Unfolded Protein Response in Lens Epithelial Cells

描述（由申请人提供）：应激通路的激活是许多类型白内障的主要原因。文献检索表明，内质网（ER）中错误折叠的蛋白质构象可诱导内质网应激，而延长的内质网应激可氧化并杀死多种细胞类型。我们的实验室先前提出，许多白内障源性应激源也表现出内质网（ER）应激，并诱导被称为未折叠蛋白反应（UPR）的明确的保护和死亡途径。UPR的第一阶段保护细胞免受应激，细胞通过泛素依赖性内质网相关降解（ERAD）消除内质网中未折叠的修饰蛋白。然而，长时间内质网应激激活了UPR的第二阶段，导致UPR依赖性死亡途径的激活。UPR死亡途径激活包括半胱天冬酶在内的特定蛋白质，刺激Ca++释放和活性氧（ROS）的过度产生，并导致LEC死亡。已知晶状体上皮细胞衍生生长因子（LEDGF）是一种转录激活因子和存活因子，通过上调应激相关基因的表达来拯救应激条件下的各种细胞类型。最近的一项研究表明，长时间的内质网应激会激活ledgf基因的表达。因此，我们认为UPR还激活了一个折衷的生存途径，该途径激活了ledgf基因和下游生存基因，以促进细胞在长时间内质网应激下的生存。据报道，化学伴侣（PBA和TMAO）和TUDCA可以抑制内质网应激。最近我们发现这些药物可以降低内质网应激，延缓LEC死亡，减轻白内障的形成。这些结果为开发LEC和白内障发展的预防性药物开辟了新的研究机会。目的1是测试A)糖尿病诱导大鼠UPR产生ROS，诱导LEC死亡，促进白内障形成，B) UPR诱导半乳糖血症大鼠LEC异常增殖，C)这些增生的LEC优先受到内质网应激的攻击。我们认为上述事件与白内障的发生有关。目的2是测试LEDGF是否是UPR的促存活下游激活因子，以及UPR通路是否调节LEDGF基因在半乳糖凝集性lec中的转录。刺激LEDGF保护通路可能是减轻慢性糖尿病白内障的另一种途径。目的3：检测化学伴侣和TUDCA是否可以作为预防药物抑制半乳凝性和糖尿病性白内障LECs的死亡。药物将被捕获在聚丙交酯-共聚物（PLGA）纳米颗粒中，并直接注射到玻璃体空间。PLGA可以输送大量的药物，并在很长一段时间内缓慢释放这些药物。公共卫生相关性：应激通路的激活是包括白内障在内的许多类型疾病的主要原因。应激的一种形式，称为内质网应激，是由错误折叠的蛋白质构象引起的。内质网应激产生活性氧（ROS）氧化游离谷胱甘肽和蛋白，诱导晶状体细胞死亡。我们将研究UPR在糖尿病和半乳糖血症大鼠晶状体上皮细胞死亡和白内障形成中的作用。此外，在内质网应激下，晶状体上皮衍生生长因子（LEDGF）在细胞中升高，我们将研究LEDGF在UPR通路中上调的基本概念，这可能会开辟另一种生存途径。最后，已知化学伴侣和TUDCA可减少内质网应激依赖性细胞死亡。我们将研究这些伴侣蛋白和TUDCA是否可以作为预防性药物来抑制内质网应激、ROS产生、LEC死亡和白内障的发生。通过这些伴侣和TUDCA调节内质网应激可能为人类白内障患者的治疗提供一种新的治疗方法。

DESCRIPTION (provided by applicant): Activation of a stress pathway is a primary cause of many types of cataracts. A search of the literatures has indicated that misfolded protein conformations in the endoplasmic reticulum (ER) induce ER stress, and prolonged ER stress oxidizes and kills numerous cell types. Our laboratory has previously proposed that many cataractogenic stressors also exhibit endoplasmic reticulum (ER) stress and induce well-defined protective and death pathways called the unfolded protein response (UPR). The first stage of the UPR protects cells against stress, and cells eliminate the unfolded modified proteins from the ER by the ubiquitin dependent ER-associated degradation (ERAD). However, prolonged ER stress activates the second stage of the UPR, which leads to activation of the UPR dependent death pathway. The UPR death pathway activates specific proteins including caspases, stimulates Ca++ release and the over production of reactive oxygen species (ROS), and results in LEC death. Lens epithelial cell derived growth factor (LEDGF) is known to be a transcriptional activator and survival factor and rescues various cell types under stress conditions by up-regulating expression of the stress associated genes. A recent study has suggested that prolonged ER stress activates the expression of the ledgf gene. Thus, we propose that the UPR also activates an eclectic survival pathway, which activates the ledgf gene and downstream survival genes to promote cell survival under prolonged ER stress. Chemical chaperones (PBA and TMAO) and TUDCA are reported to suppress ER stress. Recently we showed that these drugs reduced ER stress, delayed LEC death, and alleviated cataract formation. These results opened novel opportunities of research to develop prophylactic drugs for LEC and cataract development. Aim 1 is to test that A) diabetes induces the UPR, which produces ROS, induces LEC death, and promotes cataract formation in rats, B) the UPR induces abnormal LEC proliferation in galactosemic rat LECs, and C) these proliferative LECs are preferentially attacked by ER stress. We believe that these above events are involved in cataract development. Aim 2 is to test whether LEDGF is a prosurvival downstream activator in the UPR and if the UPR pathway regulates the transcription of the ledgf gene in galactosemic LECs. Stimulation of the LEDGF protective pathway could be another way of alleviating cataract in chronic diabetes. Aim 3 is to test whether chemical chaperones and TUDCA can be used as prophylactic agents to suppress the death of LECs in galactosemic and diabetic cataract. The drugs will be trapped in poly-lactide-co-glycolides (PLGA) nanoparticles and directly inject into the vitreal space. The PLGA can deliver larger amounts of drugs and slowly release these drugs over a long time. PUBLIC HEALTH RELEVANCE: Activation of stress pathway is a primary cause of many types of diseases including cataracts. One form of stress, so called endoplasmic reticulum (ER) stress, is caused by misfolded protein conformation. The ER stress produces reactive oxygen species (ROS) to oxidize free glutathione and proteins and induces cell death in the lens. We will study the roles of the UPR in lens epithelial cell death and cataract fromation in diabetic and galactosemic rats. Furthermore, lens epithelial derived growth factor (LEDGF) is elevated in cells under ER stress, we will study the basic notion of up-regulation of LEDGF in the UPR pathway, which may open an alternative survival pathway. Finally, chemical chaperones and TUDCA are known to reduce ER stress dependent cell death. We will study whether these chaperones and TUDCA can be used as prophylactic drugs to suppress ER stress, ROS production, LEC death, and cataract development. Modulating ER stress by these chaperones and TUDCA may offer a novel therapeutic approach to the treatment of human patients with cataract.