UVB Induced TLR3 Mediated Changes in Keratinocyte Physiology

摘要 该提案是一项指导性培训奖学金，将临床暴露和研究工作与 整合研究和临床培训计划，以确保PI过渡到以研究为重点的皮肤病学 居住权PI的临床和研究兴趣是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暴露在驱动上皮细胞 间充质（EMT）样表型在正常人角质形成细胞。该提案将使用2D和3D人体 角质形成细胞培养物、体内小鼠模型和患者样本，以检验UVB 暴露导致Toll样受体3（TLR 3）依赖性NF-κB活化，导致EMT样 通过诱导EMT相关基因和MMP-9在角质形成细胞中的表型。 在目的1中，我们假设TLR 3介导的NF-κB活化是UVB诱导的EMT样细胞凋亡所必需的。 角质形成细胞的形态学改变。为了测试这一点，将暴露于UVB或PIC的角质形成细胞与 将针对NF-κ B依赖性信号传导变化、EMT相关基因和蛋白来测定NF-κB的抑制 变化，以及角质形成细胞形态、迁移和侵袭特性的变化。我们将使用两个WT 以及皮肤鳞状细胞癌和正常皮肤的人类患者样品 探讨NF-κB和TLR 3蛋白在皮肤中的共表达。 在目标2中，我们将检验TLR 3激活和随后的NF-κB刺激是调节细胞凋亡所必需的这一假设。 UVB暴露后MMP-9的诱导和活化。暴露于UVB或PIC组合的角质形成细胞 将监测MMP-9抑制剂的形态学变化以及迁移和侵袭的改变 潜力还将使用WT和TLR 3 KO小鼠在体内研究UVB暴露对MMP-9的影响。 这一提议挑战了目前的理解，目前的理解集中在UVB作为一种DNA损伤剂， 研究TLR 3作为UVB诱导的细胞损伤的传感器和炎症途径的激活剂， 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 通过增加我们对UVB介导的先天免疫激活的理解，推进皮肤生物学领域。 免疫系统和皮肤肿瘤发生。

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.