Role of ceramide kinase in peroxisome proliferator-
activated receptor beta-induced cell survival of
mouse keratinocytes
Kiyomi Tsuji
1
, Susumu Mitsutake
2
, Urara Yokose
2
, Masako Sugiura
3
, Takafumi Kohama
4
and
Yasuyuki Igarashi
1,2
1 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Advanced Life Sciences, Hokkaido University, Sapporo, Japan
2 Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
3 Biological Research Laboratories II, Daiichi-Sankyo Co. Ltd., Tokyo, Japan
4 Exploratory Research Laboratories I, Daiichi-Sankyo Co. Ltd., Tokyo, Japan
Ceramide (Cer) has been implicated in various cellular
processes including proliferation, apoptosis and cell
signaling [1]. Intracellular Cer levels are strictly regu-
lated 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,

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 immunoprecipita-
tion 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 conclu-
sion, our results demonstrate that PPARb-mediated upregulation of CerK
gene expression is necessary for keratinocyte survival against serum starva-
tion-induced apoptosis.
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.
FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3815
NIH 3T3 fibroblasts and A549 lung cancer cells, treat-
ment 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 previ-
ous 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 super-
family. PPARs form heterodimers with the retinoid
X receptor (RXR) in a ligand-dependent manner.
Together, these heterodimers regulate the expression of

quin 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].
In this study, we examined the interaction between
CerK and PPARb, and its role in regulating keratino-
cyte survival. We report that in a mouse keratinocyte
cell line upregulation of CERK expression by activa-
tion 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 sur-
vival during skin wound healing, and PPARb expres-
sion 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 mea-
sured 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, tape-
stripping the stratum corneum significantly increased

thase 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 accumu-
lation 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 pro-
gramed cell death in plants and in cell survival in
mammalian cells [4]. Arabidopsis, carrying a CerK
mutation, exhibits a spontaneous cell-death pheno-
type 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, there-
fore, 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

4
2
0
P < 0.0001
8
6
4
2
0
P < 0.0001
a
b
c
CERK
Mrpl27
Non-
stripped
Tape -
stripped
Non-
stripped
Tape -
stripped
Non-
stripped
Tape-
stripped
TEWL (g·m
–2
·h

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.
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 time-
dependent (Fig. 3Ab) manner, indicating that activa-
tion 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
P < 0.001
250
200
150

0
C
Stress (−)
Stress (+ )
LD (−) LD (
+
)
P < 0.0001
P < 0.001
*
*
*
% Cell survival
Apoptotic cells
(% control)
Ceramide content
(% control)
a b
d
c
Fig. 2. Activation of PPARb enhances cell survival and inhibits cell death induced by serum starvation stress. (A) SP1 cells under serum star-
vation 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 l
M), in medium lacking serum (serum-starvation conditions) or in medium containing fetal bovine serum (control condi-
tions) [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 l
M), 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

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 com-
parable in the ChIP DNA obtained with acetylated
histone H4 antibodies. No PCR products with CERK-
negative 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 biotin-
labeled 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 CERK-
PPRE and PPRE-Wild, a known consensus sequence.

*
*
*
CERK mRNA
(% control)
(% control)
400
300
200
100
0
P < 0.001
P < 0.05
Stress
LD
−− ++
−+ −+
CerK activity
(% control)
CERK mRNA
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 l
M) under serum starvation conditions for 24 h
(a), or with 1 l
M LD in serum-free medium for the indicated times
(b). Total RNA was extracted from the cells, and the mRNA expres-
sion levels of CERK and Mrpl27 (a housekeeping gene) were deter-
mined by quantitative real-time PCR as described in Materials and

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
Putative CERK-PPRE
ChIP DNA
CERK-negative
LD − + − + − + − +
800
300
400
200
0
P < 0.01
LD (−) LD (+ )
Intron -1
Exon-1
Exon-2
Putative CERK-PPRE: AGGCCAcAGGCCA
IgG anti-PPARβ anti-acH4
B
C
A
SP1 cell nuclear extracts
Biotin-labeled putative CERK-PPRE
Competitors (non-labeled)

c
2P-p
utative CERK-PPRE
Relative luciferase activity
Putative CERK-PPRE
(% control)
(% control)
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 l
M 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 l
M LD in serum starvation . Nuclear extracts from the cells (5 lg) were incubated with biotin-labeled puta-
tive 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 con-
trol 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 l
M 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.
Role of CerK in PPARb-induced keratinocyte survival K. Tsuji et al.
3820 FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS

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.
Discussion
The study reported here revealed that in mouse kerati-
nocytes 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 activa-
tion improved skin wound healing by enhanced kerati-
nocyte 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 PPAR b.
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 sphingo-
lipid 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
CERK-KO keratinocytes
P < 0.005

c d
a
b
c
Apoptotic cells
(% control)
Apoptotic cells
(% control)
d
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 l
M LD. (A, B) Cells were observed
under a phase-contrast microscope. (C, D)
Cells were stained with annexin V ⁄ fluores-
cein 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.
K. Tsuji et al. Role of CerK in PPARb-induced keratinocyte survival
FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3821
glucosylceramide by glucosylceramide synthase, degra-

2
a.
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 sur-
vival 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 prolifera-
tion [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
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).

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 epi-
dermal homeostasis in stress environments.
Role of CerK in PPARb-induced keratinocyte survival K. Tsuji et al.
3822 FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 Labo-
ratory 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]. Mea-
surements 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

penicillin and 0.1 mgÆmL
)1
streptomycin). The
cultures were maintained in a humidified atmosphere of
5% CO
2
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), accord-
ing to protocols provided by the manufacturer. To remove
contaminating genomic DNA, the RNA samples were
treated with RNase-free DNase I (Qiagen) at room temper-
ature 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

tions of LD in the presence or absence of serum. After a
predetermined time, the cells were trypsinized and washed
twice with NaCl ⁄ P
i
. The dead cells were stained with
annexin V ⁄ and PI using a MEBCYTO Apoptosis kit
according to the manufacturer’s instructions. flow cyto-
metry analysis was carried out on a FACSort cell sorter
(Becton Dickinson, Mountain View, CA, USA) using
cell quest software.
K. Tsuji et al. Role of CerK in PPARb-induced keratinocyte survival
FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS 3823
Measurement of total intracellular Cer levels
Total cellular Cer levels were measured by the diacylglyc-
erol 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 mgÆmL
)1
a-dioleoylphosphatidylglycerol, then mixed
with 0.1 unit of Escherichia coli diacylglycerol kinase and
1 lCi [
32
P]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 CaCl
2
(7.5 : 4.4 : 1, v ⁄ v ⁄ v)
on Silica Gel 60 TLC plates (Merck, Darmstadt, Germany).

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 CaCl
2
,1mm cardio-
lipin, 1.5% b-octyl glucoside and 0.2 mm diethylenetri-
aminepentaacetic 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 ⁄ metha-
nol ⁄ acetic acid ⁄ water (10 : 4 : 3 : 2 : 1, v ⁄ v ⁄ v ⁄ v ⁄ v) as the
solvent system. Quantification of bands was carried out
using the Imaging Analyzer BAS2000.
ChIP assays
PPAR forms a heterodimer with RXR, and binds to PPRE
sequences of the direct repeat-1 (DR-1) type (a repeat sepa-
rated 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 pro-
vided 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,

sense 5¢-GTTTTGATCGTGTTTCGTGT-3¢) [35], were
incubated in a reaction mixture at room temperature for 20
min. The mixtures were then separated by electrophoresis
Role of CerK in PPARb-induced keratinocyte survival K. Tsuji et al.
3824 FEBS Journal 275 (2008) 3815–3826 ª 2008 The Authors Journal compilation ª 2008 FEBS
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, Swit-
zerland) and exposed to UV light (120 mJÆcm
)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 mini-
mal 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 · 10
5

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