpf) 兩者mRNA在體節位置皆有表現。在HEK-293T細胞中進行in vitro cell surface binding assay則可以偵測到Dkk3r與Itga6b的表現訊號位置在細胞膜有co-localization的情形。顯示於時間及空間上Dkk3r與Itga6b都相重疊。接下來,利用in vivo luciferase assay可以發現,於斑馬魚胚胎中過量表現dkk3r mRNA可以使得myf5啟動子所驅動的luciferase活性上升為223%,而過量表現itga6b mRNA可使得其活性上升為217%。另外,若dkk3r mRNA以及itga6b mRNA共同過量表現時,會使myf5啟動子驅動luciferase的活性有加成上升為397%的效力。上述活性上升皆有dosage-dependent的現象。而當過量表現itga6b mRNA並同時共同注射dkk3r-MO 去抑制Dkk3r的表現時,會造成myf5啟動子驅動luciferase活性下降為69%。這些證據顯示斑馬魚胚胎中Itga6b開啟下游的活性需要Dkk3r來參與。另一方面,注射itga6b-MO以抑制Itga6b的表現,可觀察到斑馬魚胚胎體節形狀異常;進而利用WISH也可以觀察到抑制Itg alpha 6b的表現,會使得myf5 mRNA於16 hpf時在體節表現剩下三節 (-II、-I、0) 且表現量較少,甚至有不表現的缺失。更進一步的證實Itga6b的缺失會使得Dkk3r下游之磷酸化p38a蛋白質的表現量下降。綜合以上實驗結果,我們認為Itga6b極可能為Dkk3r的膜蛋白受器,而兩者間會互相結合開啟下游訊息傳遞路徑,進而調控斑馬魚胚胎發育時期在體節內myf5啟動子的活性。 Myf5, one of the myogenic regulatory factors, plays important roles in the specification and differentiation of muscular cells during myogenesis. In zebrafish, an intronic microRNA (miR), miR-3906, located within myf5 intron I, has been reported to silence the translation of its target gene, dickkopf-3-related (dkk3r) gene. Dkk3r, a secretory protein, regulates the phosporylation of p38a to maintain Smad4 stability, which, in turn, enabling the Smad2/Smad3a/Smad4 complex to form and to activate the myf5 promoter in nucleus. However, the membrane receptor(s) bound by Dkk3r to control signal transduction is still unknown. After recombinant zebrafish Dkk3r tagged with Flag was produced by insect cells, we applied protein immunoprecipitation and mass spectromotry to screen the putative receptors of Dkk3r. We found that Integrin alpha 6b (Itga6b) might be one of receptors to interact with Dkk3r. To further confirm this hypothesis, we used whole-mount in situ hybridization and found that the transcripts of both dkk3r and itga6b were presented in somites at 16 hpf during myogenesis. By in vitro cell surface binding assay, we also observed that Dkk3r and Itga6b were co-expressed at the cell membrane of HEK-293T, indicating that the temporal and spatial expressions of dkk3r and itga6b are co-localized. Furthermore, in vivo luciferase assay demonstrated that the luciferase activity driven by myf5 promoter was 223% and 217% greater than that of control when the excessive dkk3r and itga6b mRNAs were injected into embryos, respectively. Interestingly, when we co-injected dkk3r and itga6b mRNAs into embryos, the luciferase activity was up-regulated as high as 397% greater than that of control embryos. This up-regulation of myf5 promoter activity mediated by interaction between dkk3r and itga6b was dosage-dependent. In contrast, when dkk3r was knockdown and co-injected with itga6b mRNA, the luciferase activity was down-regulated to 69% of control embryos, suggesting that the regulatory effect of Itga6b on the downstream activity is dependent on Dkk3r signal pathway. In addition, knockdown of itga6b by injection of itga6b-morpholinos resulted in abnormal shape of somites and weak or even absent expression of myf5 in somites at 16 hpf. Furthermore, knockdown of itga6b reduced the protein level of the phosphorylated p38a. Taken together, we concluded that it is highly likely that Itga6b functions as a receptor of Dkk3r. Their interactions drive the downstream signal transduction to regulate myf5 promoter activity in somites during the development of zebrafish embryos. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25197 |
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