Conditional knockout of C/EBP[Formula: see text] in epidermis results in dysregulated lipid biosynthesis and a defect in skin barrier function.

CCAAT/enhancer binding protein-[Formula: see text] (C/EBP[Formula: see text]) is a basic leucine zipper transcription factor that is abundantly expressed in epidermal keratinocytes of skin. In the present study, C/EBP[Formula: see text] epidermal specific conditional knockout (CKO[Formula: see text]) SKH1 mice were utilized to interrogate C/EBP[Formula: see text]'s role in lipid biosynthesis and skin barrier integrity. RNAseq data analysis and gene set enrichment analysis of RNA isolated from the epidermis of CKO[Formula: see text] and K5Cre control mice revealed that deletion of C/EBP[Formula: see text] in epidermis resulted in an enrichment of downregulated genes in gene sets associated with lipid metabolism. Further analysis showed the majority of differentially regulated genes were downregulated in gene sets related to the metabolism/biosynthesis of ceramides, fatty acids, phospholipids, sphingolipids, and cholesterol species in CKO[Formula: see text] epidermis. Ingenuity Pathway Analysis predicted inhibition of multiple pathways involving lipid biosynthesis. Lipidomic analysis of epidermis using advanced chemical separations and tandem mass spectrometry identified 470 individual lipids in epidermis with 165 significantly decreased and 82 significantly increased in CKO[Formula: see text] epidermis. The lysophospholipids were the most decreased class of lipids, and free fatty acids and ceramides important in barrier formation were also decreased. The sphingomyelin class of lipids was the most increased. High resolution mass spectrometry for cholesterol lipids revealed several cholesterol esters were also dysregulated in CKO[Formula: see text] epidermis. Finally, we assessed the functional consequences of the loss C/EBP[Formula: see text] on epidermal barrier function and found that basal permeability barrier function as measured by transepidermal water loss (TEWL) was impaired, with an approximate doubling of TEWL in CKO[Formula: see text] mice. These results indicate that C/EBP[Formula: see text] is a is a major regulator of the epidermal lipidome and the deletion of C/EBP[Formula: see text] in epidermis leads to a defect in skin barrier function.