The consequences of loricrin deficiency on epidermal barrier function

兜甲素缺乏对表皮屏障功能的影响

• 批准号: 8871513
• 负责人: YOSEF REFAELI
• 金额: $ 32.99万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8871513
• 关键词: 1q21; Accounting; Adult; Affect; Allergens; Amniotic Fluid; Animals; Antibodies; Atopic Dermatitis; Binding; Biological Assay; Birth; Cell Nucleus; Cell membrane; Cells; Chemicals; Child; Chromosomes; Chronic; Clinic; Clinical; Complex; Data; Databases; Defect; Defense Mechanisms; Developed Countries; Development; Down-Regulation; Eczema; Embryo; Ensure; Epidermis; Gene Family; General Population; Genes; Genetic; Glycine; Immune; Immune response; Immune system; In Vitro; Knock-out; Knockout Mice; Lead; Life; Lipids; Maintenance; Mass Spectrum Analysis; Microarray Analysis; Modeling; Modification; Mus; Mutate; Mutation; Pathway interactions; Patients; Phenocopy; Phenotype; Phosphorylation; Play; Pre-Clinical Model; Predisposition; Premature Infant; Protein Family; Proteins; Publishing; Reporter; Risk Factors; Role; Sampling; Serine; Signal Pathway; Site; Skin; Staging; Stress; Sulforaphane; Testing; Variant; Xenobiotics; airway hyperresponsiveness; base; cell envelope; chromatin immunoprecipitation; clinically relevant; env Gene Products; filaggrin; genetic risk factor; genome wide association study; human disease; in utero; insight; keratinocyte; loricrin; member; novel; novel therapeutic intervention; proline-rich proteins; promoter; repaired; response; skin disorder; small molecule

DESCRIPTION (provided by applicant): Loricrin is a major component of the keratinocyte cornified cell envelope (CE) that comprises >70% of the protein component of the CE. The CE is an insoluble protein/lipid matrix that replaces the keratinocyte plasma membrane at a late stage of epidermal maturation to form a functional barrier. To better understand the functional role of loricrin, we generated a germline knockout of the loricrin gene and were surprised to discover that a compensatory response was induced in utero to compensate for the loss of loricrin. Loricrin knockout (LKO) mice present with a very mild phenotype at birth that disappears in adults, who appear to have a normal epidermal barrier. We discovered that several known CE components, such as the small proline rich proteins (Sprrs) and repetin were induced to compensate for the loss of loricrin. However, none of these proteins could account for the high levels of glycine and serine present in LKO CEs, suggesting that as yet unidentified proteins must be induced. In this proposal, we present preliminary data documenting that members of the late cornified envelope (Lce) protein family, are induced in the LKO and account for the high glycine/serine content of LKO CEs. In addition, we have obtained genetic evidence suggesting that the Nrf2/Keap1 signaling pathway, one of the major cellular defense mechanisms against oxidative and xenobiotic stress, is involved in sensing the barrier defect in LKO mice, and activating the compensatory response to repair the barrier defect in utero. We propose to determine mechanistically how the lack of loricrin induces Nrf2 activation, and confirm that Nrf2 directly binds to and induces expression of both the Sprr and Lce genes. The discovery of a compensatory mechanism that evolved in terrestrial animals to ensure the formation and maintenance of a functional barrier has important clinical implications, since it may be possible to activate this signaling pathway pharmacologically to accelerate barrier maturation in premature infants. As proof-of- principle, we have obtained preliminary data documenting that sulforaphane, a naturally occurring electrophile known to activate Nrf2, can accelerate barrier repair in LKO mice in utero. Since sulforaphane is not selective for Nrf2 and has effects on other pathways, we will perform a screen to identify new compounds that may be more selective for Nrf2 and potentially safer for use in the clinic. Finally, atopic dermatitis (AD) is a chronic, reoccurring skin disease that causes dry, itchy, inflamed skin, affecting 15-30% of children in industrialized countries. Genome-wide association screens have identified linkage between AD and a region on chromosome 1q21 containing the epidermal differentiation complex (EDC), a conserved cluster of epidermal differentiation genes including loricrin (LOR) and filaggrin (FLG), both of which play important roles in the formation and maintenance of epidermal barrier function. Several groups have identified FLG as a major genetic risk factor associated with AD, and mice lacking Flg phenocopy the human disease. However, AD patients with no known FLG mutations still maintain linkage to the EDC, suggesting that mutations in other EDC genes may also result in AD. Our colleague, Dr. Irwin McLean, has now confirmed this study using a sample pool of >3000 AD patients controlled for FLG mutations, and he is confident that there is at least one additional eczema gene near FLG. In addition, published microarray analysis on affected AD skin showed significant downregulation of LOR, and in the recent SNP database release (dbSNP 131), frameshift variants in LOR have emerged in the general population, which would lead to a complete loss of LOR expression, analogous to the FLG mutations. Similar to filaggrin knockout mice, LKO mice do not display an overt phenotype. Therefore, based on the data summarized above, we decided to challenge LKO mice topically with an allergen. LKO mice produced allergen-specific antibodies, and showed an increase in interfollicular immune cells at the site of allergen administration. Additionally, treated LKO mice developed an acanthotic epidermis with hyperkeratotic foci. Thus, mutations in LOR may account for a percentage of AD cases where FLG is not mutated. We propose to further validate LKO mice as a model for predisposition to develop AD, and examine the sensitivity of LKO mice to develop airway hyper-responsiveness (AHR). If we are able to validate the LKO mouse as a clinically relevant model for AD, we will be able to further document the role that a defective epidermal barrier plays in the development of AD and use the LKO mouse as a preclinical model to test new therapeutic approaches for AD.

描述（由申请人提供）：Loricrin 是角质形成细胞角化细胞包膜 (CE) 的主要成分，其包含 >70% 的 CE 蛋白质成分。 CE是一种不溶性蛋白质/脂质基质，在表皮成熟后期取代角质形成细胞质膜，形成功能屏障。为了更好地了解兜甲蛋白的功能作用，我们对兜甲蛋白基因进行了种系敲除，并惊讶地发现在子宫内诱导了补偿反应以补偿兜甲蛋白的损失。 Loricrin 基因敲除 (LKO) 小鼠在出生时表现出非常轻微的表型，这种表型在成年后消失，成年后似乎具有正常的表皮屏障。我们发现几种已知的 CE 成分，例如富含脯氨酸的小蛋白 (Sprrs) 和 repetin 被诱导以补偿兜甲蛋白的损失。然而，这些蛋白质都不能解释 LKO CE 中存在的高水平甘氨酸和丝氨酸，这表明必须诱导尚未识别的蛋白质。在本提案中，我们提供了初步数据，记录了晚期角化包膜 (Lce) 蛋白家族的成员在 LKO 中被诱导，并解释了 LKO CE 的高甘氨酸/丝氨酸含量。此外，我们获得的遗传证据表明，Nrf2/Keap1信号通路是针对氧化和外源应激的主要细胞防御机制之一，参与感知LKO小鼠的屏障缺陷，并激活补偿反应以修复屏障子宫内缺陷。我们建议从机制上确定兜甲蛋白的缺乏如何诱导 Nrf2 激活，并确认 Nrf2 直接结合并诱导 Sprr 和 Lce 基因的表达。陆生动物中进化出的一种补偿机制的发现，确保功能屏障的形成和维持，具有重要的临床意义，因为有可能在药理学上激活这一信号通路，以加速早产儿屏障的成熟。作为原理证明，我们获得了初步数据，证明萝卜硫素（一种已知可激活 Nrf2 的天然亲电子试剂）可以加速 LKO 小鼠子宫内的屏障修复。由于萝卜硫素对 Nrf2 没有选择性，并且对其他途径有影响，因此我们将进行筛选，以确定对 Nrf2 更具选择性并且在临床使用可能更安全的新化合物。最后，特应性皮炎 (AD) 是一种慢性、复发性皮肤病，会导致皮肤干燥、发痒、发炎，影响工业化国家 15-30% 的儿童。全基因组关联筛选已经确定了 AD 与染色体 1q21 上包含表皮分化复合物 (EDC) 的区域之间的联系，EDC 是表皮分化基因的保守簇，包括兜甲蛋白 (LOR) 和聚丝蛋白 (FLG)，两者都在 AD 的形成过程中发挥着重要作用。表皮屏障功能的形成和维持。一些研究小组已将 FLG 确定为与 AD 相关的主要遗传风险因素，而缺乏 Flg 的小鼠则表现出了人类疾病。然而，没有已知FLG突变的AD患者仍然与EDC保持联系，这表明其他EDC基因的突变也可能导致AD。我们的同事 Irwin McLean 博士现已使用超过 3000 名控制 FLG 突变的 AD 患者样本库证实了这项研究，他确信 FLG 附近至少存在一个额外的湿疹基因。此外，已发表的对受影响 AD 皮肤的微阵列分析显示 LOR 显着下调，并且在最近发布的 SNP 数据库 (dbSNP 131) 中，LOR 中的移码变异已在普通人群中出现，这将导致 LOR 表达完全丧失，类似于FLG突变。与丝聚蛋白敲除小鼠类似，LKO 小鼠不表现出明显的表型。因此，根据上面总结的数据，我们决定用过敏原局部攻击 LKO 小鼠。 LKO 小鼠产生过敏原特异性抗体，并且在过敏原施用部位显示滤泡间免疫细胞增加。此外，接受治疗的 LKO 小鼠出现棘皮症表皮和角化过度灶。因此，LOR 突变可能占 FLG 未突变 AD 病例的一定比例。我们建议进一步验证 LKO 小鼠作为 AD 易感模型，并检查 LKO 小鼠发生气道高反应性 (AHR) 的敏感性。如果我们能够验证 LKO 小鼠作为 AD 临床相关模型，我们将能够进一步记录有缺陷的表皮屏障在 AD 发展中所起的作用，并使用 LKO 小鼠作为临床前模型来测试新的治疗方法AD 的方法。

项目成果

Amniotic fluid activates the nrf2/keap1 pathway to repair an epidermal barrier defect in utero.

• DOI: 10.1016/j.devcel.2012.11.002
• 发表时间: 2012-12-11
• 期刊: DEVELOPMENTAL CELL
• 影响因子: 11.8
• 作者: Huebner, Aaron J.;Dai, Daisy;Morasso, Maria;Schmidt, Edward E.;Schaefer, Matthias;Werner, Sabine;Roop, Dennis R.
• 通讯作者: Roop, Dennis R.