The epithelial zinc transporter ZIP10 epigenetically regulates human epidermal homeostasis by modulating histone acetyltransferase activity.

BACKGROUND: The skin is the first organ that manifests changes in response to zinc deficiency. However, the molecular mechanism underlying how zinc is involved in skin homeostasis, especially its epigenetic regulation, is largely unknown. OBJECTIVES: In this study we demonstrate the importance of zinc levels and the zinc transporter ZIP10 in the epigenetic maintenance of human epidermal homeostasis. METHODS: Adult human skin, including skin appendages, were stained with anti-ZIP10 antibody. Histone acetyltransferase (HAT) activity was assessed after treating human keratinocytes with ZIP10 small interfering (si)RNAs or the zinc chelator TPEN. ZIP10- or HAT-regulated genes were analysed based on limma bioinformatics analysis for keratinocytes treated with ZIP10 siRNAs or a HAT inhibitor, or using a public database for transcription factors. A reconstituted human skin model was used to validate the role of ZIP10 in epidermal differentiation and the functional association between ZIP10 and HAT. RESULTS: ZIP10 is predominantly expressed in the interfollicular epidermis, epidermal appendages and hair follicles. ZIP10 depletion resulted in epidermal malformations in a reconstituted human skin model via downregulation of the activity of the epigenetic enzyme HAT. This decreased HAT activity, resulting from either ZIP10 depletion or treatment with the zinc chelator TPEN, was readily restored by zinc supplementation. Through bioinformatics analysis for gene sets regulated by knockdown of SLC39A10 (encoding ZIP10) and HAT inhibition, we demonstrated that ZIP10 and HATs were closely linked with the regulation of genes related to epidermal homeostasis, particularly filaggrin and metallothionein. CONCLUSIONS: Our study suggests that ZIP10-mediated zinc distribution is crucial for epidermal homeostasis via HATs. Therefore, zinc-dependent epigenetic regulation could provide alternatives to maintaining healthy skin or alleviating disorders with skin barrier defects.

Interferon-inducible T-cell alpha chemoattractant (ITAC) induces the melanocytic migration and hypopigmentation through destabilizing p53 via histone deacetylase 5: a possible role of ITAC in pigment-related disorders.

BACKGROUND: Cell migration plays a major role in the immune response and in tumorigenesis. Interferon-inducible T-cell alpha chemoattractant (ITAC) elicits a strong chemotactic response from immune cells. OBJECTIVES: To examine the effect of ITAC on melanocyte migration and pigmentation and its involvement in related disorders, and to investigate potential key players in these processes. METHODS: Human melanocytes or melanoma cells were treated with ITAC and a migration assay was carried out. Global gene expression analysis was performed to find genes regulated by ITAC treatment. The function of key players involved in ITAC-induced cellular processes was addressed using knockdown or overexpression experiments in combination with ITAC treatment. ITAC expression in the inflammation-associated hypopigmentary disorder, vitiligo, was examined. RESULTS: Among CXCR3 ligands, only ITAC induced melanocyte migration. ITAC treatment upregulated the expression of histone deacetylase 5 (HDAC5) and downregulated that of p53, a known target of HDAC5. Through knockdown or overexpression of HDAC5 and p53, we confirmed that HDAC5 mediates ITAC-induced migration by decreasing levels of p53 via deacetylation. In addition, ITAC treatment could decrease pigmentation in a p53- and HDAC5-dependent manner. Finally, the increased migration of human melanoma cells by ITAC treatment and the increased ITAC expression in the epidermis of vitiligo skin were verified. CONCLUSIONS: This study provides in vitro evidence for the migratory and hypopigmentation effects of ITAC on melanocytic cells, gives translational insights into the roles of ITAC in pathological conditions, and suggests that HDAC5 and its substrate p53 are potent targets for regulating ITAC-induced cellular processes.

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.

Development of alternative loading solutions in droplet-vitrification procedures.

In plant vitrification protocols, the loading treatment, which involves treating the explants with a moderately concentrated cryoprotectant solution, precedes dehydration of explants with highly concentrated vitrification solutions in order to reduce the toxicity which can be induced by their direct exposure to such highly concentrated solutions. This study aimed at developing alternative loading solutions composed of mixtures of glycerol and sucrose at various concentrations. Differential scanning calorimetry runs of loading solutions and of loaded and dehydrated explants were performed to assay thermal events occurring during cooling and warming. These loading solutions were applied to two model species, viz. garlic and chrysanthemum which were cryopreserved using a droplet-vitrification procedure. The loading treatment proved to be beneficial to both garlic and chrysanthemum and increased recovery of cryopreserved explants. However, response to the loading solutions tested varied between the two model species employed: with garlic, all the loading solutions had a similar effect, whereas survival of chrysanthemum shoot tips was significantly influenced by the composition of the loading solution employed. A loading solution comprising 1.9 M glycerol and 0.5 M sucrose was the most effective. The loading treatment may thus act as an osmotic stress neutralizer and/or induce the physiological adaptation of tissues and cells, including membranes, to both dehydration and freezing.

Cryopreservation of potato cultivated varieties and wild species: critical factors in droplet vitrification.

The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40 degrees C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.

Identification and genetic mapping of the mouse Fkbp9 gene encoding a new member of FK506-binding protein family.

We have isolated a gene from a cDNA library generated from the thymus of a mouse with severe combined immune deficiency, termed FKBP9, that encodes a protein related to FK506-binding protein 6 (65 kDa, FKBP65). FKBP9 contains four peptidyl-prolyl cis-trans isomerase (PPIase) signature and two EF-hand domains which is identical to FKBP6/65 in overall structural organization. However, the two proteins share only 66% amino acid identity. FKBP9 is expressed at high levels in mouse heart, muscle, lung, and kidney. While FKBP6 was previously mapped to chromosome 11, the Fkbp9 gene was mapped to mouse chromosome 6 by analysis of a multilocus cross. These results identify a new member of the mouse FKBP protein family located on a separate chromosome.

N-terminal processing is essential for release of epithin, a mouse type II membrane serine protease.

Epithin was originally identified as a mouse type II membrane serine protease. Its human orthologue membrane type-serine protease 1 (MT-SP1)/matriptase has been reported to be localized on the plasma membrane. In addition, soluble forms of matriptase were isolated from human breast milk and breast cancer cell-conditioned medium. In this paper, we report a processing mechanism that appears to be required for the release of epithin. CHO-K1 or COS7 cells transfected with single full-length epithin cDNA generated two different-sized proteins in cell lysates, 110 and 92 kDa. The 92-kDa epithin was found to be an N-terminally truncated form of the 110-kDa epithin, and it was the only form detected in the culture medium. The 92-kDa epithin was also found on the cell surface, where it was anchored by the N-terminal fragment. The results of in vivo cell labeling experiments indicate that the 110-kDa epithin is rapidly processed to the 92-kDa epithin. Using site-directed mutagenesis experiments, we identified Gly(149) of the GSVIA sequence in epithin as required for the processing and release of the protein. These results suggest that N-terminal processing of epithin at Gly(149) is a necessary prerequisite step for release of the protein.

Cryopreservation of Citrus madurensis zygotic embryonic axes by vitrification: importance of pregrowth and preculture conditions.

The role of pregrowth and preculture treatments in terms of both medium composition and exposure duration on survival of embryonic axes of Citrus madurensis after cryopreservation using the vitrification procedure was investigated. The optimal pregrowth treatment for excised embryonic axes was a 3-day treatment with 0.1M sucrose. Preculture was also essential in increasing survival after cryopreservation. Among the various media and treatment durations evaluated, a 24h-preculture of embryonic axes on medium with 0.3M sucrose and 0.5M glycerol was found to be optimal. Using these pregrowth and preculture conditions followed by treatment at 25 degrees C for 20 min each with a loading solution (0.4M sucrose + 2.0M glycerol) and then the PVS2 vitrification solution, direct immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2M sucrose solution for 20 min and transfer of embryonic axes on recovery medium, 82.5% survival and regrowth without intermediary callus formation were obtained with C. madurensis embryonic axes.

Cloning and chromosomal mapping of a gene isolated from thymic stromal cells encoding a new mouse type II membrane serine protease, epithin, containing four LDL receptor modules and two CUB domains.

We cloned and sequenced a mouse gene encoding a new type of membrane bound serine protease (epithin) containing a multidomain structure. The initial cDNA clone was found previously in a polymerase chain reaction (PCR)-based subtractive library generated from fetal thymic stromal cells, and the message was shown to be highly expressed in a thymic epithelial nurse cell line. A clone isolated from a severe combined immunodeficiency (SCID) thymus library and extended to its full length at the 5' end with the RACE technique contains an open reading frame of 902 amino acids. Based on the sequence of this clone, the predicted protein structure is a type II membrane protein with a C-terminal serine protease domain linked to the membrane by four low density lipoprotein receptor modules and two CUB domains. High message expression by northern blotting was detected in intestine, kidney, lung, SCID, and Rag-2(-/-) thymus, and 2-deoxyguanosine-treated fetal thymic rudiment, but not in skeletal muscle, liver, heart, testis, and brain. Sorted MHC class II+ and II- fetal thymic stromal cells were positive for expression by reverse transcriptase-PCR, whereas CD45(+) thymocytes were not. The gene was found in chicken and multiple mammalian species under low stringency Southern hybridization conditions. Under high stringency conditions, only a single gene per haploid genome was identified in the mouse. This gene, Prss14 (protease, serine, 14), was mapped to mouse chromosome 9 and is closely linked to the Fli1 (Friend leukemia integration 1) gene.