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.

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.

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.

The Effect and Mechanism of Burnet Gels on Steroid-Dependent Dermatitis in Guinea Pig Model.

Objective: This study was designed to establish quality standards of Burnet gels and investigate the effects and mechanism of Burnet gels on steroid-dependent dermatitis (HDD) in guinea pigs. Methods: HPLC was used to determine the content of gallic acid, Gentiopicrin, and paeonol. A total of 48 male guinea pigs were recruited and randomly divided into control group, model group, tacrolimus ointment group, and Burnet gel group (Low, medium, and high concentration). The HDD guinea pig model was established by the 0.5% clobetasol propionate tincture. After HDD model establishment, control group and model group smeared normal saline and the rest of the group with corresponding drugs for three weeks. The contents of IFN-γ, IL-4, and IgE in the guinea pig serum were detected by the ELISA; the protein expression levels of FLG, LOR, and Caspase-14 in the epidermis of guinea pigs were detected by the immunohistochemical and Western blotting method. Results: The content of gallic acid, Gentiopicrin, and paeonol was 0.30 mg/g, 1.06 mg/g, and 0.56 mg/g. Compared with the normal group, the IFN-γ, IL-4, and IgE of guinea pig serum in the model group were significantly increased; the FLG, LOR, and Caspase-14 of guinea pig epidermis in the model group were significantly decreased; compared with the model group, the IFN-γ, IL-4, and IgE of guinea pig serum in the tacrolimus ointment group and Burnet gel group were significantly decreased; the FLG, LOR, and Caspase-14 of guinea pig epidermis in the tacrolimus ointment group and Burnet gel group were significantly increased. Conclusion: Burnet gels can improve guinea pig HDD model, and the mechanism may be related to inhibiting skin inflammation and promoting the formation of epidermal skin barrier.

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.

Effect of extracorporeal shock wave therapy on keratinocytes derived from human hypertrophic scars.

Hypertrophic scars represent a common complication in burn patients. In addition to cosmetic defects, they may cause serious sensory abnormalities such as pain and itching, severe dysfunction depending on the site, and emotional disorders such as anxiety and depression. The present study aimed to identify the molecular mechanisms underlying the use of extracorporeal shock wave therapy in keratinocytes. Keratinocytes derived from hypertrophic scar tissue were cultured and expression of proliferation markers (keratin 5 and 14), activation markers (keratin 6 and 17), differentiation markers (keratin 1, 10, and involucrin), apoptosis factors (Bax, Bcl2, and Caspase 14), and proliferation/differentiation regulators (p21 and p27) was investigated to compared with that of those in keratinocytes derived from normal skin tissue. Scar-derived keratinocytes were treated with extracorporeal shock waves under 1000 impulses at 0.1, 0.2, and 0.3 mJ/mm2. Shock waves altered the molecular pattern of proliferation, activation, differentiation, and apoptosis, as well as proliferation/ differentiation regulators, including Bax, Bcl2, ASK1, p21, p27, and Notch1. In summary, we show that extracorporeal shock wave therapy regulates the proliferation and differentiation of keratinocytes derived from hypertrophic scar to maintain normal epidermal integrity.

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".

Anti-Inflammatory, Barrier-Protective, and Antiwrinkle Properties of Agastache rugosa Kuntze in Human Epidermal Keratinocytes.

This work aimed to assess the skin-beneficial properties of Agastache rugosa Kuntze, an herbal medication used to treat different types of disorders in traditional folk medicine. The total phenolic compounds and total antiradical, nitrite scavenging, superoxide scavenging, antielastase, and antihyaluronidase activities of a hot water extract of A. rugosa Kuntze leaves (ARE) were spectrophotometrically determined. Intracellular reactive oxygen species (ROS) was fluorometrically quantitated using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Inducible nitric oxide synthase (iNOS) and filaggrin were evaluated using Western analysis. Real-time quantitative RT-PCR was used to measure filaggrin mRNA. Caspase-14 activity was determined using a fluorogenic substrate. ARE contained the total phenolic content of 38.9 mg gallic acid equivalent/g extract and exhibited 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide radical, and nitrite scavenging activities with the SC50 values of 2.9, 1.4, and 1.7 mg/mL, respectively. ARE exerted suppressive activities on nitric oxide (NO) and ROS levels elevated by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) in HaCaT keratinocytes. It attenuated the LPS-stimulated expression of iNOS. ARE augmented the UV-B-reduced filaggrin expression on both protein and mRNA levels and was capable of upregulating the UV-B-reduced caspase-14 activity. ARE inhibited in vitro elastase and hyaluronidase activities associated with the wrinkling process. ARE, at the concentrations used, did not interfere with the viability of HaCaT keratinocytes. These findings preliminarily imply that the leaves of A. rugosa possess desirable cosmetic potentials, such as anti-inflammatory, barrier protective, and antiwrinkle activities, which infers their skin healing potentials.

Adiponectin Promotes Caspase-14 Expression in Normal Human Epidermal Keratinocytes

No abstract available.

Ameliorative effects of a fusion protein dual targeting interleukin 17A and tumor necrosis factor α on imiquimod-induced psoriasis in mice.

In recent decades, biological agents such as tumor necrosis factor-α (TNF-α) inhibitors, have revolutionized the treatment of psoriasis. However, inhibition of a single cytokine may not achieve satisfactory therapeutic results. It is against this background that this research was undertaken to investigate the anti-psoriatic effect of a novel fusion protein (DTF) dual targeting TNF-α and interleukin-17 A (IL-17 A). Imiquimod (IMQ) was topically applied to the skin of mice to develop psoriasis-like skin and treated with etanercept or different doses of DTF. Results showed that DTF treatment (1 mg/kg, 3 mg/kg, 5 mg/kg) significantly attenuated IMQ-induced typical psoriasis-like inflammation, severity score, and epidermis thickening in a dose-dependent manner, and was again more efficient than etanercept (3 mg/kg) in alleviating all these parameters at the same dose. Furthermore, DTF was more potent than etanercept in suppressing the expression of inflammatory factors (IL-17 A, IL-6, IL-1ß, IL-23, IL-22 and IL-12) in the serum, spleen and psoriasis-like skin compared with etanercept at the same dose. In addition, DTF was more efficient than etanercept in reducing the expression of keratins, decreasing the mRNA expression of Ly-6 G and Ly-6C, and enhancing the expression of filaggrin and caspase 14 in IMQ-induced psoriasis-like skin. We conclude that DTF alleviates IMQ-induced psoriasis by attenuating inflammatory cascades, reducing keratinocytes proliferation and improving epidermal barrier function through suppressing TNF-α and IL-17 A signal pathways. These data suggest that DTF has potential to be a novel therapeutic candidate for psoriasis.

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.

Single blinded, randomized, placebo-controlled study on the effects of ciclosporin on cutaneous barrier function and immunological response in atopic beagles.

Ciclosporin (CsA) is a common treatment for canine atopic dermatitis (cAD). cAD is a very common skin disease with a multifactorial pathogenesis due to complex interactions between the host and the environment. The purpose of this study was to describe the physical and immunological effects of CsA in cAD using a canine model of AD. Fourteen beagles were enrolled; seven received CsA orally every 24 h for 28 days, and seven received placebo. All dogs were exposed to relevant allergens, house dust mite solution, one day prior to treatment and once weekly thereafter for 28 consecutive days. Canine atopic dermatitis extent and severity index-03 (CADESI-03) and skin biopsies were performed on day 0, 14, and 28. Quantitative RT-PCR was used to determine levels of cutaneous cytokines and barrier function markers. Indirect immunofluorescence was used to determine protein expression and distribution of nuclear messengers, barrier function and inflammatory [thymic stromal lymphopoietin (TSLP)] markers. The data were tested for normality and then the upaired two samples Student's t-test and the repeated measurements ANOVA, followed by the Dunnett's Multiple Comparison Test as post-hoc analysis, were performed. A P value of <0.05 was considered statistically significant. A significant decrease in CADESI-03 occurred for the treatment group compared to placebo (p = 0.023) on day 28. On day 14, a significant increase in TSLP protein expression [p = 0.019 (placebo); p = 0.02 (CsA)] and a significant decrease in Transforming Growth Factor (TGF)-ß mRNA [p = 0.01 (placebo); p = 0.015 (CsA)] were noted in both groups compared to baseline. On day 28, a significant increase in canine beta defensin (cBD)103 [p = 0.012 (placebo)] and cBD3-like mRNAs [p = 0.044 (placebo)], and filaggrin [p = 0.035 (CsA)] and TSLP protein expressions [p = 0.0092 (CsA)] were seen compared to baseline. In contrast, a significant decrease in mRNA of Tumor Necrosis factor (TNF)-α [p = 0.013 (CsA)], Interleukin (IL)-10 [p = 0.038 (CsA)], TGF-ß [p = 0.017 (CsA)], and caspase 14 [p = 0.014 (CsA)] was seen on day 28 compared to baseline. Comparison of the groups revealed no significant effect on skin immunologic milieu or barrier markers despite evident improvement of physical signs in the treatment group. Although this study confirmed the usefulness of CsA for the treatment of cAD, a clear involvement of CsA on some of the currently known immunological alterations present in cAD was not determined. However, it is important to note that there was no measurable exacerbation of skin barrier dysfunction secondary to CsA administration in this model.

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.

Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases.

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated in vivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNRHis) deiminated in vitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNRHis was shown to be cleaved by calpain-1 in vitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism.

Decreased expression of caspase-14 in an experimental model of canine atopic dermatitis.

Alterations in skin barrier function and filaggrin expression have been reported in atopic dermatitis (AD). Caspase-14, a protease important for filaggrin processing, is decreased in human AD. Atopic Beagle dogs with skin barrier alterations have been validated as model for AD. This study aimed to investigate caspase-14 in normal and atopic Beagle dogs. Skin biopsies from non-lesional and control skin were analyzed for caspase-14 by immunofluorescence. Six images/sections were blindly scored for intensity. Data were tested with unpaired Student's t test. A P value of <0.05 was considered significant. Caspase-14 was decreased in atopic compared to normal skin both quantitatively (P <0.001) and qualitatively (P = 0.006; agreement = 0.93; consistency = 0.94). In conclusion, caspase-14 is decreased in this model similarly to reports in humans, highlighting the relevance of filaggrin metabolic defects in AD.

T Helper 1 and T Helper 2 Cytokines Differentially Modulate Expression of Filaggrin and its Processing Proteases in Human Keratinocytes.

BACKGROUND: Atopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th1/Th2) cytokine expression. Filaggrin (FLG) is the key protein to maintaining skin barrier function. Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes. However, the role of Th1/Th2 cytokines on FLG processing is not substantially documented. Our aim was to investigate the impact of Th1/Th2 cytokines on FLG processing. METHODS: HaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ (IFN-γ). FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting. Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry. RESULTS: IL-4/13 significantly reduced, while IFN-γ significantly up-regulated FLG expression. IL-4/13 significantly increased, whereas IFN-γ significantly decreased the expression of kallikreins 5 and 7, matriptase and channel-activating serine protease 1. On the contrary, IL-4/13 significantly decreased, while IFN-γ increased the expression of LEKTI and caspase-14. Similar trends were observed in AD lesions. CONCLUSIONS: Our results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes. The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.