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.
The studies proposed will provide insight into a signaling pathway that may provide a safe way to accelerate skin barrier formation in premature infants. In addition, these studies may reveal a novel genetic risk factor for the development of atopic dermatitis.