Down‐regulated SHARPIN may accelerate the development of atopic dermatitis through activating interleukin‐33/ST2 signalling

Abstract

SHARPIN is an important component of the linear ubiquitin chain assembly complex (LUBAC). Loss of function of SHARPIN results in eosinophilic inflammation in multiple organs including skin with Th -dominant cytokines and dysregulated development of lymphoid tissues in mice. The clinicopathological features are similar to atopic dermatitis (AD) in humans. In order to investigate the potential role of SHARPIN in the pathogenesis of AD, we performed genetic association study of the genotypes and haplotypes as well as SHARPIN's expression between AD cases and controls. We found three mutations (g.480G>A, g.4576A>G and g.5070C>T) in patient group, and significantly decreased expression in AD lesions, suggesting a primary role of SHARPIN during AD development. Lentivirus-mediated in vitro assays identified that knockdown of SHARPIN can induce elevated expression of IL-33 and its orphan receptor ST2, FLG and STAT3 and NF-κB inactivation in HaCaT keratinocytes, which has been widely evidenced in regulating AD development. ST2 expression was highly induced in SHARPIN-silenced HaCaT keratinocytes after the combined stimulation of IL-4 and IL-13. Our in vivo and in vitro findings implicated that SHARPIN may be a novel participant in the pathogenesis and/or new therapeutic target of AD.

Downregulation of SHANK‐associated RH domain‐interacting protein elevates interleukin‐33 expression by stimulating the Janus kinase 2/signal transducer and activator of transcription signalling pathway in HaCaT cells

“…HaCaT cells were maintained in DMEM (Gibco/ThermoFisher Scientific, Waltham, MA, USA) containing L‐glutamine and supplemented with 10% fetal bovine serum (Gibco/ThermoFisher Scientific). Transduction of cells with lentivirus was performed as described by Tang et al 7 …”

“…Total RNA isolation, reverse transcription (RT), reagent volumes and evaluation methods were performed as previously described by Tang et al 7 . Quantitative (q)PCR was performed (LightCycler 96 System; Roche Diagnostics GmbH, Mannheim, Germany).…”

“…Protein concentrations were determined using a commercial kit (BCA Protein Assay Kit; Beyotime). Separation, transfer and blockage of protein lysates were performed as described by Tang et al 7 . Membranes were then incubated overnight at 4°C with primary antibodies (Table ), followed by incubation with the appropriate horseradish peroxidase‐conjugated secondary antibodies (mouse antirabbit, dilution 1 : 5000; goat antimouse 1 : 5000; rabbit antigoat 1 : 5000; Ray Antibody Biotech, Beijing, China) at room temperature (approximately 28°C) for 1 h. Finally, protein bonds were visualized by treatment with luminol (ECL Luminol Reagent; Tanon, Shanghai, China).…”

“…Subsequent in vivo genetic experiments showed that keratinocyte apoptosis induced by SHARPIN deficiency is the main driving factor for chronic proliferative dermatitis in cpdm mice 6 . Moreover, previous study 7 from our laboratory showed that silencing of SHARPIN by short hairpin RNA stimulates the expression of interleukin (IL)‐33 in HaCaT cells. Additionally, IL‐33 protein levels are significantly increased in the epidermis of patients with AD after stimulation 8 .…”

Identification of theTMEM232 gene associated with atopic dermatitis through targeted capture sequencing

“…Lacy et al [10] found IgE levels in AD patients was associated with the haplotype TGAC in the IL10 promoter region. Tang et al [11] performed genetic association study of the genotypes and haplotypes and implicated that SHARPIN may be a novel participant in the pathogenesis of AD. Our study intends to define haplotypes from the previous genotyping data in the 5q22.1 region [7] .…”