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.

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.

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.

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.

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.

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.

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.

[Human caspase-14 expression in malignant melanoma and its significance].

OBJECTIVE: To detect the caspase-14 expression in malignant melanoma cells and tumor tissues and its effect on tumor resistance to drug. METHODS: The mRNA and protein level of caspase-14 in 4 melanoma cell lines (A375, A875, M14, and SK-Mel-1) and the melanocytes, was detected by reverse transcription PCR (RT-PCR) and Western blotting. Caspase-14 expression in 34 malignant melanoma tumor tissues and 10 dermal nevus tissues was determined by in situ hybridization and immunohistochemistry. RESULTS: Caspase-14 expression was seen in melanoma cells and melanocytes. It was higher in melanoma-associated antigen 1 recognized by T cells (MART-1) positive cells than in MART-1 negative cells. The cells expressing the lower caspase-14 were more sensitive to the treatment with either chemotherapy drugs camptothecin and cisplatin or radiotherapy than the ones expressing the higher caspase-14 (P<0.01). Caspase-14 expression was observed in 70% dermal nevus, as well as 97% in malignant melanoma tissues, and the difference between them was statistically significant (P<0.05). CONCLUSION: Caspase-14 is expressed in tissues and cells of malignant melanoma. Our data indicated that the expression level of caspase-14 affected the drug sensitivity of melanoma.

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 protocols.

Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or inflammation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte formation. In previous work, we have purified active caspase-14 from human corneocyte extracts. In addition, we have clarified activation mechanism of caspase-14, where kallikrein-related peptidase 7 (KLK7) generates an intermediate form from procaspase-14 and this form finally converts procaspase-14 to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using synthetic substrate, purification of caspase-14 from corneocyte extract, preparation of constitutively active caspase-14 and specific antibody, quantification of total and active caspase-14 in corneocyte extracts using ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

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.

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.

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 Ca(2+)-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.

A novel mechanism of filaggrin induction and sunburn prevention by ß-damascenone in Skh-1 mice.

Understanding how oral administration of aroma terpenes can prevent sunburn or skin cancer in mice could lead to more effective and safer ways of blocking sun damage to human skin. To establish sunburn preventive activity, female Skh-1 mice were given oral ß-damascenone followed by irradiation with UVR from fluorescent 'sunlamps'. The following endpoints were evaluated versus controls at various times between 1 and 12 days after the terpene: whole genome gene expression and in situ immunohistochemistry of PCNA, keratin 10, filaggrin and caspase 14, and sunburn was evaluated at 5 days. UVR-induced sunburn was prevented by a single oral ß-damascenone dose as low as 20 µL (0.95 mg/g body weight). Microarray analysis showed sunburn prevention doses of ß-damascenone up-regulated several types of cornification genes, including keratins 1 and 10, filaggrin, caspase 14, loricrin, hornerin and 6 late cornified envelope genes. Immunohistochemical studies of PCNA labeling showed that ß-damascenone increased the proliferation rates of the following cell types: epidermal basal cells, follicular outer root sheath cells and sebaceous gland cells. Keratin 10 was not affected by ß-damascenone in epidermis, and filaggrin and caspase 14 were increased in enlarged sebaceous glands. The thickness of the cornified envelope plus sebum layer nearly doubled within 1 day after administration of the ß-damascenone and remained at or above double thickness for at least 12 days. ß-Damascenone protected against sunburn by activating a sebaceous gland-based pathway that fortified and thickened the cornified envelope plus sebum layer in a way that previously has been observed to occur only in keratinocytes.

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.

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.