Hypoxia downregulates farnesoid X receptor via a
hypoxia-inducible factor-independent but p38
mitogen-activated protein kinase-dependent pathway
Tomofumi Fujino
1
, Kaori Murakami
1
, Issei Ozawa
1
, Yoshie Minegishi
1
, Ryo Kashimura
1
, Toshihiro
Akita
1
, Susumu Saitou
1
, Takehisa Atsumi
1
, Takashi Sato
1
, Ken Ando
1
, Shuntaro Hara
2
, Kiyomi
Kikugawa
1
and Makio Hayakawa
1

(Received 18 September 2008, revised 28
November 2008, accepted 19 December
2008)
doi:10.1111/j.1742-4658.2009.06867.x
Farnesoid X receptor (FXR), a member of the nuclear receptor superfam-
ily, has been shown to play pivotal roles in bile acid homeostasis by regu-
lating the biosynthesis, conjugation, secretion and absorption of bile acids.
Accumulating data suggest that FXR signaling is involved in the pathogen-
esis of liver and metabolic disorders. Here we show that FXR expression is
significantly suppressed in HepG2 cells exposed to hypoxia. Concomitantly,
the expression of the bile salt export pump, known as an FXR target gene
product and responsible for the excretion of bile acids from the liver, is
also decreased under hypoxia. Overexpression of hypoxia-inducible factor
(HIF)-1a does not mimic the suppressive effect of hypoxia on FXR expres-
sion. Furthermore, simultaneous knockdown of HIF-1a, HIF-2a and
HIF-3a fails to restore the FXR expression level under hypoxia, indicating
that HIF is not involved in hypoxia-evoked FXR downregulation. Instead,
we demonstrate that p38 mitogen-activated protein kinase is an indispens-
able factor for FXR downregulation under hypoxia. Thus, we propose a
novel liver disorder model in which two signaling molecules, p38 mitogen-
activated protein kinase and FXR, may contribute to the linkage of two
pathogenic conditions, i.e. ischemia, a condition accompanying hypoxia,
and cholestasis, a condition with intrahepatic accumulation of cytotoxic
bile acids.
Abbreviations
BSEP, bile salt export pump; CDCA, chenodeoxycholic acid; ERK, extracellular signal-regulated kinase; FXR, farnesoid X receptor; FXRE,
farnesoid X receptor response element; GLUT-1, glucose transporter-1; HCC, hepatocellular carcinoma; HIF, hypoxia-inducible factor; IL,
interleukin; JNK, c-Jun N-terminal kinase; LRH-1, liver receptor homolog 1; MAPK, mitogen-activated protein kinase; MRP, multidrug
resistant-associated protein; NF-jB, nuclear factor kappaB; NR, nuclear receptor; SD, standard deviation; SHP, small heterodimer partner;
siRNA, small interfering RNA; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

and ileum bile acid-binding protein [13]. Thus, it is
clear that FXR is a key sensor for bile acids and has a
central role in maintaining bile acid homeostasis.
Bile acid homeostasis is the result of a balance
between bile acid uptake, efflux, and biosynthesis.
Maintenance of this balance is essential, as most bile
acids are cytotoxic. Cholestasis, a medical condition
characterized by the impairment of normal bile flow,
results in intrahepatic accumulation of cytotoxic bile
acids, which cause liver injury, ultimately leading to
biliary fibrosis and cirrhosis [14]. As the expression of
transporters responsible for bile acid export at the can-
alicular membrane are regulated by FXR, FXR has
been thought to be a possible pharmaceutical target
for the treatment of cholestasis [13].
In different types of clinical situations, liver ischemia
may occur and cause or contribute to hepatobiliary
dysfunction, which is most often of the cholestatic type
[15,16]. Several lines of evidence demonstrate that the
development of biliary cirrhosis is associated with the
occurrence of hepatocellular hypoxia and the induction
of hepatic angiogenesis [17–19]. Low levels of O
2
(hypoxia) are encountered by cells within rapidly
growing tissues, such as developing embryos or solid
tumors. Most vertebrates respond to this hypoxic
stress by activating the expression of a large number
of genes involved in glycolysis, angiogenesis, and
hematopoiesis [20]. This hypoxic transcriptional
response is mediated primarily by hypoxia-inducible

genesis and fibrogenesis through the inducible expres-
sion of vascular endothelial growth factor (VEGF), one
of the most representative HIF target gene products
[17–19]. In a recent report, Fouassier et al. demon-
strated that the expression of BSEP and FXR was
impaired in the ischemic rat liver or cultured hepato-
cytes exposed to hypoxia, whereas VEGF expression
was elevated under the same conditions [23]. However,
the molecular mechanism by which hepatocellular
hypoxia caused the reduced expression of the FXR and
BSEP genes has not been elucidated yet.
Bile acids are now recognized as important regula-
tory molecules, not only for their own synthesis, but
also for cholesterol synthesis, gluconeogenesis, glyco-
genesis, and apoptosis [24–26]. Among various signal-
ing pathways, mitogen-activated protein kinases
(MAPKs) play important roles in transducing or
modulating the bile acid-regulated cellular responses.
Hypoxia downregulates FXR via p38 MAPK T. Fujino et al.
1320 FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS
In mammals, four distinct subgroups of MAPKs have
been identified [27]. These include: (a) extracellular sig-
nal-regulated kinases (ERKs); (b) c-Jun N-terminal
kinases (JNKs); (c) p38 group MAPKs; and (d)
ERK5 ⁄ big MAP kinase 1 [27]. JNKs act as negative
regulators of bile acid synthesis by repressing CYP7A1
expression in SHP-independent ways [14]. Bile acid-
activated FXR induces the expression of the FGF19
gene, resulting in the suppression of CYP7A1 through
a JNK-dependent pathway [28]. Alternatively, JNK

normoxia, cells cultured under hypoxia exhibited mar-
ginal activation induced by CDCA (Fig. 1A). Similar
results were obtained when Huh7 cells were used
instead of HepG2 cells (Fig. 1B).
In order to verify whether the expression level of
BSEP, a target gene of FXR, is indeed lowered under
hypoxia, BSEP mRNA levels in cells under normoxia
or hypoxia were compared. As shown in Fig. 1C,
CDCA-induced elevation of BSEP mRNA was clearly
demonstrated under normoxia. In contrast, BSEP
induction by CDCA was greatly reduced under
hypoxia, indicating that hypoxia impaired the activity
of FXR, resulting in the lowered expression of BSEP.
Under the same hypoxic conditions, the level of
nuclear factor kappaB (NF-jB) activation induced by
TNF was comparable to that in cells under normoxia
(Fig. 1D), indicating that HepG2 cells exposed to
hypoxia maintained the physiological response in terms
of NF-jB activation. Furthermore, when viable cell
numbers were measured by 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-tetrazolium bromide assay, there was
no difference between hypoxia and normoxia (data not
shown). These results suggest that the hypoxia-induced
downregulation of FXR activity is a physiologically
regulated cellular response rather than a nonspecific
result reflecting lowered cell viability.
Next we examined whether or not FXR expression
itself is lowered in cells exposed to hypoxia. As shown
in Fig. 2A, the level of FXR protein detected by
antibody against FXR was significantly decreased in

exposed to hypoxic environments [37]. To elucidate
whether or not HIF is involved in the hypoxia-evoked
downregulation of FXR expression, we examined the
effect of ectopically overexpressed HIF-a isoforms on
T. Fujino et al. Hypoxia downregulates FXR via p38 MAPK
FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1321
FXR expression in HepG2 cells. HIF-a consists of three
isoforms, HIF-1a, HIF-2a, and HIF-3a [38]. Among
three isoforms, HIF-1a is ubiquitously expressed and
has been suggested to play a primary role in hypoxic
responses. When Pro402, Pro564 and Asn803 are
replaced by alanine residues, the HIF-1a mutant
becomes constitutively active even under normoxia,
escaping from degradation through the ubiquitin–pro-
teasome system. Indeed, we detected a significant level
of constitutively active form of HIF-a (HIF-1a)CA
expression in HepG2 cells under normoxia (Fig. 3A,
top panel). For HIF-2a and HIF-3a, overexpression
was achieved by the use of wild-type cDNAs without
Pro ⁄ Ala substitutions (Fig. 3A, middle and bottom
panels). We first examined whether or not ectopically
overexpressed HIF-a isoforms do indeed function in
HepG2 cells under normoxia. As shown in Fig. 3B,
HIF-1a CA and HIF-3a significantly induced glucose
transporter-1 (GLUT-1), a well-known HIF target gene
[39], whereas ectopically overexpressed HIF-2a failed to
elevate its level. As HIF-a isoforms are known to func-
tion in cell type-specific or target gene-specific ways [39],
HIF-2a may not be active in terms of GLUT-1 regula-
tion in HepG2 cells. Although at least HIF-1a and

cells were cultured under normoxia or hypoxia for 24 h in the pres-
ence of CDCA (100 l
M) or dimethylsulfoxide as vehicle control. The
amounts of BSEP mRNA were quantified by real-time PCR as
described in Experimental procedures. Results are calculated as
changes relative to the amount of BSEP mRNA from cells cultured
under normoxia in the absence of CDCA. Data are shown as the
mean ± SD of four determinations. Similar results were obtained in
five separate experiments. (D) HepG2 cells were cotransfected
with 3 · jB–Luc luciferase reporter vector and R. reniformis lucifer-
ase expression vector phRL–TK. After 24 h, cells were stimulated
with TNF and further cultured under normoxia or hypoxia for 24 h.
Results are calculated as changes relative to the NF-jB activity in
cells cultured under normoxia in the absence of TNF. Data are
shown as the mean ± SD of four determinations. Similar results
were obtained in three separate experiments.
Hypoxia downregulates FXR via p38 MAPK T. Fujino et al.
1322 FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS
As shown in Fig. 4A, the expression levels of endoge-
nous HIF-1a and HIF-2a were significantly elevated in
cells exposed to hypoxia. When cells were transfected
with the combined mixture of small interfering RNAs
(siRNAs) against HIF-1a, HIF-2a, and HIF-3a, the
levels of HIF-1a and HIF-2a in cells under hypoxia
were drastically decreased (Fig. 4A). In the case of
HIF-3a, endogenous HIF-3a was not significantly ele-
vated under hypoxia; therefore, knockdown efficiency
was evaluated using HepG2 cells that ectopically over-
expressed HIF-3a. As shown in Fig. 4B, the combined
transfection of three siRNAs against HIF-1a, HIF-2a

changes relative to the amount of FXR mRNA from cells cultured
under normoxia. Data are shown as the mean ± SD of four deter-
minations. Similar results were obtained in three separate experi-
ments. (C) HepG2 cells were cultured under normoxia or hypoxia
for 24 h, and the amounts of LRH-1 mRNA were quantified by real-
time PCR as in (B). Results are calculated as changes relative to
the amount of LRH-1 mRNA from cells cultured under normoxia.
Data are shown as the mean ± SD of four determinations. Similar
results were obtained in three separate experiments. (D) HepG2
cells were cultured under normoxia or hypoxia for 24 h, and the
amounts of SHP mRNA were quantified by real-time PCR as in (B).
Results are calculated as changes relative to the amount of SHP
mRNA from cells cultured under normoxia. Data are shown as the
mean ± SD of four determinations. Similar results were obtained in
three separate experiments. (E) HepG2 cells were treated with acti-
nomycin D (5 l
M) and then cultured under normoxia or hypoxia for
indicated times. The amounts of FXR mRNA were quantified by
real-time PCR as described in Experimental procedures. Results are
calculated as changes relative to the amount of FXR mRNA at 0 h.
Data are shown as the mean ± SD of four determinations. Similar
results were obtained in three separate experiments.
T. Fujino et al. Hypoxia downregulates FXR via p38 MAPK
FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS 1323
lines of evidence indicate that hypoxia induces the acti-
vation of MAPK families [33–35,40,41]. The activation
of each MAPK was examined by the use of antibodies
that detect phosphorylated forms of MAPKs (Fig. 5).
Before cells were exposed to hypoxia, the phosphory-
lated form of p38 was retained at a low level; however,

siRNAs against HIF-1a, HIF-2a, and HIF-3a, and then cultured under
normoxia or hypoxia for 24 h. Cell lysates were then prepared, and
immunoblotting analyses were then performed to detect HIF-1a
and HIF-2a. (B) In order to estimate the knockdown efficiency of
HIF-3a, HepG2 cells transfected with pcDNA3.1–HIF-3a were then
treated with the combined mixture of siRNAs against HIF-1a,
HIF-2a, and HIF-3a, and then cultured under normoxia for 24 h. Cell
lysates were then prepared, and this was followed by immunoblot-
ting analysis to detect HIF-3a. (C) As described in (A), cell lysates
were prepared, and then immunoblotting analyses were performed
to detect FXR or to detect b-actin levels as a loading control. (D)
HepG2 cells transfected with the combined mixture of siRNAs
against HIF-1a, HIF-2a and HIF-3a were then cultured under
hypoxia for 24 h. After 24 h, total RNAs were prepared, and then
real-time PCR analysis was performed to detect FXR mRNA as
described in Experimental procedures.
Hypoxia downregulates FXR via p38 MAPK T. Fujino et al.
1324 FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS
hypoxia treatment (Fig. 5). On the other hand, phos-
phorylation of ERK2 was observed before hypoxia
treatment. Hypoxia treatment for 8 h resulted in a
two-fold elevation of phospho-ERK2 level, whereas
only slight activation of ERK1 was observed under
hypoxia (Fig. 5). During this hypoxia treatment, the
expression levels of FXR were inversely lowered in a
time-dependent manner (Fig. 5). It should be noted
that we could not detect the activation of JNK using
anti-phospho-JNK antibodies in HepG2 cells exposed
to hypoxia, whereas the same antibodies can detect the
activation of JNK in L929 cells treated with TNF

rather resulted in impairment of cell viability (data not
shown). In spite of this partial knockdown efficiency,
p38a siRNA treatment sufficiently reversed the
hypoxia-dependent downregulation of FXR. As shown
in the top panel of Fig. 6A, the amount of FXR pro-
tein under hypoxia was 27% of the value of the ‘norm-
oxia’ control, and the treatment of cells with p38a
siRNA increased the FXR protein level to 42% even
under hypoxia (Fig. 6A, top panel). The restorative
effect of p38 siRNA was also confirmed by the quanti-
fication of FXR mRNA. As shown in Fig. 6B, the
lowered FXR mRNA level under hypoxia was signifi-
cantly elevated by treatment of cells with p38a siRNA.
The reduced BSEP mRNA expression under hypoxia
was also restored by the same treatment with p38a
siRNA (Fig. 6C). As a stress-activated MAPK, p38 is
known to be activated by various stimuli, including
proinflammatory cytokines. As shown in Fig. 6D,
interleukin (IL)-1b induced strong p38 activation in
HepG2 cells. Interestingly, the FXR level was reduced
by treatment with IL-1b (Fig. 6D, top panel). These
results suggest that p38 acts as an upstream signaling
molecule that responds to various environmental
stresses, including hypoxia, and downregulates FXR
transcription.
We next examined the role of ERK1 ⁄ 2 in hypoxia-
dependent downregulation of FXR. As shown in
Fig. 6E, simultaneous knockdown of ERK1 and
ERK2 reduced the ERK2 protein level to 44% of the
value of the ‘normoxia’ control (Fig. 6E, middle

by IL-1b also leads to the downregulation of FXR
(Fig. 6D).
Discussion
In the current study, we have demonstrated that FXR, a
key transcription factor that regulates bile acid meta-
bolism, is downregulated under hypoxia through a p38
MAPK-dependent mechanism. The experimental model
shown here may give an explanation of how chronic
ischemia impairs liver function by attenuating the bile
acid homeostasis regulated by FXR and possibly leads
to progressive liver disorders such as primary biliary
cirrhosis and primary sclerosing cholangitis. Indeed, an
ischemia-induced low-oxygen condition, i.e. hypoxia,
has been thought of as a possible cause of bile duct
injury, in particular after liver transplantation, hepatic
surgery, and intra-arterial chemotherapy [23,46,47].
Hypoxia is a serious stress for living organs, because
of the need to make the massive change from oxygen-
dependent to oxygen-independent energy production.
Therefore, the ability to sense and respond to changes
in oxygen is essential for the survival of multicellular
organisms. HIF is the key transcription factor for sens-
ing and responding to lowered oxygen, acting by
inducing the transcription of various genes required
C
B
D
E
A
Fig. 6. p38 but not ERK1 ⁄ 2 MAPK is involved in the hypoxia-

lysates were then prepared followed by the immunoblotting analy-
ses to detect FXR and ERK1 ⁄ 2orb-actin levels as a loading
control. Quantification of the bands was done by densitometric
analysis (
IMAGE GAUGE 4.0). Similar results were obtained in three
separate experiments.
Hypoxia downregulates FXR via p38 MAPK T. Fujino et al.
1326 FEBS Journal 276 (2009) 1319–1332 ª 2009 The Authors Journal compilation ª 2009 FEBS
for survival under conditions where the oxygen supply
is limited. VEGF is one such HIF-targeted gene, and
is known to be upregulated in the cirrhotic liver [17].
During the development of biliary cirrhosis, hepatocel-
lular hypoxia and hepatic angiogenesis induced by
VEGF are known to contribute to the progression of
liver fibrosis [18]. Moreover, enhanced proliferation of
liver tumor cells leads to local hypoxia in hepato-
cellular carcinoma (HCC) [48], and in turn, hypoxia-
induced expression of angiogenic factors such as
VEGF results in the hypervascularity of HCC.
Undoubtedly, HIF and VEGF play central roles dur-
ing new vessel formation in HCC [48]. In contrast, our
present study has revealed that HIF is not involved in
hypoxia-dependent downregulation of FXR. Thus,
hypoxia may affect liver disorders by activating two
signaling pathways: one is the HIF-dependent pathway
that leads to the induction of VEGF, resulting in
hypervascularity in the liver; and the other is the
HIF-independent but p38-mediated pathway that
causes down-egulation of FXR and BSEP, resulting in
a decreased capacity for bile acid excretion from the

the hepatic expression of human MRP3 is usually very
low; however, it is induced in patients with cholestasis
and cirrhosis [54]. Thus, the basolateral MRPs upregu-
lated during severe cholestasis may act as an alterna-
tive export system to eliminate bile acids from the liver
by elevating bile acid efflux across the basolateral
membrane of the hepatocyte, instead of their being
excreted through the bililary tract using transporters
located on the canalicular membrane.
Therefore, hypoxia-evoked downregulation of FXR
and BSEP may have opposite effects on the progress of
cholestasis, depending on whether or not bile flow is
severely obstructed. Under conditions where biliary
tract is not yet injured, the decreased bile flow that
results from the downregulation of FXR ⁄ BSEP in
response to hypoxia will lead to the accumulation of
toxic bile acids in hepatocytes, and cholestasis will con-
sequently be promoted. It should be noted that genetic
defects of BSEP cause a severe liver disease in humans
called progressive familial intrahepatic cholestasis
type 2, which leads to irreversible liver damage, owing
to intrahepatic bile acid accumulation [55]. In addition,
decreased FXR expression and activity is known to be
associated with FIC1 mutations, suggesting that FXR
itself may play an important role in the pathogenesis of
progressive familial intrahepatic cholestasis type 1
[56,57]. As we have shown in our present study, the
expression level of FXR is dynamically changed in cells
in response to extracellular stimuli, such as hypoxic
stress. Thus, at the early stage of ischemia, when bile

signaling pathways [14,28–32]. In this study, we have
highlighted p38 MAPK and FXR as the signaling mol-
ecules that may play key roles in the pathogenesis of
the liver disorders accompanying ischemia and chole-
stasis. Although the molecules that act downstream of
p38 MAPK to suppress the function of FXR remain
to be elucidated, we believe that our present study con-
tributes to the understanding of the molecular basis of
cholestasis progressing under ischemia. Further
insights into the crosstalk between p38 MAPK and
FXR will be useful in identifying a novel therapeutic
target for this type of liver disorder.
Experimental procedures
Antibodies
Antibodies specific for b-actin (C-2), FXR (D-3) and
HIF-3a (H-170) were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). Antibodies against p38,
phospho-p38 (Thr180 ⁄ Tyr182) and phospho-ERK1 ⁄ 2 were
obtained from Cell Signalling Technology (Danvers, MA,
USA). Antibody against HIF-1a (clone 54) was a product
of BD Biosciences Pharmingen (Franklin Lakes, NJ, USA).
Antibody against HIF-2a (EP-190b) was obtained from
Novus Biologicals (Littleton, CO, USA). ECL anti-mouse
IgG, horseradish peroxidase-linked whole antibody (from
sheep), and ECL anti-rabbit IgG, horseradish peroxidase-
linked whole antibody (from donkey), were purchased from
GE Healthcare (Little Chalfont, UK).
Cell culture
Human hepatocellular carcinoma cell lines HepG2 and Huh7
were cultured in DMEM containing 10% fetal bovine serum,

into cells using Lipofectamine 2000 transfection reagent
(Invitrogen, Carlsbad, CA, USA) according to the manu-
facturer’s instructions. After 24 h of incubation, cells were
treated with 100 lm CDCA, and then further incubated in
the hypoxia workstation or left under the regular culture
condition (normoxia) for 24 h. Cells were then harvested,
and cellular firefly and Renilla luciferase activities were
measured using a chemiluminescense photometer. Firefly
luciferase activity was normalized with that of Renilla
luciferase. Data were analyzed by Student’s t-test.
NF-jB activity assay
TNF-induced NF-jB activation in HepG2 cells exposed to
hypoxia was assessed by dual luciferase assay (Promega).
HepG2 cells were seeded on a 24-well culture plate at a
density of 2 · 10
5
cells per well and cultured for 24 h, and
3 · j B–Luc luciferase reporter vector (0.4 lg) and phRL–
TK (0.04 lg) were cotransfected into cells using Lipofecta-
mine 2000 transfection reagent (Invitrogen). After 24 h of
incubation, cells were treated with 1 or 10 ngÆmL
)1
TNF,
and then further incubated in the hypoxia workstation or
left under the regular culture condition (normoxia) for
24 h. Cells were then harvested, and cellular firefly and
Renilla luciferase activities were measured using a chemilu-
minescense photometer. Firefly luciferase activity was
normalized with that of Renilla luciferase.
Immunoblotting

Hs00231968 can detect all of the four isoforms of FXR [60].
Amplification and quantification were done with the
PRISM 7000 Real-Time PCR System (Applied Biosystems).
FXR, BSEP, LRH-1, SHP and GLUT-1 mRNA levels
were normalized to the levels of b-actin mRNA as an internal
control. Data were analyzed by Student’s t-test.
Construction of the constitutively active HIF-1a
expression vector
We have previously used the expression vector encoding the
mutant of HIF-1a, in which Pro564 and Asn803 are
replaced by alanine [61], as this mutant was expected to act
as a constitutively active version of HIF-1a [21]. In addi-
tion to Pro564, Pro402 in HIF-1a is now recognized as the
target residue of prolyl hydroxylase; therefore, we made an
additional mutation, replacing Pro402 with the alanine resi-
due. In brief, we performed site-directed mutagenesis by
PCR as follows. The pBluescript SK vector (Stratagene, La
Jolla, CA, USA) encompassing an HIF-1a cDNA in which
Pro564 and Asn808 were replaced with alanines was used
as a template. Two sets of primers [set 1, 5¢-TAGTCCAG
TGTGGTGGAATTCTGC-3¢ (sense) and 5¢-AAAGCATC
AGGTTCCTTCTTAAG-3¢ (antisense); set 2, 5¢-AACTTT
GCTGGCCGCCGCCGCTGG-3¢ (sense) and 5¢-GGCAAC
TAGAAGGCACAGTCGAGG-3¢ (anti-sense)] were used
to generate the substitution of Pro402 for the alanine resi-
due. The resultant cDNA was subcloned into pcDNA3.1
vector (Invitrogen) and termed pcDNA3.1–HIF-1a CA.
Transfection of HIF-a isoform
HepG2 cells were seeded on 35 mm dishes at a density of
2 · 10

(150 nm each), using Oligofectamine Reagent (Invitrogen)
according to the manufacturer’s instructions. On the day
after the first transfection, a second transfection was
performed similarly to the first [63]. Cells were then
cultured under normoxic or hypoxic conditions for 24 h,
and protein extracts or total RNAs were prepared for
immunoblotting or real-time PCR analyses. To knock
down endogenous p38a or ERK1 ⁄ 2 expression, we used the
following validated siRNAs: Hs_MAPK14_6_HP validated
siRNA against human p38a; Hs_MAPK3_7_HP validated
siRNA against human ERK1; and Hs_MAPK1_10_HP
validated siRNA against human ERK2. HepG2 cells were
seeded on 35 mm dishes at a density of 5 · 10
5
cells per
dish. Immediately after seeding, cells were transfected with
p38a siRNA (10 nm), or with the mixture of ERK1 siRNA
and ERK2 siRNA (10 nm each), using HiPerfect Transfec-
tion Reagent (Qiagen) according to the manufacturer’s
instructions. Cells were then cultured under hypoxic condi-
tions for 24 h, and protein extracts or total RNAs were
prepared for immunoblotting or real-time PCR analyses. In
the series of RNA interference experiments, ‘nonsilencing
control’ siRNA (#1022076) from Qiagen was used as a
control.
Acknowledgements
We thank R. Sato, T. Nishimaki-Mogami and H. Hay-
ashi for helpful advice and discussions. We also thank
S. Miyamoto for providing us with the 3 · jB–Luc
luciferase reporter vector. This work was supported in

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