UVB Induced TLR3 Mediated Changes in Keratinocyte Physiology

UVB 诱导 TLR3 介导角质形成细胞生理学变化

• 批准号: 10240474
• 负责人: Andrea M Schneider
• 金额: $ 4.46万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10240474
• 关键词: 3-Dimensional; Acute; Agonist; Automobile Driving; Biological Assay; Biology; CRISPR/Cas technology; Carcinoma; Cell Culture Techniques; Chemicals; Clinical; Clinical Research; DNA Damage; Data; Dermatology; Development; Double-Stranded RNA; Embryonic Development; Ensure; Environment; Epithelial; Excision; Exhibits; Exposure to; Fellowship; Gelatinase B; Gene Proteins; Genes; Human; IL8 gene; Immune; Inflammation; Inflammatory; Inflammatory Response; Injury; Innate Immune System; Interleukin-6; Malignant Neoplasms; Mediating; Mediator of activation protein; Mentors; Mesenchymal; Microscopy; Molecular; Monitor; Morphology; Mus; Pathway Analysis; Pathway interactions; Patients; Pharmacology; Phenotype; Physiology; Poly C; Poly I-C; Production; Prognosis; Property; Proteins; Reporter; Research; Residencies; Role; Sampling; Signal Pathway; Signal Transduction; Skin; Skin Aging; Skin Cancer; Skin Carcinoma; Sunburn; TLR3 gene; TWIST1 gene; Testing; Tissues; Training; Transcript; UV induced DNA damage; UVB induced; Ultraviolet B Radiation; Untranslated RNA; Up-Regulation; Western Blotting; cell injury; combat; cytokine; epithelial to mesenchymal transition; in vivo; induced pluripotent stem cell; inhibitor/antagonist; interest; keratinocyte; mRNA Expression; melanoma; migration; mouse model; novel; programs; response; sensor; skin damage; skin disorder; skin regeneration; skin squamous cell carcinoma; transcription factor; transcriptome sequencing; tumor progression; tumorigenesis; ultraviolet; wound; wound healing

Abstract This proposal is a mentored training fellowship that combines clinical exposure and research efforts with an integrated research and clinical training plan to ensure the PI’s transition to a research-focused dermatology residency. The PI’s clinical and research interests are in the inflammatory and immune-related molecular mechanisms of skin disease. The research focus of this proposal investigates the role of UVB exposure in driving an epithelial to mesenchymal (EMT)-like phenotype in normal human keratinocytes. This proposal will use 2D and 3D human keratinocyte cell cultures, in vivo mouse models, and patient samples to test the overall hypothesis that UVB exposure results in Toll-Like Receptor 3 (TLR3)-dependent activation of NF-κB, leading to an EMT-like phenotype in keratinocytes through the induction of EMT-associated genes and MMP-9. In Aim 1, we hypothesize that TLR3-mediated NF-κB activation is required for the UVB-induced EMT-like morphologic change in keratinocytes. To test this, keratinocytes exposed to UVB or PIC in combination with inhibition of NF-κB will be assayed for NF-κB-dependent signaling changes, EMT associated gene and protein changes, and changes in keratinocyte morphology, migration, and invasion properties. We will use both WT and TLR3KO mice as well as human patient samples of cutaneous squamous cell carcinoma and normal skin to investigate the co-expression of NF-κB and TLR3 proteins in skin. In Aim 2, we will test the hypothesis that TLR3 activation and subsequent NF-κB stimulation are required for MMP-9 induction and activation following UVB exposure. Keratinocytes exposed to UVB or PIC in combination with MMP-9 inhibition will be monitored for changes in morphology and alterations in migration and invasion potential. The effect of UVB exposure on MMP-9 will also be studied in vivo using WT and TLR3KO mice. This proposal challenges current understanding which focuses on UVB as a DNA-damaging agent and instead investigates TLR3 as a sensor of UVB-induced cell damage and activator of inflammatory pathways that can have profound effects on keratinocyte morphology and function. Our proposal is significant in that it will advance the field of skin biology by increasing our understanding of UVB-mediated activation of the innate immune system and skin tumorigenesis.

摘要 这项建议是一项有指导的培训奖学金，将临床暴露和研究工作与 整合研究和临床培训计划，确保PI向以研究为重点的皮肤科过渡 实习医生。PI的临床和研究兴趣在于炎症和免疫相关分子 皮肤病的机制。 这项提案的研究重点是调查UVB暴露在推动上皮细胞 正常人角质形成细胞间充质(EMT)样表型。这项提案将使用2D和3D人类 角质形成细胞培养、活体小鼠模型和患者样本来检验UVB的总体假设 暴露导致依赖Toll样受体3(TLR3)的NF-κB激活，导致EMT样 角质形成细胞的表型通过EMT相关基因和基质金属蛋白酶-9的诱导。 在目标1中，我们假设TLR3介导的NF-κB激活是UVB诱导的EMT样所必需的 角质形成细胞的形态变化。为了测试这一点，角质形成细胞暴露在UVB或PIC联合 抑制核因子-κB将检测依赖于核因子-κB的信号变化、相关基因和蛋白 角质形成细胞形态、迁移和侵袭特性的变化。我们将使用这两个WT 和TLR3KO小鼠以及人类皮肤鳞状细胞癌和正常皮肤的患者样本 目的：探讨皮肤组织中核因子-κB和TLR3蛋白的共同表达。 在目标2中，我们将测试TLR3激活和随后的NF-κB刺激是需要的假设 中波紫外线照射后基质金属蛋白酶-9的诱导和激活。UVB或PIC联合照射角质形成细胞 通过抑制基质金属蛋白酶-9，将监测形态的变化以及迁移和侵袭的变化 潜力。我们还将在WT和TLR3KO小鼠身上研究UVB辐射对基质金属蛋白酶-9的影响。 这项提议挑战了目前对UVB作为DNA破坏剂的理解，而不是 研究TLR3作为UVB诱导的细胞损伤的感受器和炎症通路的激活剂，可以 对角质形成细胞的形态和功能有深远的影响。我们的建议意义重大，因为它将 通过增加对UVB介导的天然皮肤的激活的了解来促进皮肤生物学领域的发展 免疫系统与皮肤肿瘤发生。