DOI: 10.1111/exd.
12231
www.wileyonlinelibrary.com/journal/EXD
Effects of sphingoid bases on the sphingolipidome in early
keratinocyte differentiation
Alexander Sigruener1, Victoria Tarabin1, György Paragh1,3, Gerhard Liebisch1, Tim Koehler2, Mike Farwick2
and Gerd Schmitz1
1
Institute for Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center, Regensburg, Germany; 2Evonik Industries AG,
Essen, Germany; 3Department of Dermatology, Medical College of Wisconsin, Madison, WI, USA
Correspondence: Gerd Schmitz, MD, Institute for Clinical Chemistry and Laboratory Medicine, Regensburg University Medical Center,
Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany, Tel.: +49-941-9446201, Fax: +49-941-9446202, e-mail: gerd.schmitz@ukr.de
Abstract: Keratinocyte sphingolipids are structural elements of
epidermal permeability barrier and potential regulators of
epidermal functions. We tested the influence of sphingoid bases
sphinganine, sphingosine and phytosphingosine on in vitro
keratinocyte differentiation. Lipidomic and transcriptomic
analysis after treatment emphasizes sphinganine and
phytosphingosine as potent modulators of keratinocyte
differentiation and lipid metabolism. Sphinganine treatment
regulated differentiation and sphingolipid metabolism-related
genes, and also increased all major ceramide species. Sphingosine
treatment increased ceramide and phytoceramide pools without
changes in dihydroceramides. Phytosphingosine treatment
markedly increased phytoceramide pools without raising
ceramide or dihydroceramide levels. Sphinganine treatment
Background
Ex vivo models of keratinocytes mimic homeostatic function of
proliferating cells and terminal differentiation. Characteristic
changes include altered lipid composition, autophagic elimination
of lipid droplets and formation of sphingolipid-rich lamellar
bodies (Figure S1), requirements for stratum corneum formation,
which protects against mechanical and chemical stress and trans-
epidermal water loss (1–3). Ceramides are the major component
of the lipid barrier in the stratum corneum (4) and composed of
a sphingoid base (SpB), either sphinganine/dihydrosphingosine,
sphingosine, phytosphingosine or 6-hydroxysphingosine, and an
amide-linked fatty acid (FA) (5–8). Inhibition of lipid metabolism
and transport leads to improper barrier function (9–12). Sphingo-
lipids were shown to improve barrier function, dermatitis, acne
and photodamage (13–17).
Question addressed
The effects of sphingoid bases were analysed in a confluence-
induced in vitro keratinocyte differentiation model.
Experimental design
Sphingoid bases were administered at 25 lM for 24 h at 90%
confluence to a confluence-induced in vitro keratinocyte differenti-
ation model, and lipidomic and transcriptomic changes were
analysed (Data S1).
Results
Most abundant lipid classes on day 0 in our in vitro differentiation
model were glycerophospholipids (>50% of analysed lipids). Cera-
mide and hexosylceramide represented 1.08% and 0.20% of analy-
ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2013, 22, 656–681
Letter to the Editor
increased specifically very long chain ceramides essential for
intact barrier function. In summary, sphingoid bases, especially
sphinganine, promote differentiation and ceramide production in
keratinocytes. Free sphinganine may serve as a dermatological
and cosmetic agent by enhancing formation and maintenance of
an intact epidermal lipid barrier, with beneficial effects for skin
and hair care applications.
Abbreviations: FA, fatty acid; SpB, sphingoid base.
Key words: keratinocyte differentiation – phytosphingosine – sphinganine
– sphingolipid metabolism – sphingosine
Accepted for publication 20 August 2013
sed lipids in undifferentiated cells (Table 1). After 4 days
differentiation, total levels of ceramide, free bases and hexosylcera-
mide were markedly increased (Table 1). One day incubation with
SpBs resulted in highly elevated cellular SpBs levels (probably due
to uptake) and increased total ceramide levels (Table 1).
Influence of tested SpBs on intracellular sphingolipid species
was determined. Not surprisingly, SpBs treatment increased the
relative amounts of respective base containing ceramide, hexosyl-
ceramide and sphingomyelin (Tables S1A–C). Only sphinganine
increased all analysed lipid classes (Table S1A–C). The overall
ceramide and sphingomyelin species patterns were quite similar in
all analysed groups, except for sphingosine treatment, that strongly
increased 16:1 and especially 18:1 FA containing ceramide and
sphingomyelin species (Table S1A–C). Very long chain fatty acyl
ceramide species (≥C24) were found to be significantly increased
exclusively by sphinganine (Table S1A).
We focused on the regulation of genes involved in sphingolipid
metabolic processes during keratinocyte differentiation in our
recently published data set (18). The regulation of selected genes
during early differentiation of untreated control cells after four
compared with 1 day cultivation, and after 1 day of sphingolipid
compared with vehicle treatment, is summarized in the Table S2.
All SpBs showed substantial effects regarding induction of lipid
barrier gene expression. Noteworthy, sphinganine showed the
strongest influence on transcription. Gene ontology analysis of
specific sphinganine-regulated genes revealed that mainly pathways
related to keratinocyte differentiation were affected (Table S3).
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Letter to the Editor

Table 1. Lipid class distribution

Relative lipid Fold change Fold change Fold change Fold change
composition 0d (%) SD (%) 4d vs 0d SD DS vs Ctrl SD S vs Ctrl SD P vs Ctrl SD

PC 39.41 2.66 1.05 0.04 1.10 0.03 1.20 0.10 1.08 0.05
SM 13.56 2.40 1.15 0.22 1.02 0.04 1.05 0.09 1.07 0.14
DS-SM 2.29 0.87 1.06 0.22 1.30 0.23 1.40 0.19 1.08 0.07
PE 11.05 2.60 1.17 0.17 1.02 0.09 1.14 0.17 1.08 0.13
PS 6.87 1.58 1.16 0.13 1.00 0.08 1.18 0.16 1.10 0.10
LPC 0.65 0.19 1.07 0.20 1.03 0.11 1.18 0.41 1.09 0.11
Cer 1.08 0.27 1.93 0.44 1.98 0.33 1.82 0.75 1.43 0.42
HexCer 0.20 0.10 2.20 0.72 1.50 0.15 1.69 0.58 1.41 0.30
SpB*100 0.04 0.01 1.43 0.33 96.22 58.46 179.45 169.97 39.87 23.76
CE 2.62 1.37 1.43 0.18 1.00 0.19 1.03 0.28 1.14 0.13
FC 22.22 3.84 1.29 0.08 1.02 0.07 1.05 0.19 1.04 0.06

Lipid composition of keratinocytes on day 0 expressed as % of the analysed lipids and fold changes in lipid class distribution in control cells [4d] vs [0d] and after 1 day
sphingoid base treatment. Significant changes (P < 0.05) are indicated in bold. n = 3. CE, cholesteryl ester; Cer, ceramide; DS, sphinganine; DS-SM, dihydrosphingomy-
elin; FC, free cholesterol; HexCer, hexosylceramide; LPC, lysophosphatidylcholine; P, phytosphingosine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phos-
phatidylserine; S, sphingosine; SD, standard deviation; SM, sphingomyelin; SpB, sphingoid bases.

Moreover, an upregulation of sphingolipid metabolism genes in
the microarray data by sphinganine and phytosphingosine bases
emphasizes the positive effect of these bases on barrier formation
(Table S2).
Conclusions
To date, no information is available on the internalization of SpBs
and the changes in the sphingolipid profile of cultured keratino-
cytes. A comprehensive study from our group describes the effect
of sphingolipid molecules on keratinocyte differentiation, with
focus on transcriptional changes (18,19).
Here, we focused on the effect of exogenous SpBs treatment on
lipid composition and gene expression during the early stages of
keratinocyte terminal differentiation. Keratinocytes were found to
incorporate exogenous sphingosine, sphinganine and phyto-
sphingosine, as reflected by drastically increased base levels after
exposure to the molecules (Table 1). We demonstrated that in par-
ticular treatment with sphinganine induced ceramide synthesis,
increasing all measured ceramide classes including dihydrocera-
mides and phytoceramides (Table S1A). Moreover, sphinganine
elicits the strongest response on overall sphingolipid metabolism
and keratinocyte differentiation, as reflected by microarray analysis
(Table S2). As the balance between all naturally occurring epidermal
ceramides is important for intact barrier formation (20), the posi-
tive influence of exogenous sphinganine on total ceramide produc-
tion may be valuable for dermatological or cosmetic applications.
Phytosphingosine showed distinct effects. Compared with the
broad activity of sphinganine, phytosphingosine specifically
increases phytoceramide pools (Table S1A). This superior stimula-
tory effect on phytoceramide levels (Table S1A) is noteworthy, as
phytoceramides are known to be particularly reduced in condi-
tions leading to dry and atopic skin (21,22). Similarly to sphinga-
nine, phytosphingosine had substantial positive effects on
induction of sphingolipid gene expression, although the effects
were less pronounced (Table S2).
Sphingosine led to a moderate induction of ceramide and phy-
toceramide levels, but had no impact on dihydroceramide levels
(Table S1A). The upregulation of ceramide synthase expression in
the microarray data further supported the results of lipid analysis
(Table S2).
Epidermal ceramides not only contain a unique variety of SpBs,
but their FA chain length makes them indispensable for the
maintenance of the intact epidermal lipid barrier (23,24). We
found that very long chain ceramide species specifically increased
upon sphinganine treatment (Table S1A). Consistently, certain
elongase genes were upregulated after sphinganine treatment
(Table S2). Deficient elongase activity in mice was shown to lead
to severe skin permeability barrier defects (24,25); deficient elong-
ase activity can also be suspected in atopic dermatitis (26). There-
fore, sphinganine-induced elongase activity may serve as a
therapeutic tool.
In spite of clear similarities, apoptosis and terminal differentia-
tion of epidermal keratinocytes are considered separate processes
following distinct regulatory mechanisms (27–29). Previously,
sphingolipids have been shown to induce differentiation and/or
apoptosis (30). Here, gene ontology analysis of sphinganine-
specific-regulated genes revealed that mainly pathways related to
keratinocyte differentiation were affected (Table S3).
In summary, exogenous SpBs differentially influence the forma-
tion of lipid barrier by inducing the production of specific subsets
of ceramide species. Phytosphingosine had a superior stimulatory
effect on the phytoceramide pool, while sphinganine stimulated
total ceramide production. In particular, the synthesis of long
chain ceramide was induced. Our results suggest that phyto-
sphingosine as well as sphinganine may be valuable active com-
pounds with distinct benefits for the maintenance and repair of
barrier function, and therefore in dermatologic or cosmetic skin
and hair care applications.
Acknowledgements
AS wrote the manuscript. VT and GP performed the study and analysed
the data. GL performed the lipid analyses. TK and MF contributed the
compounds and designed the study. GS designed the study, reviewed
the results and wrote the manuscript. All authors revised the final form of
the manuscript. The authors thank Susanne Ohmayer for her contribution
and Harry Isslinger for his excellent technical help.
Funding sources
This work was supported by grants from Evonik Industries AG, the Euro-
pean Community’s Seventh Framework Programme (FP7/2007-2013) under
grant agreement n 202272, IP-Project LipidomicNet and BMBF (‘SysMBo’,
sponsorship number 0315494C).
Conflict of interests
Compounds and part of the funding were provided by Evonik Industries
AG.

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678 Experimental Dermatology, 2013, 22, 656–681
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Letter to the Editor

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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Model of lipid metabolic processes in
differentiating keratinocytes.
Table S1. Distribution of sphingolipid species in
human keratinocytes after sphingoid base treatment.
Table S2. Regulation of selected genes by sphingoid
bases.
Table S3. Selected biological processes to which
sphinganine-specific genes could be dedicated in Gen-
omatix Pathway System analysis.
Data S1. Material and methods.

DOI: 10.1111/exd.12233
www.wileyonlinelibrary.com/journal/EXD
Letter to the Editor

Deletion of the activating NKG2C receptor and a functional

polymorphism in its ligand HLA-E in psoriasis susceptibility
Xue Zeng1,2, Haoyan Chen1,3, Rashmi Gupta1, Oscar Paz-Altschul1, Anne M. Bowcock4 and Wilson Liao1
1
Department of Dermatology, University of California San Francisco, San Francisco, CA, USA; 2Department of Dermatology, Guang’anmen
Hospital, China Academy of Chinese Medical Sciences, Beijing, China; 3Department of Gastroenterology, Ren Ji Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai Institute of Digestive Diseases, Shanghai, China; 4Cancer Genomics and National Heart and Lung
Institute, Imperial College, London, UK
Correspondence: Wilson Liao, Department of Dermatology, University of California San Francisco, San Francisco, CA, USA, Tel.: 415-476-8364,
Fax: 415-476-8837, e-mail: LiaoWi@derm.ucsf.edu

Abstract: Psoriasis is an inflammatory, immune-mediated disease
of the skin. Several studies have suggested that natural killer
(NK) cells and their receptors may be important for its
pathogenesis. Here, we examined whether deletion of the
activating natural killer receptor gene NKG2C, which has a
frequency of 20% in the European population, was associated
with psoriasis susceptibility. The NKG2C deletion and a
functional polymorphism in its ligand HLA-E were genotyped in
a Caucasian cohort of 611 psoriasis cases and 493 controls. We
found that the NKG2C deletion was significantly increased in
cases compared with controls [0.258 vs 0.200, P = 0.0012,
OR = 1.43 (1.15–1.79)]. The low-expressing HLA-E*01:01 allele
was associated with psoriasis (P = 0.0018), although this
association was dependent on HLA-C. Our findings support a
potential immunoregulatory role for NK cells in psoriasis and
suggest the importance of future studies to investigate the
contribution of NK cells and their regulatory receptors to the
pathogenesis of psoriasis.
Key words: HLA-E – KLRC2 – natural killer – NKG2C – psoriasis
Accepted for publication 21 August 2013

Background (4). Moreover, the psoriasis susceptibility gene HLA-C*06:02 con-

Psoriasis is a common chronic inflammatory skin disease affecting
2–3% of the population. Both T cells (1) and keratinocytes (2) are
thought to play a central role in the initiation and maintenance of
psoriasis. Natural killer (NK) cells may also play a role in the
pathogenesis of psoriasis (3). NK cells in psoriatic lesional skin
secrete excessive amounts of the Th1 cytokine interferon gamma
tains the C2 epitope which binds the activating NK cell receptor
KIR2DS1, which has been genetically associated with psoriasis (5–
7). Recently, we found that another activating NK cell receptor,
KIR3DS1, was associated with psoriasis (8). The work of Gladman
and others has described a role for the interaction of KIR recep-
tors with HLA in psoriatic arthritis (9). Thus, several studies have

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Experimental Dermatology, 2013, 22, 656–681 679