斑馬魚Dickkopf-3-related gene (Dkk3a) 藉由膜蛋白受器 Integrinα6b 調控肌肉調控蛋白myf5 基因啟動子的活性

Abstract

肌肉調控因子Myf5在肌肉發育中扮演了重要的角色，在斑馬魚Myf5中具有一個 microRNA(miR), 稱為miR3906 或miR-I300 ， 目前已經被報導能夠來抑制 dickkopf-3-related gene (dkk3r 或 dkk3a)，並進一步來調控myf5啟動子活性。然而會與 Dkk3a結合經由訊息傳遞來調控myf5的膜接受器依然是未知的。 為了探討會與Dkk3a結合的膜接受器，我們利用免疫沉澱的實驗與LC-MS/MS質譜 分析來篩選可能與Dkk3a結合的膜接受器，最後篩選到了Integrin α6b (Itgα6b)為可能與 Dkk3a結合的膜蛋白受器。為進一步確認，我們利用cell-surface binding assays來證實 Dkk3a會與Itgα6b在HEK-293T細胞膜上結合，同時Crosslinking immunoprecipitation實驗 也顯示出Itgα6b對於Dkk3a結合具有高親和力，我們進一步證實Itgα6b的β-propeller repeated domains是與Dkk3a結合的主要位置，並且當dkk3a mRNA與itgα6b mRNA注射到 胚胎中，myf5啟動子luciferase的活性會上升4倍。相反的當分別共同注射dkk3a mRNA與 itgα6b MO或itgα6b mRNA與dkk3a MO，其luciferase會下降至與控制組胚胎類似。另一方 面當itgα6b knockdown後會對胚胎造成不正常的體節形狀、較少的體節細胞數、減少或 消失的myf5表現與p38a磷酸化下降的缺失性狀，而這些在體節肌肉發育的缺失性狀與 dkk3a knockdown的胚胎是相類似的。 綜合上述實驗結果，我們證實了外泌型的Dkk3a會藉由與膜蛋白受器Itgα6b結合來 增加p38a蛋白的磷酸化進而在斑馬魚肌肉發育過程中來活化myf5啟動子的活性。

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repressing myf5 promoter activity. However, the membrane receptor which interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS mass spectrometry to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we employed cell-surface binding assays which showed that Dkk3a and Itgα6b co-expressed at the cell membrane of HEK-293T cells. Crosslinking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeated domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was upregulated four-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a-knockdown embryos co-injected with itgα6b mRNA and itgα6b-knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker, or absent, myf5 expression, and reduced protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a-knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.