Epidermal keratinocytes form a functional skin barrier in the absence of Atg7 dependent autophagy.

BACKGROUND: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects. OBJECTIVE: This study tested whether autophagy is essential for epidermal barrier formation and function. METHODS: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7Δepi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays. RESULTS: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7Δepi epidermis. Atg7Δepi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7Δepi mice. CONCLUSION: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin.

The antimicrobial heterodimer S100A8/S100A9 (calprotectin) is upregulated by bacterial flagellin in human epidermal keratinocytes.

Antimicrobial peptides (AMPs) have a central role in the innate immune system of the skin. Epidermal keratinocytes (KCs) express numerous such peptides either constitutively or in response to exposure to microbial compounds. Here, we investigated the regulation of S100A8 (calgranulin A) and S100A9 (calgranulin B), which form an antimicrobial heterodimeric complex also known as calprotectin, in KCs. Culture supernatants of gram-negative bacteria, but not of gram-positive bacteria nor of the yeast Candida albicans, triggered the expression of S100A8 and S100A9. To identify pathogen-associated molecular patterns (PAMPs) responsible for the upregulation of S100A8 and S100A9, KCs were stimulated with ligands for Toll-like receptors (TLRs). Quantitative real-time PCR (qRT-PCR) analysis revealed that the TLR5 ligand flagellin increased the mRNA expression of both S100A8 and S100A9. Supernatant from wild-type (WT) Escherichia coli, but not from a flagellin-deficient E. coli strain (ΔFliC), induced S100A8 and S100A9 protein production in KCs. Moreover, small interfering RNA-mediated knockdown of TLR5 expression suppressed the ability of KCs to upregulate S100A8 and S100A9 mRNA expression in response to E. coli supernatant. Like in cell culture, stimulation of human skin explants with E. coli induced the expression of S100A8 and S100A9. Our data suggest that bacterial flagellin induces the upregulation of S100A8/S100A9 via a TLR5-dependent mechanism in epidermal KCs.

Gene silencing in a human organotypic skin model.

Here we present a simple and highly reproducible method which allows the study of the effects of a single gene knockdown in an organotypic skin model. Human keratinocytes (KC) were transfected with backbone-modified short interfering RNAs (siRNAs) specific for vascular endothelial growth factor (VEGF) and matriptase-1. Twenty-four hours later the transfected cells were seeded onto fibroblast collagen suspensions and allowed to build up a multilayered epidermis by culture at the air/medium interface for 7 days. Protein expression of both targeted genes remained down-regulated by more than 80% up to 8 days after transfection. As expected, VEGF knockdown by siRNA did not alter epidermis formation in our organotypic skin model. By contrast ablation of matriptase-1 led to aberrant KC differentiation and impaired filaggrin processing and resulted in an epidermal phenotype closely resembling that of matriptase-1 deficient mouse skin. Our results suggest that siRNA-mediated gene silencing is highly efficient in an organotypic skin model and readily allows the assessment of the roles of individual genes during terminal KC differentiation.