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）： 兜甲蛋白是角质形成细胞皮质细胞包膜（CE）的主要组分，其占CE的蛋白质组分的>70%。CE是一种不溶性蛋白质/脂质基质，在表皮成熟的后期取代角质形成细胞质膜以形成功能屏障。为了更好地理解兜甲蛋白的功能作用，我们产生了兜甲蛋白基因的种系敲除，并惊讶地发现，在子宫内诱导的补偿反应，以补偿兜甲蛋白的损失。兜甲蛋白敲除（LKO）小鼠在出生时表现出非常轻微的表型，在成年后消失，成年后似乎具有正常的表皮屏障。我们发现，几个已知的CE组件，如小脯氨酸丰富的蛋白质（Sprrs）和repetin的诱导，以弥补损失的兜甲蛋白。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鉴定的蛋白质。在这个建议中，我们提出了初步的数据记录，晚期皮质包膜（LCE）蛋白家族的成员，诱导在LKO和帐户的高甘氨酸/丝氨酸含量的LKO CE。此外，我们已经获得的遗传学证据表明，Nrf 2/Keap 1信号通路，对氧化和外源性应激的主要细胞防御机制之一，涉及在LKO小鼠中感知屏障缺陷，并激活补偿反应，以修复子宫内的屏障缺陷。我们建议确定机械缺乏兜甲蛋白如何诱导Nrf 2激活，并确认Nrf 2直接结合并诱导Sprr和Lce基因的表达。在陆生动物中进化的一种补偿机制的发现，以确保功能屏障的形成和维持具有重要的临床意义，因为它可能会激活这一信号通路，从而加速早产儿的屏障成熟。作为原理证明，我们已经获得了初步数据，证明萝卜硫素（一种天然存在的亲电体，已知可激活Nrf 2）可加速子宫内LKO小鼠的屏障修复。由于萝卜硫素对Nrf 2没有选择性，并且对其他途径有影响，我们将进行筛选，以确定可能对Nrf 2更具选择性并且在临床上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，可导致皮肤干燥、瘙痒、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AD与染色体1 q21上含有表皮分化复合物（EDC）的区域之间的连锁，所述表皮分化复合物是表皮分化基因的保守簇，包括兜甲蛋白（LOR）和聚丝蛋白（FLG），这两者在表皮屏障功能的形成和维持中起重要作用。几个研究小组已经确定FLG是与AD相关的主要遗传风险因素，缺乏Flg的小鼠表现出人类疾病。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，欧文姆克林博士，现在已经证实了这项研究使用的样本池>3000 AD患者控制的FLG突变，他相信，有至少一个额外的湿疹基因附近的FLG。此外，已发表的对受影响的AD皮肤的微阵列分析显示LOR的显著下调，并且在最近的SNP数据库发布（dbSNP 131）中，LOR中的移码变体已经出现在一般人群中，这将导致LOR表达的完全丧失，类似于FLG突变。类似于丝聚蛋白敲除小鼠，LKO小鼠不显示明显的表型。因此，基于以上总结的数据，我们决定用过敏原局部激发LKO小鼠。LKO小鼠产生过敏原特异性抗体，并显示在过敏原管理网站的滤泡间免疫细胞的增加。此外，经治疗的LKO小鼠出现棘皮表皮伴角化过度病灶。因此，LOR中的突变可能占FLG未突变的AD病例的百分比。我们建议进一步验证LKO小鼠作为易患AD的模型，并检查LKO小鼠发展气道高反应性（AHR）的敏感性。如果我们能够验证LKO小鼠作为AD的临床相关模型，我们将能够进一步记录缺陷的表皮屏障在AD发展中的作用，并使用LKO小鼠作为临床前模型来测试AD的新治疗方法。

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.