热休克转录因子4（Hsf4）对晶状体纤维细胞分化的双刃剑调控

Genetic mutation of Heat shock factor 4 (Hsf4) causes the family inherited autosome dominant lamellar cataract with the deregulation of neonatal lens fiber cell denucleation, a key process of lens organelle-free zone (OFZ) formation. However, the molecular mechanism underlying Hsf4 is still not completely clear. Our preliminary data demonstrate that Hsf4 plays multiple roles in modulating the homeostasis of lens epithelial and fiber cells, for examples, (1) For the first time our immunohistochemistry results indicate that Hsf4 is differently expressed in the cytoplasm of anterior and equator epithelial cells and the nuclear of secondary and primary fiber cells in the neonatal lens tissue; (2) Hsf4 can significantly inhibit the caspase3 activation induced by apoptotic drugs (cisplatin, staurosporine and CCCP); (3) Hsf4 can upregulate lysosome-mediated hydrolysis of GFP-LC3 and exogenous DNA in vitro, and increase the expression of lysosomal H+ bump V-ATPase, lamp-1 and DNase IIβ; The dynamic cycles of caspase 3’s activation and inactivation and the lysosome-mediated autophagy are two important pathways in modulating lens fiber cell differentiation including the process of fiber cell denucleation. Therefore, we hypothesize accordingly that Hsf4 plays the double-edge sword roles in the regulation of lens fiber cell differentiation by simultaneously activating lysosome-mediated denucleation and inhibiting the over-activation of caspase3 induced apoptosis. The dynamic balances of fiber cell nuclear degradation, caspase 3 activation and anti-apoptosis are important for the differentiated fiber cell homeostasis. In this grant application, our study will focus on the following topics: 1. Dissection of the molecular mechanism underlying Hsf4’s antiapoptosis; 2. Investigation of the signal pathways by which the Hsf4 modulates the expression of V-ATPase, lamp1, DNase IIβ and lysosome activity; 3. How does lysosome work on the denucleation of lens fiber cells? The studying results of this grant will provide more novel evidences for early diagnosis and prevention of the congenital cataract associated to Hsf4-mutants.

晶状体中心透明区纤维细胞脱核障碍是Hsf4基因突变诱发遗传性白内障的主要病因。但其调控脱核的机制不清楚。我们发现：1. Hsf4在新生晶状体上皮细胞胞质，初级和次级纤维细胞核内表达；2. Hsf4抑制顺铂等药物诱导的caspase3活化和细胞凋亡；3. Hsf4能上调溶酶体Lamp1、V-ATPase、DNaseIIβ的基因表达。Caspase3和溶酶体是负责水解细胞核碎片的主要场所。由此推测纤维细胞分化过程中，Hsf4具有上调溶酶体-自噬介导的脱细胞核和抑制caspase3过度活化诱发凋亡的双刃剑作用。本课题将利用晶状体上皮细胞系，转基因模式生物（斑马鱼和小鼠）和分子生物学技术揭示：1. Hsf4在晶状体组织内抗凋亡的分子机制；2. Hsf4上调溶酶体-自噬通路的分子机理及其对纤维细胞脱核的调控机制。意义：揭示Hsf4突变致白内障的分子机理，为Hsf4缺陷型白内障的早期诊断提供理论依据。

热休克转录因子4（HSF4）基因突变是先天性遗传性白内障致病基因之一。该项目以小鼠晶状体上皮细胞系，HSF4基因缺失斑马鱼转基因模型等为研究材料，研究了HSF4在晶状体发育过程的双刃剑调控机制，并在以下几方面取得研究成果和进展：1）确定了HSF4在晶状体发育过程中的表达分布特点。在新生小鼠晶状体内，HSF4表达在上皮和次级纤维细胞的胞质和胞核内，随年龄增加，HSF4主要分布在细胞核内，胞质内表达量减少；2）揭示了HSF4的抗凋亡机制。HSF4通过调控下游小分子热休克蛋白HSP25 和aB-crystallin的表达，抑制顺铂和H2O2诱导晶状体上皮细胞凋亡。Alpha B-crystallin结合线粒体释放的细胞色素C，抑制线粒体膜电位激活和caspase 3活化，降低细胞内ROS的产生是HSF4抗凋亡的主要机制；3）首次报道HSF4具有调控溶酶体内稳态的作用。aB-crystallin作为HSF4下游，能结合溶酶体膜上氢离子泵复合体中的ATP6V1A，并能抑制ATP6V1A蛋白的泛素化修饰和蛋白酶体降解，继而稳定溶酶体酸性微环境.这一研究成果揭示了HSF4调控晶状体蛋白内稳态的新机制；4）揭示DNase1L1L是斑马鱼晶状体纤维细胞分化脱核的关键脱氧核酸内切酶。HSF4通过上调DNase1L1L的转录表达，调控纤维细胞脱核。这一研究再次证明HSF4在不同种属动物晶状体中，通过调控脱氧核糖核酸内切酶的表达参与晶状体纤维细胞分化。在该课题支持下发表8篇SCI论文，获得河南省科技进步二、三等奖各一项，培养3名青年教师获得国家自然科学基金支持，培养10名硕士研究生。