Dermal-Epidermal Junction Disruptors: Mechanistic Insights

项目摘要/摘要 一系列不同的化学品，包括某些化学战剂(例如硫磺芥末)、某些静脉注射 化疗药物(如甲氧氯乙胺、MEC、紫杉醇、脂质体阿霉素、异环磷酰胺、苯达莫司汀)，以及 某些环境毒素(例如，蛇毒金属蛋白酶、斑腿甲虫)会引起急性和严重的 皮肤毒性包括真皮-表皮交界处(DEJ)破裂和起泡(水泡)。皮肤 这些类型的干扰物对化学诱导的DEJ干扰的机制仍然不清楚 旨在减少DEJ干扰的治疗措施仍然严重不足。本提案的主要目的是，具体 目的1，研究皮肤对DEJ干扰物反应的新机制。最近，监管失灵 雷帕霉素复合体1的哺乳动物靶点(MTORC1)与皮肤损伤有关；据观察 MTORC1的一种成分Raptor的表皮表达不足的小鼠皮肤表现出表皮脱落 与水泡一致。基于这一重要信息，我们将在这里检验DEJ干扰通过 MEC，一个DEJ干扰物的原型，涉及到时间和剂量依赖的Raptor表皮表达的减少 以及S6K和4E-BP-1的磷酸化形式。为此，我们将使用两个相关和互补的皮肤模型： (A)小鼠耳部发泡剂模型(MEVM；体内模型)和(B)人皮肤全层3D培养(体外模型)。 不断增加剂量的DEJ干扰物MEC原型将应用于小鼠耳朵皮肤或人类皮肤和穿孔活检 样本将在暴露后0.5、1、2、4、8、12和24小时进行皮肤反应评估。由以下物质引起皮肤毒性 将对MEC进行组织学研究(通过对水肿/增生/炎症/ 泡化)和机械(通过使用免疫组织化学分析Raptor和 表皮中S6K和4E-BP1的磷酸化形式)。这种高度创新的方法将使我们能够破译 DEJ干扰物引起的组织水泡与mTORC1信号通路组件之间的关系。此外 对于具体目标1，有两个与本提案相关的子目标(分别为子目标1和子目标2)。在子- 目的1、几种关键炎症标志物(肿瘤坏死因子α、白介素1β、诱导型一氧化氮合酶、白介素6、基质金属蛋白酶9)和miR-132在皮肤中的表达。 一种在皮肤中丰富并参与表皮细胞增殖和伤口愈合的microrna将在所有组织中进行评估。 采用定量聚合酶链式反应和免疫组织化学(IHC)方法检测样本(小鼠和人)。在次级目标2中，时间和剂量依赖 将研究MEC对皮肤DNA损伤感知和修复反应的影响。MEC对DNA的损伤将 通过使用免疫荧光对所有组织样本(小鼠和人)中是否存在DNA损伤灶进行评估 含有磷酸化的γH_2AX(次要的H2组蛋白变异体X)和修复酶FEN1(FLAW核酸内切酶1) 和APE1(无嘌呤/脱嘧啶核酸内切酶1)。 QPCR.总而言之，这里提出的工作将为皮肤对MEC的反应建立重要的基础，并将 帮助破译未来药物开发的新目标，旨在消除其他药物/化学品对DEJ的干扰。

Project Summary/Abstract A diverse array of chemicals including certain chemical warfare agents (e.g., sulfur mustard), certain intravenous chemotherapy drugs (e.g., mechlorethamine, MEC; paclitaxel, liposomal doxorubicin, ifosfamide, bendamustine), and certain environmental toxins (e.g., viper snake venom metalloproteinases, blister beetle cantharidin) cause acute and severe cutaneous toxicity including dermal-epidermal junction (DEJ) disruption and blistering (vesication). The cutaneous mechanisms involved in chemically-induced DEJ disruption by these types of disruptors remain obscure and current treatments aimed at reducing DEJ disruption remain grossly inadequate. The primary aim of the present proposal, Specific Aim 1, is to investigate novel mechanisms involved in cutaneous responses to DEJ disruptors. Recently, dysregulation of the mammalian target of rapamycin complex 1 (mTORC1) has been implicated in cutaneous injury; with the observation that mouse skin deficient in epidermal expression of Raptor, a component of mTORC1, exhibits epidermal detachments consistent with vesication. Based on this important information, here we will test the hypothesis that DEJ disruption by MEC, a prototype DEJ disruptor, involves both time- and dose- dependent decreases in the epidermal expression of Raptor and phosphorylated forms of S6K and 4E-BP-1. To this end, we will utilize two relevant and complementary skin models: (a) the mouse ear vesicant model (MEVM; in vivo model) and (b) a human skin full thickness 3D culture (in vitro model). Increasing doses of the prototype DEJ disruptor MEC will be applied to mouse ear skin or human skin and punch biopsy samples will be evaluated for cutaneous responses at 0.5, 1, 2, 4, 8, 12 and 24 h after exposure. Dermatotoxicity caused by MEC will be investigated both histologically (by microscopic analyses of edema/ hyperplasia/ inflammation/ vesication) and mechanistically (through the use of immunohistochemical analyses of tissue expression of Raptor and phosphorylated forms of S6K and 4E-BP1 in the epidermis). This highly innovative approach will allow us to decipher the relationship between tissue blistering by DEJ disruptors and components of the mTORC1 signaling pathway. In addition to Specific Aim 1, there are two sub-aims associated with this proposal (Sub-Aim 1 and Sub-Aim 2, respectively). In Sub- Aim 1, the cutaneous expression of several key inflammatory markers (TNFα, IL-1β, iNOS, IL-6, MMP-9) and mIR-132, a microRNA abundant in skin and involved in epidermal cell proliferation and wound healing, will be evaluated in all tissue samples (mouse and human) using qPCR and immunohistochemistry (IHC). In Sub-Aim 2, the time- and dose-dependent effects of MEC on DNA damage sensing and repair responses of the skin will be investigated. DNA damage by MEC will be assessed in all tissue samples (mouse and human) by using immunofluorescence for the presence of DNA damage foci containing phosphorylated γH2AX (the minor H2 histone variant X), and the repair enzymes FEN1 (flap endonuclease 1) and APE1 (apurinic/apyrimidinic endonuclease 1. Mitochondrial DNA (mtDNA) damage by MEC will be investigated by qPCR. All in all, the work proposed here will establish an important foundation of cutaneous responses to MEC and will aid in deciphering novel targets for future drug development designed to eliminate DEJ disruption by other drugs/chemicals.