Analysis of temporal mRNA expression changes in cultured keratinocytes from Casp-8F/–K5-Cre and Casp-8F/+K5-Cre mice. Mus musculus

Expression of enzymatically inactive caspase-8, or deletion of caspase-8 from basal epidermal keratinocytes, triggers chronic skin inflammation in mice. Unlike similar inflammation resulting from arrest of NF-kB activation in the epidermal cells, the effect induced by caspase-8 deficiency did not depend on TNF, IL1, dermal macrophage function, or expression of the Toll-like receptor adapter proteins MyD88 or TRIF. Both interferon regulatory factor (IRF)3 and TANK-binding kinase were constitutively phosphorylated in the caspase-8-deficient epidermis, and knockdown of IRF3 in the epidermis-derived cells from these mice abolished the expression of upregulated genes. Temporal and spatial analyses of the alterations in gene expression that result from caspase-8 deficiency reveal that the changes are initiated before birth, around the time that cornification develops, and occur mainly in the suprabasal layer. Finally, we found that caspase-8-deficient keratinocytes display an enhanced response to gene activation by transfected DNA. Our findings suggest that an enhanced response to endogenous activators of IRF3 in the epidermis, presumably generated in association with keratinocyte differentiation, contributes to the skin inflammatory process triggered by caspase-8 deficiency. Overall design: Epidermal keratinocytes were isolated from the skin of Casp-8F/–K5-Cre or Casp-8F/+K5-Cre mice. Keratinocyte samples from two mice of each genotype were isolated and subsequently cultured. Total RNA was extracted at the first and the third passage of cultivation (8 RNA samples originated in total). The appropriate pairs of RNA samples suited for a direct comparison of the two different genotypes under examination were differently labeled and subsequently co-hybridized onto one microarray (dual color design).