Tài liệu Báo cáo khoa học: Role of ceramide kinase in peroxisome proliferatoractivated receptor beta-induced cell survival of mouse keratinocytes ppt

Role of ceramide kinase in peroxisome proliferatoractivated receptor beta-induced cell survival of
mouse keratinocytes
Kiyomi Tsuji1, Susumu Mitsutake2, Urara Yokose2, Masako Sugiura3, Takafumi Kohama4 and
Yasuyuki Igarashi1,2
1
2
3
4

Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan
Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
Biological Research Laboratories II, Daiichi-Sankyo Co. Ltd., Tokyo, Japan
Exploratory Research Laboratories I, Daiichi-Sankyo Co. Ltd., Tokyo, Japan

Keywords
cell survival; ceramide; ceramide
1-phosphate; CerK; PPARb
Correspondence
Y. Igarashi, Laboratory of Biomembrane and
Biofunctional Chemistry, Faculty of
Pharmaceutical Sciences and Faculty of
Advanced Life Sciences, Hokkaido
University, Nishi 6, Kita 12, Kita-ku, Sapporo
060-0812, Japan
Fax: +81 11 706 4986
Tel: +81 11 706 3970
E-mail:
Website: />english/index.html
(Received 5 April 2008, revised 26 May
2008, accepted 29 May 2008)
doi:10.1111/j.1742-4658.2008.06527.x

Ceramide (Cer) is known to be a lipid mediator in apoptosis and to have
an important role in cell fate, via control of intracellular Cer levels.
Recently, ceramide kinase (CerK) was identified as an enzyme that converts
Cer to ceramide 1-phosphate (C1P). We examined potential functions of
CerK in the regulation of keratinocyte survival, and the possible involvement of peroxisome proliferator-activated receptor beta (PPARb). PPARb
is known to be a nuclear receptor acting as a ligand-inducible transcription
factor and has been implicated in the control of keratinocyte survival. In
the mouse keratinocyte cell line SP1, serum starvation induced cell death
and the accumulation of intracellular Cer, an apoptotic event. However,
apoptosis was inhibited by activation of PPARb. Interestingly, activation
of PPARb enhanced the mRNA expression of CerK and CerK activity.
Furthermore, the cell survival effect of PPARb was greatly diminished in
keratinocytes isolated from CerK-null mice. Chromatin immunoprecipitation revealed that, in vivo, PPARb binds to the CerK gene via a sequence
located in the first intron. Electrophoretic mobility-shift assays confirmed
that PPARb associates with this sequence in vitro. These findings indicated
that CerK gene expression was directly regulated by PPARb. In conclusion, our results demonstrate that PPARb-mediated upregulation of CerK
gene expression is necessary for keratinocyte survival against serum starvation-induced apoptosis.

Ceramide (Cer) has been implicated in various cellular
processes including proliferation, apoptosis and cell
signaling [1]. Intracellular Cer levels are strictly regulated by several enzymes, including ceramide kinase
(CerK), which converts Cer to ceramide 1-phosphate
(C1P) [2]. Previous studies have suggested that CerK
and C1P are involved in many cell functions, including

membrane fusion, phagocytosis and degranulation in
mast cells, among others [3]. Recently, several studies
have established a function for CerK in cell growth
and apoptosis. For example, in Arabidopsis plants,

mutation of CerK was associated with an accumulation of Cer and enhanced symptoms during pathogen
attack [4]. In addition, in mammalian cells such as

Abbreviations
ABC, ATP-binding cassette; C1P, ceramide 1-phosphate; Cer, ceramide; CerK, ceramide kinase; ChIP, chromatin immunoprecipitation;
EMSA, electrophoretic mobility shift assays; LD, L-165,041; PI, propidium iodide; PPARb, peroxisome proliferator-activated receptor beta;
PPRE, PPAR response element; RXR, retinoid X receptor; TEWL, transepidermal water loss.

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Role of CerK in PPARb-induced keratinocyte survival

K. Tsuji et al.

NIH 3T3 fibroblasts and A549 lung cancer cells, treatment with exogenous C1P at low concentrations
enhanced cell survival, whereas high concentrations of
C1P reduced cell survival and enhanced apoptosis
induced by serum starvation [5]. Although these previous reports suggest that CerK and C1P are involved in
the regulation of cell survival or cell proliferation, the
molecular mechanisms involved remain largely
unknown.
Peroxisome proliferator-activated receptors (PPARs)
are members of the nuclear hormone receptor superfamily. PPARs form heterodimers with the retinoid
X receptor (RXR) in a ligand-dependent manner.
Together, these heterodimers regulate the expression of
target genes by binding to their PPAR response elements (PPREs) [6,7]. Three subtypes of PPARs have
been identified, PPARa ⁄ NR1C1, PPARc ⁄ NR1C3 and

PPARb ⁄ PPARd ⁄ NR1C2, each with reportedly unique
tissue distribution and distinct cellular functions in
lipid metabolism, diabetes, inflammation and tumor
progression [8]. PPARb is ubiquitously expressed and
has been implicated by recent studies in tumor-promotion in various cell types [9]. PPARb has also been
suggested to have an important role in skin wound
healing. After a skin injury, PPARb expression is rapidly elevated in the epidermis at the wound edges; deletion of a single PPARb allele results in delayed wound
healing [10]. Furthermore, inflammation-induced apoptosis was found to be enhanced in keratinocytes
isolated from PPARb-null mice [11]. These studies suggest a role for PPARb in the regulation of keratinocyte
survival and apoptosis. Interestingly, the promoter
activity of PPARb was reported to be increased by the
treatment of exogenous Cer [11]. However, any interactions between PPAR and sphingolipids remain to be
further defined.
Skin epidermis contains abundant lipids, including
cholesterol, fatty acids and Cer, each of which plays a
critical role in water retention and epidermal permeability barrier functions [12]. Recently, an abnormal
sphingolipid distribution pattern was found in the
keratinocytes of patients suffering from harlequin
ichthyosis, a genetic disorder in which the keratinocytes carry abnormal lamellar granules [13]. In harlequin ichthyosis, mutations have been identified in the
ABCA12 gene, which encodes a member of the lipid
transporter ATP-binding cassette (ABC) family that is
also part of the ABCA subfamily. ABCA12 is thought
to function in keratinocytes as a glucosylceramide
transporter to lamellar bodies [13]. Interestingly,
expression of ABCA12 mRNA has been shown to be
induced by activation of PPARb or PPARc in cultured
human keratinocytes [14].
3816

In this study, we examined the interaction between

CerK and PPARb, and its role in regulating keratinocyte survival. We report that in a mouse keratinocyte
cell line upregulation of CERK expression by activation of PPARb results in a decrease in intracellular
Cer levels and enhancement of cell survival.

Results
Skin barrier disruption in hairless mice induces
mRNA expressions for both PPARb and CerK
PPARb plays an important role in keratinocyte survival during skin wound healing, and PPARb expression is elevated at injury sites [10,15]. We investigated
whether skin barrier disruption by tape-stripping
would induce PPARb expression. To quantify the skin
barrier disruption, transepidermal water loss (TEWL),
an indicator of skin barrier function [16], was measured in the dorsal skin of hairless mice treated with
or without tape-stripping. With normal skin barrier
function, low levels of water loss from epidermal tissue
and low TEWL rates were evident. However, tapestripping the stratum corneum significantly increased
the TEWL rate and resulted in skin barrier disruption
(Fig. 1A). PPARb mRNA expression was determined
in keratinocytes isolated from the epidermal layer of
these skins, and was found to be significantly increased
in skin barrier disruption following tape-stripping
(Fig. 1Ba,b). Furthermore, CERK mRNA expression
was also elevated following tape-stripping (Fig. 1Ba,c).
This is the first evidence that CERK expression
increases in response to skin barrier disruption by
tape-stripping, and that it is accompanied by increases
in PPARb expression.
Stress-induced cell death is inhibited by
the treatment of specific PPARb ligand:
L-165,041 (LD)
Cer has emerged as an apoptotic lipid mediator, and

accumulation of Cer and apoptosis are known to be
induced under various stimuli such as tumor necrosis
factor-alpha, serum deprivation and c-radiation [17].
Previous studies have provided evidence that control
of intracellular Cer levels, through their modulation by
enzymes active in sphingolipid metabolism, is important in drug resistance and cancer cell survival [18,19].
Liu et al. [18] reported that the activity of glucosylceramide synthase, the enzyme that converts Cer to
glucosylceramide, and glucosylceramide levels were
increased in adriamycin-resistant breast cancer cells.
Uchida et al. [19] reported that in HL-60 ⁄ ADR cells

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Role of CerK in PPARb-induced keratinocyte survival

A
TEWL (g·m–2·h–1)

100

P< 0.0001

80
60
40
20
0

NonTape stripped stripped

b

PPARβ
CERK
Mrpl27
Non- Tapestripped stripped

c

8

P < 0.0001

CERK mRNA
(fold increase)

a

PPARβ mRNA
(fold increase)

B

6
4
2
0

8
P < 0.0001

6
4
2
0

NonTape stripped stripped

Non- Tape stripped stripped

Fig. 1. Skin barrier disruption in hairless mice induces mRNA expression for both PPARb and CerK. (A) Quantification of TEWL indicates skin
barrier disruption. The dorsal skin of each mouse was tape-stripped five to eight times to perturb the skin barrier function. TEWL was measured in tape-stripped mice skin (tape-stripped) or control mice skin (not stripped) as described in Materials and methods. Each column represents the mean ± SD of three animals for each group. (B) Expression levels of PPARb and CERK mRNA in skin barrier disruption. Total
RNA was extracted from the epidermis of tape-stripped mice skin (tape-stripped) or control mice skin (not stripped), and the expression levels of PPARb, CERK and Mrpl27 mRNA were determined by RT-PCR as described in Materials and methods. (a) Agarose gel electrophoresis
of the products of PCR using specific primers for PPARb, CERK or Mrpl27 mRNA. (b, c) Density of the mRNA expression of PPARb (b) or
CERK (c). The results shown are normalized to the mRNA level of Mrpl27, and are relative to control mice skin (not stripped), and the
mean ± SD of three animals for each group.

transcriptional upregulation of glucosylceramide synthase via doxorubicin-induced activation of Sp1 results
in a decreased Cer level and obtained drug resistance.
Moreover, we previously reported that exogenous Cer
is incorporated, hydrolyzed to sphingosine and then
recycled into intracellular Cer itself, and that accumulation of Cer contributes to the induction of apoptosis
[20]. However, the signaling mechanism involved in
Cer-mediated apoptosis remains to be further defined.
CerK is an enzyme that converts Cer to C1P.
Recently, CERK was cloned [2]. C1P has been
reported to be involved in the regulation of programed cell death in plants and in cell survival in

mammalian cells [4]. Arabidopsis, carrying a CerK
mutation, exhibits a spontaneous cell-death phenotype and accumulates Cer late in development [4]. In
A549 human lung adenocarcinoma cells transfected
with CERK siRNA, downregulation of CerK
reduced cellular proliferation [5]. We thought, therefore, that CerK could be involved in cell survival
promoted by PPARb, and that a decrease in Cer
content following activation of CerK would cause

suppression of cell death. In order to investigate this
possibility, we examined whether CerK could be
affected by activation of PPARb, using the mouse
keratinocyte cell line SP1. Enhanced cell survival has
previously been reported in the human keratinocyte
cell line HaCat following treatment with the specific
PPARb ligand L-165,041 (LD) [21].
We confirmed that SP1 cell survival could be
enhanced by PPARb. As shown in Fig. 2A, serum
starvation stress induced limited cell growth and death
in SP1 cells (Fig. 2Ac), yet treatment with 1 lm LD
inhibited this cell death (Fig. 2Ad). Furthermore, as
determined using a cell proliferation assay (Fig. 2B),
the rate of cell survival in SP1 cells treated with serum
starvation stress was reduced in a time-dependent
manner. However, treatment with 1 lm LD inhibited
the reduction of cell survival induced by serum
starvation stress. Flow cytometry analysis further
demonstrated that treatment with LD (1 or 10 lm)
suppressed the number of annexin V ⁄ propidium iodide
(PI)-positive cells, representing late apoptotic or necrotic cells, which had increased upon serum starvation

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Role of CerK in PPARb-induced keratinocyte survival

A

K. Tsuji et al.

B

LD ( + )

LD (−)

140

b
% Cell survival

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Stress (−)

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LD (−)

*

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24 (h)

Stress (+)

C

D
Ceramide content
(% control)

Apoptotic cells
(% control)

300
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LD (μM)

P < 0.001
250

P < 0.0001

400

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1

10

Stress (− )

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1

10

Stress (+ )

P < 0.001

200
150
100
50
0

Stress
LD

−
−

−
+

+
−

+
+

Fig. 2. Activation of PPARb enhances cell survival and inhibits cell death induced by serum starvation stress. (A) SP1 cells under serum starvation stress treated with LD. SP1 cells were cultured for an additional 24 h in the presence [LD (+)] or absence [LD ())] of the specific
PPARb ligand LD (1 lM), in medium lacking serum (serum-starvation conditions) or in medium containing fetal bovine serum (control conditions) [Stress (+) or Stress ()), respectively] for 24 h. SP1 cells were observed under a phase-contrast microscope. (B) Effect of LD on the
cell survival rate of mouse keratinocyte cells. SP1 cells were cultured in the presence [LD (+)] or absence [LD ())] of LD (1 lM), in serum
starvation conditions [Stress (+)]. After treatment for the indicated times, the cell survival rate was determined by Cell Counting Kit-8 as
described in Materials and methods. Significant difference from the corresponding LD ()) time point (*P < 0.05). (C) Inhibition by LD of
serum starvation stress-induced cell death. SP1 cells were cultured in serum starvation conditions [Stress (+)] or in control conditions [Stress
())], in the presence of LD (0, 1 or 10 lM) for 24 h. Cells were then stained with annexin V ⁄ fluorescein isothiocyanate and PI, and analyzed
by flow cytometry. The results shown are relative to untreated, unstressed cells the mean ± SD of three wells, and each experiment was
repeated three times. (D) Activation of PPARb inhibits the generation of cellular Cer induced by serum starvation stress. SP1 cells were left
untreated or treated with 1 lM LD under control conditions [Stress ())] or serum starvation conditions [Stress (+)] for 24 h. Total cellular
lipids were extracted using the standard Bligh–Dyer protocol. Total cellular Cer levels were measured by diacylglycerol kinase assay as
described under Materials and methods and quantified using an Image Analyzer BAS2000. The results shown are relative to untreated,
unstressed cells [Stress ()) ⁄ LD ())], and are the mean ± SD of three experiments.

stress (Fig. 2C). These findings demonstrate that SP1
cells are sensitive to ligand activation of PPARb, which
enhanced cell survival or inhibited cell death induced
by serum starvation.
Next, we investigated the participation of Cer in
cell survival promoted by PPARb, by examining
intracellular Cer levels. Total cellular lipids were
extracted from serum-starved or non-starved SP1
cells cultured for 24 h in the presence or absence of
1 lm LD, and the levels of endogenous Cer were
determined by diacylglycerol kinase assay [22]. Cer
levels were significantly increased in cells stressed by

serum starvation, compared with control cells
(Fig. 2D). However, similar to results observed in
the survival studies, treatment with LD inhibited the
increase in Cer levels induced by serum starvation
stress. These findings suggest that regulation of Cer
levels is involved in enhanced cell survival resulting
from activation of PPARb.
3818

Activation of PPARb by LD induces CERK mRNA
expression and increases CerK activity in SP1
cells
To investigate the role of CerK and its regulation of
Cer levels in the effect of PPARb activation on cell
survival, the expression of CERK mRNA in SP1 cells
treated with LD was determined using real-time PCR.
CERK mRNA expression was increased by LD
treatment in a dose-dependent (Fig. 3Aa) and timedependent (Fig. 3Ab) manner, indicating that activation of PPARb is involved in the gene transcription
of CERK. Furthermore, an in vitro kinase assay
determined that CerK activities in whole-cell lysates
were significantly increased in SP1 cells treated for
24 h with 1 lm LD, compared with untreated cells
(Fig. 3B). Although serum starvation stress did not
affect CerK activity, the rate of increase in CerK
activity in cells treated with LD was larger in cells

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Role of CerK in PPARb-induced keratinocyte survival

A
a

the effect of PPARb activation on cell survival in
mouse keratinocytes.

CERK mRNA
(% control)

300
250
200

*

150
100
50
0

b

*

*

0

0.1

0.5 1.0 5.0
LD (μ M)

10

CERK mRNA
(% control)

300
200

*

*

150
100
50
0

B

*

250

0

3

6 12 24
Time (h)

48

400

CerK activity
(% control)

P < 0.001

300

P < 0.05

200
100

0
Stress
LD

−
−

−

+

+
−

+
+

Fig. 3. Activation of PPARb induces CERK mRNA expression and
increased CerK activity in SP1 cells. (A) CERK mRNA expression is
induced by treatment with LD. SP1 cells were treated with LD (0,
0.1, 0.5, 1, 5 or 10 lM) under serum starvation conditions for 24 h
(a), or with 1 lM LD in serum-free medium for the indicated times
(b). Total RNA was extracted from the cells, and the mRNA expression levels of CERK and Mrpl27 (a housekeeping gene) were determined by quantitative real-time PCR as described in Materials and
methods. The results shown are the mean ± SD of three wells,
and each experiment was repeated three times. *P < 0.05 compared with the controls at 0 lM LD (a) or 0 h (b). (B) CerK activity is
enhanced in cells treated with LD. SP1 cells were left untreated
[LD ())] or treated [LD (+)] with 1 lM LD in serum starvation conditions or control conditions [Stress (+) or Stress ()), respectively].
After treatment for 24 h, cell lysates were collected and analyzed
for in vitro CerK activities, as described in Materials and methods
using C18-Ceras a substrate with [32P]ATP[cP]. [32P]C1P was quantified using an Image Analyzer BAS2000 (Fuji Film). The results
shown are relative to untreated, unstressed cells, are the
mean ± SD of three experiments.

undergoing serum starvation stress than in unstressed
cells [23]. These findings suggest that CerK, through
its regulation of Cer levels, plays an important role in

PPARb binds to a putative PPRE in CERK and
transactivates the CERK gene

The results above suggest that PPARb can regulate
expression of the CERK gene. PPARs bind to
response elements (PPREs) in target genes and regulate transcription. Functional PPREs should reside in
the regulatory region of the target gene. Although the
promoter region in the mouse CERK gene has not
been identified, transcription start sequences at the
5¢-end of the gene reside  100 bp upstream of the
protein-coding sequence, as identified by the Database
of Transcriptional Start Sites (DBTSS) [24] and our
experimental results (data not shown). Analysis of the
region near the protein-coding sequence in the mouse
CERK gene sequence (GenBank accession number
NC_000081) was performed using nubiscan, an
in silico tool for predicting nuclear receptor binding
sites [25]. This analysis revealed a putative PPRE,
which we refer to as putative CERK-PPRE, in
intron 1 of the mouse CERK gene (Fig. 4A). In order
to determine whether PPARb binds to the mouse
CERK gene in vivo, chromatin immunoprecipitation
(ChIP) was carried out using SP1 cells, untreated [LD
())] or treated [LD (+)] with 1 lm LD for 24 h.
Using immunoprecipitated chromatin, the CERK gene
sequence containing putative CERK-PPRE was
analyzed by PCR. In the ChIP DNA obtained with
the anti-PPARb IgG, the amount of DNA containing
putative CERK-PPRE was significantly greater in the
chromatin of cells treated with LD compared with
untreated controls (Fig. 4B,C). The results were comparable in the ChIP DNA obtained with acetylated
histone H4 antibodies. No PCR products with CERKnegative were obtained in ChIP DNA, indicating that
the binding of putative CERK-PPRE to PPARb is

specific. Furthermore, the interaction between PPARb
and CERK was confirmed by an EMSA using biotinlabeled putative CERK-PPRE and nuclear extract
from SP1 cells. As shown in Fig. 4D, the binding of
biotin-labeled putative CERK-PPRE to nuclear extract
from SP1 cells was detected as shift bands. The levels
of these shift bands were reduced in the presence of
competitors, including unlabeled putative CERKPPRE and PPRE-Wild, a known consensus sequence.
However, no reduction in shift band levels was
observed in the presence of PPRE-Mutant, a mutation
sequence of PPRE-Wild. These results demonstrate
specific binding of putative CERK-PPRE to PPARb
in nuclear extracts of SP1 cells, and provide further

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Role of CerK in PPARb-induced keratinocyte survival

K. Tsuji et al.

A
CERK gene

Exon-1

Exon-2

Intron -1

Putative CERK-PPRE: AGGCCAcAGGCCA

ChIP DNA
IgG
anti-PPARβ anti-acH4
−
+ −
+
−
+

LD

Input
DNA
−
+

C
Putative CERK-PPRE
(% control)

B

Putative CERK-PPRE
CERK-negative

P < 0.01

800
300
400
200
0

LD (−) LD (+ )

D

0

R
PP

K-

K-

ER
C

iv

e

e
tiv
ta

in
in

P-

Lu
c2

P-

pu

m

-Δ
c2
P
Lu
m

Pin
m

E

E

−
−
+

ve
ct
o

−
+
−

at

+
−
−

P-

−
−
−

ER

−
−
−

500

r

+
+

pu
t

+
+

c2

+
+

C

+
+

P-

Competitors (non-labeled)
putative CERK-PPRE
PPRE-Wild
PPRE-Mutant

−
+

Lu

SP1 cell nuclear extracts
Biotin-labeled putative CERK-PPRE

1000

R

Free

P < 0.0001
1500

PP

Relative luciferase activity
(% control)

E

Shift band

Fig. 4. PPARb binds to putative CERK-PPRE and transactivates the CERK gene. (A) Schematic representation of the CERK gene illustrating the
position (bar) and sequence of the putative PPRE (putative CERK-PPRE). (B, C) ChIP demonstrates PPARb binding to putative CERK-PPRE
in vivo. SP1 cells were untreated or treated with 1 lM LD in serum starvation medium for 24 h. Nuclear extracts were collected and subjected
to a ChIP assay, as described in Materials and methods, using antibodies against PPARb or acetylated histone H4, or IgG as a negative control.
ChIP DNA and aliquot of pre-immunoprecipitation samples of nuclear extracts (Input DNA) were analyzed by PCR with primers for putative
CERK-PPRE or CER-negative as PCR negative control with primers for unrelated putative PPRE, as described in Materials and methods. PCR
products of ChIP DNA and Input DNA were analyzed with 1% agarose gel electrophoresis (B). The bands of PCR products corresponding to the

binding of PPARb to putative CERK-PPRE were normalized to the bands of PCR product of Input DNA, and are relative to untreated sample [LD
())] (C). The results shown are the means ± SD of three experiments. (D) EMSAs indicate that PPARb binds to putative CERK-PPRE in vitro.
SP1 cells were treated for 24 h with 1 lM LD in serum starvation . Nuclear extracts from the cells (5 lg) were incubated with biotin-labeled putative CERK-PPRE oligonucleotide (20 fmol). Competition assays were performed with non-biotinylated oligonucleotides (4 pmol) of putative
CERK-PPRE, a specific DNA binding consensus sequence for PPARs (PPRE-Wild), or a mutant sequence of PPRE-Wild (PPRE-Mutant). Arrows
indicate the labeled putative CERK-PPRE oligonucleotide in specific complex with SP1 nuclear extracts (Shift band) and unbound (Free).
(E) Transfection assays indicate that PPARb transactivates the CERK gene. SP1 cells were transiently cotransfected with pCMX–mPPARb,
pCMX–mRXRa, the luciferase reporter constructs: minP-Luc2P-putative CERK-PPRE or minP-Luc2P-D putative CERK-PPRE, and pRL-SV40 control vector. Transfected cells were treated for 24 h in Phenol Red-free Dulbecco’s modified Eagle’s medium containing with 10% charcoal
stripped fetal bovine serum and 1 lM LD. Results were normalized with Renilla luciferase activity to correct for variability in transfection
efficiency. Values represent the means ± SD of three wells, and each experiment was repeated twice.

evidence that PPARb directly regulates expression of
the CERK gene through PPRE.
Next, we assessed the function of putative CERKPPRE using the Dual-Luciferase Reporter Assay
System. In keeping with the genomic organization of
the CERK gene, the 1807 bp fragment containing
putative CERK-PPRE, or the 1007 bp fragmant not
containing putative CERK-PPRE, in the CERK
intron 1 region was subcloned downstream of the
3820

minimal promoter and the Luc2P reporter gene in
pGL4.27[luc2P ⁄ minP ⁄ Hygro],
respectively
named
minP-Luc2P-putative CERK-PPRE or minP-Luc2P-D
putative CERK-PPRE. Reporter gene transfection
studies showed that the region containing putative
CERK-PPRE has the capacity to significantly influence
transcriptional activity. By contrast, constructs not
containing putative CERK-PPRE were approximately

equal to the control luciferase activity from cells

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Role of CerK in PPARb-induced keratinocyte survival

transfected with an empty vector (minP-Luc2P-vector).
These findings suggest that putative CERK-PPRE is
the functional PPRE in mouse CERK gene.

keratinocyte cultures, however, the inhibitory effect of
LD was once again diminished (Fig. 5D). These findings
indicate that CerK is necessary for the enhanced cell
survival associated with activation of PPARb. Taken
together, the data demonstrate that upregulation of
CERK by PPARb suppresses Cer accumulation induced
by serum starvation stress, which results in cell survival
and inhibition of cell death in mouse keratinocytes.

Enhanced cell survival associated with activation
of PPARb is diminished in keratinocytes from
CerK-null mice
The role of CerK in cell survival associated with PPARb
activation was further examined using primary keratinocytes isolated from CerK-null mice [26]. These mice
lack exon 6 of the mouse CERK gene, which encodes a
region of the protein that is known to be essential for
kinase activity and contains a diacylglycerol kinase-like

catalytic domain [2]. As shown in Fig. 5A, cell death
and limited cell growth following serum starvation
stress for 24 h were apparent in primary keratinocytes
isolated from wild-type mice. In wild-type cells treated
with 1 lm LD under serum starvation conditions, cell
death was inhibited, similar to results observed in SP1
cells (Fig. 2A). However, in primary keratinocytes isolated from CerK-null mice, the inhibitory effect of LD
on cell death induced by serum starvation stress was
diminished (Fig. 5B). To confirm this finding, serum
deprivation-induced cell death was investigated by flow
cytometry analysis with annexin V and PI staining. A
significant increase in the percentages of annexin V ⁄ PIpositive cells was observed in cultures of wild-type
keratinocytes undergoing serum starvation stress for
24 h, but, as expected, treatment with 1 lm LD
suppressed this number (Fig. 5C). In CERK-KO

A

Discussion
The study reported here revealed that in mouse keratinocytes upregulation of CERK through activation of
PPARb results in decreased intracellular Cer levels and
increased cell survival with less cell death (Fig. 6).
There have been previous reports that PPARb activation improved skin wound healing by enhanced keratinocyte survival ⁄ anti-apoptosis [10,15]. In this study,
the effects on cell survival of PPARb were diminished
in CERK-KO keratinocytes (Fig. 5), suggesting the
biological importance of CerK in PPARb functions.
This study provides the first evidence for the necessity
of CerK in mouse keratinocyte survival associated with
activation of PPARb.
Cer is known to have an important role in apoptosis

and cell-cycle arrest induced by various stressors.
Intracellular Cer levels are adjusted by several sphingolipid production pathways, such as de novo ceramide
synthesis by serine palmitoyltransferase and cleavage
of sphingomyelin by sphingomyelinase, as well as by
Cer metabolism pathways, including conversion to

Wild-type keratinocytes
LD (−)
LD( +)

a

CERK-KO keratinocytes
LD (−)
LD( +)

a

b

c

b

d

Stress (−)

Stress (−)

c

d
Stress (+)

Stress (+)

C

Wild-type keratinocytes
300

200

100

0
Stress
LD

D

CERK-KO keratinocytes
300

P < 0.005

Apoptotic cells
(% control)

Apoptotic cells
(% control)

Fig. 5. Enhanced cell survival associated
with activation of PPARb is diminished in
keratinocytes from CerK-null mice. (A–D)
Mouse primary keratinocytes isolated from
wild-type mice (A, C) or CerK-null mice
(B, D) were grown to 80% confluence then
grown for 24 h under serum starvation
[Stress (+)] or control [Stress ())] conditions,
in the presence [LD (+)] or absence [LD ())]
of 1 lM LD. (A, B) Cells were observed
under a phase-contrast microscope. (C, D)
Cells were stained with annexin V ⁄ fluorescein isothiocyanate and PI, and analyzed by
flow cytometry. Results shown are relative
to untreated, unstressed cells, are the
means ± SD of three wells, and each
experiment was repeated three times.

B

−
−

−
+

FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS

+
−

+
+

P > 0.08

200

100

0
Stress
LD

−
−

−
+

+
−

+
+

3821

Role of CerK in PPARb-induced keratinocyte survival

K. Tsuji et al.

Fig. 6. Schematic model illustrating the role
of CerK in cell survival mediated by PPARb
in mouse keratinocytes. Under injury stress,
endogenous ligands activate PPARb. Subsequently, the activated PPARb promotes transcription of the CERK gene. The conversion
of Cer to C1P by the produced CerK results
in decreased levels of intracellular Cer, and
the inhibition of stress-induced cell death.
The interaction between CerK and PPARb
may play an important role in regulating epidermal homeostasis in stress environments.

glucosylceramide by glucosylceramide synthase, degradation to sphingosine and conversion to C1P by CerK.
In some cancer cells, decreases in Cer levels by amplified activation of glucosylceramide synthase result in
drug resistance [18,19]. Mitra et al. [5] reported that
siRNA-induced downregulation of CerK reduced cell
proliferation and promoted apoptosis. Previous studies
have suggested that metabolism of eicosanoids is regulated by feedback pathways through PPARs. Recently,
Xu et al. [27] reported a positive feedback loop
between PPARb and prostaglandin E2 through which
PPARb promotes COX-2 expression and prostaglandin E2 synthesis, and subsequent activation of cystolic
phospholipase A2a, which is responsible for arachidonic acid release, through mitogen-activated protein
kinase and phosphatidylinositol 3-kinase-mediated
phosphorylation. This pathway results in cholangiocarcinoma cell growth. CerK has also been reported to
act as an upstream modulator of cystolic phospholipase A2a in the inflammatory response [28,29]. CerK
may also be implicated in the regulation of PPARb
activation through inflammatory factors such as cystolic

phospholipase A2a.
In this study, the skin of CerK-null mice appeared
normal under specific pathogen-free conditions (data
not shown), yet examinations of cultured CERK-KO
keratinocytes showed a diminished effect on cell survival by activation of PPARb compared with that in
wild-type keratinocytes (Fig. 5). PPARb expression has
been reported to be undetectable in the epidermal tissue
of adult mice; however, it is apparently upregulated
in various stress conditions, such as skin wound
healing, that result in enhanced keratinocyte proliferation [10,15]. Reportedly, CerK is also expressed highly
at embryonic day 7 but decreases rapidly thereafter [2].
In the Arabidopsis plant, expression of CERK mRNA
is induced after infection with a bacterial pathogen [4].
Considering all this information, it appears that CerK
3822

function may be upregulated under stress conditions,
such as those that induce PPARb expression. Future
studies will be required into the role of CerK using an
animal model.
In conclusion, we have shown that PPARb-mediated
upregulation of CerK gene expression is necessary for
keratinocyte survival against serum starvation-induced
apoptosis. The interaction between CerK and PPARb
may play an important role in regulating epidermal
homeostasis in stress environments.

Materials and methods
Materials
Dispase was obtained from Godo Shusei (Tokyo, Japan).

Keratinocyte serum-free medium, epidermal growth factor,
bovine pituitary extract, Phenol Red-free Dulbecco’s modified Eagle’s medium, and charcoal stripped fetal bovine
serum were obtained from GIBCO BRL (Gaithersburg,
MD, USA). Minimum essential medium without calcium
chloride was obtained from Cambrex (Walkersville, MD,
USA). Penicillin–streptomycin, 0.125% trypsin–0.01%
EDTA and LD were from Sigma (St Louis, MO, USA).
Rabbit anti-PPARb IgG was from Santa Cruz Biotechnology Inc, (Santa Cruz, CA, USA) and the anti-acetylHistone H4 serum was from Upstate Biotechnology (Lake
Placid, NY, USA). A MEBCYTO Apoptosis Kit was
purchased from Medical and Biological Laboratories
(Nagoya, Japan), and a Nuclear Extract Kit was from
Active Motif (Carlsbad, CA, USA). The ChIP Assay Kit
was also a product of Upstate Biotechnology. Biotin
3¢-end DNA Labeling Kit and LightShift Chemiluminescent EMSA Kit were purchased from Pierce (Rockford,
IL, USA). CulturPlate-96, White was purchased from
Perkin-Elmer (Boston, MA, USA) and a lipofectamine
2000 reagent was from Invitrogen (Carlsbad, CA, USA).
Dual-Luciferase reporter assay system was purchased from
Promega (Madison, WI, USA).

FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS


K. Tsuji et al.

Animals
Hairless mice (HR-1), 4-week-old males, were purchased
from Hoshino Experimental Animal Center (Saitama,
Japan). C57BL ⁄ 6J mice were purchased from Clea Japan
(Tokyo, Japan). All animal experiments were performed in

accordance with the Guide for the Care and Use of Laboratory Animals (Hokkaido University Graduate School of
Medicine, Japan). Animals were housed in plastic cages
with metal lids at a temperature of 22 ± 3°C, with
50 ± 20% relative humidity, and were exposed daily to
12 h of light and 12 h of darkness.

Skin barrier disruption by tape-stripping
In the dorsal skins of hairless mice, skin barrier disruption
was performed by stripping with adhesive tape (P.P.S.
Nichiban, Tokyo, Japan: 2.5 · 3.0 cm) repeatedly, five to
eight times. An Evaporimeter AS-TW1 (Asahi Biomed, Co.
Ltd, Yokohama, Japan) was used to measure TEWL, in
accordance with the ventilated chamber method [30]. Measurements were carried out at a temperature of 22 ± 3°C,
with 50 ± 20% humidity, and were performed in triplicate
at each treatment skin spot.

Harvest and culture of keratinocytes
Mouse keratinocytes were isolated from the epidermis of
hairless mice or newborn of C57BL ⁄ 6J mice. Briefly, the
epidermis was separated from the dermis following an overnight incubation at 4°C in 2.5 mL)1 Dispase. Keratinocytes isolated from the epidermis were harvested after
treatment with 0.125% trypsin and 0.01% EDTA at 37 °C
for 5 min. Keratinocytes isolated from the epidermis of newborn mice were incubated in a humidified atmosphere of 5%
CO2 in air at 37 °C with keratinocyte serum-free medium
containing 0.02 mm Ca2+, 5 ngỈmL)1 epidermal growth
factor and 50 lgỈmL)1 bovine pituitary extract. The isolated
keratinocytes were used at a subconfluent state (80% confluency). The mouse keratinocyte cell line SP1 cells, a kind gift
from S.H. Yuspa (National Cancer Institute, Bethesda, MD,
USA) [31], was cultured in minimum essential medium without calcium chloride supplemented with 0.02 mm Ca2+, 8%
chelexed fetal bovine serum [32] and antibiotics (100
unitsỈmL)1 penicillin and 0.1 mgỈmL)1 streptomycin). The

cultures were maintained in a humidified atmosphere of
5% CO2 in air at 37 °C.

Total RNA isolation
The total RNA from each sample was isolated using an
RNeasy Mini Kit (Qiagen, Chatsworth, CA, USA), according to protocols provided by the manufacturer. To remove
contaminating genomic DNA, the RNA samples were

Role of CerK in PPARb-induced keratinocyte survival

treated with RNase-free DNase I (Qiagen) at room temperature for 30 min.

RT-PCR
RT-PCR of each mRNA was performed with Omniscript
RT Kit (Qiagen) following the manufacturer’s instructions
using oligo(dT) primers and Taq DNA polymerase (Qiagen)
with specific primers. Sequences of the specific primers
included, for PPARb forward 5¢-GCAGCCTCTTCCTCA
ATGAC-3¢, for reverse 5¢-GTACTGGCTGTCAGGGTG
GT-3¢; CERK forward 5¢-TCTGCAAGGACAGACCCT
CT-3, reverse 5¢-CAAGTGCCATTTGCTGAGAA-3¢; and
mitochondrial ribosomal protein L27 (Mrpl27) forward
5¢-GGGATAGTCCGCTACACGAA-3¢, reverse 5¢-ACCA
TGTGGTTGTTGGGAA-3¢. The PCR condition of
PPARb was as follows: 95 °C for 30 s, 60 °C for 30 s and
72 °C for 60 s, and 35 cycles were used. The PCR condition
of CERK was as follows: 95 °C for 30 s, 55 °C for 30 s
and 72 °C for 60 s, and 35 cycles were used. The PCR
condition of Mrpl27 was as follows: 95 °C for 30 s, 60 °C
for 30 s and 72 °C for 60 s, and 28 cycles were used. PCR

products were separated by electrophoresis on 2% agarose
gels and visualized by ethidium bromide staining. The
relative intensity of the gel bands was measured using
nih image software, and results were normalized to the
mRNA level of Mrpl27, a housekeeping enzyme [33]. We
performed these experiments using samples from three
animal preparations.

Assessment of cell survival and cell death
The rate of cell survival was determined using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), following the manufacturer’s instructions. Briefly, the cells
were seeded onto a six-well plate (1.0 · 105 cellsỈmL)1),
incubated at 37 °C for 3 days, then, in the presence or
absence of serum, cells were treated with or without LD
(1 lm). After treatment, a solution of Cell Counting Kit-8,
in 1 ⁄ 10 volume of the culture medium, was added to each
well, and the culture continued at 37 °C for 4 h. Each well
was then assessed at D450 using an automatic enzyme-linked
immunosorbent assay plate reader.
Cell death was quantified by flow cytometry analysis.
Cells were seeded onto six-well plates, grown to 80% confluence, then treated with or without varying concentrations of LD in the presence or absence of serum. After a
predetermined time, the cells were trypsinized and washed
twice with NaCl ⁄ Pi. The dead cells were stained with
annexin V ⁄ and PI using a MEBCYTO Apoptosis kit
according to the manufacturer’s instructions. flow cytometry analysis was carried out on a FACSort cell sorter
(Becton Dickinson, Mountain View, CA, USA) using
cell quest software.

FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS

3823

Role of CerK in PPARb-induced keratinocyte survival

K. Tsuji et al.

solvent system. Quantification of bands was carried out
using the Imaging Analyzer BAS2000.

Measurement of total intracellular Cer levels
Total cellular Cer levels were measured by the diacylglycerol kinase method [22]. Briefly, total cellular lipids were
extracted using the Bligh–Dyer protocol as previously
described [34]. Extracts were suspended in micelle buffer
containing 7.5% n-b-d-octyl glucopyranoside and
19.4 mgỈmL)1 a-dioleoylphosphatidylglycerol, then mixed
with 0.1 unit of Escherichia coli diacylglycerol kinase and
1 lCi [32P]ATP[cP], and incubated for 1 h at 37 °C. After
the reaction, lipids were separated by a solvent system of
chloroform ⁄ methanol ⁄ 15 mm CaCl2 (7.5 : 4.4 : 1, v ⁄ v ⁄ v)
on Silica Gel 60 TLC plates (Merck, Darmstadt, Germany).
Bands corresponding to C1P derived from intracellular Cer
were quantified using an Imaging Analyzer BAS2000 (Fuji
Film, Tokyo, Japan).

Quantitative real-time PCR
Quantification of CERK mRNA was performed using an
ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). RNA samples were reverse
transcribed to synthesize first-strand cDNA using the
Omniscript RT kit (Qiagen), then analyzed by real-time
PCR using specific primers and the double-stranded DNA

dye SYBR Green I (Qiagen), according to protocols provided by the manufacturer. Specific primer sequences used
included for CERK forward 5¢-GAGTGGCAAGTGACA
TGTGG-3¢ and for reverse 5¢-GCACTTCCGGATAAG
GATGA-3¢; those for Mrpl27 were for forward 5¢-CTGC
CCAAGGGTGCTGTGCTC-3¢ and for reverse 5¢-TTGTT
CTCACCAGACCCTTGAC-3¢. All reactions were run with
a hot-start preincubation step of 10 min at 95 °C, followed
by cycles of 15 s at 95 °C and 1 min at 60 °C. The amount
of template was quantified using the comparative cycle
threshold method as outlined in the manufacturer¢s technical bulletin. Quantified CERK mRNA levels were normalized to the Mrpl27 mRNA level for reporting.

Quantification of in vitro CerK activity
CerK activity assays were performed as described by
Bajjalieh et al. [3]. Briefly, cells were washed three times
with ice-cold NaCl ⁄ Pi, then lysed in a buffer containing
10 mm Hepes, 2 mm EGTA, 1 mm dithiothreitol, 40 mm
KCl and complete protease inhibitor mixture (Roche,
Basel, Switzerland). The enzyme reactions were performed
for 30 min at 30 °C in a reaction mixture containing
20 mm Hepes, 80 mm KCl, 3 mm CaCl2, 1 mm cardiolipin, 1.5% b-octyl glucoside and 0.2 mm diethylenetriaminepentaacetic acid, with 40 mm Cer (C18:0, d18:1) as
a substrate. After the reaction, lipids were extracted and
separated on Silica Gel 60 HPTLC plates (Merck,
Darmstadt, Germany) in chloroform ⁄ acetone ⁄ methanol ⁄ acetic acid ⁄ water (10 : 4 : 3 : 2 : 1, v ⁄ v ⁄ v ⁄ v ⁄ v) as the

3824

ChIP assays
PPAR forms a heterodimer with RXR, and binds to PPRE
sequences of the direct repeat-1 (DR-1) type (a repeat separated by one nucleotide) on DNA. Using the nubiscan
program [25], putative PPRE elements were identified

within the first intron of the mouse CERK gene (putative
CERK-PPRE). ChIP was performed using a ChIP Assay
Kit (Upstate Biotechnology), according to protocols provided by the manufacturer, with some modifications.
Briefly, cells were seeded onto six-well plates, grown to
80% confluence, and then treated with or without LD
(1 lm) in serum starvation medium for 24 h. To cross-link
the DNA, cells were fixed with 1% formaldehyde at 37 °C
for 15 min, then sonicated to fragments ranging in size
from 200 to 500 bp. ChIP was carried out using PPARb
antibodies and acetylated histone H4-specific antibodies,
with normal rabbit IgG used as a negative control. Reverse
cross-linking of DNA fragments was achieved at 65 °C for
6 h. After phenol ⁄ chloroform treatment of purified DNA,
the DNA was amplified by PCR using primers, for putative
CERK-PPRE forward 5¢-GTAGGCATGAGAACGGGA
AG-3 and for reverse 5¢-GGGGGTAAGAGGAGGAGA
AA-3¢ and for CERK-negative forward 5¢-CCGCAAG
AGGCTTTATTGTC-3 and reverse 5¢-TATGCCAAGGA
CACGGAGAT-3¢, as a negative control PCR primer. The
condition for PCR amplification was as follows: 95 °C for
30 s, 60 °C for 30 s and 72 °C for 60 s; 32 cycles were used,
depending on the abundance of DNA.

Electrophoretic mobility shift assays
PPRE studies were performed using biotin-labeled oligonucleotides and nuclear extracts from SP1 cells. The nucleotide sequences of putative CERK-PPRE, including the
sense 5¢-CTCTCCAGGCCACAGGCCAGAGCGG-3¢ and
anti-sense 5¢-GAGAGGTCCGGTGTCCGGTCTCGCC-3¢
sequences, were biotin-labeled using a Biotin 3¢-end DNA
Labeling Kit (Pierce). Nuclear extracts from SP1 cells were
prepared using a Nuclear Extract Kit (Active Motif).

EMSA was performed using a LightShift Chemiluminescent
EMSA Kit (Pierce), following the manufacturer’s protocols,
with some modifications. Briefly, nuclear extracts (5 lg)
from SP1 cells and 20 fmol biotin-labeled putative
CERK-PPRE, alone or with 4 pmol unlabeled probe
oligonucleotides (putative CERK-PPRE, PPRE-Wild, sense
5¢-CAAAACTAGGTCAAAGGTCA-3¢ and anti-sense
5¢-GTTTTGATCCAGTTTCCAGT-3¢; or PPRE-Mutant
sense 5¢-CAAAACTAGCACAAAGCACA -3¢ and antisense 5¢-GTTTTGATCGTGTTTCGTGT-3¢) [35], were
incubated in a reaction mixture at room temperature for 20
min. The mixtures were then separated by electrophoresis

FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS


K. Tsuji et al.

on a 6% polyacrylamide gel at 4°C in 0.5· TBE at 100 V
for 2–2.5 h. The samples were subsequently transferred to
Nylon Membranes, Positively Charged (Roche, Basel, Switzerland) and exposed to UV light (120 mJỈcm)2, 1 min) to
cross-link the DNA to the membrane. In accordance with
the manufacturer’s protocol, detection of Biotin-labeled
DNA probe was performed.

Plasmid constructs
The expression plasmids pCMX–mouse PPARb and pCMX
were a kind gift from R. M. Evans (Salk Institute, San
Diego, CA, USA) [36]. The cDNA encoding mouse RXR a
was subcloned into pCMX (pCMX–RXRa). The 1807
bp

fragment
containing
putative
CERK-PPRE
(NC_000081:c86013544-86011738), or the 1007 bp fragmant
non-containing putative CERK-PPRE (NC_000081:c86013544-86012538) in the CERK intron 1 region were
PCR amplified, and subcloned downstream of the minimal promoter and the Luc2P reporter gene in
pGL4.27[luc2P ⁄ minP ⁄ Hygro] (Promega). The pRL-SV40
control vector was purchased from Promega.

Transfection and luciferase reporter assays
SP1 cells (1.0 · 105 cellsỈmL)1) were trypsinized and replated into CulturPlate-96, White (Perkin-Elmer), and incubated for 3 days. The cells were cotransfected with 100 ng
of either reporter plasmid: minP-Luc2P-vector, minPLuc2P-D putative CERK-PPRE or minP-Luc2P-D putative
CERK-PPRE, 100 ng of pRL-SV40 control vector, 12.5 ng
of pCMX–mRXRa and 12.5 ng of pCMX–mPPARb using
lipofectamine 2000 reagent (Invitrogen). At 24 h after
transfection, cells were rinsed with phosphate buffer and
replaced with Phenol Red-free Dulbecco’s modified Eagle’s
medium containing with 10% charcoal-stripped fetal bovine
serum and 1 lm LD. After treatment for 24 h, luciferase
activities were assayed using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturer’s
protocol. The levels of Photinus pyralis (firefly) luciferase
activity were normalized to the levels of Renilla luciferase
activity of the cotransfected pRL-SV40 control plasmid.

Statistics
Results are expressed as the mean ± SD for each sample.
Statistical analysis was performed using the unpaired
Student’s t-test. Statistical significance was defined as

P < 0.05, 0.01 or 0.001.

Acknowledgements
This work was supported by the Sapporo Biocluster
Bio-S, the Knowledge Cluster Initiative of the Ministry

Role of CerK in PPARb-induced keratinocyte survival

of Education, Sports, Science and Technology (MEXT),
Japan.

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