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Cutaneous permeability barrier function in signal transducer and activator of transcription 6-deficient mice is superior to that in wild-type mice
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Cutaneous permeability barrier function in signal transducer and activator of transcription 6-deficient mice is superior to that in wild-type mice Wei Zhang, Takashi Sakai, Haruna Matsuda-Hirose, Mizuki Goto, Tomoko Yamate, Yutaka Hatano* Department of Dermatology, Faculty of Medicine, Oita University, Oita, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2018 Received in revised form 18 June 2018 Accepted 31 July 2018
Background: Th2 cytokines exhibit a variety of inhibitory effects on permeability barrier function via signal transducer and activator of transcription 6 (STAT6). However, the role of STAT6 signaling on the construction and/or homeostasis of permeability barrier function in the physiological state has not been fully assessed. Objective: We compared permeability barrier function between Stat6-deficient and wild-type C57BL/6 mice at steady state. Methods and results: Measurement of transepidermal water loss and quantitative penetration assay revealed that permeability barrier function was superior in Stat6-deficient mice. Accordingly, expressions of loricrin, acidic sphingomyelinase (aSMase) and β-glucocerebrosidase (β-GlcCer’ase) in epidermis and ceramide levels in stratum corneum were elevated in STAT6-deficient mice. On the other hands, up-regulations of loricrin, aSMase and β-GlcCer’ase were not observed in 3-dimensionally cultured human keratinocytes transfected with siRNA for STAT6. Meanwhile, number of mast cells in the dermis was decreased in Stat6-deficient mice. Conclusions: These results suggest that STAT6 signaling negatively affects permeability barrier function in vivo, even in the physiological state. However, the superior permeability barrier function in Stat6deficient mice may be a secondary effect exerted via cells other than keratinocytes, such as mast cells, since mast cells are known to influence permeability barrier function in vivo. Blockade of STAT6 signaling might be a strategy to augment the permeability barrier function. © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: Permeability barrier STAT6 Steady states
1. Introduction Numerous negative effects of Th2 cytokines, IL-4 and IL-13 on cutaneous permeability barrier functions have been reported. Production of ceramide, an integral part of the extracellular lipid bilayer of the stratum corneum (SC) that forms the permeability barrier in the skin [1], and expressions of epidermal differentiation-related molecules such as filaggrin (FLG), loricrin (LOR) and
Abbreviations: aCEase, acid-ceramidase; β-GlcCer’ase, β-glucocerebrosidase; EDTA, ethylenediaminetetraacetic acid; FLG, filaggrin; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IL, interleukin; IVL, involucrin; LOR, loricrin; mRNA, messenger RNA; NHKs, normal human keratinocytes; PCR, polymerase chain reaction; SC, stratum corneum; siRNA, small interfering RNA; SMase, sphingomyelinase; STAT, signal transducer and activator of transcription; 3D, 3dimensionally; TEWL, transepidermal water loss; Th, T helper. * Corresponding author at: Department of Dermatology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan. E-mail address: [email protected] (Y. Hatano).
involucrin (IVL), are negatively regulated by Th2 cytokines [2–6]. Those deleterious effects of Th2 cytokines on the permeability barrier are thought to be involved in the vicious cycle of allergic inflammation and cutaneous permeability barrier dysfunction in the pathogenesis of atopic dermatitis [7,8]. However, the role of Th2 cytokines in the homeostasis and/or construction of the cutaneous permeability barrier in the steady state has not been fully assessed, although Sehra et al. demonstrated that SC integrity was increased with elevated expressions of IVL and FLG but not LOR in IL-4-deficient mice [9]. Functions of IL-4 and IL-13 are well known to be mediated by a signal transduction molecule, signal transducer and activator of transcription 6 (STAT6) [10]. The possibility of alternative pathways of STAT6 activation have also been observed [10]. As for the regulation of expressions of epidermal differentiationrelated molecules, loricrin, involucrin, and filaggrin expression have been demonstrated to be regulated by Th2 cytokines through STAT-6 [6,11].
https://doi.org/10.1016/j.jdermsci.2018.07.008 0923-1811/ © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: W. Zhang, et al., Cutaneous permeability barrier function in signal transducer and activator of transcription 6deficient mice is superior to that in wild-type mice, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.07.008
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The present study examined the cutaneous permeability barrier function of Stat6-deficient mice to assess the role of STAT6 in cutaneous permeability barrier homeostasis in the steady state.
performed using electron microscopic images taken by an investigator blinded to the source of the specimen. 2.6. Counts of mast cells in murine dermis
2. Materials and methods 2.1. Animals Female C57BL/6 mice (Japan SLC, Hamamatsu, Japan) were purchased for use as wild-type mice. Female Stat6-knockout mice were donated by Prof. Yokozeki (Tokyo Medical and Dental University, Tokyo, Japan). Background strain of Stat6-knockout used here was C57BL/6 mice. The mice were used at 9–10 weeks old. All animals were housed under conventional conditions and had ad libitum access to commercial diet and water. All experiments with mice were approved by the Ethics of Animal Experimentation Committee at Oita University. 2.2. Physiological assessments TEWL, SC hydration and SC surface pH on flanks were measured using a Tewameter (TM300; Courage & Khazaka, Cologne, Germany), Corneometer (CM825; Courage & Khazaka) and skin pH meter (PH 905; Courage & Khazaka), as described previously [12], 2 days after hair shaving. 2.3. Skin sample preparation Skin samples were collected 3 days after hair shaving. Whole skin samples for penetration assay, hematoxylin and eosin stain, Giemsa stain, and electromicroscopic analysis were collected from flanks. Epidermal sheet for real-time PCR and Western blotting was collected from each flank by 1-h incubation at 37 C with 1000 IU/ ml of dispase (Godo Shusei, Tokyo, Japan). SC for lipid analysis was collected from 2 cm2 of each abdomen by incubation for 2.5 h with 0.5% trypsin (Difco Laboratories, Detroit, MI) at 37 C. 2.4. Penetration assay Quantitative evaluation of outside-to-inside penetration of the skin was assessed with Evans blue dye, as described previously [13]. Each sample was floated on MCDB 153 medium (SigmaAldrich, St. Louis, MO) containing 1.8 mM of CaCl2 with the outer epidermal surface of each sample exposed to the air. Next, 50 ml of 2% Evans blue in phosphate-buffered saline was pipetted onto the outer epidermal surface of each skin explant. The dye was allowed to penetrate the skin for 2 h at room temperature, then the surface of the skin was washed with phosphate-buffered saline and gently wiped with a Kimwipe (Nippon Paper Crecia, Tokyo, Japan). After washing procedures had been repeated three times, the center of each explant was biopsied with a 4-mm punch and each 4-mm disk was placed into 100 ml of 1 N KOH. After overnight incubation at 37 C, each sample was neutralized by the addition of 900 ml of a mixture of 0.6 N H3PO4 and acetone (5:13, v/v). After vigorous vortexing for a few seconds, the mixture was centrifuged at 3000 rpm for 15 min in an RA-150AM centrifuge (Kubota, Tokyo, Japan), and absorbance of supernatants was measured at 360 nm. 2.5. Quantitative morphology under electron microscopy Skin biopsies from mice were fixed in Karnovsky’s fixative, and post-fixed with either 1% aqueous osmium tetroxide, containing 1.5% potassium ferrocyanide. Ultrathin sections were examined using an electron microscope (JEM-1200EX II; JEOL, Tokyo, Japan) operated at 80 kV. Measurement of the number of SC layers was
Mast cells in Giemsa-stained murine dermis were counted under high-power magnification. Counts were performed in 9–16 areas in each skin sample. The average number in each sample was analyzed. 2.7. Culture of keratinocytes transfected with siRNA at the air-liquid interface Culture of normal human keratinocytes (NHKs) transfected with siRNA was performed as previously reported [14]. Third- or fourth-passage NHKs (Cell Applications, San Diego, CA) were cultured in Keratinocyte Basal Medium 2 with Supplement Pack Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) at 37 C in a humidified atmosphere of 5% CO2 in air. Stealth siRNA transfection was performed once cells reached 70–90% confluence. The Stealth siRNA against human Stat6 (HSS110291, Thermo Fisher Scientific, Lafayette, CO) and Stealth RNAi Negative Control (Thermo Fisher Scientific) were used. Cells were transfected with 100 nM siRNAs in Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and OPTI-MEM (Invitrogen), according to the instructions from the manufacturer. Transfected cells were harvested with ethylenediaminetetraacetic acid (EDTA)-trypsin (DS Pharma Biomedical, Osaka, Japan), then seeded into Cell Culture Inserts (pore size, 0.4 mm; BD Biosciences, Durham, NC) with Keratinocyte Basal Medium 2, which were placed into a Companion Plate (BD Biosciences) containing the same medium 24 h after the transfection. The medium in Cell Culture Inserts was aspirated 24 h later, and the medium in the Companion Plate was changed to assay medium (EPI-MODEL; Japan Tissue Engineering, Aichi, Japan). Keratinocytes transfected with siRNA were grown at the air-liquid interface. The medium in the plate was changed every three day, and keratinocytes were cultured for 7 days. Then, stratified keratinocytes and conditioned medium were harvested for realtime PCR or Western blotting. 2.8. Real-time PCR Total RNA was isolated from epidermal sheets and cultured NHKs using an RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany), and reverse transcription was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the instructions from the manufacturer. Complementary DNA products were amplified in a LightCycler 480 System (Roche Diagnostics GmbH), as described previously [14]. The primers used for real-time PCR are shown in Supplementary Table S1. Product specificity was evaluated by melting curve analysis, and relative gene expression was calculated from a standard curve included in each run. Relative mRNA expression levels were normalized with the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or mouse β-actin. 2.9. Western blotting Western blotting was performed as previously described [12,14]. Each epidermal sheet and cultured NHKs were homogenized in Pierce RIPA buffer (Thermo Scientific, Rockford, lL) with Halt Protease Inhibitor Cocktail (Thermo Scientific). These samples were separated by 10% SDS-PAGE before transfer to Immobilon-P TM Transfer Membrane (Millipore, Billerica, MA) or Hybond -P (GE Healthcare, Buckinghamshire, UK). We used antibodies against FLG
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(COVANCE, Emeryville, CA), IVL (COVANCE), LOR (COVANCE), STAT6 (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Cell Signaling Technology, Danvers, MA) as primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) as secondary antibodies, then visualized using ECLTM Western Blotting Detection Reagents (GE Healthcare).
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Amounts of ceramide were normalized by reference to the area of the sample skin (i.e., 2 cm2). 2.11. Statistical analysis All experiments were analyzed using two-tailed Student’s ttest. All results are presented as mean standard error of the mean. Values of P < 0.05 were considered statistically significant.
2.10. Analysis of SC ceramides 3. Results SC lipids were extracted by Bligh-Dyer solvents and the extracted fraction was suspended in a mixture (1:1, vol/vol) of chloroform and methanol after evaporation under a stream of nitrogen gas. This suspension was applied to a thin-layer chromatography plate, as described previously [15]. The plate was developed with a mixture (190:9:1, vol/vol) of chloroform, methanol, and acetic acid. After solvent development, the chromatogram was air-dried, soaked in an aqueous solution of 10% CuSO3 and 8% H3PO4, and charred at 180 C. Charred lipids were quantitated by Image J (National Institutes of Health, Bethesda, ML). Ceramides were quantitated by reference to appropriate commercial standards as follows: non-hydroxy fatty acid ceramide from bovine brain (Sigma-Aldrich) and ceramides (hydroxy) (Matreya, State College, PA) were used as standards for ceramides 1 and 2 and for ceramides 3, 4, 5, and 6, respectively.
3.1. Lack of morphological difference between Stat6-deficient and wild-type mice Hematoxylin and eosin staining revealed no significant difference in skin between Stat6-deficient mice and wild-type mice (Fig. S1a). Electron microscopy also revealed no significant difference in thickness of the SC (Fig. S1b, S1c). 3.2. Cutaneous permeability barrier in Stat6-deficient mice was superior to that in wild-type mice Values of transepidermal water loss (TEWL) and SC pH were lower in Stat6-deficient mice than in wild-type mice (Fig. 1a and c). Values of SC hydration in Stat6-deficient mice were higher than
Fig. 1. Cutaneous permeability barrier function in Stat6-deficient mice is superior to that in wild-type mice. The results show cutaneous permeability function in Stat6-deficient mice (STAT6-/-) is superior to that in wild-type mice (WT). Measurement of transepidermal water loss (TEWL) (a; n = 6), SC hydration (b; n = 6), and SC pH (c; n = 6), and a penetration assay using Evans blue (d; n = 11 in WT, n = 12 in STAT6-/-) were performed as described in the Materials and Methods section. Error bars indicate mean SEM. P-value vs. WT.
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those in wild-type mice (Fig. 1b). Penetration assay revealed that penetration of Evans blue was suppressed in Stat6-deficient mice, compared to wild-type mice (Fig. 1d). 3.3. Expression of loricrin was up-regulated in Stat6-deficient mice Real-time polymerase chain reaction (PCR) revealed that expression of LOR in epidermis was up-regulated in Stat6-deficient mice, compared to that in wild-type mice (Fig. 2a). Western blotting supported up-regulation of LOR in Stat6-deficient mice (Fig. 2d). Expression of IVL in Stat6-deficient mice was broadly equivalent to that in wild-type mice (Fig. 2b). Expression of FLG tended to be up-regulated in Stat6-deficient mice, but this upregulation was not statistically significant (Fig. 2c). Western blotting detected no significant difference in the expressions of IVL and FLG between Stat6-deficient and wild-type mice (data not shown). 3.4. Ceramide levels in SC were larger in Stat6-deficient mice than in wild-type mice SC ceramides 2 and 5 are produced by the hydrolysis of sphingomyelin by sphingomyelinase (SMase), particularly acidSMase (aSMase) [16,17], while other groups of SC ceramides are
produced by the degradation of glucosylceramide by βglucocerebrosidase (β-GlcCer’ase) [18]. Ceramide hydrolysis is catalyzed by acid-ceramidase (aCEase) in the SC under physiological conditions [19–21]. Real-time PCR revealed that expressions of aSMase and β-GlcCer’ase in epidermis were upregulated in Stat6-deficient mice, compared to those in wildtype mice (Fig. 3a and b), while expression of aCEase in Stat6deficient mice was comparable to that in wild-type mice (Fig. 3c). Thin-layer chromatography revealed that amounts of ceramide 1, ceramide 2, ceramide 3, ceramide 4–6, and total ceramides in SC were higher in Stat6-deficient mice than in wild-type mice (Fig. 4). 3.5. Effects of Stat6 knockdown in 3-dimensionally (3D) cultured normal human keratinocytes were not identical to those in STAT6deficient mice Real-time PCR and Western blotting revealed that transfection of small interfering RNA (siRNA) for Stat6 successfully knocked down expression of Stat6 in 3D-cultured normal human keratinocytes (NHKs; Fig. S2). Real-time PCR revealed that expressions of LOR, IVL, FLG, aSMase, and aCEase were not affected in 3D-cultured NHKs, while expressions of β-GlcCer’ase were reduced by knockdown of Stat6 (Fig. 5).
Fig. 2. Expression of loricrin is elevated in Stat6-deficient mice, compared with that in wild-type mice. The results show expressions of loricrin (LOR; a), but not involucrin (IVL; b) or filaggrin (FLG; c) in epidermis were elevated in Stat6-deficient mice (STAT6-/-), compared to levels in wild-type mice (WT) for either mRNA (a–c; n = 6 in WT and n = 6 in STAT6-/-) or protein (d). Sample preparation, real-time RT-PCR and Western blotting were performed as described in the Materials and Methods section. Error bars indicate mean SEM. P-value vs. WT. Values of P < 0.05 are shown.
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Fig. 3. Expressions of acid-sphingomyelinase and β-glucocerebrosidase, but not acid-ceramidase are elevated in Stat6-deficient mice, compared with those in wild-type mice. The results show expressions of acid-sphingomyelinase (aSMase; a) and β-glucocerebrosidase (β-GlcCer’ase; b), but not acid-ceramidase (aCEase; c) in epidermis are elevated in Stat6-deficient mice (STAT6-/-), compared with those in wild-type mice (WT). Sample preparation and real-time RT-PCR were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
Fig. 4. Ceramides in SC are elevated in Stat6-deficient mice, compared with those in wild-type mice. (a) Thin-layer chromatography (TLC). Quantitative analysis reveals that levels of ceramide 1 (b), ceramide 2 (c), ceramide 3 (d), ceramide 4–6 (e) and total ceramides (f) in SC are elevated in Stat6-deficient mice (STAT6-/-), compared with those in wild-type mice (WT). Sample preparation and TLC were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
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Fig. 5. Effects of knockdown of Stat6 in 3D-cultured human keratinocytes are inconsistent with those in STAT6-knockout mice. The results show expressions of loricrin (LOR; a), involucrin (IVL; b), filaggrin (FLG; c), acid-sphingomyelinase (aSMase; d) and acid-ceramidase (aCEase; f) in 3D-cultured human keratinocytes are not influenced and expression of β-glucocerebrosidase (β-GlcCer’ase; e) is down-regulated following transfection of siRNA against Stat6 (STAT6 siRNA). Three-dimensional culturing, sample preparation and real-time RT-PCR were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 8 in control siRNA, n = 8 in STAT6 siRNA). P-value vs. control siRNA. Values of P < 0.05 are shown.
Fig. 6. The number of mast cells in dermis is reduced in Stat6-deficient mice. The results show the number (b) of mast cells identified by Giemsa stain (a) is reduced in Stat6-deficient mice (STAT6-/-), compared with wild-type mice (WT). Sample preparation and Giemsa stain were performed as described in the Materials and Methods section. Scale bar = 100 mm in (a). Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
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3.6. Numbers of mast cells in dermis but not expressions of histamine receptors in epidermis were reduced in Stat6-deficient mice Mast cells and products of mast cells, such as histamine, reportedly modulate the permeability barrier [22,23]. We therefore counted numbers of mast cells in dermis. Numbers of mast cells in dermis were reduced in Stat6-deficient mice, compared to numbers in wild-type mice (Fig. 6). On the other hands, real-time PCR revealed that expressions of histamine H1, H2, H3, and H4 receptors in epidermis in Stat6-deficient mice were identical to those in wild-type mice (Fig. S3). 4. Discussion Although no morphological abnormalities were apparent in the skin, values of TEWL in Stat6-deficient mice were decreased, suggesting that the permeability barrier function in Stat6-deficient mice is superior to that in wild-type mice. Penetration assay strongly supported the augmented permeability barrier function in Stat6-deficient mice. Elevated SC hydration and the lack of change in SC thickness suggest that the augmented permeability barrier function in Stat6-deficient mice is primarily due to the upregulated SC function, rather than to compensatory accumulation of SC. In line with accumulated knowledge regarding the importance of SC acidity in permeability barrier homeostasis/ construction [7], the decrease in SC pH in Stat6-deficient mice not only supports the augmentation of SC permeability barrier function, but also might represent one of the underlying mechanisms. LOR is the most abundant protein in the cornified envelope [24]. In addition, it has been reported that permeability barrier formation, although transiently, delays in LOR-deficient mice [25]. Therefore, up-regulation of LOR expression might be one of the bases of the augmented permeability barrier function in Stat6deficient mice, although there has not been clear evidences showing that increase in LOR could augment permeability barrier. Meanwhile, expressions of IVL and FLG were unaffected in Stat6deficient mice, although expressions of IVL and FLG (but not LOR) were demonstrated to be elevated in IL-4-deficient mice [9]. These discrepancies in the regulation of LOR, IVL, and FLG suggest that the role of STAT6 in keratinocyte differentiation is not necessarily identical to that of IL-4 in vivo. The reasons for the difference between Stat6-deficient and IL-4 deficient mice remain unclear, although STAT6 signaling independent of IL-4 and IL-13 receptors and/or some specific functions of IL-13 might be involved in this discrepancy, particularly in vivo. Elevated amounts of ceramides in SC and mRNA expressions of aSMase and β-GlcCer’ase (but not aCEase) in epidermis might be involved in the up-regulation of cutaneous permeability barrier in Stat6-deficient mice, and again support the importance of SC ceramides in permeability barrier homeostasis/construction. These results seem consistent with the findings of previously reported studies demonstrating the negative effects of Th2 cytokines on the production of ceramide in keratinocytes, which were accompanied by down-regulation of mRNA expressions of aSMase and β-GlcCer’ase, while no changes were seen in aCEase expression [2,4]. However, whether Th2 cytokines could be produced and secreted and what kinds of cells could be sources of Th2 cytokines in the steady state remain unclear. Effects of Stat6 knockdown in cultured keratinocytes were not identical to the observations in STAT6-deficient mice. No effects of Stat6 knockdown in expressions of LOR, IVL, and FLG in cultured keratinocytes were consistent with those reported by Amano et al. [26]. These discrepancies between the effects of Stat6 knockdown in cultured keratinocytes and observations in Stat6-deficient mice might suggest that down-regulations of LOR, aSMase and β-
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GlcCer’ase in the epidermis of wild-type mice, compared to those of Stat6-deficient mice, might represent indirect effects on keratinocytes through STAT6 signaling in cells other than keratinocytes. For example, mast cells are one candidate for contributing to the down-regulation of permeability barrier function in wild-type mice, not only because mast cell maturation has been reported to be reduced in Stat6-deficient mice in an allergic inflammatory model [27], but also because products from mast cells, such as histamine, affect permeability barrier functions with reducing the expression of the differentiation-associated proteins, such as keratin 1/10, filaggrin, and loricrin [22]. In the present study, numbers of mast cells in dermis but not expressions of histamine receptors in epidermis were reduced in Stat6deficient mice, compared to those in wild-type mice. Decreased numbers of dermal mast cells might thus be involved in the augmented permeability barrier function in Stat6-deficient mice identified in the present study, although which factors from mast cells affect the barrier function and how functions of mast cells are affected by STAT6 depletion remain unclear. Meanwhile, the decrease in numbers of dermal mast cells in the present study supports that STAT6 contributes to the proliferation and growth of mast cells [28]. On the other hand, mast cell-deficient mice were recently reported to exhibit a dysfunction of permeability barrier accompanied by decreased expressions of FLG, LOR, and IVL [23], suggesting that decreased numbers of dermal mast cells are not necessarily involved in the augmented permeability barrier function seen in Stat6-deficient mice. Meanwhile, differences in study species (i.e., human or mouse) and/or in degree of modulation of the STAT6 gene (i.e., knockdown or knockout) might have contributed to the discrepancies between effects of Stat6 knockdown in cultured keratinocytes and the findings in Stat6-deficient mice. In conclusion, the present study suggests that STAT6 signaling negatively affects cutaneous permeability barrier homeostasis in vivo and inhibition of this signaling might be useful to augment permeability barrierfunction even in the steady state. However, the mechanisms involved in these findings remain unclear. Funding Trust Accounts in Oita University. This study was partly supported by grants from Japan Society for the Promotion of Science (no.26461662 and 17K10214). Conflict of interest All authors have no conflict of interest. Acknowledgements We wish to express our thanks to Mr. Hiroaki Kawazato and Ms. Aiko Yasuda (Research Promotion Project, Faculty of Medicine, Oita University, Oita, Japan) for their excellent work on ultrastructural analyses. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j. jdermsci.2018.07.008. References [1] P.M. Elias, K.R. Feingold, Lipids and the epidermal water barrier: metabolism, regulation, and pathophysiology, Semin. Dermatol. 11 (1992) 176–182. [2] Y. Hatano, H. Terashi, S. Arakawa, K. Katagiri, Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier
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Journal of Dermatological Science xxx (2018) xxx–xxx
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Cutaneous permeability barrier function in signal transducer and activator of transcription 6-deficient mice is superior to that in wild-type mice Wei Zhang, Takashi Sakai, Haruna Matsuda-Hirose, Mizuki Goto, Tomoko Yamate, Yutaka Hatano* Department of Dermatology, Faculty of Medicine, Oita University, Oita, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 February 2018 Received in revised form 18 June 2018 Accepted 31 July 2018
Background: Th2 cytokines exhibit a variety of inhibitory effects on permeability barrier function via signal transducer and activator of transcription 6 (STAT6). However, the role of STAT6 signaling on the construction and/or homeostasis of permeability barrier function in the physiological state has not been fully assessed. Objective: We compared permeability barrier function between Stat6-deficient and wild-type C57BL/6 mice at steady state. Methods and results: Measurement of transepidermal water loss and quantitative penetration assay revealed that permeability barrier function was superior in Stat6-deficient mice. Accordingly, expressions of loricrin, acidic sphingomyelinase (aSMase) and β-glucocerebrosidase (β-GlcCer’ase) in epidermis and ceramide levels in stratum corneum were elevated in STAT6-deficient mice. On the other hands, up-regulations of loricrin, aSMase and β-GlcCer’ase were not observed in 3-dimensionally cultured human keratinocytes transfected with siRNA for STAT6. Meanwhile, number of mast cells in the dermis was decreased in Stat6-deficient mice. Conclusions: These results suggest that STAT6 signaling negatively affects permeability barrier function in vivo, even in the physiological state. However, the superior permeability barrier function in Stat6deficient mice may be a secondary effect exerted via cells other than keratinocytes, such as mast cells, since mast cells are known to influence permeability barrier function in vivo. Blockade of STAT6 signaling might be a strategy to augment the permeability barrier function. © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
Keywords: Permeability barrier STAT6 Steady states
1. Introduction Numerous negative effects of Th2 cytokines, IL-4 and IL-13 on cutaneous permeability barrier functions have been reported. Production of ceramide, an integral part of the extracellular lipid bilayer of the stratum corneum (SC) that forms the permeability barrier in the skin [1], and expressions of epidermal differentiation-related molecules such as filaggrin (FLG), loricrin (LOR) and
Abbreviations: aCEase, acid-ceramidase; β-GlcCer’ase, β-glucocerebrosidase; EDTA, ethylenediaminetetraacetic acid; FLG, filaggrin; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IL, interleukin; IVL, involucrin; LOR, loricrin; mRNA, messenger RNA; NHKs, normal human keratinocytes; PCR, polymerase chain reaction; SC, stratum corneum; siRNA, small interfering RNA; SMase, sphingomyelinase; STAT, signal transducer and activator of transcription; 3D, 3dimensionally; TEWL, transepidermal water loss; Th, T helper. * Corresponding author at: Department of Dermatology, Faculty of Medicine, Oita University, 1-1 Idaigaoka, Hasama-machi, Yufu, Oita, 879-5593, Japan. E-mail address: [email protected] (Y. Hatano).
involucrin (IVL), are negatively regulated by Th2 cytokines [2–6]. Those deleterious effects of Th2 cytokines on the permeability barrier are thought to be involved in the vicious cycle of allergic inflammation and cutaneous permeability barrier dysfunction in the pathogenesis of atopic dermatitis [7,8]. However, the role of Th2 cytokines in the homeostasis and/or construction of the cutaneous permeability barrier in the steady state has not been fully assessed, although Sehra et al. demonstrated that SC integrity was increased with elevated expressions of IVL and FLG but not LOR in IL-4-deficient mice [9]. Functions of IL-4 and IL-13 are well known to be mediated by a signal transduction molecule, signal transducer and activator of transcription 6 (STAT6) [10]. The possibility of alternative pathways of STAT6 activation have also been observed [10]. As for the regulation of expressions of epidermal differentiationrelated molecules, loricrin, involucrin, and filaggrin expression have been demonstrated to be regulated by Th2 cytokines through STAT-6 [6,11].
https://doi.org/10.1016/j.jdermsci.2018.07.008 0923-1811/ © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.
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The present study examined the cutaneous permeability barrier function of Stat6-deficient mice to assess the role of STAT6 in cutaneous permeability barrier homeostasis in the steady state.
performed using electron microscopic images taken by an investigator blinded to the source of the specimen. 2.6. Counts of mast cells in murine dermis
2. Materials and methods 2.1. Animals Female C57BL/6 mice (Japan SLC, Hamamatsu, Japan) were purchased for use as wild-type mice. Female Stat6-knockout mice were donated by Prof. Yokozeki (Tokyo Medical and Dental University, Tokyo, Japan). Background strain of Stat6-knockout used here was C57BL/6 mice. The mice were used at 9–10 weeks old. All animals were housed under conventional conditions and had ad libitum access to commercial diet and water. All experiments with mice were approved by the Ethics of Animal Experimentation Committee at Oita University. 2.2. Physiological assessments TEWL, SC hydration and SC surface pH on flanks were measured using a Tewameter (TM300; Courage & Khazaka, Cologne, Germany), Corneometer (CM825; Courage & Khazaka) and skin pH meter (PH 905; Courage & Khazaka), as described previously [12], 2 days after hair shaving. 2.3. Skin sample preparation Skin samples were collected 3 days after hair shaving. Whole skin samples for penetration assay, hematoxylin and eosin stain, Giemsa stain, and electromicroscopic analysis were collected from flanks. Epidermal sheet for real-time PCR and Western blotting was collected from each flank by 1-h incubation at 37 C with 1000 IU/ ml of dispase (Godo Shusei, Tokyo, Japan). SC for lipid analysis was collected from 2 cm2 of each abdomen by incubation for 2.5 h with 0.5% trypsin (Difco Laboratories, Detroit, MI) at 37 C. 2.4. Penetration assay Quantitative evaluation of outside-to-inside penetration of the skin was assessed with Evans blue dye, as described previously [13]. Each sample was floated on MCDB 153 medium (SigmaAldrich, St. Louis, MO) containing 1.8 mM of CaCl2 with the outer epidermal surface of each sample exposed to the air. Next, 50 ml of 2% Evans blue in phosphate-buffered saline was pipetted onto the outer epidermal surface of each skin explant. The dye was allowed to penetrate the skin for 2 h at room temperature, then the surface of the skin was washed with phosphate-buffered saline and gently wiped with a Kimwipe (Nippon Paper Crecia, Tokyo, Japan). After washing procedures had been repeated three times, the center of each explant was biopsied with a 4-mm punch and each 4-mm disk was placed into 100 ml of 1 N KOH. After overnight incubation at 37 C, each sample was neutralized by the addition of 900 ml of a mixture of 0.6 N H3PO4 and acetone (5:13, v/v). After vigorous vortexing for a few seconds, the mixture was centrifuged at 3000 rpm for 15 min in an RA-150AM centrifuge (Kubota, Tokyo, Japan), and absorbance of supernatants was measured at 360 nm. 2.5. Quantitative morphology under electron microscopy Skin biopsies from mice were fixed in Karnovsky’s fixative, and post-fixed with either 1% aqueous osmium tetroxide, containing 1.5% potassium ferrocyanide. Ultrathin sections were examined using an electron microscope (JEM-1200EX II; JEOL, Tokyo, Japan) operated at 80 kV. Measurement of the number of SC layers was
Mast cells in Giemsa-stained murine dermis were counted under high-power magnification. Counts were performed in 9–16 areas in each skin sample. The average number in each sample was analyzed. 2.7. Culture of keratinocytes transfected with siRNA at the air-liquid interface Culture of normal human keratinocytes (NHKs) transfected with siRNA was performed as previously reported [14]. Third- or fourth-passage NHKs (Cell Applications, San Diego, CA) were cultured in Keratinocyte Basal Medium 2 with Supplement Pack Keratinocyte Growth Medium 2 (PromoCell, Heidelberg, Germany) at 37 C in a humidified atmosphere of 5% CO2 in air. Stealth siRNA transfection was performed once cells reached 70–90% confluence. The Stealth siRNA against human Stat6 (HSS110291, Thermo Fisher Scientific, Lafayette, CO) and Stealth RNAi Negative Control (Thermo Fisher Scientific) were used. Cells were transfected with 100 nM siRNAs in Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and OPTI-MEM (Invitrogen), according to the instructions from the manufacturer. Transfected cells were harvested with ethylenediaminetetraacetic acid (EDTA)-trypsin (DS Pharma Biomedical, Osaka, Japan), then seeded into Cell Culture Inserts (pore size, 0.4 mm; BD Biosciences, Durham, NC) with Keratinocyte Basal Medium 2, which were placed into a Companion Plate (BD Biosciences) containing the same medium 24 h after the transfection. The medium in Cell Culture Inserts was aspirated 24 h later, and the medium in the Companion Plate was changed to assay medium (EPI-MODEL; Japan Tissue Engineering, Aichi, Japan). Keratinocytes transfected with siRNA were grown at the air-liquid interface. The medium in the plate was changed every three day, and keratinocytes were cultured for 7 days. Then, stratified keratinocytes and conditioned medium were harvested for realtime PCR or Western blotting. 2.8. Real-time PCR Total RNA was isolated from epidermal sheets and cultured NHKs using an RNeasy Fibrous Tissue Mini Kit (QIAGEN, Hilden, Germany), and reverse transcription was performed using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the instructions from the manufacturer. Complementary DNA products were amplified in a LightCycler 480 System (Roche Diagnostics GmbH), as described previously [14]. The primers used for real-time PCR are shown in Supplementary Table S1. Product specificity was evaluated by melting curve analysis, and relative gene expression was calculated from a standard curve included in each run. Relative mRNA expression levels were normalized with the housekeeping gene, human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or mouse β-actin. 2.9. Western blotting Western blotting was performed as previously described [12,14]. Each epidermal sheet and cultured NHKs were homogenized in Pierce RIPA buffer (Thermo Scientific, Rockford, lL) with Halt Protease Inhibitor Cocktail (Thermo Scientific). These samples were separated by 10% SDS-PAGE before transfer to Immobilon-P TM Transfer Membrane (Millipore, Billerica, MA) or Hybond -P (GE Healthcare, Buckinghamshire, UK). We used antibodies against FLG
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(COVANCE, Emeryville, CA), IVL (COVANCE), LOR (COVANCE), STAT6 (Santa Cruz Biotechnology, Santa Cruz, CA) and β-actin (Cell Signaling Technology, Danvers, MA) as primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) as secondary antibodies, then visualized using ECLTM Western Blotting Detection Reagents (GE Healthcare).
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Amounts of ceramide were normalized by reference to the area of the sample skin (i.e., 2 cm2). 2.11. Statistical analysis All experiments were analyzed using two-tailed Student’s ttest. All results are presented as mean standard error of the mean. Values of P < 0.05 were considered statistically significant.
2.10. Analysis of SC ceramides 3. Results SC lipids were extracted by Bligh-Dyer solvents and the extracted fraction was suspended in a mixture (1:1, vol/vol) of chloroform and methanol after evaporation under a stream of nitrogen gas. This suspension was applied to a thin-layer chromatography plate, as described previously [15]. The plate was developed with a mixture (190:9:1, vol/vol) of chloroform, methanol, and acetic acid. After solvent development, the chromatogram was air-dried, soaked in an aqueous solution of 10% CuSO3 and 8% H3PO4, and charred at 180 C. Charred lipids were quantitated by Image J (National Institutes of Health, Bethesda, ML). Ceramides were quantitated by reference to appropriate commercial standards as follows: non-hydroxy fatty acid ceramide from bovine brain (Sigma-Aldrich) and ceramides (hydroxy) (Matreya, State College, PA) were used as standards for ceramides 1 and 2 and for ceramides 3, 4, 5, and 6, respectively.
3.1. Lack of morphological difference between Stat6-deficient and wild-type mice Hematoxylin and eosin staining revealed no significant difference in skin between Stat6-deficient mice and wild-type mice (Fig. S1a). Electron microscopy also revealed no significant difference in thickness of the SC (Fig. S1b, S1c). 3.2. Cutaneous permeability barrier in Stat6-deficient mice was superior to that in wild-type mice Values of transepidermal water loss (TEWL) and SC pH were lower in Stat6-deficient mice than in wild-type mice (Fig. 1a and c). Values of SC hydration in Stat6-deficient mice were higher than
Fig. 1. Cutaneous permeability barrier function in Stat6-deficient mice is superior to that in wild-type mice. The results show cutaneous permeability function in Stat6-deficient mice (STAT6-/-) is superior to that in wild-type mice (WT). Measurement of transepidermal water loss (TEWL) (a; n = 6), SC hydration (b; n = 6), and SC pH (c; n = 6), and a penetration assay using Evans blue (d; n = 11 in WT, n = 12 in STAT6-/-) were performed as described in the Materials and Methods section. Error bars indicate mean SEM. P-value vs. WT.
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those in wild-type mice (Fig. 1b). Penetration assay revealed that penetration of Evans blue was suppressed in Stat6-deficient mice, compared to wild-type mice (Fig. 1d). 3.3. Expression of loricrin was up-regulated in Stat6-deficient mice Real-time polymerase chain reaction (PCR) revealed that expression of LOR in epidermis was up-regulated in Stat6-deficient mice, compared to that in wild-type mice (Fig. 2a). Western blotting supported up-regulation of LOR in Stat6-deficient mice (Fig. 2d). Expression of IVL in Stat6-deficient mice was broadly equivalent to that in wild-type mice (Fig. 2b). Expression of FLG tended to be up-regulated in Stat6-deficient mice, but this upregulation was not statistically significant (Fig. 2c). Western blotting detected no significant difference in the expressions of IVL and FLG between Stat6-deficient and wild-type mice (data not shown). 3.4. Ceramide levels in SC were larger in Stat6-deficient mice than in wild-type mice SC ceramides 2 and 5 are produced by the hydrolysis of sphingomyelin by sphingomyelinase (SMase), particularly acidSMase (aSMase) [16,17], while other groups of SC ceramides are
produced by the degradation of glucosylceramide by βglucocerebrosidase (β-GlcCer’ase) [18]. Ceramide hydrolysis is catalyzed by acid-ceramidase (aCEase) in the SC under physiological conditions [19–21]. Real-time PCR revealed that expressions of aSMase and β-GlcCer’ase in epidermis were upregulated in Stat6-deficient mice, compared to those in wildtype mice (Fig. 3a and b), while expression of aCEase in Stat6deficient mice was comparable to that in wild-type mice (Fig. 3c). Thin-layer chromatography revealed that amounts of ceramide 1, ceramide 2, ceramide 3, ceramide 4–6, and total ceramides in SC were higher in Stat6-deficient mice than in wild-type mice (Fig. 4). 3.5. Effects of Stat6 knockdown in 3-dimensionally (3D) cultured normal human keratinocytes were not identical to those in STAT6deficient mice Real-time PCR and Western blotting revealed that transfection of small interfering RNA (siRNA) for Stat6 successfully knocked down expression of Stat6 in 3D-cultured normal human keratinocytes (NHKs; Fig. S2). Real-time PCR revealed that expressions of LOR, IVL, FLG, aSMase, and aCEase were not affected in 3D-cultured NHKs, while expressions of β-GlcCer’ase were reduced by knockdown of Stat6 (Fig. 5).
Fig. 2. Expression of loricrin is elevated in Stat6-deficient mice, compared with that in wild-type mice. The results show expressions of loricrin (LOR; a), but not involucrin (IVL; b) or filaggrin (FLG; c) in epidermis were elevated in Stat6-deficient mice (STAT6-/-), compared to levels in wild-type mice (WT) for either mRNA (a–c; n = 6 in WT and n = 6 in STAT6-/-) or protein (d). Sample preparation, real-time RT-PCR and Western blotting were performed as described in the Materials and Methods section. Error bars indicate mean SEM. P-value vs. WT. Values of P < 0.05 are shown.
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Fig. 3. Expressions of acid-sphingomyelinase and β-glucocerebrosidase, but not acid-ceramidase are elevated in Stat6-deficient mice, compared with those in wild-type mice. The results show expressions of acid-sphingomyelinase (aSMase; a) and β-glucocerebrosidase (β-GlcCer’ase; b), but not acid-ceramidase (aCEase; c) in epidermis are elevated in Stat6-deficient mice (STAT6-/-), compared with those in wild-type mice (WT). Sample preparation and real-time RT-PCR were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
Fig. 4. Ceramides in SC are elevated in Stat6-deficient mice, compared with those in wild-type mice. (a) Thin-layer chromatography (TLC). Quantitative analysis reveals that levels of ceramide 1 (b), ceramide 2 (c), ceramide 3 (d), ceramide 4–6 (e) and total ceramides (f) in SC are elevated in Stat6-deficient mice (STAT6-/-), compared with those in wild-type mice (WT). Sample preparation and TLC were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
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Fig. 5. Effects of knockdown of Stat6 in 3D-cultured human keratinocytes are inconsistent with those in STAT6-knockout mice. The results show expressions of loricrin (LOR; a), involucrin (IVL; b), filaggrin (FLG; c), acid-sphingomyelinase (aSMase; d) and acid-ceramidase (aCEase; f) in 3D-cultured human keratinocytes are not influenced and expression of β-glucocerebrosidase (β-GlcCer’ase; e) is down-regulated following transfection of siRNA against Stat6 (STAT6 siRNA). Three-dimensional culturing, sample preparation and real-time RT-PCR were performed as described in the Materials and Methods section. Error bars indicate mean SEM (n = 8 in control siRNA, n = 8 in STAT6 siRNA). P-value vs. control siRNA. Values of P < 0.05 are shown.
Fig. 6. The number of mast cells in dermis is reduced in Stat6-deficient mice. The results show the number (b) of mast cells identified by Giemsa stain (a) is reduced in Stat6-deficient mice (STAT6-/-), compared with wild-type mice (WT). Sample preparation and Giemsa stain were performed as described in the Materials and Methods section. Scale bar = 100 mm in (a). Error bars indicate mean SEM (n = 6 in WT, n = 6 in STAT6-/-). P-value vs. WT. Values of P < 0.05 are shown.
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3.6. Numbers of mast cells in dermis but not expressions of histamine receptors in epidermis were reduced in Stat6-deficient mice Mast cells and products of mast cells, such as histamine, reportedly modulate the permeability barrier [22,23]. We therefore counted numbers of mast cells in dermis. Numbers of mast cells in dermis were reduced in Stat6-deficient mice, compared to numbers in wild-type mice (Fig. 6). On the other hands, real-time PCR revealed that expressions of histamine H1, H2, H3, and H4 receptors in epidermis in Stat6-deficient mice were identical to those in wild-type mice (Fig. S3). 4. Discussion Although no morphological abnormalities were apparent in the skin, values of TEWL in Stat6-deficient mice were decreased, suggesting that the permeability barrier function in Stat6-deficient mice is superior to that in wild-type mice. Penetration assay strongly supported the augmented permeability barrier function in Stat6-deficient mice. Elevated SC hydration and the lack of change in SC thickness suggest that the augmented permeability barrier function in Stat6-deficient mice is primarily due to the upregulated SC function, rather than to compensatory accumulation of SC. In line with accumulated knowledge regarding the importance of SC acidity in permeability barrier homeostasis/ construction [7], the decrease in SC pH in Stat6-deficient mice not only supports the augmentation of SC permeability barrier function, but also might represent one of the underlying mechanisms. LOR is the most abundant protein in the cornified envelope [24]. In addition, it has been reported that permeability barrier formation, although transiently, delays in LOR-deficient mice [25]. Therefore, up-regulation of LOR expression might be one of the bases of the augmented permeability barrier function in Stat6deficient mice, although there has not been clear evidences showing that increase in LOR could augment permeability barrier. Meanwhile, expressions of IVL and FLG were unaffected in Stat6deficient mice, although expressions of IVL and FLG (but not LOR) were demonstrated to be elevated in IL-4-deficient mice [9]. These discrepancies in the regulation of LOR, IVL, and FLG suggest that the role of STAT6 in keratinocyte differentiation is not necessarily identical to that of IL-4 in vivo. The reasons for the difference between Stat6-deficient and IL-4 deficient mice remain unclear, although STAT6 signaling independent of IL-4 and IL-13 receptors and/or some specific functions of IL-13 might be involved in this discrepancy, particularly in vivo. Elevated amounts of ceramides in SC and mRNA expressions of aSMase and β-GlcCer’ase (but not aCEase) in epidermis might be involved in the up-regulation of cutaneous permeability barrier in Stat6-deficient mice, and again support the importance of SC ceramides in permeability barrier homeostasis/construction. These results seem consistent with the findings of previously reported studies demonstrating the negative effects of Th2 cytokines on the production of ceramide in keratinocytes, which were accompanied by down-regulation of mRNA expressions of aSMase and β-GlcCer’ase, while no changes were seen in aCEase expression [2,4]. However, whether Th2 cytokines could be produced and secreted and what kinds of cells could be sources of Th2 cytokines in the steady state remain unclear. Effects of Stat6 knockdown in cultured keratinocytes were not identical to the observations in STAT6-deficient mice. No effects of Stat6 knockdown in expressions of LOR, IVL, and FLG in cultured keratinocytes were consistent with those reported by Amano et al. [26]. These discrepancies between the effects of Stat6 knockdown in cultured keratinocytes and observations in Stat6-deficient mice might suggest that down-regulations of LOR, aSMase and β-
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GlcCer’ase in the epidermis of wild-type mice, compared to those of Stat6-deficient mice, might represent indirect effects on keratinocytes through STAT6 signaling in cells other than keratinocytes. For example, mast cells are one candidate for contributing to the down-regulation of permeability barrier function in wild-type mice, not only because mast cell maturation has been reported to be reduced in Stat6-deficient mice in an allergic inflammatory model [27], but also because products from mast cells, such as histamine, affect permeability barrier functions with reducing the expression of the differentiation-associated proteins, such as keratin 1/10, filaggrin, and loricrin [22]. In the present study, numbers of mast cells in dermis but not expressions of histamine receptors in epidermis were reduced in Stat6deficient mice, compared to those in wild-type mice. Decreased numbers of dermal mast cells might thus be involved in the augmented permeability barrier function in Stat6-deficient mice identified in the present study, although which factors from mast cells affect the barrier function and how functions of mast cells are affected by STAT6 depletion remain unclear. Meanwhile, the decrease in numbers of dermal mast cells in the present study supports that STAT6 contributes to the proliferation and growth of mast cells [28]. On the other hand, mast cell-deficient mice were recently reported to exhibit a dysfunction of permeability barrier accompanied by decreased expressions of FLG, LOR, and IVL [23], suggesting that decreased numbers of dermal mast cells are not necessarily involved in the augmented permeability barrier function seen in Stat6-deficient mice. Meanwhile, differences in study species (i.e., human or mouse) and/or in degree of modulation of the STAT6 gene (i.e., knockdown or knockout) might have contributed to the discrepancies between effects of Stat6 knockdown in cultured keratinocytes and the findings in Stat6-deficient mice. In conclusion, the present study suggests that STAT6 signaling negatively affects cutaneous permeability barrier homeostasis in vivo and inhibition of this signaling might be useful to augment permeability barrierfunction even in the steady state. However, the mechanisms involved in these findings remain unclear. Funding Trust Accounts in Oita University. This study was partly supported by grants from Japan Society for the Promotion of Science (no.26461662 and 17K10214). Conflict of interest All authors have no conflict of interest. Acknowledgements We wish to express our thanks to Mr. Hiroaki Kawazato and Ms. Aiko Yasuda (Research Promotion Project, Faculty of Medicine, Oita University, Oita, Japan) for their excellent work on ultrastructural analyses. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j. jdermsci.2018.07.008. References [1] P.M. Elias, K.R. Feingold, Lipids and the epidermal water barrier: metabolism, regulation, and pathophysiology, Semin. Dermatol. 11 (1992) 176–182. [2] Y. Hatano, H. Terashi, S. Arakawa, K. Katagiri, Interleukin-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier
Please cite this article in press as: W. Zhang, et al., Cutaneous permeability barrier function in signal transducer and activator of transcription 6deficient mice is superior to that in wild-type mice, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.07.008
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Please cite this article in press as: W. Zhang, et al., Cutaneous permeability barrier function in signal transducer and activator of transcription 6deficient mice is superior to that in wild-type mice, J Dermatol Sci (2018), https://doi.org/10.1016/j.jdermsci.2018.07.008