Human keratinocyte caspase-14 expression is altered in human epidermal 3D models by dexamethasone and by natural products used in cosmetics

Caspase-14 is a cysteinyl-aspartate-specific proteinase that is specifically expressed in epidermal keratinocytes. Dysregulation of caspase-14 expression is implicated in impaired skin barrier formation. To elucidate the regulation of caspase-14 in differentiated keratinocytes, we characterized the expression of caspase-14 in normal human epidermal keratinocytes (NHEKs) and two types of three-dimensional (3D) human epidermis culture models, EPI-200 and EPI-201, via RT-PCR and immunoblot analyses. Caspase-14 expression was absent in subconfluent NHEKs, but was present in confluent NHEKs as well as those induced to differentiate by calcium. Caspase-14 expression levels in the 3D epidermis models were almost equal to that in the Ca²⁺-treated differentiated NHEKs. Despite the presence of caspase-14 expression in these models, caspase-14 activity was found only in the mature 3D skin model, EPI-200. This was confirmed by detection of a 17 kDa cleaved fragment of caspase-14 present only in the EPI-200 model. Since glucocorticoid (GC) receptor is required for skin barrier competence, we investigated whether the GC dexamethasone (Dex) and various natural components of common skin moisturizers affect caspase-14 expression in keratinocytes. Dex decreased caspase-14 expression in undifferentiated, but not differentiated, NHEKs. Conversely, Dex increased caspase-14 expression in both 3D skin models, although it did not alter caspase protease activity. Similar to treatment with Dex, treatment of the premature 3D skin mode, EPI-201 with a Galactomyces ferment filtrate markedly increased expression of caspase-14. Further, these results suggest that the effect of Dex, or lack thereof, on caspase-14 expression is dependent on the stage of keratinocyte differentiation.     

Cuts by caspase-14 control the proteolysis of filaggrin

Although mutations in the filaggrin gene (FLG) have been shown to be associated with ichthyosis vulgaris and atopic dermatitis, the function and regulation of filaggrin remain incompletely understood. In this issue, Hoste et al. report that filaggrin is directly cleaved by caspase-14. Acting in concert with other proteases, caspase-14 controls the breakdown of filaggrin to free amino acids and amino acid derivatives that contribute to the hydration and UVB absorption capacity of the stratum corneum. These findings identify a new layer of complexity in the regulation of epidermal barrier function.     

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.     

Caspase-14 expression by epidermal keratinocytes is regulated by retinoids in a differentiation-associated manner

Caspase-14 is the only member of the caspase family that shows a restricted tissue expression. It is mainly confined to epidermal keratinocytes and in contrast to other caspases, is not activated during apoptosis induced by ultraviolet irradiation or cytotoxic substances. As it is cleaved under conditions leading to terminal differentiation of keratinocytes we suggested that caspase-14 plays a part in the physiologic cell death of keratinocytes leading to skin barrier formation. Here we show that retinoic acid, at concentrations inhibiting terminal differentiation of keratinocytes, strongly suppressed caspase-14 mRNA and protein expression by keratinocytes in monolayer culture and in a three-dimensional in vitro model of differentiating human epidermis (skin equivalent). By contrast, the expression of the caspases 3 and 8, which are both activated during conventional apoptosis, was increased and unchanged, respectively, after retinoic acid treatment. In addition to inhibition of differentiation in skin equivalents, retinoic acid treatment led to keratinocyte apoptosis and activation of caspase-3, both of which were undetectable in differentiated control skin equivalents. As this occurred in the absence of detectable caspase-14, our data demonstrate that caspase-14 is dispensable for keratinocyte apoptosis. The fact that in contrast to caspase-3 and caspase-8, caspase-14, similarly to other keratinocyte differentiation-associated proteins, is downregulated by retinoids, strongly suggests that this caspase, but not caspase-3 and -8, plays a part in terminal keratinocyte differentiation and skin barrier formation.     

Skin presenting a higher level of caspase-14 is better protected from UVB irradiation according to in vitro and in vivo studies

Caspase-14, a cysteine endoproteinase belonging to the conserved family of aspartate-specific proteinases, was shown to play an important role in the terminal differentiation of keratinocytes and barrier function of the skin. In the present study, we developed a biofunctional compound that we described as a modulator of caspase-14 expression. Using normal human keratinocytes (NHK) in culture and human skin biopsies, this compound was shown to increase caspase-14 expression and partially reverse the effect of caspase-14-specific siRNA on NHK. Moreover, the increase in filaggrin expression visualized on skin biopsies and the recovery of the barrier structure after tape-stripping indicated that this compound could exhibit a beneficial effect on the skin barrier function. Considering the possible link between caspase-14 and the barrier function, a UVB irradiation on NHK and skin biopsies previously treated with the caspase-14 inducer, was performed. Results indicated that pretreated skin biopsies exhibited less signs of UV damage such as active caspase-3 and cyclobutane pyrimidine dimers (CPDs). Likewise, pretreated NHK were protected from UV-induced genomic DNA damage, as revealed by the Comet Assay. Finally, a clinical test showed a reduction of transepidermal water loss (TEWL) on the treated skin compared with placebo, under UV stress condition, confirming a protecting effect. Taken together, these results strongly suggest that, by increasing caspase-14 expression, the biofunctional compound could exhibit a protective effect on the skin barrier function, especially in case of barrier damage and UV irradiation.     

Loss-of-function mutations in the filaggrin gene and allergic contact sensitization to nickel

Allergic contact dermatitis is one of the most frequent dermatological problems affecting 7% of the general population. Impaired skin barrier function facilitates the penetration of contact allergens and irritants into the epidermal layer and is regarded as an important cofactor promoting the process of allergic contact sensitization. Filaggrin is crucial for the maintenance of the skin barrier function. Loss-of-function mutations within the filaggrin (FLG) gene are associated with skin barrier diseases such as ichthyosis vulgaris and atopic eczema (AE). To assess the impact of FLG on allergic contact sensitization and plausible intermediate traits, the two prevalent FLG mutations R501X and 2282del4 were typed in 1,502 individuals of the KORA C population-based cohort with extensive dermatologic phenotyping. Associations of FLG mutations with AE could be replicated. Strong associations were seen with dry skin, palmar hyperlinearity, and keratosis pilaris. In addition, an association with contact sensitization to nickel and contact sensitization to nickel combined with intolerance to fashion jewelry, but not with other contact allergens, was observed. From these data, we conclude that a genetically determined FLG deficiency manifests as dry skin and features of ichthyosis vulgaris. In addition, FLG deficiency may also represent a risk factor for contact sensitization to allergens.     

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin

Background

Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function.

Objective

To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems.

Methods

We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99.

Results

The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis.

Conclusion

Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.     

TNF-α downregulates filaggrin and loricrin through c-Jun N-terminal kinase: role for TNF-α antagonists to improve skin barrier

Filaggrin (FLG), loricrin (LOR), and involucrin are important epidermal barrier proteins. As psoriasis is characterized by overexpression of tumor necrosis factor-α (TNF-α) and impaired skin barrier, we investigated the expression of skin barrier proteins in psoriasis patients and whether their expression was modulated by TNF-α. The expression of FLG and LOR was found to be decreased in lesional and non-lesional skin of psoriasis patients. A correlation was found between the expression of TNF-α and epidermal barrier proteins in psoriasis. TNF-α was found to modulate the expression of FLG and LOR via a c-Jun N-terminal kinase-dependent pathway. Importantly, we report that clinical treatment of psoriasis patients with a TNF-α antagonist results in significant enhancement of epidermal barrier protein expression. Our current study suggests that TNF inhibits barrier protein expression, and TNF-α antagonists may contribute to clinical improvement in patients with psoriasis by improving barrier protein expression.     

Expression of caspase-14 and keratin-19 in the human epidermis and appendages during fetal skin development

Caspase-14 is a seemingly non-apoptotic caspase involved in keratinocyte differentiation and cornification of the skin. Keratin-19 is an epithelial marker and a potential marker of epidermal stem cells that is expressed during human fetal skin development. We examined the immunohistochemical expression of caspase-14 in relation to CK-19 in the human fetal skin during development and perinatally, to assess their role in human skin maturation. Skin samples were received at autopsy. In the fetal epidermis, caspase-14 was predominantly expressed in the more differentiated layers, gradually disappearing from the basal layer toward term. By contrast, keratin-19 expression gradually decreased with epidermal maturation through gestation (rho = ?0.949; p = 0.0001) and was a marker of the germinative layers. Keratin-19 was preserved in scarce basal cell nests at term and postnatally. Caspase-14 and keratin-19 were inversely expressed in the differentiating epidermal layers through gestation (p < 0.0001). Concerning the appendages, in hair follicles and sebaceous glands, caspase-14 located preferentially in the more differentiated layers of the inner root sheath, whereas keratin-19 was expressed in the outer sheath. Eccrine sweat glands showed a variable pattern of caspase-14 and keratin-19 expression. In conclusion, caspase-14 emerged as a marker of human skin differentiation during development, while keratin-19 marked the germinative epithelial layers in the fetal epidermis and appendages and possibly the nests of epidermal stem cells.     

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.     

Unique and recurrent mutations in the filaggrin gene in Singaporean Chinese patients with ichthyosis vulgaris

Filaggrin is an abundant protein of the outer epidermis that is essential for terminal differentiation of keratinocytes and formation of an effective barrier against water loss and pathogen/allergen/irritant invasion. Recent investigations in Europe and Japan have revealed null mutations in the filaggrin gene (FLG) as the underlying cause of ichthyosis vulgaris (IV), a common skin disorder characterised by dry skin, palmar hyperlinearity and keratosis pilaris. Following the development of a strategy for the comprehensive analysis of FLG, we have identified five unique mutations and one recurrent mutation in Singaporean Chinese IV patients. Mutation 441delA is located in the profilaggrin S100 domain, whereas two additional frameshift mutations, 1249insG and 7945delA, occur in the first partial filaggrin repeat ("repeat 0") and in filaggrin repeat 7, respectively. Both nonsense mutations Q2147X and E2422X are found in filaggrin repeat 6, whereas R4307X was found on one of the longer size variant alleles of FLG, within duplicated repeat 10.2. Mutation E2422X, previously found in a single Dutch patient, was found in one Singaporean IV patient and at a low frequency in Asian population controls. Our study confirms the presence of population-specific as well as recurrent FLG mutations in Singapore.     

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.     

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。     

The skin microbiome of caspase‐14‐deficient mice shows mild dysbiosis

Caspase‐14, an important proteinase involved in filaggrin catabolism, is mainly active in terminally differentiating keratinocytes, where it is required for the generation of skin natural moisturizing factors (NMFs). Consequently, caspase‐14 deficient epidermis is characterized by reduced levels of NMFs such as urocanic acid and 2‐pyrrolidone‐5‐carboxylic acid. Patients suffering from filaggrin deficiency are prone to develop atopic dermatitis, which is accompanied with increased microbial burden. Among several reasons, this effect could be due to a decrease in filaggrin breakdown products. In this study, we found that caspase‐14^?/? mice show enhanced antibacterial response compared to wild‐type mice when challenged with bacteria. Therefore, we compared the microbial communities between wild‐type and caspase‐14^?/? mice by sequencing of bacterial 16S ribosomal RNA genes. We observed that caspase‐14 ablation leads to an increase in bacterial richness and diversity during steady‐state conditions. Although both wild‐type and caspase‐14^?/? skin were dominated by the Firmicutes phylum, the Staphylococcaceae family was reduced in caspase‐14^?/? mice. Altogether, our data demonstrated that caspase‐14 deficiency causes the imbalance of the skin‐resident bacterial communities.     

Comparison of barrier function and lipids in psoriasis and essential fatty acid-deficient rats

Elevated rates of transepidermal water loss (TEWL) in plaques of human psoriasis and in the skin of essential fatty acid (EFA)-deficient rats were compared. Cutaneous application of sunflower seed oil to EFA-deficient rats lowered the rate of TEWL to normal, healed the characteristic scaly skin of this condition and increased the incorporation of linoleic acid of the sunflower seed oil into epidermal phospholipid. Application of sunflower seed oil to psoriatic skin did not lower the TEWL, nor produce clinical improvement, but the linoleic acid of epidermal phospholipid was increased. Local application of a steroid ointment, clobetasol propionate (Dermovate) reduced the elevated TEWL in psoriasis and produced clinical improvement, but had no effect upon skin or plasma lipids. Application of this steroid to EFA-deficient rat skin cleared the skin scaliness but did not restore barrier function or change the composition of the skin lipids. It is concluded that the impaired barrier function in psoriasis is not due to EFA-deficiency.     

Filaggrin and the great epidermal barrier grief

One of the principal functions of human skin is to form an effective mechanical barrier against the external environment. This involves the maturation and death of epidermal keratinocytes as well as the assembly of a complex network of differentially and spatially expressed proteins, glycoproteins and lipids into the keratinocyte cell membrane and surrounding extracellular space. In 2006, the key role of the granular cell layer protein filaggrin (filament-aggregating protein) in maintaining the skin barrier was determined with the identification of loss-of-function mutations in the profilaggrin gene (FLG). These mutations have been shown to be the cause of ichthyosis vulgaris and a major risk factor for the development of atopic dermatitis, asthma associated with atopic dermatitis as well as systemic allergies. Mutations in the FLG gene are extremely common, occurring in approximately 9% of individuals from European populations. The remarkable discovery of these widespread mutations is expected to have a major impact on the classification and management of many patients with ichthyosis and atopic disease. It is also hoped that the genetic discovery of FLG mutations will lead to the future development of more specific, non-immunosuppressive treatments capable of restoring effective skin barrier function and alleviating or preventing disease in susceptible individuals.     

Mite and cockroach allergens activate protease-activated receptor 2 and delay epidermal permeability barrier recovery

Protease-activated receptor-2 (PAR-2) is known to be involved in epidermal permeability barrier function homeostasis. PAR-2 activation occurs after barrier disruption and further activation of PAR-2 by activating peptide significantly delays barrier recovery rate. Cockroach and house dust mite allergens, both known to be associated with the development of asthma, allergic rhinitis, and atopic dermatitis, have protease activity, which can activate PAR-2. In this study, we investigated the effects of both allergens on the epidermal barrier function as well as on the epidermal calcium gradient. Both allergens, when topically applied on the barrier-disrupted site, increased protease activities in the epidermis and delayed barrier recovery and lamellar body secretion in murine skin. The topical application of PAR-2-specific antagonist or protease inhibitors normalized the barrier recovery. Cockroach allergens induced intracellular calcium oscillations in cultured human keratinocytes through PAR-2-involved pathway, which was confirmed by desensitization protocol. Abnormal calcium ion distribution after barrier disruption was also observed in allergens-applied skin. These results suggest that allergens with protease activity can influence the epidermal permeability barrier homeostasis through PAR-2 activation and consequent modulation of the calcium ions in skin.