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.

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.

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.

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.

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.

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.

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.

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.

Dietary silk protein, sericin, improves epidermal hydration with increased levels of filaggrins and free amino acids in NC/Nga mice.

Epidermal hydration is maintained primarily by natural moisturising factors (NMF), of which free amino acids (AA) are major constituents that are generated by filaggrin degradation. To identify dietary sources that may improve skin dryness of atopic dermatitis (AD), we investigated dietary effects of silk proteins, sericin and fibroin, on epidermal levels of hydration, filaggrins and free AA, as well as PPARγ, peptidylarginine deiminase-3 (PAD3) and caspase-14 proteins involved in filaggrin expression and degradation processes. NC/Nga mice, an animal model of AD, were fed a control diet (group CA: atopic control) or diets with 1 % sericin (group S) or fibroin (group F) for 10 weeks. In group S, epidermal levels of hydration, total filaggrins and total free AA, as well as PPARγ, PAD3 and caspase-14, which were reduced in group CA, were increased to higher or similar levels of a normal control group of BALB/c mice (group C). Furthermore, profilaggrin, a precursor with multiple filaggrin repeats, and three repeat intermediates were increased, while two repeat intermediates and filaggrin were decreased in parallel with increased levels of glutamate and serine, major AA of NMF in group S. Despite increased levels of total filaggrins, total free AA, PPARγ and PAD3, epidermal levels of hydration, glutamate, serine and caspase-14 were not increased, but other minor AA of NMF were highly detected in group F. Dietary sericin improves epidermal hydration in parallel with enhancing profilaggrin expression and degradation into free AA that is coupled with elevated levels of PPARγ, PAD3 and caspase-14 proteins.

Function of caspase-14 in trophoblast differentiation.

BACKGROUND: Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. METHODS: Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. RESULTS: Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. CONCLUSION: Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

Glucocorticoid receptor is required for skin barrier competence.

To investigate the contribution of the glucocorticoid receptor (GR) in skin development and the mechanisms underlying this function, we have analyzed two mouse models in which GR has been functionally inactivated: the knockout GR(-/-) mice and the dimerization mutant GR(dim/dim) that mediates defective DNA binding-dependent transcription. Because GR null mice die perinatally, we evaluated skin architecture of late embryos by histological, immunohistochemical, and electron microscopy studies. Loss of function of GR resulted in incomplete epidermal stratification with dramatically abnormal differentiation of GR(-/-), but not GR(+/-) embryos, as demonstrated by the lack of loricrin, filaggrin, and involucrin markers. Skin sections of GR(-/-) embryos revealed edematous basal and lower spinous cells, and electron micrographs showed increased intercellular spaces between keratinocytes and reduced number of desmosomes. The absent terminal differentiation in GR(-/-) embryos correlated with an impaired activation of caspase-14, which is required for the processing of profilaggrin into filaggrin at late embryo stages. Accordingly, the skin barrier competence was severely compromised in GR(-/-) embryos. Cultured mouse primary keratinocytes from GR(-/-) mice formed colonies with cells of heterogeneous size and morphology that showed increased growth and apoptosis, indicating that GR regulates these processes in a cell-autonomous manner. The activity of ERK1/2 was constitutively augmented in GR(-/-) skin and mouse primary keratinocytes relative to wild type, which suggests that GR modulates skin homeostasis, at least partially, by antagonizing ERK function. Moreover, the epidermis of GR(+/dim) and GR(dim/dim) embryos appeared normal, thus suggesting that DNA-binding-independent actions of GR are sufficient to mediate epidermal and hair follicle development during embryogenesis.

Transcription of the caspase-14 gene in human epidermal keratinocytes requires AP-1 and NFkappaB.

Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.

Terminal differentiation of nail matrix keratinocytes involves up-regulation of DNase1L2 but is independent of caspase-14 expression.

Terminal differentiation of keratinocytes in the epidermis and in epidermal appendages results in specialized forms of cell death. Keratinocytes of the nail matrix differentiate into nail corneocytes, the building blocks of the nail plate. Here, we show that, in contrast to the abrupt breakdown of the nucleus during corneocyte formation of epidermal keratinocytes, chromatin undergoes progressive condensation over several nail matrix cell layers below the transition zone to the nail plate, where nuclear DNA disappears. Virtually all keratinocytes in the cell layer immediately beneath the nail plate contained terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling-positive DNA fragments. Nail matrix keratinocytes lacked processed caspase-3, a marker of apoptosis, and did not express caspase-14, a protease up-regulated during terminal differentiation of epidermal keratinocytes. By contrast, DNase1L2, which is also up-regulated during the differentiation of epidermal keratinocytes and plays an essential role in differentiation-associated degradation of nuclear DNA in epidermal keratinocytes, was strongly expressed in the nail matrix-nail plate transition layer. Our results show that caspase-14 is not strictly, if at all, required for differentiation-associated keratinocyte cell death and implicates DNase1L2 in terminal differentiation of nail matrix keratinocytes.

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.

Mutational analysis of caspase-14 gene in common carcinomas.

AIMS: Whereas most caspases play roles in apoptosis and/or inflammation, caspase-14 plays a main role in epithelial differentiation. In cancers, expression of caspase-14 is frequently altered and is associated with some clinicopathological characteristics, suggesting caspase-14 might contribute to the pathogenesis of cancers. As a potential mechanism of caspase-14 alterations in cancers, we explored the possibility that mutation of caspase-14 gene is a characteristic of human cancers. METHODS: We analysed the entire coding region and all splice sites of human caspase-14 gene for the detection of somatic mutations in a series of 345 cancers, including 105 colorectal, 60 gastric, 60 hepatocellular, 60 breast and 60 lung carcinomas, by a single-strand conformation polymorphism (SSCP) assay. RESULTS: Overall, we detected two somatic mutations of caspase-14 gene, which consisted of one missense mutation (S32Y) and one silent mutation (T234T). The caspase-14 mutations were detected in two of 105 colorectal carcinomas (1.9%), but not in other carcinomas. CONCLUSION: These data indicate that caspase-14 gene is rarely mutated in colorectal carcinomas, but not mutated in gastric, lung, breast and hepatocellular carcinomas. The data also suggest that the caspase-14 mutation may not be a direct target of inactivation in tumorigenesis of common carcinomas.

Expression of caspase-14 reduces tumorigenicity of skin cancer cells.

BACKGROUND: The green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) possesses anti-carcinogenic properties and was found to induce terminal differentiation in epidermal keratinocytes. Caspase-14, a member of the caspase family associated with epithelial cell differentiation, planned cell death, and barrier formation, is induced by EGCG in normal human epidermal keratinocytes but not in cancer cells. MATERIALS AND METHODS: A human epidermoid cancer cell line, A431, was co-transfected with a caspase-14-expressing pCMV vector and a GFP/neo-etpressingpCMVvector. Cell growth and tumorigenicity of the stable transfectant were determined in comparison to cells transfected with the control GFP/neo-expressing pCMV vector. RESULTS: Expression of exogenous caspase-14 led to growth inhibition and reduced the tumorigenicity of A431 cells. CONCLUSION: Pending future studies, caspase-14 could be used as a novel approach to skin cancer therapy via gene delivery systems.