A modified protocol for studying filaggrin degradation using a reconstructed human epidermis model under low and high humidity.

BACKGROUND: Filaggrin (FLG) is an essential protein that plays a vital role in maintaining skin barrier function and moisture levels, allowing the skin to adapt to dry environments. However, the precise temporal dynamics of FLG metabolism in the human epidermis remain poorly understood, and suitable tools to study these time-dependent effects are currently lacking. OBJECTIVE: To investigate the molecular mechanisms and time course of FLG metabolism and skin barrier function under high- and low-humidity conditions, utilizing a reconstructed epidermis model. METHODS: EpiSkin specimens cultured under humid or dry conditions for varying durations (2-48 h) were compared by assessing FLG degradation and skin barrier formation using immunofluorescence staining and western blotting. RESULTS: Under conditions of low humidity, the proteolysis of FLG in EpiSkin increased between 4 and 12 h and was accompanied by elevated levels of cysteine-aspartic protease (caspase)-14. The expression of peptidyl arginine deiminase 1 and calpain 1 also increased at 4 h. However, after 24 h, the expression of these three FLG-degrading proteins significantly decreased. Conversely, the levels of pyrrolidone-5-carboxylic acid and urocanic acid initially decreased at 2 h and then increased between 12 and 24 h. Additionally, the expression of skin barrier proteins, such as FLG, transglutaminase 5, loricrin and zonula occludens-1, decreased starting from 12 h. Notably, epidermal cell viability and activity were also inhibited. CONCLUSION: We propose a reliable and ethical model to study the temporal dynamics of FLG metabolism and its role in skin barrier function. Using a commercially reconstructed epidermis to mimic dry skin formation obviates the need for animal and human testing.

CONTEXTE: la filaggrine (FLG) est une protéine essentielle qui joue un rôle vital dans le maintien de la fonction de barrière cutanée et des taux d'humidité, permettant à la peau de s'adapter aux environnements secs. Cependant, la dynamique temporelle précise du métabolisme de la FLG dans l'épiderme humain reste mal comprise, et des outils appropriés pour étudier ces effets dépendant du temps font actuellement défaut. OBJECTIF: étudier les mécanismes moléculaires et l'évolution dans le temps du métabolisme de la FLG et de la fonction de barrière cutanée en milieux à humidité élevée et faible, en utilisant un modèle d'épiderme reconstruit. MÉTHODES: les échantillons EpiSkin cultivés en milieux humides ou secs pendant des durées variables (2 à 48 h) ont été comparés en évaluant la dégradation de la FLG et la formation d'une barrière cutanée à l'aide d'une coloration par immunofluorescence et d'un Western blot. RÉSULTATS: en milieux à faible humidité, la protéolyse de la FLG dans EpiSkin a augmenté entre 4 et 12 h et s'est accompagnée de taux élevés de cystéine­protéase aspartique (caspase)­14. L'expression du peptidyl arginine déiminase 1 et de la calpaïne 1 a également augmenté à 4 h. Cependant, après 24 h, l'expression de ces trois protéines de dégradation de la FLG a significativement diminué. Inversément, les taux d'acide pyrrolidone­5­carboxylique et d'acide urocanique ont initialement diminué au bout de 2 h, puis ont augmenté entre 12 et 24 h. En outre, l'expression des protéines de la barrière cutanée, telles que la FLG, la transglutaminase 5, la loricrine et le zonula occludens­1, a diminué à partir de 12 h. Notamment, la viabilité et l'activité des cellules épidermiques ont également été inhibées. CONCLUSION: nous proposons un modèle fiable et éthique pour étudier la dynamique temporelle du métabolisme de la FLG et son rôle dans la fonction de barrière cutanée. L'utilisation d'un épiderme reconstitué commercialement pour imiter la formation d'une peau sèche élimine la nécessité de réaliser des examens sur des animaux et des humains.

Biophysical insights into OR2T7: Investigation of a potential prognostic marker for glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive and prevalent form of brain cancer, with an expected survival of 12-15 months following diagnosis. GBM affects the glial cells of the central nervous system, which impairs regular brain function including memory, hearing, and vision. GBM has virtually no long-term survival even with treatment, requiring novel strategies to understand disease progression. Here, we identified a somatic mutation in OR2T7, a G-protein-coupled receptor (GPCR), that correlates with reduced progression-free survival for glioblastoma (log rank p-value = 0.05), suggesting a possible role in tumor progression. The mutation, D125V, occurred in 10% of 396 glioblastoma samples in The Cancer Genome Atlas, but not in any of the 2504 DNA sequences in the 1000 Genomes Project, suggesting that the mutation may have a deleterious functional effect. In addition, transcriptome analysis showed that the p38α mitogen-activated protein kinase (MAPK), c-Fos, c-Jun, and JunB proto-oncogenes, and putative tumor suppressors RhoB and caspase-14 were underexpressed in glioblastoma samples with the D125V mutation (false discovery rate < 0.05). Molecular modeling and molecular dynamics simulations have provided preliminary structural insight and indicate a dynamic helical movement network that is influenced by the membrane-embedded, cytofacial-facing residue 125, demonstrating a possible obstruction of G-protein binding on the cytofacial exposed region. We show that the mutation impacts the "open" GPCR conformation, potentially affecting Gα-subunit binding and associated downstream activity. Overall, our findings suggest that the Val125 mutation in OR2T7 could affect glioblastoma progression by downregulating GPCR-p38 MAPK tumor-suppression pathways and impacting the biophysical characteristics of the structure that facilitates Gα-subunit binding. This study provides the theoretical basis for further experimental investigation required to confirm that the D125V mutation in OR2T7 is not a passenger mutation. With validation, the aforementioned mutation could represent an important prognostic marker and a potential therapeutic target for glioblastoma.

Caspase-14-From Biomolecular Basics to Clinical Approach. A Review of Available Data.

Caspase-14 is a unique member of the caspase family-a family of molecules participating in apoptosis. However, it does not affect this process but regulates another form of programmed cell death-cornification, which is characteristic of the epidermis. Therefore, it plays a crucial role in the formation of the skin barrier. The cell death cycle has been a subject of interest for researchers for decades, so a lot of research has been done to expand the understanding of caspase-14, its role in cell homeostasis and processes affecting its expression and activation. Conversely, it is also an interesting target for clinical researchers searching for its role in the physiology of healthy individuals and its pathophysiology in particular diseases. A summary was done in 2008 by Denecker et al., concentrating mostly on the biotechnological aspects of the molecule and its physiological role. However, a lot of new data have been reported, and some more practical and clinical research has been conducted since then. The majority of studies tackled the issue of clinical data presenting the role of caspase in the etiopathology of many diseases such as retinal dysfunctions, multiple malignancies, and skin conditions. This review summarizes the available knowledge on the molecular and, more interestingly, the clinical aspects of caspase-14. It also presents how theoretical science may pave the way for medical research. Methods: The authors analyzed publications available on PubMed until 21 March 2021, using the search term "caspase 14".

Impaired Skin Barrier Function and Downregulated Expression of Caspase-14 in Moderate to Severe Chronic Hand Eczema.

AIM: To investigate whether the skin barrier function is impaired with regard to the pH value, water content, transepidermal water loss (TEWL), and the integrity of the stratum corneum, and whether the expression of caspase-14 is altered in moderate to severe chronic hand eczema (CHE). METHODS: Thirty patients with moderate to severe CHE treated at our institute and 30 healthy volunteers were included in this study. The pH value, water content, TEWL, and the integrity of the stratum corneum were measured in all subjects. RESULTS: Significantly increased pH value, decreased water content, elevated TEWL, and impaired integrity of the stratum corneum were observed in the lesional skin of CHE patients compared with the nonlesional skin of CHE patients and the normal skin of healthy volunteers. The expression of caspase-14 decreased in the lesional and nonlesional skin of CHE patients compared with the normal skin of healthy volunteers, especially prominent in the nonlesional skin. The mean optical density (OD) value of immunohistochemical staining for caspase-14 was significantly lower in the nonlesional skin than in the lesional skin and normal skin (p < 0.01 for both). Although the mean OD value was lower in the lesional skin than in the normal skin, the difference was not statistically significant (p > 0.05). CONCLUSION: Skin barrier dysfunction indeed occurs in CHE patients, which may be related to mechanisms associated with a downregulated expression of caspase-14.

Sphingoid Base-Upregulated Caspase-14 Expression Involves MAPK.

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Especially, sphingoid base-backbones of sphingolipids (sphingosine, sphinganine, and phytosphingosine) and their metabolites N-acyl-sphingoid bases (ceramides) are highly bioactive. In skin, one of the caspases, caspase-14, is expressed predominantly in cornifying epithelia, and caspase-14 plays an important role in keratinocyte differentiation. As ceramides were surrounding lipids in the keratinocytes and ceramides stimulate keratinocyte differentiation, we therefore examined the upregulation of caspase-14 by various sphingoid bases and ceramide. Sphingosine, sphinganine, phytosphingosine, and C2-ceramide treatment at the doses not damaging cells significantly increased caspase-14 mRNA and protein expression in dose-dependent manner on human keratinocyte HaCaT cells. These results indicated that sphingoid bases and ceramide upregulated caspase-14 mRNA to increase intracellular caspase-14 protein level. We next examined the caspase-14 upregulation mechanism by sphingoid bases. We used the most effective sphingoid base, phytosphingosine, and revealed that specific inhibitors of the mitogen-activated protein kinase, p38 and c-jun N-terminal protein kinase (JNK), blocked caspase-14 expression. This indicates that phytosphingosine upregulation of caspase-14 is involved of p38 and JNK activation. Moreover, phytosphingosine induced caspase-14 upregulation in vivo, suggesting that sphingoid bases were involved in keratinocyte differentiation by affecting caspase-14.

Blocking mTOR Signalling with Rapamycin Ameliorates Imiquimod-induced Psoriasis in Mice.

The mTOR (mechanistic target of rapamycin) inhibitor rapamycin has long been known for its immune suppressive properties, but it has shown limited therapeutic success when given systemically to patients with psoriasis. Recent data have shown that the mTOR pathway is hyperactivated in lesional psoriatic skin, which probably contributes to the disease by interfering with maturation of keratinocytes. This study investigated the effect of topical rapamycin treatment in an imiquimod-induced psoriatic mouse model. The disease was less severe if the mice had received rapamycin treatment. Immunohistological analysis revealed that rapamycin not only prevented the activation of mTOR signalling (P-mTOR and P-S6 levels), but almost normalized the expression of epidermal differentiation markers. In addition, the influx of innate immune cells into the draining lymph nodes was partially reduced by rapamycin treatment. These data emphasize the role of mTOR signalling in the pathogenesis of psoriasis, and support the investigation of topical mTOR inhibition as a novel anti-psoriatic strategy.

Increased filaggrin-metabolizing enzyme activity in atopic skin: a pilot study using a canine model of atopic dermatitis.

BACKGROUND: Filaggrin (FLG) and its metabolites are essential for skin barrier function and hydration of the stratum corneum. Alteration of the FLG metabolism could be the basis for an abnormal skin barrier in allergic dogs. OBJECTIVES: To investigate the expression and distribution of calpain-1, caspase-14, furin and matriptase, four enzymes involved in FLG metabolism, in the skin of atopic and healthy beagles. METHODS: Skin biopsies were collected from four healthy and four atopic beagles before and after allergen exposure. The dogs were challenged for three consecutive days to mimic an acute exposure, or once weekly to mimic a chronic exposure to allergens. Skin biopsies were taken on days 0 (nonlesional), 3 and 10 in the "acute" model and on days 0 (nonlesional), 14 and 28 in the "chronic" model. Four healthy dogs were used as controls. Indirect immunofluorescence was used to analyse the distribution and the expression of FLG enzymes in a semi-quantitative manner. Five consecutive pictures/section were taken and the intensity analysed tracing the epidermis and using ImageJ on the traced areas. The enzymes' expression was compared between healthy and atopic nonlesional skin (Day 0) and over time in each group. RESULTS: All enzymes were expressed in all layers of the epidermis. A significantly higher expression of calpain-1 (P = 0.028), caspase 14 (P = 0.028) and matriptase (P = 0.028) was evident in atopic compared to control dogs on Day 0. No differences over time were seen for any enzyme analysed. CONCLUSIONS AND CLINICAL IMPORTANCE: This preliminary study suggests an abnormal catabolism of FLG in canine atopic skin.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

Comparative genomics reveals conservation of filaggrin and loss of caspase-14 in dolphins.

The expression of filaggrin and its stepwise proteolytic degradation are critical events in the terminal differentiation of epidermal keratinocytes and in the formation of the skin barrier to the environment. Here, we investigated whether the evolutionary transition from a terrestrial to a fully aquatic lifestyle of cetaceans, that is dolphins and whales, has been associated with changes in genes encoding filaggrin and proteins involved in the processing of filaggrin. We used comparative genomics, PCRs and re-sequencing of gene segments to screen for the presence and integrity of genes coding for filaggrin and proteases implicated in the maturation of (pro)filaggrin. Filaggrin has been conserved in dolphins (bottlenose dolphin, orca and baiji) but has been lost in whales (sperm whale and minke whale). All other S100 fused-type genes have been lost in cetaceans. Among filaggrin-processing proteases, aspartic peptidase retroviral-like 1 (ASPRV1), also known as saspase, has been conserved, whereas caspase-14 has been lost in all cetaceans investigated. In conclusion, our results suggest that filaggrin is dispensable for the acquisition of fully aquatic lifestyles of whales, whereas it appears to confer an evolutionary advantage to dolphins. The discordant evolution of filaggrin, saspase and caspase-14 in cetaceans indicates that the biological roles of these proteins are not strictly interdependent.

Hairless is a histone H3K9 demethylase.

The hairless (HR) protein contains a Jumonji C (JmjC) domain that is conserved among a family of proteins with histone demethylase (HDM) activity. To test whether HR possesses HDM activity, we performed a series of in vitro demethylation assays, which demonstrated that HR can demethylate monomethylated or dimethylated histone H3 lysine 9 (H3K9me1 or me2). Moreover, ectopic expression of wild-type HR, but not JmjC-mutant HR, led to pronounced demethylation of H3K9 in cultured human HeLa cells. We also show that two missense mutations in HR, which we and others described in patients with atrichia with papular lesions, abolished the demethylase activity of HR, demonstrating the role of HR demethylase activity in human disease. By ChIP-Seq analysis, we identified multiple new HR target genes, many of which play important roles in epidermal development, neural function, and transcriptional regulation, consistent with the predicted biological functions of HR. Our findings demonstrate for the first time that HR is a H3K9 demethylase that regulates epidermal homeostasis via direct control of its target genes.

Topical application of delphinidin reduces psoriasiform lesions in the flaky skin mouse model by inducing epidermal differentiation and inhibiting inflammation.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and aberrant keratinocyte differentiation. We have shown that treatment of reconstituted human skin with delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, increased the expression and processing of caspase-14, which is involved in cornification. Delphinidin also increases the expression of epidermal differentiation marker proteins. OBJECTIVES: To determine whether topical application of delphinidin can modulate pathological markers of psoriasiform lesions in flaky skin mice and if this is associated with increased epidermal differentiation and a reduction in proliferation and inflammation. METHODS: Five-week-old female homozygous flaky skin mice (fsn/fsn) were treated topically with delphinidin (0·5 mg cm(-2) and 1 mg cm(-2) skin areas, respectively), five times a week, up to 14 weeks of age. RESULTS: Treatment of flaky skin mice with delphinidin resulted in a reduction in (i) pathological markers of psoriasiform lesions; (ii) infiltration of inflammatory cells; and (iii) mRNA and protein expression of inflammatory cytokines. Delphinidin treatment also increased the expression and processing of caspase-14, and expression of filaggrin, loricrin, keratin-1 and keratin-10. Furthermore, there was a decrease in the expression of markers for cell proliferation (proliferating cell nuclear antigen and keratin-14) and modulation of tight junction proteins (occludin and claudin-1). In addition, delphinidin treatment increased the expression of activator protein-1 transcription factor proteins (JunB, JunD, Fra1 and Fra2). CONCLUSIONS: Delphinidin could be a promising agent for treatment of psoriasis and other hyperproliferative skin disorders.

Aberrant expression of caspase 14 in salivary gland carcinomas.

OBJECTIVES: Caspase 14 is reduced in adenocarcinomas of the stomach and colon. In contrast, breast and lung adenocarcinomas frequently show an overexpression of caspase 14. Salivary gland adenocarcinomas have not been evaluated for potential aberrant caspase 14 expression. MATERIALS AND METHODS: Samples from salivary gland carcinomas (n = 43) were analysed by immunohistochemistry (caspase 14, filaggrin, GATA3 and Ki67) and fluorescence in situ hybridization. RESULTS: Caspase 14 is not expressed in normal salivary glands, while in a subfraction of carcinomas (32%) an aberrant expression was found. Filaggrin could not be detected. Caspase 14 staining was not associated with tumour dedifferentiation, GATA3 expression or amplification of gene locus 19p13. CONCLUSION: In summary, aberrant expression of caspase 14 can be found in a subfraction of salivary gland carcinomas but could not be used as a biomarker for a specific carcinoma subtype of the salivary gland.

Skin dryness in apparently healthy human skin is associated with decreased expression of bleomycin hydrolase in the stratum corneum.

BACKGROUND: Maintenance of water balance in the stratum corneum (SC) is determined by the content of intercellular lipids and natural moisturizing factors (NMFs) in corneocytes. AIM: To investigate the association between the NMFs and (pro)filaggrin and the proteases responsible for the processing of (pro)filaggrin to NMFs in the SC of hydrated and dry skin areas of healthy human subjects. METHODS: The SC hydration state and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter, respectively. Proteases, (pro)filaggrin and NMFs were extracted from SC samples obtained by tape-stripping of the tested skin. Expression levels of (pro)filaggrin were determined by dot blotting and western blotting, and total NMFs by ultra-high performance liquid chromatography. Expression of the proteases caspase-14, calpain-1 and bleomycin hydrolase was measured by western blotting. RESULTS: The levels of (pro)filaggrin were not significantly different between hydrated and dry skin, whereas the level of total NMFs was significantly reduced in dry skin. A negative correlation between (pro)filaggrin and NMFs was found in dry skin (Pearson correlation coefficient r = - 0.57, *P < 0.05). Bleomycin hydrolase expression was significantly decreased in the SC of dry skin. CONCLUSIONS: These results suggest that the low hydration state of dry skin may be due to the reduction in (pro)filaggrin degradation caused by decreased bleomycin hydrolase expression.

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.

Caspase-14 suppresses GCM1 acetylation and inhibits placental cell differentiation.

Glial cell missing 1 (GCM1) transcription factor regulates placental cell fusion into the syncytiotrophoblast. Caspase-14 is proteolytically activated to mediate filaggrin processing during keratinocyte differentiation. Interestingly, altered expression of nonactivated caspase-14 proenzyme is associated with tumorigenesis and diabetic retinopathy, suggesting that caspase-14 may perform physiological functions independently of its protease activity. Here, we performed tandem affinity purification coupled with mass spectrometry analysis to identify caspase-14 proenzyme as a GCM1-interacting protein that suppresses GCM1 activity and syncytiotrophoblast differentiation. Immunohistochemistry revealed that caspase-14 and GCM1 colocalize to placental cytotrophoblast cells at 8 wk of gestation and syncytiotrophoblast layer at term. Further, we demonstrated that caspase-14 mRNA level is decreased by 40% in placental BeWo cells treated with forskolin (FSK). To the contrary, stimulation of GCM1-regulated placental cell fusion and human chorionic gonadotropin ß (hCGß) expression by FSK is enhanced by caspase-14 knockdown. Indeed, GCM1 protein level is increased by 40% in the caspase-14-knockdown BeWo cells. Because GCM1 is stabilized by acetylation, we subsequently showed that caspase-14 impedes the interaction between GCM1 and cAMP response element-binding protein (CREB)-binding protein (CBP) to suppress CBP-mediated acetylation and transcriptional coactivation of GCM1. Therefore, caspase-14 can suppress placental cell differentiation through down-regulation of GCM1 activity.

Comprehensive evaluation of caspase-14 in vulvar neoplasia: an opportunity for treatment with black raspberry extract.

OBJECTIVE: The aim of this study is to determine the expression of caspase-14, a key protein in maturation of squamous epithelia, in archival malignant and premalignant vulvar squamous lesions and examine in-vitro effects of a black raspberry extract (BRB-E) on a vulvar squamous cell carcinoma (VSCC) cell line. METHODS: VSCC cell cultures were exposed to different BRB-E concentrations and used to create cell blocks. Immunohistochemistry for caspase-14 was performed on cell block sections, whole tissue sections, and a tissue microarray consisting of normal vulvar skin, lichen sclerosus (LS), classic and differentiated vulvar intraepithelial neoplasia (cVIN and dVIN respectively), and VSCC. RESULTS: LS demonstrated abnormal full thickness (5/11) or absent (1/11) caspase-14 staining. dVIN often showed markedly reduced expression (4/7), and cVIN occasionally demonstrated either absent or reduced caspase-14 (6/22). VSCC predominantly had absent or markedly reduced caspase-14 (26/28). VSCC cell cultures demonstrated a significant increase in caspase-14 (p=0.013) after BRB-E treatment: 7.3% (±2.0%) of untreated cells showed caspase-14 positivity, while 21.3% (±8.9%), 21.7% (±4.8%), and 22.6% (±5.3%) of cells were positive for caspase-14 after treatment with 200, 400, and 800 µg/mL BRB-E, respectively. Pair-wise comparisons between the treatment groups and the control demonstrated significant differences between no treatment with BRB-E and each of these treatment concentrations (Dunnett's adjusted p-values: 0.024, 0.021, and 0.014, respectively). CONCLUSIONS: Caspase-14 is frequently decreased in premalignant and malignant vulvar squamous lesions, and is upregulated in VSCC cell culture by BRB-E. BRB-E should be further explored and may ultimately be incorporated in topical preparations.

Association of caspase-14 and filaggrin expression with keratinization of the oral mucosa and reconstruction culture rat models.

BACKGROUND AND OBJECTIVE: Keratinization of the oral mucosa, such as the gingiva, has been shown to be important for periodontal health. Caspase-14 is a protease that plays a role in keratinization of the epidermis. The objective of this study was to investigate whether the expression of caspase-14 is intimately linked with keratinization and to examine the effect of the main component of green tea on the improvement of keratinization in rat oral mucosal preparations. MATERIAL AND METHODS: Histological and immunohistochemical analyses and quantitative mRNA measurements of caspase-14 and its substrate filaggrin were performed using different types of rat epithelial tissue and organotypic reconstruction culture models derived from epithelial cells and fibroblasts taken from the rat oral mucosa. RESULTS: In the skin, palate, buccal mucosa and esophagus, the degree of keratinization appeared to be associated with expression of cytokeratin 10. The relative protein and mRNA expression levels of caspase-14 and filaggrin were consistent with the degree of keratinization in the following order: skin > palate > buccal mucosa > esophagus. The culture models of palatal and buccal mucosa retained a stratified epithelial structure. Expression of caspase-14 appeared to be stronger in the palatal model than in the buccal model. Remarkably, epigallocatechin-3-gallate (EGCG) improved the localization of cytokeratins and increased the expression of caspase-14 and filaggrin. This expression was more intense in the palatal model than in the buccal model, indicating that both models maintain the intrinsic properties of keratinization of the mucosa from where the cultured cells were derived. CONCLUSIONS: These results suggest that keratinization is closely associated with expression of caspase-14 and filaggrin. Our reconstruction models are promising tools for drug evaluation and show that EGCG is beneficial for improving both keratinization and expression of the linked protease in the oral mucosa.

Comparative genomics of sirenians reveals evolution of filaggrin and caspase-14 upon adaptation of the epidermis to aquatic life.

The mammalian epidermis has evolved to protect the body in a dry environment. Genes of the epidermal differentiation complex (EDC), such as FLG (filaggrin), are implicated in the barrier function of the epidermis. Here, we investigated the molecular evolution of the EDC in sirenians (manatees and dugong), which have adapted to fully aquatic life, in comparison to the EDC of terrestrial mammals and aquatic mammals of the clade Cetacea (whales and dolphins). We show that the main subtypes of EDC genes are conserved or even duplicated, like late cornified envelope (LCE) genes of the dugong, whereas specific EDC genes have undergone inactivating mutations in sirenians. FLG contains premature stop codons in the dugong, and the ortholog of human CASP14 (caspase-14), which proteolytically processes filaggrin, is pseudogenized in the same species. As FLG and CASP14 have also been lost in whales, these mutations represent convergent evolution of skin barrier genes in different lineages of aquatic mammals. In contrast to the dugong, the manatee has retained functional FLG and CASP14 genes. FLG2 (filaggrin 2) is truncated in both species of sirenians investigated. We conclude that the land-to-water transition of sirenians was associated with modifications of the epidermal barrier at the molecular level.

The moisturizing effect of Capparis spinosa fruit extract targeting filaggrin synthesis and degradation.

BACKGROUND: Small molecular natural products, such as betaine, have unique moisturizing advantages. Capparis spinosa L. fruit is rich in quaternary ammonium alkaloids such as betaine and stachydrine. However, few studies investigated its efficacy and mechanism on human skin. OBJECTIVE: Polysaccharides-free C. spinosa fruit extract (CS) was obtained to study its moisturizing effect and mechanisms focusing on filaggrin (FLG) synthesis and degradation. METHODS: The clinical moisturizing test was carried out on human arms, calves, and faces after CS treatment for 0.5-6 h. The change in the level of FLG, caspase 14, loricrin, and transglutaminase 5 (TGM 5) was measured by immunofluorescence after CS treatment for 4 and 24 h in a reconstructed epidermis model. Also, the content of pyrrolidone carboxylic acid (PCA) in the stratum corneum was tested by high-performance liquid chromatography (HPLC) both in the epidermis model and human calves. RESULTS: Compared with glycerin (positive control), 5% CS showed a strong skin hydration effect on arms and calves when applied for 0.5-6 h. Also, the face hydration increased at 0.5 and 4 h. In addition, 3% CS applied to the recombinant epidermis model under low humidity promoted the immunodetected levels of caspase 14 and PCA content but reduced the levels of FLG at 4 h, however, the levels of FLG, loricrin, and TGM 5 were promoted at 24 h. Meanwhile, CS treatment for 4 h in human calves increased the PCA content in the stratum corneum by 29.9%. CONCLUSIONS: Topical application of CS on human skin showed an instant and long-lasting increase in skin hydration by regulating the FLG network. It promoted FLG degradation to form PCA at 4 h both in vivo and in vitro, increasing FLG synthesis after 24 h, potentially reforming the FLG monomer reservoir to alleviate the skin's dry condition.

Caspase-14: a novel caspase in the retina with a potential role in diabetic retinopathy.

PURPOSE: The purpose of this study was to evaluate caspase-14 expression in the retina under normal and diabetic conditions, and to determine whether caspase-14 contributes to retinal microvascular cell death under high glucose conditions. METHODS: Quantitative real-time polymerase chain reaction and western blot analysis were used to evaluate caspase-14 expression in retinal cells, including pericytes (PCs), endothelial cells (ECs), astrocytes (ACs), choroidal ECs, and retinal pigment epithelium (RPE) cells. We also determined caspase-14 expression in the retinas of human subjects with or without diabetic retinopathy (DR) and in experimental diabetic mice. Retinal ECs and PCs were infected with adenoviruses expressing human caspase-14 or green fluorescent protein. Caspase-14 expression was also assessed in retinal vascular cells cultured under high glucose conditions. The number of apoptotic cells was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling staining and confirmed by determining the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3. RESULTS: Our experiments demonstrated that retinal ECs, PCs, ACs, choroidal ECs, and RPE cells expressed caspase-14, and DR was associated with upregulation and/or activation of caspase-14 particularly in retinal vasculature. High glucose induced marked elevation of the caspase-14 level in retinal vascular cells. There was a significant increase in the apoptosis rate and the levels of cleaved poly (ADP-ribose) polymerase-1 and caspase-3 in retinal ECs and PCs overexpressing caspase-14. CONCLUSIONS: Our findings indicate that caspase-14 might play a significant role in the pathogenesis of DR by accelerating retinal PC and EC death. Further investigations are required to elaborate the underlying mechanisms.