模式生物研究Hsf4b/T472磷酸化修饰障碍诱发白内障的分子机理

Cataract is the predominant cause of blindness worldwide. It consists of aging cataract and congenital cataract. Aging cataract is mainly associated with peroxidation, while the congenital cataract is closely related to the genetic mutations. Exploration of the molecular mechanism underlying cataract is important for therapeutic prevention of cataract. Hsf4 is a family member of heat shock transcriptional factors that are known to regulate the transcription of heat shock proteins in response to the variety of stresses. Impairment of Hsf4b transcriptional activities e.g. germline mutations, has been reported to be closely associated with the human congenital cataract and aging cataract. However, the signaling pathways that regulate Hsf4b transcriptional activities are not completely understood. Our previous results indicated that Hsf4b transcription activities are tightly regulated by posttranslational phosphorylation. Our in vitro data show that phosphorylation of Hsf4b/T472 can regulate Hsf4b nucleus translocation and transcription activity by interacting with the nuclear membrane transporter protein complex importin β-1/Hsc70. MEK6 is a potential kinase for the phosphorylation of Hsf4b/T472. However, it is still not clear whether this phosphorylation site of Hsf4b participates in the regulation of lens development or aging cataract in vivo. In this grant application, we will: 1. Determine the phosphorylation effects of MEK6 pathways on Hsf4b’ transcription activities; 2. Establish the transgenic mouse and zebrafish models to study the roles of phosphorylation of Hsf4b/T472 on Hsf4b transcriptional activity in vivo and during lens development. Our studying results will demonstrate for the first time the regulatory effects of Hsf4b phosphorylation on cataract formation in vivo.

Hsf4是白内障致病基因，其转录活性障碍导致白内障发生。研究发现Hsf4b的磷酸化修饰调控其转录活性。通过体外实验我们证明，Hsf4b/T472的磷酸化修饰调控Hsf4b与核膜转运蛋白importin β-1/Hsc70复合体的结合，进而调控Hsf4b的核内转运和转录活性。但Hsf4b/T472的磷酸化修饰在整体动物体内是否调控Hsf4b的转录活性，进而调控晶状体发育还不清楚。因此，本课题中我们首先揭示直接调控Hsf4b/T472磷酸化的激酶（MEK6，ERK1/2），进而探究晶状体发育中Hsf4b/T472的磷酸化状态及参与的上游信号通路。同时，构建Hsf4b/T472突变的转基因动物模型，体内实验阐明Hsf4b/T472的磷酸化修饰调控晶状体发育的分子机理。该课题将建立一套体内研究Hsf4b转录活性的动物模型，为靶向防治Hsf4b转录活性下降诱发的白内障提供理论依据和药物筛选平台。

Hsf4是先天性白内障致病基因，其转录活性的失调导致晶状体发育异常。Hsf4转录活性如何被调控的分子机制并不清楚。本项目研究发现，模式动物斑马鱼晶状体中的Hsf4为一全新的剪切本，但其T472磷酸化位点缺失。经过序列比对，Hsf4/S299磷酸化位点在所有物种间高度保守。通过构建Hsf4/S299磷酸化位点突变的晶状体上皮细胞稳定株，我们发现Hsf4/S299磷酸化与Hsf4下游基因Hsp25和Cryab启动子结合能力减弱，进而抑制Hsf4的转录活性。通过制备特异性识别Hsf4/S299位点的磷酸化抗体，我们明确了晶状体分化过程中Hsf4/S299位点磷酸化的时空表达情况。同时我们发现EGF，cisplatin和MG132等刺激可以诱导Hsf4/S299位点的磷酸化修饰，进一步的研究表明MEK1-ERK1/2信号通路参与修饰Hsf4/S299位点的磷酸化，JNK和p38通路并不参与这一过程。去磷酸酶PP2A也介导该位点的去磷酸化过程。MEK1-ERK1/2通路调控Hsf4/S299位点的磷酸化在体外培养的晶状体中也得到验证。成功构建了晶状体中特异性表达Hsf4/S299磷酸化位点突变的斑马鱼模型，F0和F1代斑马鱼晶状体中可以明显观察到野生型和突变型Hsf4的表达，F2代Hsf4/S299突变斑马鱼正在筛选中，其表型需进一步观察。此外，Hsf4缺失的斑马鱼模型，其白内障表型明显，Hsf4已知下游基因变化明显。利用转录组学技术，我们鉴定出一新的Hsf4下游基因Aim1。染色质免疫沉淀和Luciferase报告实验证实Hsf4通过直接结合在其启动子上调控其表达。总之，利用晶状体上皮细胞稳定细胞系和斑马鱼模型，我们阐述了Hsf4/S299位点磷酸化修饰的信号通路，拓展了目前对Hsf4转录活性调控的认识，为靶向Hsf4相关白内障提供了线索。