Modulation of function and structure of stratum corneum in sphingomyelin synthase 2-deficient mice

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Modulation of function and structure of stratum corneum in sphingomyelin synthase 2-deficient mice

https://doi.org/10.21203/rs.3.rs-1627640/v1

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The effects of sphingomyelin synthase (SMS) deficiency on stratum corneum (SC) barrier function (skin permeability of the hydrophilic and lipophilic compounds) and SC lamellar structure are unknown. Skin permeability of the hydrophilic compound increased significantly for SMS2-KO mice when compared with that of WT mice. In contrast, no difference was observed in the penetration of lipophilic compounds in the skin of both SMS2-KO and WT mice. In SC of SMS2-KO mice, two sharp SAXS peaks were observed due to the lamellar structure with a repetition period of 4.8 nm. The WAXS revealed that the intensity ratio R_0.42/0.37 of the 0.42 nm peak at 2.4 nm^− 1 to the 0.37 nm peak at 2.7 nm^− 1 was smaller in the SMS2-KO mouse than in the WT mouse. Due to the temperature dependence of the WAXS, the peaks of 2.4 and 2.7 nm^− 1 remained until the higher temperatures in SMS2-KO mouse SC than those in WT mouse SC. The results of X-ray diffraction suggest that deficiency of SMS2 may cause the appearance of highly ordered structures of SC, which in turn may reduce the barrier function of SC.

The outermost stratum corneum (SC) is composed of corneocytes and SC intercellular lipids and functions as both a barrier and water retention. Ceramide (Cer) is present in SC intercellular lipids and plays an important role in the barrier function of the SC by forming a lamellae structure with free fatty acids and cholesterol (Chol). Cer is a major component, accounting for approximately 50% of intercellular lipids in the SC [1, 2]. Cer levels have been reported to be lower in the skin of patients with skin diseases such as atopic dermatitis and psoriasis when compared with that of healthy individuals [3, 4], further confirming the important role of Cer in the barrier function of these SCs. SC intercellular lipids are important for the barrier function and prevent evaporation of water from the body and entry of foreign substances by forming a layered lamellar structure. Importantly, the barrier function depends on the formation of the lamellar structures, but the relation between the lamellar structures and the barrier function has not been fully elucidated. In general, the lamellar structure of the SC has a long-period lamellar structure and a short-period lamellar structure [5–7]. In the lateral direction, the structure of the hydrocarbon chain packing can be orthorhombic (ORTHO), hexagonal (HEX) and liquid phase (LIQUID) [8–10]. The structure of the hydrocarbon chain packing is closely associated with skin penetration; the highly ordered structure has low skin permeability, whereas the poorly ordered structure has high skin permeability. However, the present research suggests another structure that there is a new permeation mechanism for water-soluble compounds.

Cer has multiple metabolic pathways [11]. In the sphingomyelin pathway, Cer is metabolized to sphingomyelin (SM) by sphingomyelin synthase (SMS). SMS exists as three isozymes, SMS1, SMS2 and SMSr, with SMS1 and SMS2 having synthetic catalytic activity [12]. Conversely, SM is converted to Cer by sphingomyelinase. Therefore, because sphingomyelin is a precursor to ceramide, there is a close relationship between SM and Cer content. SMS1 has higher catalytic activity for SM synthesis when compared with that of SMS2, therefore SMS1 knockout mice are not readily available because of embryonic lethality.

We have reported previously that SMS2-knockout (SMS2-KO) mice have lower Cer in the SC and lower SM in the epidermis when compared with those of wild-type (WT) mice, which leads to greater transepidermal water loss (TEWL) than that observed for WT mice [13]. In SMS2-KO mice, we showed that the SMS2-KO mouse SC had reduced ceramide NS and AS contents with relatively short fatty acid side chains when compared with those of WT mice [13]. However, skin permeability of compounds through the skin of SMS2-KO mice and the intercellular lipid lamellar structures in the SMS2-KO mouse SC have not been investigated yet. In this paper, we evaluated the skin permeability of lipophilic and hydrophilic model compounds in SMS2-KO mice to elucidate the effects of SMS2 on the barrier function of skin. Furthermore, we investigated the structure formed by the intercellular lipids in the SMS2-KO mouse SC to elucidate the role of SMS2 in the barrier function of the SC.

Skin cross-section image and epidermis thickness measurement in SMS2-KO mice.

The skin cross-sectional structure and epidermal thickness of SMS2-KO mice were not significantly different from those of WT mice (Fig. 1a, b).

Figure 1a and 1b

Skin permeability of sodium fluorescein (hydrophilic model compound) and Nile Red (lipophilic model compound) through full-thickness skin and stripped skin.

Permeation of the hydrophilic compound Fl-Na through skin of SMS2-KO mice was significantly higher than in WT mice (Fig. 2a). In addition, the flux of SMS2-KO mouse skin increased by ~2.5-fold when compared with that of WT mouse skin (Table 1). In contrast, permeation of the lipophilic compound Nile Red through the skin of SMS2-KO mice was similar to the permeation of Nile Red through the skin of WT mice (Fig. 2b). No difference in the amount of Fl-Na that permeated through stripped skin of both mice was observed, that is, the flux also did not differ significantly (Fig. 2c, Table 1).

Figure 2a, 2b and 2c

Table 1

Parameters of skin permeation through full-thickness and stripped skin.

Skin Type	Compounds (Molecular weight, LogKow)	Mouse Type	Flux (nmol/cm²/h)	Flux ratio (SMS2-KO/WT)
Full-thickness skin	Fl-Na (376.3, –1.52)	WT	0.16 ± 0.01	1.00
	Fl-Na (376.3, –1.52)	SMS2-KO	0.40 ± 0.06**	2.52
Full-thickness skin	Nile Red (318.4, 3.20)	WT	0.08 ± 0.01	1.00
	Nile Red (318.4, 3.20)	SMS2-KO	0.08 ± 0.02	0.94
Stripped skin	Fl-Na (376.3, –1.52)	WT	21.36 ± 3.05	1.00
	Fl-Na (376.3, –1.52)	SMS2-KO	24.41 ± 6.10	1.14

**, p < 0.01 vs WT mouse, Student’s t-test.

A new highly ordered lamellar structure of the SC observed by SAXS.

The lamellar structures in the SC were examined by SAXS (Fig. 3a, b). As shown in Fig. 3b, new sharp peaks denoted by K1 and K2 were observed for the SC of SMS2-KO mice, but as shown in Fig. 3a were not observed for the WT SC. These results suggest that in SMS2-KO mice a highly ordered lamellar structure with repetition period of 4.8 nm appeared.

Figure 3a and 3b

Ratio of 0.42- and 0.37-nm peak of hydrocarbon chain packing in the SC.

Peaks due to the hydrocarbon chain packing structures were observed in the wide-angle profiles (Fig. 4a, b). The peak was fitted with a Gauss function to calculate the peak intensity. The integrated intensity ratio (R_0.42/0.37) of the peaks at S = 2.4 nm^–1 (0.42 nm) and S = 2.7 nm^–1 (0.37 nm) for SMS2-KO mouse was 4.03, while the value for the WT mouse was 4.36. The R_0.42/0.37 was reduced significantly in the SMS2-KO mouse when compared with that of the WT mouse (Fig. 4c). This result indicates whether the SMS2-KO mouse have a large percentage of the ORTHO structure or the WT mice have a small percentage of the HEX structure.

Figure 4a, 4b and 4c

Temperature-dependent changes in the X-ray scattering peaks for the hydrocarbon chain packing structures of the SC.

In the wide-angle profiles of the SC from WT and SMS2-KO mice, the HEX and ORTHO peak appeared at 2.4 nm^–1, and the ORTHO peak appeared at 2.7 nm^–1. The ORTHO peak (2.7 nm^–¹) shifted to a slightly lower angle as the temperature increased, and the ORTHO structure transformed to

the high-temperature HEX structure at temperatures at ~45 °C in the WT sample and at ~50 °C in the SMS2-KO sample (Fig. 5a, b). Thus, the ORTHO structure transformed to the high-temperature HEX structure of the SMS2-KO sample at higher temperature. The peak at 2.4 nm^–1 disappeared at ~45 °C in the WT sample and at ~50 °C in the SMS2-KO sample (Fig. 5a, b). The peak which comes from the ORTHO appeared above ~50 °C shifted to a lower angle with increasing temperature and disappeared at 75 °C in the WT mouse sample, whereas the corresponding peak in the SMS2-KO mice sample disappeared at 79 °C. This indicates that the peaks derived from the SC hydrocarbon chain packing structures of SMS2-KO mice were present at higher temperatures when compared with that of WT mice. Thus, the peak representing the high-temperature HEX disappeared at higher temperatures in the SMS2-KO mice.

Figure 5a and 5b

In this paper, the effects of SMS2 activity on the barrier function and SC structure of skin were investigated. The skin conditions of WT and SMS2-KO mice were examined in advance, and genotypes of both mice were confirmed (Supplementary Information). We measured TEWL as an in vivo assessment of the skin barrier function. The TEWL of SMS2-KO mice increased significantly when compared with that of WT mice [13]. It has been known that patients with skin diseases such as atopic dermatitis and ichthyosis have been reported to have higher TEWL with reduced barrier function when compared with that of healthy subjects [3, 14, 15]. The present findings suggest that SMS2-KO mice developed a skin barrier dysfunction that is similar to that of atopic dermatitis and ichthyosis patients. Skin permeability of two compounds was evaluated using full-thickness and stripped skin. Fl-Na permeation and flux through the full-thickness skin of SMS2-KO mice increased significantly when compared with those of WT mice (Fig. 2a, Table 1). In contrast, there was no difference between SMS2-KO and WT mice for Nile Red permeation and flux (Fig. 2b, Table 1). There was also no difference in the amount of Fl-Na permeation and flux through stripped skin of both mice (Fig. 2c, Table 1). These results suggest that the polarity of the compound governs the permeability of the compound through SMS2-KO mouse skin. Generally, there are two routes of penetration into the skin: the skin appendage pathway, such as hair follicles and sweat glands, and the stratum corneum pathway [16, 17]. The stratum corneum pathway is classified into the intercellular lipid route and the transcellular route. It is well known that in many cases, lipophilic compounds penetrate the skin through the intercellular route and hydrophilic compounds through the transcellular route. The results presented herein indicate that SMS2 activity affects the water-soluble permeation route. Fick's first and second laws have been used to define an inverse relationship between skin thickness and the penetration rate of compounds. Microscopic examination of skin freeze sections from WT and SMS2-KO mice revealed that the morphology of the skin was not noticeably different (Fig. 1b). There was also no difference in the thickness of the epidermis between the WT and SMS2-KO mice (Fig. 1c). From these results, it is assumed that differences in the structure of the stratum corneum affected the permeability of the hydrophilic compounds.

To characterize this structural difference, we carried out a detailed structural analysis of the SC by SAXS and WAXS. The SAXS detected first- and second-order diffraction peaks K1 and K2 with a lattice constant of 4.8 nm in the lamellar structure of SMS2-KO mice, respectively, and both of them were not observed in the WT mouse SAXS profile. Hatta et al. reported that the short-period lamellar structure and the ORTHO are derived from to the same single-domain lamellar structure [18]. Our results suggest that the new lamellar structure is strongly associated with the short lamellar structure having the small spacing in which the water layer is very small, since the new lamellar structure is so close to the spacing without water. On the other hand, the new lamellar structure seems to be deficient in some components of intercellular lipids, therefore the lamellar structure cannot retain sufficient water molecules. As a result, this short-period lamellar structure may be dysfunctional and unable to regulate water content in the SC, although the very narrow layer might be associated with penetration of hydrophilic molecules. In the paper of Hatta et al. also suggests that the LIQUID is a major pathway for low molecular weight hydrophobic compounds [18]. This pathway might be concerned in penetration of the hydrophobic molecule in the WT mice and the SMS2-KO mice, since the LIQUID exists in both mice. The contribution of the LIQUID to the penetration of the compound is important, but the details are unknown and require further investigation.

The WAXS analysis revealed that SMS2-KO mice have significantly reduced R_0.42/0.37 values when compared with that of WT mice (Fig. 4). As mentioned above, this might come from the existence of the strong short period lamellar structure that is related to the high percentage of the ORTHO. While, the low percentage of the HEX is supported by the fact that might be prominent in model lipid membranes prepared with Cer and cholesterol, however the addition of free fatty acids suppresses the formation of the HEX [6, 19]. Thus, the lower percentage of the HEX in the SC of SMS2-KO mice when compared with that of WT mice may be because of low levels of Cer and Chol in the SC of SMS2-KO mice. These hypotheses require further examination, but it is possible that the decrease in NS may have resulted in a decrease in the HEX.

The thermodynamical properties of the SC phases were verified by temperature scanning X-ray diffraction. The peaks representing hydrocarbon chain packing structures in SMS2-KO mice was present at higher temperatures than that for the WT mouse sample (Fig. 5). This indicates that the hydrocarbon chain packing structures is highly ordered in SMS2-KO mice up to higher temperatures. However, in general, higher ordering of the hydrocarbon chain packing in the SC might give rise to a strong skin barrier against the hydrophobic components [20]. The present results indicate that the penetration of the hydrophobic compound took place in WT mice and SMS2-KO mice in the same mechanism proposed before, however the penetration of the hydrophilic compounds cannot be explained in this mechanism. One of the mechanisms of penetration enhancement of hydrophilic molecules might be explained by the fact that the highly ordered short period lamellar structure is responsible to penetrate hydrophilic molecules. In connection with the other mechanism, Forslind reported a domain mosaic model [21], arguing that the distribution of intercellular lipids in skin is not uniform and there are regions where particular lipids are localized and form domain structures. Forslind argued that each domain consisting of an ordered structure of hydrocarbon chains yields large distortion at their boundaries (domain boundaries) which has a disordered structure. This distorted region is the penetration pathway in intercellular lipids of the SC. In the SMS2-KO mouse SC, the highly ordered lamellar structure related the short lamellar structure was formed, as a result the penetration of the hydrophilic compounds is enhanced. Therefore, we hypothesize that the distortion between the highly ordered lamellar and the control lamellar structures plays an important role in the penetration enhancement of the hydrophilic compounds. Ceramides with short-chain fatty acids such as NS and AS in SMS2KO mice are reduced. Other cholesterol and fatty acid contents have not been measured and are unknown. Although the details are unknown, it is hypothesized that changes in this ratio are involved in the development of the highly ordered short lamellar structure. Several researchers have reported that the relationship between transdermal absorption and stratum corneum structure requires consideration of not only the crystalline structure but also the amorphous region [19, 22]. Future studies should include amorphous regions of intercellular lipids in stratum corneum. The findings in this paper provide a better understanding of barrier dysfunction in conditions with reduced ceramide content, such as atopic dermatitis and senile xeroderma.

Materials.

Sodium fluorescein (Fl-Na), Nile Red, 10% formalin solution, Mayer's hematoxylin solution and Eosin Y were obtained from Fuji Film Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Trypsin from bovine pancreas and trypsin inhibitor from Glycine max (soybean) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Glass capillaries (φ = 1.5 mm) were obtained from Toho Co., Ltd. (Tokyo, Japan).

Animals.

SMS2-KO mice with a C57BL/6 background were generated by homologous recombination using targeted vectors. The mice were kept under a 12-h light/dark cycle in a conventional animal room maintained at 22 ± 2°C and 55 ± 5% humidity. A total of 28 SMS-KO mice and 24 WT mice (Seven-to-nine-week-old male and female) were used for all experiments. All procedures were approved by the Ethics Committee of Josai University (approval Nos. 2013-4, 2017-7), and all methods were implemented in accordance with Josai University's relevant animal care and use guidelines and regulations (approval Nos. H25089, JU18095). In addition, P1A diffusion prevention measures were used in all experiments. All experimental were performed in accordance with the ARRIVE guidelines (http://arriveguidelines.org).

Hematoxylin eosin (HE) staining.

The skin was collected, cut into 5 ´ 10 mm pieces, placed in a plastic dish and embedded with the O.C.T. (optimal cutting temperature) compound (Sakura Finetech Japan Co., Ltd., Tokyo, Japan). Frozen tissue sections 10 µm thick were prepared by using a Leica CM3050 S cryostat (Leica Biosystems, Nußloch, Germany) at in-house temperature (CT) of − 25°C and sample table temperature (OT) of − 25°C. Thin sliced sections were immersed in a 10% formalin solution for 30 min and then immersed in the order of 100, 90, 80 and 70% ethanol for hydrophilic treatment. After washing with water, the sections were immersed in Mayer's hematoxylin solution for 6 min and washed with water. The sections were then immersed in Eosin Y solution containing acetic acid. For dehydration treatment, thin sliced sections were soaked in the order of 70, 80, 90 and 100% ethanol and then soaked in xylene for clearing. The sections were observed with an all-in-one fluorescence microscope (BZ-X710, Keyence Corporation, Osaka, Japan).

In vitro permeation study.

Full-thickness skin or stripped skin by the tape stripping technique was collected from WT and SMS2-KO mice. The modified Franz cells (effective permeation area: 1.77 cm²) were mounted with the epidermis on the donor side and the dermis on the receiver side. One-and-a-half milliliters 1 mM Fl-Na aqueous solution or 1 mM Nile Red in methanol was placed on the donor side, and 4.5 mL of PBS (–) solution or 50% methanol solution was placed on the receiver side. Receiver solutions were taken at 1, 2, 3, 4, 5 and 6 h of full thickness skin, or 10, 20, 30, 40, 50 and 60 min of stripped skin. The amount of Fl-Na (Ex: 495 nm/Em: 520 nm) or Nile Red (Ex: 553 nm/Em: 637 nm) that had permeated samples were measured using a microplate reader. The cumulative amount of permeation and the skin permeation rate (flux) were calculated.

Preparation of stratum corneum.

The mouse skin was peeled off, immersed in a PBS (–) solution containing 0.1% trypsin and incubated at 4°C for 21 h. The skin sample was then immersed at 37°C for a further 3 h. The reaction was stopped by immersing the skin sample in a PBS (–) solution of 0.1% trypsin inhibitor for 5 min. The SC was collected, washed in pure water and dried. The SC was stored in an environment where the humidity was controlled by a saturated aqueous solution of potassium sulfate (room temperature, relative humidity 97.6 ± 0.6%).

Small- and wide-angle X-ray scattering.

X-ray scattering experiments on the SC were carried out using the beam line BL40B2 (Structural Biology II) at a large synchrotron radiation facility (SPring-8, Sayocho, Hyogo, Japan). The SC samples were filled in glass capillaries, and small- and wide-angle X-ray scattering were measured simultaneously. An imaging plate was used as the detector. The measurement conditions were set as follows: reference material, silver behenate (AgBh); wavelength, 0.08 nm; X-ray energy, 15 keV; camera length, 550 mm; and exposure time, 30 s. The obtained intensity profile was plotted against the scattering vector S (nm^− 1) in the small-angle and wide-angle regions, where S = (2/λ) sin θ, λ is the X-ray wavelength and 2θ is the scattering angle. The observed wide-angle peaks were analyzed by fitting to a Gaussian function and a straight line. The peak intensity was calculated based on the analyzed results.

The X-ray scattering peaks of the hydrocarbon chain packing structure of intercellular lipids in the SC appeared at S = 2.4 nm^− 1 (0.42 nm) in the hexagonal and orthorhombic structures and at S = 2.7 nm^− 1 (0.37 nm) in the orthorhombic structure. The integrated intensity ratio (R_0.42/0.37) of the 0.41-nm peak and the 0.37-nm peak was calculated from the intensities obtained above. Further, the sample temperature was increased from 10 to 120°C at a rate of 2°C/min, and during heating the wide-angle X-ray scattering experiment was performed. The measurement conditions in this experiment were set as: reference material, silver behenate; wavelength, 0.07 nm; camera length, 2195 mm; exposure time, 5 s.

Statistical analysis.

Data are shown as the mean and standard deviation (mean ± S.D.) and statistically processed using JMP Pro version 13.0 (SAS Institute, Cary, NC, USA). For statistical tests, the Student's t-test was used after confirming equal variance by the F-test.

Funding

This research was supported by JSPS KAKENHI Grant Number JP18K06604 to Prof. Yoshihiro Tokudome.

Acknowledgements

The authors would like to thank Dr. Ken Watanabe (National Center for Geriatrics and Gerontology) and Prof. Tadashi Yamashita (Azabu University) for providing the SMS2-deficient mice.

The synchrotron radiation experiments were performed at the BL40B2 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2016B1357, 2017A1413, 2017B1315, 2017B1320, 2018A1473, 2018A1478, 2018B1254 and 2018B1257). We thank Edanz Group (https://en-author-services.edanz.com/ac) for editing a draft of this manuscript.

Author contributions

Designed research; NH, IH, TO and YT. Performed the experiments; NH and YT. Data analysis; NH. Project administration, supervision; YT. Writing- original draft; NH, IH and YT. Writing- review& editing; NH, IH, TO and YT. All authors have read and approved the final manuscript.

Data availability statement

The datasets used in this study are not publicly available due to the privacy policy of the data provider. However, the data are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Modulation of function and structure of stratum corneum in sphingomyelin synthase 2-deficient mice

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