772 Development of a zebrafish model for CARD14-associated human disorders (original) (raw)
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Filaggrin expression in normal and pathological skin
Virchows Archiv A Pathological Anatomy and Histopathology, 1988
Filaggrin is a basic, histidine-rich protein synthesized by cells of keratinizing epithelia, that subserves major physiological functions in maturating epidermis. Its precursor, profilaggrin, constitutes a major component of keratohyalin granules. In this work the expression of profilaggrin/filaggrin was studied in 140 specimens of normal human adult and fetal skin, cultured epithelia before and after allo-and xeno-grafting, and several cases of keratinization disorders and tumours. An avidin-biotin-alkaline phosphatase technique was applied on deparaffinized tissue sections, by using a specific monoclonal antibody (AKHI) to profilaggrin/filaggrin. The results show that the expression of profilaggrin/filaggrin is variously altered in keratinization disorders, whereas in epithelial proliferations it seems to decrease with an increasing degree of dedifferentiation of the tumour. Therefore the expression of these antigens can be considered to be a sensitive marker of maturation of normal epidermis and provides a new tool for the study of differentiation of the surface epithelium in skin diseases.
Cell death & disease, 2015
Atopic dermatitis is a chronic inflammatory skin disease with defects in the epidermal barrier. In a cohort of African-American children, a FLG2 nonsense mutation has been associated with the disease. In the epidermis of European patients, the expression of filaggrin-2, the filaggrin-related protein encoded by FLG2, is decreased. To describe the function of filaggrin-2 and evaluate the impact of its deficiency, its expression was downregulated using lentivirus-mediated shRNA interference in a three-dimensional reconstructed human epidermis (RHE) model. This resulted in parakeratosis and a compact stratum corneum, presence of abnormal vesicles inside the corneocytes, increased pH and reduced amounts of free amino acids at the RHE surface, leading to increased sensitivity to UVB radiations. The expression of differentiation markers was slightly modified. However, we observed reduced proteolytic processing of corneodesmosin, hornerin and filaggrin in parallel with reduced amounts of ca...
British Journal of Dermatology, 2010
Background: Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (AD FLG ) compared with that seen in patients with AD without these mutations (AD NON-FLG ). Objectives: We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with AD FLG versus patients with AD NON-FLG . We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flg ft /Flg ft [Flg delAPfal ] mice).
Defects of filaggrin-like proteins in both lesional and nonlesional atopic skin
Journal of Allergy and Clinical Immunology, 2013
Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by a disturbed epidermal barrier. In a subset of patients, this is explained by nonsense mutations in the gene encoding filaggrin (FLG). Objectives: We sought to evaluate the respective role of FLG mutations and proinflammatory cytokines and to assess the expression of FLG, hornerin (HRNR), and FLG2, 2 FLG-like proteins, which are involved in epidermal barrier functions, in normal skin and both lesional and nonlesional skin of patients with AD. Methods: An FLG-genotyped cohort of 73 adults with AD and 73 aged-matched control subjects was analyzed by using immunohistochemistry and immunoblotting. Normal primary human keratinocytes were differentiated in either the absence or presence of IL-4, IL-13, and IL-25. Results: Compared with control subjects, FLG, HRNR, and FLG2 were detected at significantly lower levels in the skin of patients with AD, irrespective of their FLG genotype. The reduction was greater in lesional compared with nonlesional skin. In addition, the proFLG/FLG ratio was found to be higher in the skin of wild-type patients than in control subjects. Cytokine treatment of keratinocytes induced a dramatic reduction in FLG, FLG2, and HRNR expression both at the mRNA and protein levels. Conclusion: The stratum corneum of lesional but also clinically unaffected skin of adults with AD is abnormal, with reduced expression of FLG and FLG-like proteins. In addition to nonsense mutations, proinflammatory cytokines and some defects in the proFLG processing can contribute to the FLG downregulation. Our study suggests that skin inflammation reduces the expression of FLG-like proteins, contributing to the AD-related epidermal barrier dysfunction. (J Allergy Clin Immunol 2013;131:1094-102.)
Experimental Dermatology, 2012
The S100 fused-type proteins (SFTPs) are thought to be involved in the barrier formation and function of the skin. Mutations in the profilaggrin gene, one of the best investigated members of this family, are known to be the major risk factors for ichthyosis vulgaris and atopic dermatitis. Recently, we identified human filaggrin-2 as a new member of the SFTP family. To achieve further insight into its function, here the murine filaggrin-2 was analysed as a possible orthologue. The 5¢ and 3¢ ends of the mouse filaggrin-2 cDNA of the BALB ⁄ c strain were sequenced and confirmed an organization typical for SFTPs. Murine filaggrin-2 showed an expression pattern mainly in keratinizing epithelia in the upper cell layers on both mRNA and protein levels. The expression in cultured mouse keratinocytes was increased upon elevated Ca 2+ levels. Immunoblotting experiments indicated an intraepidermal processing of the 250-kDa full-length protein. In metabolically (essential fatty acid-deficient diet) induced skin barrier dysfunction, filaggrin-2 expression was significantly reduced, whereas filaggrin expression was up-regulated. In contrast, mechanical barrier disruption with acetone treatment did not affect filaggrin-2 mRNA expression. These results suggest that filaggrin-2 may contribute to epidermal barrier function and its regulation differs, at least in parts, from that of filaggrin.
2012
Background: Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (AD FLG) compared with that seen in patients with AD without these mutations (AD NON-FLG). Objectives: We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with AD FLG versus patients with AD NON-FLG. We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flg ft /Flg ft [Flg delAPfal ] mice). Methods: One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1a, IL-1b, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flg ft /Flg ft (Flg delAPfal) mice, separated from maFlg ft /maFlg ft (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1a, IL-1b, and IL-1RA. Results: SC IL-1 levels were increased in patients with AD FLG ; these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flg ft /Flg ft mice had upregulated expression of IL-1b and IL-1RA mRNA. Conclusions: AD FLG is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.
Experimental Cell Research, 2001
Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.
Functional and proteomic analysis of a full thickness filaggrin-deficient skin organoid model
Wellcome Open Research, 2019
Background:Atopic eczema is an itchy inflammatory disorder characterised by skin barrier dysfunction. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a major risk factor, but the mechanisms by which filaggrin haploinsufficiency leads to atopic inflammation remain incompletely understood. Skin as an organ that can be modelled using primary cellsin vitroprovides the opportunity for selected genetic effects to be investigated in detail.Methods:Primary human keratinocytes and donor-matched primary fibroblasts from healthy individuals were used to create skin organoid models with and without siRNA-mediated knockdown ofFLG. Biological replicate sets of organoids were assessed using histological, functional and biochemical measurements.Results:FLGknockdown leads to subtle changes in histology and ultrastructure including a reduction in thickness of the stratum corneum and smaller, less numerous keratohyalin granules. Immature organoids showed some limited evidence of ba...
Revisiting the Roles of Filaggrin in Atopic Dermatitis
International Journal of Molecular Sciences
The discovery in 2006 that loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and can predispose to atopic dermatitis (AD) galvanized the dermatology research community and shed new light on a skin protein that was first identified in 1981. However, although outstanding work has uncovered several key functions of filaggrin in epidermal homeostasis, a comprehensive understanding of how filaggrin deficiency contributes to AD is still incomplete, including details of the upstream factors that lead to the reduced amounts of filaggrin, regardless of genotype. In this review, we re-evaluate data focusing on the roles of filaggrin in the epidermis, as well as in AD. Filaggrin is important for alignment of keratin intermediate filaments, control of keratinocyte shape, and maintenance of epidermal texture via production of water-retaining molecules. Moreover, filaggrin deficiency leads to cellular abnormalities in keratinocytes and induces subtle epidermal barri...