Increasing effect of ceramides in skin by topical application of sphingosine

Introduction and objective: Ceramidase metabolizes ceramide and generates sphingoid and fatty acid. Alkaline ceramidase has been involved in reduction of the ceramide level in atopic dermatitis or in aged dry skin. We hypothesized that the ceramide content can be increased by inhibition of the ceramidase activity. In this study, we aimed to examine the effect of alkaline ceramidase inhibitor on the amount of the ceramide in mice skin and in three-dimensional cultured epidermis model. Material and methods: 4-Nitrobenzo-2-oxa-1,3-diazole-labeled ceramide was used as a substrate and was incubated with skin homogenate at 37 ̊C for 1 or 10 h. Fluorescent-labeled fatty acid, enzymatic reaction product was detected by fluorescence high performance liquid chromatography to determine the alkaline ceramidase activity. The ceramide content was quantitatively analyzed by high performance thin layer chromatography. Results: 50% Inhibitory concentration of sphingosine for alkaline ceramidase in mice skin and three-dimensional cultured epidermis model was >70-fold lower than the corresponding oleoylethanolamide. Ceramide [AS] and ceramide [AP] contents of sphingosine-applied group of mice stratum corneum were significantly higher than in the normal group. Compared with the normal group, ceramide [NDS] in three-dimensional cultured epidermis model was also significantly increased in sphingosine-applied group. Conclusion: This study demonstrated that sphingosine inhibited alkaline ceramidase in mice skin and three-dimensional cultured epidermis model and ceramide contents was increased by application of sphingosine. These results suggest that sphingosine was beneficial compound to increase skin ceramide, though sphingosine may also be affected by another pathway.


Introduction
Ceramide (CER) in the stratum corneum (SC) has an important role in water retention as well as skin barrier function. Ceramidase (CDase) hydrolyzes the N-acyl linkage of CER and converts CER to sphingoid and fatty acid (FA). Houben and Lin reported that the human epidermis had acidic CDase (aCDase) and alkaline CDase (alkCDase) activities [1,2]. It was also demonstrated that alkCDase 1 mRNA expression level in human epidermis was higher than other tissues; total alkCDase and aCDase were activated, and mRNA expression of alkCDase 1 and aCDase was increased by differentiation of keratinocyte [3].
Skin barrier function is altered by skin conditions such as aging and atopic dermatitis (AD). Imokawa and Ghadially revealed a decrease in the CER content in SC in aged human and hairless mice [4,5]. Jin et al. demonstrated that the alkCDase activity (pH 9.0) was elevated in aged human SC compared with young [6]. AD patients showed high transepidermal water loss (TEWL) and low total CER content in SC [4]. Sphingomyelin deacylase and glucosylceramide deacylase were closely related to AD [7]. However, Okino found that alkCDase was secreted from Pseudomonas aeruginosa in skin of AD patients [8,9] and considered that it subsequently decreased the CER content [10]. But little has been reported on regulation of the CER content in SC by inhibition of the CDase.
We then hypothesized that CDase inhibitor may increase the CER content in SC. Yada and Mao reported that sphingosine (SPH) inhibited mouse alkaline ceramidase (maCER1) and guinea pig epidermal alkCDase activity [11,12]. In this study, we used hairless mice and three-dimensional cultured epidermis model, and reported the effects of SPH on the alkCDase activity and the CER content.

Animals and three-dimensional cultured epidermis model
Male HOS:HR-1 mice, 4 and 7 week sold, were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and were kept under a 12-h light and 12-h dark cycle in a conventional animal room maintained at 22 ± 2°C; 55% ± 5% and free access to food and water. All procedures were approved by the Ethics Committee of Josai University (Sakado, Saitama, Japan). Six-day cultured LabCyte EPI-MODEL was obtained from Japan Tissue Engineering Co., Ltd. (Gamagori, Aichi, Japan).

Preparation of homogenate
Mice were killed by cervical dislocation and dorsal skin was obtained. Subcutaneous tissue was removed by scissors. After weighing, the skin was minced using scissors for 3 min on ice and was homogenized in 0.25 M sucrose, supplemented with 1% Triton X-100 using Polytron PT 1200 E homogenizer (Kinematica AG, Luzernerstrasse, Lucern, Switzerland) at 25,000 rpm for 1 min on ice. The homogenate was centrifuged at 500×g at 4°C for 1 min and the supernatant was stored at −30°C until use. Three-dimensional cultured epidermal models were collected from transwell by spatula and homogenized, as described above. Protein concentration was determined by a modified Lowry method.

Measurement of the alkCDase and aCDaseactivity
The CDase activity was measured by the method of Houben et al., with some modifications [1]. NBD-C 12 -CER was used as substrate of CDase. An appropriate amount of NBD-C 12 -CER, with final concentration of 250 μM, was dissolved in methanol and allowed to dry under nitrogen gas. The substrate was redissolved by sonication (50°C, 10 min) in 12.5 μl of detergent mixture containing 40 mg/ml Triton X-100, 16 mg/ml Tween 80, and 25 μl of 250 mM Tris-HCl buffer (for alkCDase, pH 8.5) or acetate buffer (for aCDase, pH 4.2). Mice skin or three-dimensional cultured epidermis model homogenate was preincubated at 37°C for 2 min. Enzymatic reactions were initiated by the addition of 12.5 μl of 60 ng protein/ml homogenate, and the final concentration was 15 ng protein/ml to the substrate solution at 37°C. Incubation time was 1 h for mice skin or 10 h for three-dimensional cultured epidermis model. The reaction was stopped by addition of 300 μl of chloroform/methanol (2:1) that contained 0.26 μM NBD-C 6 -FA as internal standard for HPLC analysis. The reaction mixture was dried under nitrogen gas and dissolved in 100 μl of methanol. HPLC system (SHIMADZU Prominence HPLC system, SHIMADZU Corp., Kyoto, Kyoto, Japan), equipped with a reversed phase L-Column 2 ODS (5 μm, 2.1×150 mm; Chemicals Evaluation and Research Institute, Tokyo, Japan), and a fluorescence detector (RF-10AXL, SHIMADZU) set to excitation and emission wavelengths of 460 and 534 nm, respectively, were used to determine NBD-C 12 -FA in the enzymatic reaction sample. Column was maintained at 30°C. The mobile phases were 0.1% formic acid in acetonitrile (A) and 0.1% formic acid in water (B). A binary mobile phase at a flow rate of 0.4 ml/min was used under liner gradient conditions as follows: 0 min, 30% A; 0-5 min, 30%-100% A; 5-14.5 min, 100% A; 14.5-14.51 min, 30% A; 14.51-30 min, 30% A. Standard curve of peak height ratio (NBD-C 12 -FA vs. internal standard) was used to determine NBD-C 12 -FA that was formed during the enzymatic reaction. To correct the CDase activity values, the NBD-C 12 -FA content in an incubated reaction mixture without protein was subtracted from that in a reaction mixture with protein. To assess inhibitory effect, SPH dissolved in methanol was added to the substrate and dried. The inhibitory activity was measured as described above. OEA, which has the inhibitory activity for alkCDase and aCDase [1], was used as a positive control of CDase inhibitor.

Application of SPH and extract of SC lipids in mice
SPH and OEA were dissolved in propyleneglycol/ethanol (7:3; concentration, 40 mM) [13]. Samples of 50 μl were applied twice a day for three days to the dorsal skin (area, 5-6 cm 2 ) of mice. Control group was exposed to propyleneglycol/ethanol (7:3). SC sheet was collected by stripping the dorsal skin area with applied sample with cyanoacrylate resin and was stored frozen at −80°C until use. Lipids were extracted by the method of Imokawa et al. [4]. SC sheets were immersed in hexane/ethanol (95:5) and sonicated for 20 min. Solutions of extracted lipids were filtered (MILLEX-GV Syringe Driven Filter Unit, Merck Millipore, MA, USA) and transferred to test tubes. Organic solvent was dried under nitrogen gas stream at 50°C. Lipid in test tube was dissolved in chloroform/methanol (2:1) and sonicated at 37°C for 5 min. Extract was transferred to a 1.5 ml tube and dried. SC lipid samples were stored frozen at −80°C until use.

Application of SPH and extraction of epidermal lipids for three-dimensional cultured epidermis model
Liposome constructed SPH/DPPC/DPPG (molar ratio 4:4:1) was prepared by the thin layer method previously described [14]. SPH concentration in a liposome was 10 mM. Three-dimensional cultured epidermis model was cultured at 37°C under 5% CO 2 . A 50 μl solution of liposome containing SPH was applied to the SC side of three-dimensional cultured epidermis model for 6 days and was changed every 2 days. Medium was changed every day for 6 days and was supplied with 25 μg/ml ascorbic acid on the seventh day. After 24 h, three-dimensional cultured epidermis model was collected from transwell by scalpel. Control group was applied with SPH-free liposome (DPPC/DPPG, 4:1). Three-dimensional cultured epidermal samples were immersed in chloroform/methanol (2:1) and sonicated by SONIFIRE 250 (Branson, Danbury, CT, USA) on ice. Lipid collection was conducted as described above.

Quantification of the CER
The CER was quantitated by the method of Imokawa et al. [4]. Extracted lipids were redissolved in chloroform/methanol (2:1) and 10 μl of extract was spotted by capillary on the high-performance thinlayer chromatography (HPTLC) Silica gel 60, measuring 20×10 cm (Merck Millipore). The CER was isolated by chloroform/methanol/ acetic acid (190:9:1 to 9 cm) and was developed twice. The HPTLC plate was sprayed with coloring reagent (10% cupric sulfate, 8% phosphoric acid) and heated at 180°C for 10 min by TLC Plate Heater III (CAMAG, Muttenz, Switzerland). HPTLC plate was imaged by LAS1000-plus (Fujifilm Corporation, Tokyo, Japan) and the CER was quantitated from band density by Multi Gauge Version 3.0 (Fujifilm Corporation).

Data analysis
Data were expressed as the mean ± S.D. (n=4) and compared by Turkey's test, Dunnett's test, or Student's t-test using SAS version 9.2 (SAS Institute ltd., Cary, NC, USA).

The CDase activity in mice skin
Activity of alkCDase in the presence of 10 mM SPH on mice skin homogenate was measured. Compared with the normal group, the alkCDase activity was significantly reduced by 46% in OEA (positive control) group (p<0.05) and 88% in SPH group (p<0.001). The alkCDase activity in the SPH group was significantly decreased by 78% (p<0.05) relative to the OEA group ( Figure 1A). Similarly, the aCDase activity in the SPH group had a tendency to be lower than that in normal group, in spite of no significant ( Figure 1B). Subsequently, the inhibitory activity of SPH on the alkCDase was determined. SPH and OEA dose-dependently inhibited the alkCDase activity ( Figure 2). The IC 50 value of SPH was significantly lower than that of OEA (p<0.001) ( Table 1).

The CER content in mice SC
The CER content in mice SC was measured after application of 40 mM SPH or OEA. CER [NS] and CER [NP] contents were not changed by the application of OEA or SPH ( Figure 3A and 3B). In contrast, CER [AS] in the SPH group was significantly increased relatively to the normal group (p<0.05) ( Figure 3C). CER [AP] content was also significantly increased by application of SPH when compared with the normal group (p<0.001) ( Figure 3D).

The alkCDase activity in three-dimensional cultured epidermis model
Activity of alkCDase in the presence of 10 mM SPH on the threedimensional cultured epidermis model homogenate was measured. Compared with the normal group, the alkCDase activity was significantly decreased by 56% in OEA group and 83% in SPH group (p<0.001). The alkCDase activity in the SPH group was significantly reduced by 62% (p<0.01) relatively to the OEA group ( Figure 4). SPH and OEA inhibited alkCDase in a dose-dependent manner ( Figure  5). In agreement with the animal experiment, the IC 50 value of SPH for alkCDase was significantly reduced compared with that of OEA (p<0.01) ( Table 2).

The CER content in three-dimensional cultured epidermis model
The CER content was measured after application of liposome containing 10 (Figure 6A-6D). However, CER [NDS] was significantly increased in the SPH group in comparison with the normal group (p<0.001) ( Figure 6E).

Discussion
First, the inhibitory activity of SPH for alkCDase was assessed by comparison with OEA (positive control). The IC 50 values of SPH were lower than OEA (Tables 1 and 2     value of SPH for maCER1 (pH 8.0) was −0.08 mM [12]. The mice skin IC 50 values of SPH reported in this paper were similar to the previous study. In addition, the IC 50 values of SPH and OEA were similar between mice skin and three-dimensional cultured epidermis model (Tables 1 and 2). These data indicated that SPH is a stronger inhibitor of alkCDase than OEA. Subsequently, mice SC CER after application of SPH or OEA was determined. SPH concentration of 40 mM was higher than the intracellular level. However, we considered that the SPH amount in the viable epidermis was low because SPH penetration was prevented by the SC barrier. that maCER1 (pH 8.0) had specificity for CER containing D-erythrosphingosine, but CER had D-ribo-phytosphingosine and D-erythrodihydrosphingosine was not hydrolyzed [12]. Thus, it is likely that degradation of CER [AS], which contains D-erythro-sphingosine, was suppressed by inhibition of alkCDase. However, CER [NS] that also contained D-erythro-sphingosine, was not increased ( Figure  3A); whereas, CER [AP] that contained D-ribo-phytosphingosine was increased ( Figure 3D). Therefore, we considered that a mechanism other than alkCDase inhibition is also related to the increase of the CER. Further, the CER content in three-dimensional cultured epidermis model after application of SPH was also quantitated. We prepared a liposome containing SPH for application to three-dimensional cultured epidermis model because SPH was a lipophilic compound. The CER content of the control group with SPH-free liposome (DPPC/ DPPG, 4:1) was unchanged ( Figure 6). Unlike the results on mice SC, CER [AS] and CER [AP] were unchanged; however, CER [NDS] was increased ( Figure 6C-6E). Density of CER [NDS] in HPTLC analysis     [20]. Thus, it is probable that de novo synthesis of CER was stimulated by SPH in our experiment. Sphingosine-1-phosphate (S1P) is generated from SPH by sphingosine kinase (SPHK) and induces keratinocyte differentiation [21]. Hong et al. demonstrated that specific differentiation-associated maker proteins in intact mice epidermis and HaCaT cells were increased by application of SPHK activator [22]. Thus, it is likely that SPH applied to mice or three-dimensional cultured epidermis model was metabolized to S1P and differentiation of keratinocyte was promoted; consequently, the CER was increased. It was known that a salvage pathway reconverted SPH to CER [23], but it was still unclear whether it contributed to skin barrier.

Conclusion
We revealed that SPH was a stronger inhibitor than OEA to increase the CER contents, indicating that SPH suppresses degradation of CER in SC by inhibition of alkCDase. However, SPH may also be affected by another pathway, like de novo synthesis. Thus, SPH may be a beneficial compound to increase skin CER.