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是角质形成细胞蜂蜜细胞包膜（CE）的主要组成部分，占CE的蛋白质成分的70％。 CE是一种不溶性蛋白/脂质基质，在表皮成熟的后期替代角质形成细胞质膜以形成功能性屏障。为了更好地了解洛里克林的功能作用，我们产生了洛里克林基因的种系敲除，并惊讶地发现，在子宫内诱导了补偿性反应以补偿洛里克林的损失。 Loricrin敲除（LKO）小鼠出生时出现非常温和的表型，在成年人中消失，他们似乎具有正常的表皮屏障。我们发现，诱导了几种已知的CE成分，例如小脯氨酸蛋白（SPRR）和repetin来补偿洛里克林的损失。但是，这些蛋白质都无法解释LKO CES中存在的甘氨酸和丝氨酸的高水平，这表明必须诱导未识别的蛋白质。在此提案中，我们介绍了初步数据，该数据记录了LKO中诱导的晚晶状膜（LCE）蛋白质家族的成员，并解释了LKO CES的高甘氨酸/丝氨酸含量。此外，我们还获得了遗传证据，表明NRF2/KEAP1信号通路是针对氧化和异种生物胁迫的主要细胞防御机制之一，参与感知LKO小鼠中的屏障缺陷，并激活补偿性响应以修复子宫内的屏障缺陷。我们建议从机械上确定缺乏洛里克林如何诱导NRF2激活，并确认NRF2直接结合并诱导SPRR和LCE基因的表达。在陆地动物中进化以确保形成和维持功能性障碍的补偿机制具有重要的临床意义，因为有可能在药理上激活该信号通路以加速早产儿的屏障成熟。作为原则证明，我们获得了初步数据，证明了一种自然存在的电力硫素可以激活NRF2，可以加速子宫内LKO小鼠的屏障修复。由于Sulforaphane对NRF2不是选择性的，并且对其他途径具有影响，因此我们将执行一个屏幕以识别可能对NRF2更具选择性的新化合物，并可能更安全地用于诊所。最后，特应性皮炎（AD）是一种慢性，重复发生的皮肤疾病，会引起干燥，发痒，发炎的皮肤，影响工业化国家的15-30％的儿童。全基因组关联筛查已经确定了含有表皮分化复合物（EDC）的AD和一个区域之间的联系，这是一个保守的表皮分化基因簇，包括Loricrin（Lor）（LOR）和Filaggrin（FLG），这两种基因在表皮形成和维护中都起着重要作用。几个小组已经将FLG鉴定为与AD相关的主要遗传危险因素，而缺乏FLG表征的小鼠是人类疾病。但是，没有已知FLG突变的AD患者仍然保持与EDC的联系，这表明其他EDC基因中的突变也可能导致AD。我们的同事欧文·麦克莱恩（Irwin McLean）博士现在使用了受FLG突变的> 3000名AD患者的样本库证实了这项研究，他相信FLG附近至少还有一个额外的湿疹基因。此外，对受影响的AD皮肤的发表微阵列分析显示了LOR的明显下调，在最近的SNP数据库释放（DBSNP 131）中，LOR中的移码变体在一般种群中出现了，这将导致LOR表达完全丢失，类似于FLG突变。与Filaggrin敲除小鼠类似，LKO小鼠没有显示明显的表型。因此，根据上述总结的数据，我们决定用过敏原局部挑战LKO小鼠。 LKO小鼠产生了过敏原特异性抗体，并显示过过敏原部位的流体界面免疫细胞增加。此外，经过处理的LKO小鼠以高乳突灶形成了棘皮表皮。因此，LOR中的突变可能占未突变的AD病例的百分比。我们建议进一步验证LKO小鼠作为发展AD的模型，并检查LKO小鼠对发展气道高反应性（AHR）的敏感性。如果我们能够将LKO小鼠作为AD的临床相关模型验证，我们将能够进一步记录表皮屏障有缺陷在AD开发中发挥作用的作用，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.