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

Abstract

BackgroundAtopic eczema (AE) is characterized by skin barrier and immune dysfunction. Null mutations in filaggrin (FLG), a key epidermal barrier protein, strongly predispose to AE; however, the precise role of FLG deficiency in AE pathogenesis remains incompletely understood.ObjectivesWe sought to identify global proteomic changes downstream of FLG deficiency in human epidermal living skin–equivalent (LSE) models and validate findings in skin of patients with AE.MethodsDifferentially expressed proteins from paired control (nontargeting control short hairpin RNA [shNT]) and FLG knockdown (FLG knockdown short hairpin RNA [shFLG]) LSEs were identified by means of proteomic analysis (liquid chromatography–mass spectrometry) and Ingenuity Pathway Analysis. Expression of key targets was validated in independent LSE samples (quantitative RT-PCR and Western blotting) and in normal and AE skin biopsy specimens (immunofluorescence).ResultsProteomic analysis identified 17 (P ≤ .05) differentially expressed proteins after FLG knockdown, including kallikrein-7 (KLK7; 2.2-fold), cyclophilin A (PPIA; 0.9-fold), and cofilin-1 (CFL1, 1.3-fold). Differential protein expression was confirmed in shNT/shFLG LSEs; however, only KLK7 was transcriptionally dysregulated. Molecular pathways overrepresented after FLG knockdown included inflammation, protease activity, cell structure, and stress. Furthermore, KLK7 (1.8-fold) and PPIA (0.65-fold) proteins were differentially expressed in lesional biopsy specimens from patients with AE relative to normal skin.ConclusionsFor the first time, we show that loss of FLG in the absence of inflammation is sufficient to alter the expression level of proteins relevant to the pathogenesis of AE. These include proteins regulating inflammatory, proteolytic, and cytoskeletal functions. We identify PPIA as a novel protein with levels that are decreased in clinically active AE skin and show that the characteristic upregulation of KLK7 expression in patients with AE occurs downstream of FLG loss. Importantly, we highlight disconnect between the epidermal proteome and transcriptome, emphasizing the utility of global proteomic studies.

Advances in atopic dermatitis in 2017

“…These findings help to further characterize molecular patterns in patients with atopic disease. 25 These concepts tie into the notion of FLGdriven skin changes, and FLG-determined predisposition to AD as the first step in the atopic march toward development of food allergy, allergic rhinitis, and asthma.…”

“…24 LOF mutations in FLG are enough to alter expression of proteins involved in regulation of skin inflammation. 25 Mutational variants can contribute to differences in response to topical therapeutics. 27…”

ADAM17-Deficient Mice Model the Transcriptional Signature of Human Atopic Dermatitis

“…Critically, the absence of any A17 hDEG in MADAD may also reflect low sensitivity of MADAD itself to detect subtle transcriptional change in human AD, because failure to detect low-expression genes on whole-tissue microarrays would carry through the meta-analysis approach. Indeed, the human orthologs of several nonoverlapping A17 hDEGs, including filaggrin (FLG), inflammasome regulator NLRP10 (NLRP10), putative antioxidant CCDC80 (CCDC80), and IL-2 receptor alpha (IL2RA), were missing from MADAD but have been implicated in human AD genome-wide association studies (Paternoster et al, 2015) and other work (Elias et al, 2017;Furue et al, 1994;Miyai et al, 2016).…”

Filaggrin and childhood eczema

“…Bringing these ideas together, a recent paper examining proteomic changes downstream of filaggrin deficiency concludes that loss of FLG is itself an important primary pathogenic factor in the development of AD 18. The authors underscore that the mechanism of FLG reduction—be it via genotype, inflammatory cytokine effects, or some combination—may not be important, and that all can lead to the development of AD.…”