Laminin 332 is an essential component of the dermal-epidermal junction, acting as an anchoring protein attaching the epidermis to the dermis. It is decreased in ageing and is decreased or absent in the rare skin disease junctional epidermolysis bullosa (JEB). The epidermal barrier requires the synthesis and secretion of lipids by epidermal keratinocytes. The lipid matrix has a unique lamellar organization consisting of ceramides, cholesterol, and free fatty acids. Neonates with severe JEB are susceptible to infection suggesting that there is an impaired skin barrier. In order to understand the role of Laminin 332 in normal keratinocyte biology, the transcriptome of primary keratinocytes transfected with siRNA targeting Lama3 or non-targeting control was determined using RNASeq. Analysis of differentially expressed genes identified a subset involved in lipid synthesis that were significantly upregulated in the siLama3 cells. To study this further, lentiviral shRNA was used to stably silence each individual subunit of laminin 332 (a3, b3 & g2) in N-TERT keratinocytes. An increase in cholesterol biosynthesis pathway genes (HMGCS1, HMGCR, MVD, FDPS, LSS, NSHDL, DHCR7 and DHCR24) with each laminin subunit knockdown was confirmed by qRT-PCR and western blotting compared to control. 3D organotypic skin equivalents generated from knockdown cells showed defective lipid expression compared to control. Staining and lipidomic analysis of sections from a LamA3 knockout mouse (Lama3fl/fl/K14-Cre-ERT) confirmed altered lipid levels in the knockout mice compared to control. Analysis of cholesterol regulating transcription factor localisation (SREBP2) and filipin staining of free cholesterol in knockdown cells identified a failure to traffic cholesterol to the plasma membrane with loss of laminin 332. These findings suggest a role for Laminin 332 in the regulation of keratinocyte lipid synthesis and epidermal barrier formation. 681Aryl hydrocarbon receptor activation upregulates a battery of antimicrobial genes The Aryl Hydrocarbon Receptor (AHR) is a multifaceted transcription factor involved in xenobiotic metabolism, immune cell development, and epidermal differentiation. AHR is activated by aryl hydrocarbons, of which TCDD is well-known. Coal tar (CT) is an activator of AHR and is used in the clinic as a treatment for atopic dermatitis. To better understand AHR activation and downstream effects, we studied ligand-mediated DNA binding by AHR, its target gene transcription, and overall differentially expressed genes in treated keratinocytes. TCDD and CT treated human primary keratinocytes were subjected to chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) and RNA-sequencing to study genomewide effects of AHR activation by both AHR ligands. TCDD and CT, as AHR ligands, show early (after 2 hours) upregulation of genes involved in detoxification pathways. Also, we found an upregulation of important genes encoding terminal differentiation proteins (IVL, FLG), antimicrobial proteins (e.g. PI3, SLPI, S100A8), and ti...

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Pityriasis rubra pilaris (PRP) is a rare chronic inflammatory skin disorder clinically characterized by keratotic follicular papules, well-demarcated scaly erythematous plaques interspersed with distinct islands of uninvolved skin, and palmoplantar keratoderma. The autosomal dominant subtype, which is classified as PRP type V (PRPV), is associated with gain-of-function mutations in CARD14. Although around 30 cases with PRPV have been reported in the literature, diagnosis and treatment remain difficult. In this study, we analysed a 60-year-old Japanese male with erythroderma, palmoplantar keratoderma, ectropion and joint contractures of the fingers, all of which had appeared since birth. Notably, he has experienced multiple skin tumours including malignant melanoma and squamous cell carcinoma. Although the patient was initially diagnosed with congenital ichthyosiform erythroderma (CIE), mutation analysis unexpectedly revealed a heterozygous mutation c.356T>C (p.Met119Thr) in CARD14, whereas no pathogenic mutations were detected in any of the 12 genes responsible for CIE. This de novo mutation was absent from available databases. Overexpression of this mutant CARD14 formed aggregations in the cytoplasm, which exhibited a completely different distribution from that of the wild type. Furthermore, the NF-kB luciferase reporter assay revealed that mutant CARD14 showed a 2-fold increase in NF-kB activity compared with the wild type, suggesting the pathogenicity of the mutation. These findings led to the final diagnosis of PRPV. We have treated the present case with ustekinumab, which we have found partially effective. This study further expands our understanding of clinical and genetic features of PRPV. The multiple occurrences of skin tumours with this patient may suggest potential tumorigenesis associated with the disease.