A Novel Effect of Lipids Extracted from Vernix Caseosa on Regulation of Filaggrin Expression in Human Epidermal Keratinocytes

BackgroundVernix caseosa (VC), which is known as a unique human substance, is a biofilm that covers the skin of most human newborns. VC has many biological functions including anti-infective, skin cleansing and skin barrier repair.ObjectiveIn the study, we purpose to investigate the novel effect of lipids extracted from VC on the regulation of filaggrin (FLG) expression and anti-inflammation in normal human epidermal keratinocyte (NHEK) cells.MethodsThe lipids were extracted by chloroform/methanol (Folch method) and the major properties of fatty acid methyl esters were determined with gas chromatography-mass spectrometer. The relative viability of NHEK cells was evaluated by Cell Counting Kit 8 assay. The related expression of skin barrier protein was accessed with real-time quantitative polymerase chain reaction, Western blot and Immunofluorescence in NHEK cells with or without poly (I:C). Meanwhile, the changes of thymic stromal lymphopoietin (TSLP) and tumor necrosis factor alpha (TNF-α) are analyzed by enzyme-linked immunosorbent assay.ResultsVC lipids mostly contained saturated and branched chains fatty acids. The expression of mRNA and protein of FLG were significantly increased after the supplement with lipid in NHEK cells. Meanwhile, lipids reversed the inhibition of poly (I:C) on FLG. Moreover, lipids suppressed the over secretion of TSLP and TNF-α induced by poly (I:C).ConclusionThese results indicate that lipids extracted from VC has positive effects on the expression of FLG and anti-inflammation, suggesting that lipids of VC may be used for a reference for novel therapeutic method in reducing and remedying skin disease like atopic disease.     

On the role of the epidermal differentiation complex in ichthyosis vulgaris, atopic dermatitis and psoriasis

Undisturbed epidermal differentiation is crucial for an intact skin barrier function. The epidermal differentiation complex (EDC) is a cluster of genes on chromosome 1q21 encoding proteins that fulfil important functions in terminal differentiation in the human epidermis, including filaggrin, loricrin, S100 proteins and others. Recently, evidence emerged that variation within EDC genes plays an important role in the pathogenesis of three common skin disorders, ichthyosis vulgaris, atopic dermatitis (AD) and psoriasis. Two loss-of-function mutations in the filaggrin (FLG) gene, R501X and 2282del4, were identified as causative for ichthyosis vulgaris in 15 affected European families, and the mode of inheritance was found to be semidominant. As ichthyosis vulgaris and AD often occur concomitantly in affected individuals, these two mutations were subsequently investigated in AD patients and found to be strongly associated with the disease. Following this first report, seven replication studies have been performed that all confirm an association of these two mutations with AD (or AD subtypes) in several European cohorts. Additionally, two unique loss-of-function mutations in the FLG gene were identified in Japanese ichthyosis vulgaris families and found to be associated with AD in a Japanese cohort. Thus, the FLG mutations are among the most consistently replicated associations for AD. Additionally, linkage analysis has suggested that variation within the EDC might also predispose for psoriasis but the exact susceptibility variation(s) have not yet been elucidated. Taken together, these findings convincingly demonstrate the important role of barrier dysfunction in various common skin disorders.     

Epigenetic control of the S100A6 (calcyclin) gene expression

S100A6 (calcyclin) is a calcium-binding protein of cell-specific expression whose gene is clustered with other S100 genes within the epidermal differentiation complex, on human chromosome 1q21. Many S100 proteins, including S100A6, are expressed in human epidermis at various stages of differentiation and their expression is often deregulated in skin and epithelial cancers. To gain insight into the mechanism of regulation of S100A6 expression, we examined epigenetic marks, that is DNA methylation and histone modifications along the S100A6 gene. Sequencing of bisulfite-modified DNA within a 3,247 bp long genomic region encompassing the promoter/first exon CpG island, the coding sequence of the S100A6 gene and a downstream region showed that it is almost entirely methylation-free in S100A6 expressing human epidermoid carcinoma (Hep-2) cells and lymphocytes and methylated in S100A6-negative embryonic epithelial (HEK293) cells. Chromatin immunoprecipitation revealed profound differences in the level of histone H3 acetylation and methylation and in the in vivo binding of upstream regulatory factor (USF), to the S100A6 gene promoter in S100A6-negative and -positive cells. These data demonstrate that cell-specific S100A6 expression is under control of epigenetic mechanisms.     

特应性皮炎与丝聚蛋白研究进展

丝聚蛋白(FLG)基因的失功能突变可使表皮FLG含量减少或缺失,进而引起表皮屏障功能发生改变,增加特应性皮炎(AD)的患病风险。本文对FLG在表皮屏障形成中的作用、FLG基因结构、FLG基因在AD患者中的突变情况及FLG基因突变与AD临床表型的相关性进行综述。     

Skin DNA methylation profile in atopic dermatitis patients: A case–control study

Atopic dermatitis (AD) is a worldwide disease with a complex aetiology. Both genetic and environmental factors cause a predisposition to AD. DNA methylation may be an additional predisposing factor. The goal of our study was to investigate genome-wide methylation profiles of skin from patients with AD and healthy persons. This case–control study included 12 AD patients and 6 healthy volunteers. DNA methylation levels in skin samples were analysed using the Illumina Infinium HumanMethylation450 BeadChip. We found that the methylation profile of skin from patients with AD was significantly different from that of healthy controls. Differential DNA methylation was observed for genes involved in a number of AD-related processes including regulation of the immune response, activation of lymphocytes, cell proliferation, apoptosis and differentiation of the epidermis. Our study indicates the involvement of epigenetic regulation in the development of atopic dermatitis.     

Knock‐down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT‐based 3D‐skin equivalent (NSE) after knock‐down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock‐down (FLG‐KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG‐KD alone does not necessarily affect the functionality of a proper barrier function.     

Silencing of homeobox A5 gene in the stratum corneum of psoriasis

Analysis of psoriatic parakeratotic cells is helpful for understanding the pathogenesis of psoriasis. Methylation analysis can be performed on psoriatic scales, but it is unclear whether genes can be silenced by DNA methylation in psoriatic stratum corneum. The present study was conducted to detect genes silenced in psoriatic stratum corneum. Methylation array analysis with 485 577 probes, quantitative real‐time methylation‐specific PCR (RT‐MSP) and bisulphite sequencing were performed for 30 psoriatic scale samples, 6 fully developed psoriatic skin samples and 12 normal skin samples. Immunohistochemical staining of HOXA5 was performed for 29 psoriatic epidermal samples and 13 normal epidermal samples. The genome‐wide methylation array detected two CpG sites within CpG islands (CGIs) located in promoter regions of HOXA5 and LIAS that had methylation levels of >0.6 in at least one of the three psoriatic scale samples and of <0.2 in all three normal skin tissue samples (methylation rate range, 0.0‐1.0). RT‐MSP for HOXA5CGI, in which the primers were successfully developed, revealed that the average methylation level of 27 psoriasis scales (60.2%) is significantly higher than that of 9 normal skin samples (34.6%) (P=.013). Immunohistochemical staining revealed that HOXA5 protein was not expressed in the stratum corneum of fully developed psoriatic epidermis, but the protein was expressed in the stratum corneum of incompletely developed epidermis and normal epidermis. In conclusion, HOXA5 can be silenced in the stratum corneum of psoriasis. The silenced gene was identified by non‐invasive methylation analysis of psoriatic scales.     

Adiponectin Attenuates the Inflammation in Atopic Dermatitis-Like Reconstructed Human Epidermis

BackgroundAtopic dermatitis (AD) is a chronic disorder, with a vicious cycle of repetitive inflammation and deterioration of the epidermal barrier function. Adiponectin, an adipokine, has anti-inflammatory effects on various metabolic and inflammatory disorders. Recently, its level was found to be reduced in serum and tissue samples from AD patients.ObjectiveWe aimed to investigate the effects of adiponectin on epidermal inflammation and barrier structures in AD skin.MethodsA three-dimensional in vitro epidermal equivalent model mimicking AD was obtained by adding an inflammatory substance cocktail to normal human epidermal equivalents (HEEs). The expression of epidermal differentiation markers, primary inflammatory mediators, and lipid biosynthetic enzymes was compared between adiponectintreated AD-HEEs, untreated control AD-HEEs, and normal HEEs.ResultsAdiponectin co-treatment 1) inhibited the increase in mRNA expression of major inflammatory mediators (carbonic anhydrase II, neuron-specific NEL-like protein 2, thymic stromal lymphopoietin, interleukin-8, tumor necrosis factor-alpha, and human beta-defensin-2) from keratinocytes in AD-inflammatory HEEs, 2) enhanced the expression of lipid biosynthetic enzymes (fatty acid synthase, HMG CoA reductase, and serine-palmitoyl transferase), and 3) promoted the expression of differentiation factors, especially filaggrin. We also found that the expression of adiponectin receptor-1 and -2 decreased in the epidermis of chronic AD lesion.ConclusionActivation of the adiponectin pathway is expected to enhance epidermal differentiation and barrier function as well as attenuate inflammatory response to AD as a therapeutic approach.     

Expression profile of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins during skin barrier repair

Background Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed. Objectives To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation‐regulating proteins. Methods We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls. Results Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS‐instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD. Conclusions Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification‐related proteins, and decreased expression of some structural and desquamation‐related proteins.     

UV irradiation‐induced DNA hypomethylation around WNT1 gene: Implications for solar lentigines

Wnt/β‐catenin signalling promotes melanogenesis in melanocytes and also induces melanocytogenesis from melanocyte stem cells (McSCs). Previous study reported that WNT1, a ligand which activates Wnt/β‐catenin signalling pathway, was more highly expressed in the epidermis at SLs than in normal skin areas, suggesting that WNT1 causes hyperpigmentation. To elucidate the mechanism by which WNT1 expression is increased in SLs, we examined the methylation of 5‐carbon of cytosine (5mC), that is 5‐methylcytosine (5mC) level, in a region within the WNT1 promoter; the methylation of the region was known to negatively regulate WNT1 gene expression. We used an immortalized cell line of human interfollicular epidermal stem cells to analyse the effect of UVB irradiation on DNA methylation level of WNT1 promoter and found that UVB irradiation caused demethylation of WNT1 promoter and promoted WNT1 mRNA expression. It was also found that UVB irradiation reduced the expression of DNA methyltransferase 1 (DNMT1), an enzyme responsible for maintaining methylation patterns during cell division. Pathological analysis of SLs and non‐SL regions in the human skin revealed that both DNMT1 expression and 5mC level were decreased at SLs compared to non‐SL skins. Furthermore, bisulphite sequencing showed that the methylated CpG level in WNT1 promoter was also lower at SLs than in non‐SL skins. Thus, in the skin exposed to a high amount of UV rays, excessive expression of WNT1 is thought to be caused by the demethylation of WNT1 promoter, and the upregulated WNT1 promotes melanocytogenesis and melanogenesis, then resulting in SL formation.     

丝聚蛋白与相关皮肤病发病机制的研究进展

丝聚蛋白通过参与角质形成细胞的终末角化过程,在表皮屏障中发挥重要作用.中间丝聚合蛋白基因突变引起蛋白表达下降,导致多种过敏性疾病.丝聚蛋白基因突变导致特应性皮炎,也是欧洲和亚洲人种寻常型鱼鳞病的主要诱发因素.丝聚蛋白基因与特应性皮炎发病的婴儿期或早期儿童期关系密切,而与晚期儿童期和成人期无关.有研究发现,小鼠体内缺氧诱导因子引起丝聚蛋白表达降低,提示丝聚蛋白可能不是疾病的单一致病因素.     

Filaggrin mutations in children with severe atopic dermatitis

Atopic dermatitis (AD) results from strong genetic and environmental interactions. AD shows genetic linkage to Chromosome 1q21. This region contains the epidermal differentiation complex (EDC), which consists of genes that form essential components of epidermal surfaces. Filaggrin (FLG) is one of these. Mutations in FLG/(R501X and 2282del4) are reported to be strongly associated with AD and to influence asthma accompanying AD. We investigated these effects in families recruited through a child with severe AD. We genotyped two panels of families, totalling 426, containing 990 affected and unaffected children. We found significant associations with AD (P=0.0001), asthma (P=0.006), and atopy (P=0.002). The FLG mutations were present in 26.7% of patients with AD, but were also present in 14.4% of children without AD. They were weakly associated with disease severity. The variants were not independently associated with asthma. The overall LOD score for genetic linkage of markers to the region was 3.57. This fell to 2.03 after accounting for the FLG mutations, indicating the presence of other genetic variants influencing AD at this locus. Our results provide further confirmation of the importance of mutations in FLG and the skin barrier in AD pathogenesis. The results indicate that investigations of other genes within the EDC should be undertaken.     

Calmodulin‐like skin protein level increases in the differentiated epidermal layers in atopic dermatitis

In atopic dermatitis (AD), the skin barrier is disturbed, and the expression of calcium‐dependent S100 proteins and the calcium gradient is also altered in the epidermis. The calmodulin‐like skin protein (CLSP), which is expressed in the differentiated epidermis, is believed to modulate the function of calcium‐dependent proteins involved in barrier formation and is significantly increased in the epidermis of psoriatic patients. We, therefore, investigated the CLSP level in skin biopsies taken from patients with acute exacerbated and non‐exacerbated AD as well as from healthy control subjects. Immunohistochemical, Western blot and ELISA analyses showed significant increases (P < 0.03) in CLSP level in the epidermis from patients with acute exacerbated AD as compared to that from patients with non‐exacerbated AD and from control subjects. Such increased expression of CLSP may help re‐establish a functional epidermal barrier in acute AD.     

Kontaktallergie und Atopie

Retrospective studies demonstrate that the prevalence of skin sensitization does not significantly differ between atopic and non-atopic patients. In children and adolescents the risk for sensitization seems to occur independently from AD. According to the results of a recent study, AD patients are overrepresented in the group of polysensitized patients. IgE-mediated sensitization as well as an early onset of AD and duration of the disease have been identified as possible risk factors for skin sensitization to contact allergens. A defective permeability barrier with increased epidermal water loss is a hallmark of AD and contributes to sensitization against common allergens. A highly significant association between FLG mutations and the risk of early onset, severe, persistent AD and an increased risk for asthma has been shown in several studies. A more recent study revealed an association between FLG mutations and increased nickel sensitization, but not other contact allergens. However, further large prospective studies with well-characterized patients are necessary to clarify the correlation between impaired skin barrier, atopic dermatitis and allergic contact dermatitis.     

Accelerated human epidermal turnover driven by increased hyaluronan production

BackgroundHyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive.ObjectiveWe examined the interaction of increased HA production mediated by 1-ethyl-β-N-acetylglucosaminide (β-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation.MethodsThe amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in β-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined.Resultsβ-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that β-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in β-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by β-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of β-NAG2 to UDP-NAG abolished β-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes.ConclusionThese results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.     

Role of YAP-related T cell imbalance and epidermal keratinocyte dysfunction in the pathogenesis of atopic dermatitis

BackgroundAtopic dermatitis (AD) is characterized by impaired skin barrier function and immune system dysfunction. The expression and role of Yes-associated protein (YAP) in AD are unclear.ObjectiveTo characterize the role of the YAP in T cell imbalance and epidermal keratinocyte dysfunction in the pathogenesis of AD.MethodsWe included 35 patients with AD (21 acute and 14 chronic). An AD mouse model was constructed using 2,4-dinitrofluorobenzene, and AD-like inflammatory cell model was constructed using TNF-α/IFN-γ-activated HaCaT cells. The proportion of Th1/Th2/Th17/Treg cells was detected using flow cytometry. After mononuclear cells were obtained from human peripheral blood or mouse spleen and induced to differentiate into different T cell subsets, YAP mRNA and protein expression were analyzed. Up-regulation of YAP was induced by lentivirus and down-regulation of YAP was induced by its specific inhibitor verteporfin (VP). The expression of YAP in skin lesions and infiltrating T cell subsets was detected using immunohistochemistry and double immunofluorescence staining, respectively.ResultsWe found differing degrees of Th1/Th2/Th17/Treg imbalance in acute and chronic AD. YAP expression was downregulated in Treg cells and upregulated in Th17 cells; YAP expression was downregulated in the AD epidermis. After YAP overexpression, the proportion of both Th17 and the Treg cells differentiated from mouse spleen mononuclear cells increased. There was an opposite trend after YAP inhibition. The proliferation and migration decreased and apoptosis increased after YAP inhibition in HaCaT cells.ConclusionChange of YAP expression may cause T cell imbalance and hamper the healing of the epidermis in AD.