Regulation of cutaneous wound healing by GCN2

GCN2 对皮肤伤口愈合的调节

• 批准号: 10651695
• 负责人: DAN F SPANDAU
• 金额: --
• 依托单位: RLR VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10651695
• 关键词: 3-Dimensional; Acceleration; Acute; Address; Affect; Aging; American; Biochemical; Biological Assay; Biological Models; Biopsy; CRISPR/Cas technology; Caring; Cell Culture Techniques; Cells; Cellular Stress; Clinical Research; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Compensation; Cutaneous; Data; Debridement; Diabetes Mellitus; Diagnosis; Disease; Disease Progression; EIF-2alpha; Elderly; Environment; Epithelium; Eukaryotic Initiation Factor-2; Exons; Failure; Genes; Genetic; Goals; Granulation Tissue; Growth; Health Care Costs; Healthcare Systems; Homeostasis; Human; Human Volunteers; Immune; In Vitro; Individual; Infection; Inflammatory; Initiator tRNA; Intervention; KRP protein; Knock-out; Laboratories; Malnutrition; Medical; Messenger RNA; Molecular; Nutrient; Obesity; Organ; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Phosphorylation; Phosphotransferases; Protein Biosynthesis; Protein Inhibition; Protein Kinase; Protein Subunits; Proteins; Quality of life; Regulation; Resolution; Ribosomes; Role; Signal Pathway; Site; Skin; Skin injury; Skin wound healing; Specimen; Stasis Ulcer; Stress; Substance abuse problem; System; Therapeutic; Tissues; Translations; United States; Venous; Veterans; Wound healing therapy; acute wound; biological adaptation to stress; body system; cell motility; chronic wound; cost effectiveness; decubitus ulcer; diabetic ulcer; environmental stressor; epithelial wound; flexibility; genome-wide analysis; human old age (65+); improved; in vivo; inhibitor; keratinocyte; keratinocyte differentiation; mRNA Translation; migration; military veteran; novel therapeutics; pharmacologic; polysome profiling; recruit; response; seal; skin wound; tool; transcriptome sequencing; two-dimensional; wound; wound bed; wound closure; wound healing; wound response

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.

每年约有 650 万美国人被诊断患有皮肤慢性伤口，造成的损失 美国医疗保健系统每年花费超过 200 亿美元。 在老年人中，高达 85% 的慢性伤口患者年龄超过 65 岁，或者患有以下疾病的患者： 类似的并发疾病，如糖尿病、肥胖、营养不良或药物滥用。 正确治愈伤口是退伍军人面临的一个主要问题，因为目前有超过 900 万退伍军人 65 岁以上的退伍军人，约占退伍军人总数的 38%，这些患者通常会受到影响。 一系列不同的治疗方法，通常没有机械上的合理性，而且它们仍然缺乏足够的证据 因此，可以更好地了解慢性伤口的具体原因。 一旦出现任何类型的慢性伤口，就需要一系列治疗方案来解决这一未满足的需求。 无论是糖尿病性溃疡、褥疮性溃疡还是静脉瘀血性溃疡，都是主要限制因素之一 伤口闭合是指表皮细胞无法使伤口床重新上皮化，从而保留慢性伤口。 停滞在炎症阶段，几乎没有角质形成细胞穿过伤口床。 数据表明，一种进化保守的机制，称为综合应激反应 (ISR)，对于人类皮肤的正常上皮化至关重要 ISR 由四个不同但相关的部分组成。 蛋白激酶被各种类型的环境应激激活，导致蛋白激酶磷酸化 相同的靶蛋白，真核起始因子2对其α亚基（eIF2α~P）的影响。 eIF2α~P 包括对蛋白质翻译的整体抑制，从而保存重要的细胞能量储存，并且 重要的是，最近出现了与应激反应相关的基因的选择性翻译。 ISR 在正常皮肤的角质形成细胞分化过程中被激活。此外， 角质形成细胞缺乏一种 eIF2α~P 蛋白激酶（GCN2，它在生长过程中磷酸化 eIF2α~P） 表皮分化）形成异常、角化不良的皮肤，缺乏正常的屏障功能。 人角质形成细胞中的表达被敲除（使用 CRISPR/CAS9 删除外显子 12）、角质形成细胞 上皮层迁移存在缺陷，但不影响单个细胞的运动性。 假设 GCN2 是皮肤受伤反应的关键组成部分，并且操纵 通过药物进行 ISR 将改善伤口愈合。 几种调节 ISR（积极或消极）的药物已经进入人体临床试验。 不相关的疾病可能会加速任何有希望的药物迅速进入人体试验。 本提案中对 ISR 对伤口愈合作用的初步研究使用模型系统，包括两个： 三维角质形成细胞体外生长以及三维器官皮肤系统和人体临床 体内研究将确定 GCN2 受伤后激活的机制。 角质形成细胞细胞培养物，确定角质形成细胞片迁移中的哪些功能受到损害 GCN2失活，并分析哪些基因和信号通路被增强蛋白激活 结合多核糖体分析和 RNA-seq 分析进行翻译将确定。 ISR 特异性激活剂/抑制剂对皮肤伤口愈合的效用，确定正常 ISR 的失活是否 老年患者的 ISR 改变正常伤口愈合，并检查慢性伤口的病理活检 确定 ISR 在该疾病的进展过程中是否失活。 伤口以及加速解决急性皮肤损伤的潜力对于 退伍军人，既是退伍军人患者的生活质量问题，也是退伍军人管理局医疗护理的成本效益问题 该提案的目的是确定可以的特定治疗或机制途径。 减轻慢性伤口的后果。

项目成果

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids.

eIF2 激酶 GCN2 通过活性氧和氨基​​酸的协调来指导伤口愈合过程中角质形成细胞集体细胞的迁移。

• 发表时间: 2021
• 作者: Miles, Rebecca R;Amin, Parth H;Diaz, Miguel Barriera;Misra, Jagannath;Aukerman, Erica;Das, Amitava;Ghosh, Nandini;Guith, Tanner;Knierman, Michael D;Roy, Sashwati;Spandau, Dan F;Wek, Ronald C
• 通讯作者: Wek, Ronald C

Wounding Therapies for Prevention of Photocarcinogenesis.

预防光癌发生的创伤疗法。

• 发表时间: 2021
• 作者: Frommeyer, Timothy C;Rohan, Craig A;Spandau, Dan F;Kemp, Michael G;Wanner, Molly A;Tanzi, Elizabeth;Travers, Jeffrey B
• 通讯作者: Travers, Jeffrey B

Randomized controlled trial of fractionated laser resurfacing on aged skin as prophylaxis against actinic neoplasia.

对老化皮肤进行分段激光换肤作为预防光化性肿瘤的随机对照试验。

• 发表时间: 2021-10-01
• 作者: Spandau, Dan F;Chen, Roy;Wargo, Jeffrey J;Rohan, Craig A;Southern, David;Zhang, Angela;Loesch, Mathew;Weyerbacher, Jonathan;Tholpady, Sunil S;Lewis, Davina A;Kuhar, Matthew;Tsai, Kenneth Y;Castellanos, Amber J;Kemp, Michael G;Markey, Michael
• 通讯作者: Markey, Michael

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses.

真核起始因子 2 α 亚基的磷酸化是响应不同细胞应激而激活 NF-κB 所必需的。

• DOI: 10.4142/jvs.2018.19.5.667
• 发表时间: 2018-09-30
• 期刊: Molecular and cellular biology
• 影响因子: 1.8
• 作者: Prete CD;Ciani F;Tafuri S;Pasolini MP;Valle GD;Palumbo V;Abbondante L;Calamo A;Barbato V;Gualtieri R;Talevi R;Cocchia N
• 通讯作者: Cocchia N