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Phytosphingosine enhances moisture level in human skin barrier through stimulation of the filaggrin biosynthesis and degradation leading to NMF formation

DOI:10.1007/s00403-017-1782-8
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Hyun Kyung Choi
Hyun Kyung Choi
Hyun Kyung Choi
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Young Hoon Cho
Young Hoon Cho
Young Hoon Cho
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Eun Ok Lee
Eun Ok Lee
Eun Ok Lee
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Jin Wook Kim
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Abstract and Figures

Phytosphingosine (PHS) is a sphingoid that is a key component of phytoceramides NP, AP and EOP. PHS has been known to have anti-inflammation and antimicrobial activities and to stimulate epidermal differentiation. In addition, it is reported that PHS treatment notably increased phytoceramide content in keratinocytes. In this study, we tried to investigate whether PHS has any effect on the maturation of corneocytes such as formation of cornified envelope and natural moisturizing factor (NMF) that is also an essential event during the formation of skin barrier, stratum corneum. Special focus was made on the filaggrin (FLG) metabolism that is directly responsible for NMF production. PHS increased the expression of essential keratinocyte differentiation genes such as involucrin and transglutaminase 1 in cultured human keratinocytes. Interestingly, the expressions of FLG, caspase 14 and bleomycin hydrolase, all of which involved in NMF production in corneocytes, were significantly induced by PHS treatment in vitro. The effect of PHS on FLG metabolism was manifested as the increase of pyrrolidone carboxylic acid and skin hydration in vivo human skin. Results showed PHS had skin moisturizing effect by modulating FLG metabolic pathways and suggested to be an essential role in coordinated formation of the corneocyte envelope and NMF within.
PHS increases the expression of TGase-1 and FLG in cultured keratinocytes. Cultured human epidermal keratinocytes were treated with 1, 5 μM of PHS and 1.2 mM calcium. Calcium was used as a positive control for keratinocyte differentiation. The protein expression of TGase-1 (a) and FLG (c) was evaluated by immunoblotting. The relative mRNA level of TGase-1 (b) and pro-FLG (d) was evaluated by quantitative RT-PCR. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
PHS increases the expression of TGase-1 and FLG in cultured keratinocytes. Cultured human epidermal keratinocytes were treated with 1, 5 μM of PHS and 1.2 mM calcium. Calcium was used as a positive control for keratinocyte differentiation. The protein expression of TGase-1 (a) and FLG (c) was evaluated by immunoblotting. The relative mRNA level of TGase-1 (b) and pro-FLG (d) was evaluated by quantitative RT-PCR. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
… 
Immunohistochemical staining of FLG and INV using a 3D human skin epidermal equivalent. a, d H&E staining, b, e INV, c, f FLG immunohistochemical staining. a–c Untreated control and d–f 10 μM PHS was topically applied. After incubation for 48 h, H&E staining and immunohistochemistry were conducted to analyze the FLG and INV expression
Immunohistochemical staining of FLG and INV using a 3D human skin epidermal equivalent. a, d H&E staining, b, e INV, c, f FLG immunohistochemical staining. a–c Untreated control and d–f 10 μM PHS was topically applied. After incubation for 48 h, H&E staining and immunohistochemistry were conducted to analyze the FLG and INV expression
… 
Effects of PHS on the expression FLG-degrading enzymes in keratinocytes. Human epidermal keratinocytes were treated with PHS at the indicated concentration for 72 h. The relative mRNA level of caspase 14 was evaluated by RT-PCR (a). β-Actin was used as a loading control. Cellular proteins were prepared and protein expression of caspase 14 (b) and BLMH (c) was assessed by western blotting. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
Effects of PHS on the expression FLG-degrading enzymes in keratinocytes. Human epidermal keratinocytes were treated with PHS at the indicated concentration for 72 h. The relative mRNA level of caspase 14 was evaluated by RT-PCR (a). β-Actin was used as a loading control. Cellular proteins were prepared and protein expression of caspase 14 (b) and BLMH (c) was assessed by western blotting. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
… 
The effects of PHS on the epidermal barrier functions in human skin. Subject’s forearms were treated with CON (0.5% lactic acid solution) or PHS (1% phytosphingosine in 0.5% lactic acid solution) for 2 weeks. a TEWL and b SC hydration, respectively. UNT untreated control. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
The effects of PHS on the epidermal barrier functions in human skin. Subject’s forearms were treated with CON (0.5% lactic acid solution) or PHS (1% phytosphingosine in 0.5% lactic acid solution) for 2 weeks. a TEWL and b SC hydration, respectively. UNT untreated control. The data were expressed as mean ± SEM (*p < 0.05, **p < 0.01)
… 
The quantity of PCA and trans-UCA in PHS-treated human skin. After 2 weeks of topical application of CON (0.5% lactic acid solution) or PHS (1% phytosphingosine in 0.5% lactic acid solution), PCA (a) and trans-UCA (b) was determined by HPLC. UNT untreated control. The data were expressed as mean ± SEM (*p < 0.05)
The quantity of PCA and trans-UCA in PHS-treated human skin. After 2 weeks of topical application of CON (0.5% lactic acid solution) or PHS (1% phytosphingosine in 0.5% lactic acid solution), PCA (a) and trans-UCA (b) was determined by HPLC. UNT untreated control. The data were expressed as mean ± SEM (*p < 0.05)
… 
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Vol.:(0123456789)
1 3
Arch Dermatol Res (2017) 309:795–803
DOI 10.1007/s00403-017-1782-8
ORIGINAL PAPER
Phytosphingosine enhances moisture level inhuman skin barrier
throughstimulation ofthefilaggrin biosynthesis anddegradation
leading toNMF formation
HyunKyungChoi1· YoungHoonCho1· EunOkLee2· JinWookKim2·
ChangSeoPark1
Received: 8 March 2017 / Revised: 7 September 2017 / Accepted: 13 September 2017 / Published online: 21 September 2017
© Springer-Verlag GmbH Germany 2017
Keywords Phytosphingosine· Filaggrin· Natural
moisturizing factor· Pyrrolidone carboxylic acid· Skin
barrier
Introduction
The epidermis provides physical and physiological protec-
tive functions such as permeability barrier to prevent water
loss from inside and invasion of harmful external factors
such as chemicals, microbial pathogens, allergens and UV
radiation [4, 9, 12]. The skin barrier function is located in the
outermost layer of the epidermis, the stratum corneum (SC),
which consists of corneocytes and intercellular lipid lamellar
organization. These two structural compartments have been
depicted as ‘bricks and mortar model’ [10, 32, 35]. Formation
of normal corneocytes (bricks) requires two separate events
to be completed: first, maturation of cornified envelope (CE)
that replaces cell membrane of viable keratinocytes with the
aggregates of proteins such as involucrin and loricrin tightly
cross-linked each other by transglutaminase-1 (TGase-1).
Subsequently, CE becomes corneocyte lipid envelope (CLE)
through covalent attachment of ω-hydroxyceramide. Second,
anucleated corneocytes, flattened by the compressive action
of filaggrin (FLG) on keratin intermediate filaments (KIF),
should be filled with natural moisturizing factors (NMF) such
as PCA, UCA and other amino acids derived from degrada-
tion of FLG through hydrolyzing processes involved calpain
1, caspase 14 and bleomycin hydrolase (BLMH) [4, 11, 40].
The importance of CE maturation has been well documented.
TGase-1 is essential to maintain epidermal homeostasis and
healthy skin conditions [4, 19]. Rawlings etal. have reported
that reduced TGase activity can lead to immaturity of CEs and
this has been specially represented in dry skin [8]. Further-
more, dysregulated differentiation process is closely connected
Abstract Phytosphingosine (PHS) is a sphingoid that is a
key component of phytoceramides NP, AP and EOP. PHS
has been known to have anti-inflammation and antimicro-
bial activities and to stimulate epidermal differentiation. In
addition, it is reported that PHS treatment notably increased
phytoceramide content in keratinocytes. In this study, we
tried to investigate whether PHS has any effect on the matu-
ration of corneocytes such as formation of cornified enve-
lope and natural moisturizing factor (NMF) that is also an
essential event during the formation of skin barrier, stratum
corneum. Special focus was made on the filaggrin (FLG)
metabolism that is directly responsible for NMF produc-
tion. PHS increased the expression of essential keratinocyte
differentiation genes such as involucrin and transglutami-
nase 1 in cultured human keratinocytes. Interestingly, the
expressions of FLG, caspase 14 and bleomycin hydrolase,
all of which involved in NMF production in corneocytes,
were significantly induced by PHS treatment invitro. The
effect of PHS on FLG metabolism was manifested as the
increase of pyrrolidone carboxylic acid and skin hydration
invivo human skin. Results showed PHS had skin mois-
turizing effect by modulating FLG metabolic pathways and
suggested to be an essential role in coordinated formation of
the corneocyte envelope and NMF within.
* Chang Seo Park
dgucsp@dongguk.edu
1 Department ofChemical andBiochemical Engineering,
Dongguk University, 3-26, Pil-dong, Chung-gu,
Seoul100-715, RepublicofKorea
2 LCS Biotech, Seodun-dong 103-2, Gwonseon-gu, Suwon-si,
Gyeonggi-do441-857, RepublicofKorea
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
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Full-text available
Background Maintenance of water balance in the stratum corneum (SC) is determined by the content of intercellular lipids and natural moisturizing factors (NMFs) in corneocytes.AimTo investigate the association between the NMFs and (pro)filaggrin and the proteases responsible for the processing of (pro)filaggrin to NMFs in the SC of hydrated and dry skin areas of healthy human subjects.Methods The SC hydration state and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter, respectively. Proteases, (pro)filaggrin and NMFs were extracted from SC samples obtained by tape-stripping of the tested skin. Expression levels of (pro)filaggrin were determined by dot blotting and western blotting, and total NMFs by ultra-high performance liquid chromatography. Expression of the proteases caspase-14, calpain-1 and bleomycin hydrolase was measured by western blotting.ResultsThe levels of (pro)filaggrin were not significantly different between hydrated and dry skin, whereas the level of total NMFs was significantly reduced in dry skin. A negative correlation between (pro)filaggrin and NMFs was found in dry skin (Pearson correlation coefficient r = − 0.57, *P < 0.05). Bleomycin hydrolase expression was significantly decreased in the SC of dry skin.Conclusions These results suggest that the low hydration state of dry skin may be due to the reduction in (pro)filaggrin degradation caused by decreased bleomycin hydrolase expression.
Phytosphingosine, sphingosine and dihydrosphingosine ceramides in model skin lipid membranes: permeability and biophysics
Article
Ceramides based on phytosphingosine, sphingosine and dihydrosphingosine are essential constituents of the skin lipid barrier that protects the body from excessive water loss. The roles of the individual ceramide subclasses in regulating skin permeability and the reasons for C4-hydroxylation of these sphingolipids are not completely understood. We investigated the chain length-dependent effects of dihydroceramides, sphingosine ceramides (with C4-unsaturation) and phytoceramides (with C4-hydroxyl) on the permeability, lipid organization and thermotropic behavior of model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesteryl sulfate. Phytoceramides with very long C24 acyl chains increased the permeability of the model lipid membranes compared to dihydroceramides or sphingosine ceramides with the same chain lengths. Either unsaturation or C4-hydroxylation of dihydroceramides induced chain length-dependent increases in membrane permeability. Infrared spectroscopy showed that C4-hydroxylation of the sphingoid base decreased the relative ratio of orthorhombic chain packing in the membrane and lowered the miscibility of C24 phytoceramide with lignoceric acid. The phase separation in phytoceramide membranes was confirmed by X-ray diffraction. In contrast, phytoceramides formed strong hydrogen bonds and highly thermostable domains. Thus, the large heterogeneity in ceramide structures and in their aggregation mechanisms may confer resistance towards the heterogeneous external stressors that are constantly faced by the skin barrier.
A fundamental investigation into aspects of the physiology and biochemistry of the stratum corneum in subjects with sensitive skin
Article
  • Apr 2016
Background: Sensitive skin is a poorly understood skin condition. Defects in stratum corneum (SC) barrier function and/or extrasensory neuronal networks in the epidermis are believed to be involved in the problem. Objectives: This study aimed to unravel the relationships between bleomycin hydrolase (BH) and calpain-1 (C-1), pyrrolidone carboxylic acid (PCA) levels, corneocyte maturation, transglutaminase (TG) and plasmin activities on the cheeks of subjects with sensitive skin. Methods: Forty-eight female Caucasian subjects, Fitzpatrick skin phototypes II-III, with self - perceived sensitive facial skin were assessed and underwent a capsaicin reactivity test. Expert grading of skin condition was conducted as well as measurement of transepidermal water loss (TEWL), skin capacitance, SC cohesion and SC integrity. BH, C-1 and plasmin activities were measured as well as PCA levels, plasmin and TG activity. Differential Nile red and involucrin immunostaining was performed to assess corneocyte maturation and size. Results: 52% of the subjects reacted to capsaicin. There were no significant differences between the capsaicin-sensitive and non-capsaicin-sensitive subjects with reference to skin grading, TEWL, skin capacitance and SC cohesion. PCA levels and BH activity were lowest in the capsaicin-sensitive panel (p<0.05) and were correlated in non-capsaicin-sensitive subjects (r = 0.72). The activity of TG was significantly lower (48%) in the capsaicin-sensitive subjects (p<0.001) and their corneocytes were less mature and smaller (p ≤ 0.03). SC was estimated to be thinner (6.87 ± 0.28 vs. 8.68 ± 0.26 μm; p=0.001) in the capsaicin-sensitive subjects with a corresponding shorter SC path length (83.2± 4.4 μm and. 113.1 ± 4.5 μm; p=0.001). Conclusions: Despite the physiological similarities between the two groups of sensitive skin subjects, differences in their biochemistry were clearly evident. Lower levels of PCA, BH and TG activities together with a greater number of smaller and immature corneocytes indicate inferior SC maturation in the capsaicin-sensitive subjects. The reduced maturation of corneocytes and thinner SC likely contributes to a greater penetration of capsaicin and the associated increased skin sensitivity. This article is protected by copyright. All rights reserved.
Variation in the activities of late stage filaggrin processing enzymes, calpain-1 and bleomycin hydrolase, together with pyrrolidone carboxylic acid levels, corneocyte phenotypes and plasmin activities in non-sun exposed and sun-exposed facial stratum corneum of different ethnicities
Article
  • Mar 2016
Background: Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin (FLG) processing enzymes in facial stratum corneum (SC) is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. Methods: Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and Albino African (AA) female subjects living in South Africa. Results: PCA concentration levels were of the order AA > BA > C subjects and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA) whereas in the AA subjects the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects but the activity was low in the BA subjects, In both sites. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. Conclusion: Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest that they can be used as markers for epidermal photodamage. This article is protected by copyright. All rights reserved.
Stimulation of PPARα normalizes the skin lipid ratio and improves the skin barrier of normal and filaggrin deficient reconstructed skin
Article
Background: Therapeutic options for atopic dermatitis mostly address the symptoms but causal therapies are still missing. Peroxisome proliferator activated receptor (PPAR) agonists exert beneficial effects in patients suffering this disease, whereas the stimulation of PPARα and γ seemed most promising. Objectives: To elucidate the effects of the PPARα specific agonist WY14643, the PPARγ agonist ciglitazone, and the dual PPARα+γ agonist docosahexaenoic acid (DHA) on the homeostasis and barrier function of filaggrin deficient skin. Methods: The effects of the PPAR agonists on skin differentiation were evaluated via qPCR, Western blot, histological or immunofluorescence staining. Skin lipid organization was determined by ATR-FTIR and lipid composition was analyzed by HPTLC. Ultimately, the skin barrier function was assessed by skin absorption studies using the radioactively labeled compound testosterone. Results: Significant upregulation of filaggrin after DHA and WY14643 supplementation, but no effect of ciglitazone, on protein and mRNA level was detected. DHA and WY14643, but not ciglitazone, normalized the molar ratio of the main skin barrier lipids to 1:1:1 (free fatty acids:ceramides:cholesterol). Furthermore, DHA and WY14643 supplementation normalized the skin lipid profile in filaggrin deficient skin, but only WY14643 significantly improved the skin barrier function. Conclusion: Supplementation particularly with the PPARα agonist WY14643 improved the homeostasis and barrier function of filaggrin deficient skin models by normalization of the free fatty acid profile underlining the potential of PPAR agonists for the treatment of filaggrin-associated skin diseases.
Candi E, Schmidt R, Melino GThe cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol 6:328-340
Article
  • Apr 2005
The epidermis functions as a barrier against the environment by means of several layers of terminally differentiated, dead keratinocytes - the cornified layer, which forms the endpoint of epidermal differentiation and death. The cornified envelope replaces the plasma membrane of differentiating keratinocytes and consists of keratins that are enclosed within an insoluble amalgam of proteins, which are crosslinked by transglutaminases and surrounded by a lipid envelope. New insights into the molecular mechanisms and the physiological endpoints of cornification are increasing our understanding of the pathological defects of this unique form of programmed cell death, which is associated with barrier malfunctions and ichthyosis.
Keratinocyte differentiation and upregulation of ceramide synthesis induced by an oat lipid extract via the activation of PPAR pathways
Article
Activation of peroxisome proliferator-activated receptors (PPARs) has been shown to have an important role in skin barrier function by regulating differentiation and lipid synthesis in keratinocytes. Oat (Avena sativa) has long been used as a soothing agent to relieve skin irritations and the clinical benefits of topical oat formulations have been proven; however, the mechanistic understanding of oat's mode of action remains unknown. We investigated whether an oat lipid extract could activate PPARs and subsequently increase epidermal lipid synthesis and differentiation markers. Primary human epidermal keratinocytes and transformed cell lines were treated with PPAR agonists and oat lipid extracts to investigate the PPAR agonism. PPAR target genes and epidermal differentiation markers were analyzed by using quantitative real time PCR and HPTLC analysis. Oat lipid extract demonstrated robust dual agonism for PPARα and β/δ, and increased direct PPAR target gene induction in primary human keratinocytes. In addition, oat oil treatment increased both receptor expression and, consistent with the literature on PPARs, oat oil treatment resulted in a significant up-regulation of differentiation genes (Involucrin, SPRRs and transglutaminse-1) and ceramide processing genes (β-glucocerebrosidase, sphingomyelinases 3 and ABCA12). Further, oat oil treatment in keratinocytes significantly increased ceramide levels (70%), suggesting a functional translation of PPAR activation by oat oil in keratinocytes. Taken together, these results demonstrate that oat lipids possess robust dual agonistic activities for PPARα and β/δ, increase their gene expression, and induce differentiation and ceramide synthesis in keratinocytes, which can collectively improve skin barrier function.This article is protected by copyright. All rights reserved.
Various peroxisome proliferator-activated receptor (PPAR)-γ agonists differently induce differentiation of cultured human keratinocytes
Article
Peroxisome proliferator-activated receptors (PPARs) are potentially useful for treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti-inflammatory effects and improve barrier function. We examined five PPAR-γ agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin-II receptor blocker (telmisartan), for their ability to up-regulate filaggrin and loricrin expression at both mRNA and protein levels in cultured normal human keratinocytes (NHKs). Troglitazone, rosiglitazone, pioglitazone and telmisartan significantly increased filaggrin expression at both mRNA and protein levels in calcium-induced differentiated NHKs. Rosiglitazone and pioglitazone, but not troglitazone nor telmisartan, also significantly increased loricrin expression at both mRNA and protein levels in differentiated NHKs. These effects were not found in undifferentiated NHKs nor differentiated NHKs treated with ciglitazone. This study revealed differential effects of various PPAR-γ agonists on epidermal differentiation, and the most potent of those are rosiglitazone and pioglitazone.This article is protected by copyright. All rights reserved.