Knockdown of Filaggrin Impairs Diffusion Barrier Function and Increases UV Sensitivity in a Human Skin Model

Abstract

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

“…Moreover, keratin intermediate filament integrity, assessed by susceptibility to urea extraction, remains unchanged in the organotypic, siRNA knock-down cultures that we used in these studies. 53 This could be because low amounts of filaggrin are sufficient to aggregate keratin filaments, as seen previously in IV, or because other filaggrin-like proteins present in the epidermis can replace the keratin binding function of filaggrin. Yet, on an ultrastructural level, the same perinuclear retraction halos are present in these cultures as in FLG-deficient subjects (data not shown).…”

“…53 The knock-down efficiency was Ն90% for both siRNAs (data not shown). Whereas control cultures again excluded tracer from the SC (Figure 4C), FLGdeficient cultures displayed lanthanum movement into and across the SC, but again solely via the extracellular (paracellular) pathway (Figure 4, D and E).…”

“…Although these alterations could be due to an altered lipid composition of FLG-deficient SC, epidermal lipid profiles in IV in the absence of skin inflammation have not yet been assessed, and in organotypic siRNA knock-down cultures, epidermal lipid profiles reportedly are unchanged. 53 Thus, the extracellular lipid abnormalities in IV likely are due to altered lipid organization rather than to altered lipid composition. As we show here, defective lamellar bilayer structure appears to result from altered loading of lamellar body contents, with defective, postsecretory lipid processing and organization (Figure 8).…”

“…52 Stealth small interfering RNAs (siRNAs) specific for FLG and a scrambled control were obtained from Invitrogen. 53 siRNA transfection and preparation of organotypic skin cultures were performed as described previously. 52 Briefly, thirdpassage keratinocytes were transfected using Lipofectamine 2000 (Invitrogen).…”

127 Proteomic analysis of filaggrin deficiency identifies molecular signatures characteristic of atopic eczema

“…[19][20][21] In many regards these mirror changes observed in AE skin, supporting their utility as a disease model. However, to the best of our knowledge, no systematic transcriptomic or proteomic analysis has been performed after FLG knockdown in human epidermis.…”