Regulation of cutaneous wound healing by GCN2

每年大约有650万美国人被诊断出患有皮肤慢性伤口， 美国的医疗保健系统每年花费超过200亿美元。通常这些慢性伤口 在老年人中，高达85%的慢性伤口患者年龄超过65岁，或 并发疾病，如糖尿病、肥胖症、营养不良或药物滥用。同样， 妥善愈合伤口是退伍军人的一个主要问题，因为目前有900多万 65岁以上的退伍军人，约占退伍军人总人口的38%。这些患者往往会受到 一系列的各种治疗，往往没有机械的理由，他们仍然缺乏足够的 解决他们的伤口。因此，更好地了解慢性伤口的具体原因， 需要一系列治疗方案来解决这一未满足的需求。一旦出现任何类型的慢性伤口， 无论是糖尿病性溃疡、褥疮性溃疡还是静脉淤滞性溃疡， 伤口闭合是表皮细胞不能使伤口床再上皮化。慢性伤口仍然存在 在炎症阶段停止，几乎没有角质形成细胞穿过伤口床的迁移。最近 数据表明，一种进化上保守的机制，称为综合应激反应， (ISR)对于人皮肤的正常上皮再生至关重要。ISR由四个不同但相关的 蛋白激酶被各种类型的环境应激激活，导致蛋白激酶的磷酸化。 真核细胞起始因子2 α亚基（eIF 2 α~P）。的后果 eIF 2 α~P包括对蛋白质翻译的全面抑制，这保存了重要的细胞能量储存， 选择性翻译与应激反应相关的基因。重要的是， 证明ISR在正常皮肤中分化角质形成细胞中被激活。此外，委员会认为， 角质形成细胞缺乏eIF 2 α~P蛋白激酶之一（GCN 2，其在角质形成过程中磷酸化eIF 2 α~P）， 表皮分化）形成缺乏正常屏障功能的异常角化障碍皮肤。如果GCN 2 在人角质形成细胞中敲除表达（使用CRISPR/CAS9的外显子12缺失）， 缺乏上皮片迁移而不影响单个细胞的运动性。这些数据导致了 假设GCN 2是皮肤创伤反应的关键组分，并且 通过药剂的ISR将改善伤口愈合。最后一点特别有趣，因为 几种调节ISR（积极或消极）的药物已经在人体临床试验中， 这些疾病可能会加速任何有希望的药物迅速进入人体试验。 在本提案中，对ISR在伤口愈合中的作用的初步研究使用模型系统，包括两个- 体外三维角质形成细胞生长以及三维器官皮肤系统和人类临床 体内研究。体外研究将确定GCN 2激活的机制， 角质形成细胞培养，确定角质形成细胞片层迁移中哪种功能受到损害， GCN 2失活，并分析哪些基因和信号通路被增强的蛋白激活 使用多核糖体谱分析和RNA-seq分析的组合进行翻译。体内研究将确定 ISR特异性激活剂/抑制剂对皮肤伤口愈合的效用，确定是否正常的细胞的失活， 老年患者的ISR改变了正常的伤口愈合，并检查了慢性伤口的病理活检 以确定ISR在疾病进展过程中是否失活。管理慢性 伤口，以及加速急性皮肤损伤的解决的潜力，对于 退伍军人，无论是退伍军人患者的生活质量问题，还是退伍军人管理局医疗保健的成本效益问题 系统本提案的目的是确定特定的治疗或机制途径， 减轻慢性创伤的后果。

Approximately 6.5 million Americans are diagnosed with a cutaneous chronic wound each year, costing the healthcare system in the United States over 20 billion dollars annually. Often these chronic wounds are found in geriatric individuals, up to 85% of patients with chronic wounds are over 65 years old, or in patients with concurrent diseases such as diabetes, obesity, malnutrition, or substance abuse. Similarly, the failure to properly heal wounds is a major problem for the Veteran population as presently there are over 9 million Veterans over the age of 65, or about 38% of the total Veteran population. Often these patients are subjected to a litany of various treatments, frequently without mechanistic justification, and they still lack adequate resolution of their wounds. Therefore, a better understanding of specific causes of chronic wounds and a better menu of treatment options is needed to address this unmet need. Once any type of chronic wound appears, whether it is a diabetic ulcer, decubitus ulcer, or venous stasis ulcer, one of the primary limiting factors in wound closure is the inability of epidermal cells to re-epithelialize the wound bed. Chronic wounds remain arrested in the inflammatory phase with little to no migration of keratinocytes across the wound bed. Recent data have demonstrated that an evolutionarily-conserved mechanism, called the Integrated Stress Response (ISR), is crucial for normal re-epithelialization in human skin. The ISR consists of four distinct but related protein kinases are activated by various types of environmental stress leading to the phosphorylation of the same target protein, eukaryotic initiation factor 2 on its alpha subunit (eIF2α~P). The consequences of eIF2α~P include a global inhibition of protein translation, which conserves vital cellular energy stores, and the selective translation of genes associated with responding to the stress. Importantly, it was recently demonstrated that the ISR is activated in differentiating keratinocytes in normal skin. Furthermore, keratinocytes deficient for one of the eIF2α~P protein kinases (GCN2, which phosphorylates eIF2α~P during epidermal differentiation) form abnormal, dyskeratotic skin lacking a normal barrier function. If GCN2 expression is knocked-out in human keratinocytes (an exon 12 deletion using CRISPR/CAS9), keratinocytes are deficient in epithelial sheet migration without affecting individual cell motility. These data led to the hypothesis that GCN2 is a critical component of the cutaneous wounding response, and that manipulation of the ISR via pharmaceutical agents will improve wound healing. This last point is particularly intriguing, because several drugs that regulate the ISR (either positively or negatively) are already in human clinical trials for unrelated diseases which could accelerate bringing any drugs that showed promise rapidly into human trials. The initial studies into the role of the ISR on wound healing in this proposal use model systems including two- dimensional keratinocyte growth in vitro as well as three-dimensional organ skin systems and human clinical studies in vivo. In vitro studies will define the mechanism of GCN2 activation following wounding of the keratinocyte cell culture, determine which function in keratinocyte sheet migration is compromised following GCN2 inactivation, and analyze which genes and signaling pathways are activated by enhanced protein translation using a combination of polysome profiling and RNA-seq analyses. In vivo studies will determine the utility of ISR-specific activators/inhibitors on cutaneous wound healing, determine if the inactivation of normal ISR in geriatric patients alters normal wound healing, and examine pathological biopsies from chronic wounds to determine is the ISR is inactivated during the progression of this disease. The management of chronic wounds, as well as the potential to accelerate resolution of acute cutaneous injuries, is of critical importance to Veterans, both as quality of life issues for Veteran patients and cost-effectiveness of medical care for the VA system. It is the objective of this proposal to identify specific therapeutic or mechanistic pathways that can alleviate the consequences of chronic wounds.