The consequences of loricrin deficiency on epidermal barrier function

描述（由申请人提供）：Loricrin是角质形成细胞角化包膜（CE）的主要成分，占CE蛋白质成分的70%。CE是一种不溶性蛋白/脂质基质，在表皮成熟后期取代角质细胞质膜形成功能屏障。为了更好地了解loricrin的功能作用，我们对loricrin基因进行了种系敲除，并惊讶地发现在子宫内诱导了代偿反应来补偿loricrin的损失。Loricrin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，似乎具有正常的表皮屏障。我们发现，一些已知的CE成分，如小脯氨酸富蛋白（Sprrs）和重复蛋白（repetin）被诱导来补偿loricrin的损失。然而，这些蛋白质都不能解释LKO ce中存在高水平的甘氨酸和丝氨酸，这表明尚未确定的蛋白质必须被诱导。在这篇论文中，我们提供了初步的数据，证明LKO中诱导了晚期糖化包膜（Lce）蛋白家族的成员，并解释了LKO ce中高甘氨酸/丝氨酸含量的原因。此外，我们获得的遗传学证据表明，Nrf2/Keap1信号通路是LKO小鼠抗氧化和外源应激的主要细胞防御机制之一，参与感知屏障缺陷，并在子宫内激活代偿反应来修复屏障缺陷。我们拟确定loricrin缺乏诱导Nrf2激活的机制，并证实Nrf2直接结合并诱导Sprr和Lce基因的表达。在陆生动物中进化出一种代偿机制，以确保功能性屏障的形成和维持，这一发现具有重要的临床意义，因为有可能从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，我们已经获得了初步数据，证明萝卜硫素是一种天然存在的亲电剂，已知可以激活Nrf2，可以加速子宫内LKO小鼠的屏障修复。由于萝卜硫素对Nrf2没有选择性，并且对其他途径有影响，我们将进行筛选，以确定可能对Nrf2更具选择性的新化合物，并可能更安全地用于临床。最后，特应性皮炎（AD）是一种慢性、反复发作的皮肤病，可导致皮肤干燥、发痒、发炎，影响工业化国家15-30%的儿童。全基因组关联筛选发现AD与染色体1q21上包含表皮分化复合体（EDC）的区域存在关联，EDC是一个保守的表皮分化基因簇，包括loricrin （LOR）和聚丝蛋白（FLG），两者在表皮屏障功能的形成和维持中发挥重要作用。一些研究小组已经确定FLG是与AD相关的主要遗传风险因素，缺乏FLG的小鼠会出现人类疾病。然而，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基因的突变也可能导致AD。我们的同事，Irwin McLean博士，现在已经通过控制FLG突变的3000名AD患者的样本库证实了这项研究，他相信FLG附近至少有一个额外的湿疹基因。此外，已发表的对受影响的AD皮肤的微阵列分析显示LOR显著下调，并且在最近发布的SNP数据库（dbSNP 131）中，在一般人群中出现了LOR移码变体，这将导致LOR表达的完全丧失，类似于FLG突变。与聚丝蛋白敲除小鼠类似，LKO小鼠不表现出明显的表型。因此，基于以上总结的数据，我们决定用过敏原局部刺激LKO小鼠。LKO小鼠产生过敏原特异性抗体，并在过敏原给药部位显示滤泡间免疫细胞增加。此外，经治疗的LKO小鼠出现棘层表皮伴角化灶。因此，在FLG未突变的AD病例中，LOR突变可能占一定比例。我们建议进一步验证LKO小鼠作为AD易感性的模型，并检测LKO小鼠对气道高反应性（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，我们将能够进一步证明有缺陷的表皮屏障在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。

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.