Molecular and Cellular Pharmacology
Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice
under non-hypoxic conditions in a μ-opioid receptor-dependent manner
Hiroki Daijo a, Shinichi Kai a, Tomoharu Tanaka a, Takuhiko Wakamatsu a, Shun Kishimoto a,
Kengo Suzuki a,b, Hiroshi Harada c,d, Satoshi Takabuchi a, Takehiko Adachi e,
Kazuhiko Fukuda a, Kiichi Hirota a,⁎
a Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan
b Department of Anesthesiology, Graduate School of Medicine Tohoku University, Sendai, Japan
c Group of Radiation and Tumor Biology, Career-Path Promotion Unit for Young Life Scientists, Kyoto University, Kyoto, Japan
d Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, Kyoto, Japan
e Department of Anesthesia, Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan
a b s t r a c ta r t i c l e i n f o
Article history:
Received 27 October 2010
Received in revised form 20 April 2011
Accepted 6 June 2011
Available online 17 June 2011
Keywords:
Fentanyl
Opioid
Hypoxia-inducible factor 1 (HIF-1)
SH-SY5Y cells
Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene
expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However,
the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of
fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells,
lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was
examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression
was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase
assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was
investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other
opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target
gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24 h.
Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays
and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did
not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of
mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that
fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases
in expression of genes associated with adaptation to hypoxia.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Opioids are among the most powerful analgesic drugs used in
pain treatment (Fukuda, 2009). In addition to their well recognized
analgesic effects, various studies suggest that opioids elicit a variety of
biological effects that appear to be independent of their analgesic
properties, and may affect cell survival or proliferation (Martin-Kleiner
et al., 2006; Tegeder and Geisslinger, 2004). With regard to cell
protective effects, recentdata indicate that activation of opioid receptors
preserves cellular status following hypoxic insults and immunomodula-
tion (Martin-Kleiner et al., 2006; Peart et al., 2005).
Oxygen (O2) deprivation in human tissue leads to the up-
regulation of a complex array of genes that exert adaptive functions,
particularly with regard to cellular metabolism and improvement in
O2 delivery (Hirota, 2002; Semenza, 2001). One of the most important
transcription factors controlling O2-tension regulated gene expression
is hypoxia-inducible factor 1 (HIF-1) (Semenza, 2001). HIF-1 is a
heterodimer containing the constitutively expressed HIF-1β subunit
and the inducible HIF-1α subunit (Wang et al., 1995). Regulation of
HIF-1 activity occurs at multiple levels in cells, and mechanisms
regulating HIF-1α protein expression and transcriptional activity have
been extensively analysed. The von Hippel–Lindau tumour suppressor
protein has been identified as a HIF-1α-binding component of an
ubiquitin–protein ligase that targets HIF-1α for proteasomal degra-
dation in non-hypoxic cells (Hirota and Semenza, 2005; Huang et al.,
1996; Kallio et al., 1997). Under hypoxic conditions, hydroxylation of
European Journal of Pharmacology 667 (2011) 144–152
⁎ Corresponding author at: Department of Anesthesia, Kyoto University Hospital, 54
Shogoin-Kawaracho, Sakyo-Ku, Kyoto 606-8507, Japan. Fax: +81 75 752 3259.
E-mail address: hif1@mac.com (K. Hirota).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.06.014
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HIF-1-specific proline and asparagine residues is inhibited due to
substrate (O2) limitation, resulting in HIF-1α protein stabilization and
transcriptional activation (Hirota and Semenza, 2005; Huang et al.,
1998). Physiological stimuli other than hypoxia can also induce HIF-1
activation and transcription of hypoxia-inducible genes. Signalling via
the HER2/neu or IGF-1 receptor tyrosine kinase induces HIF-1
expression in an oxygen-independent mechanism. HER2/neu or
IGF-1 stimulation increases the rate of HIF-1α protein synthesis via
phosphatidylinositol 3-kinase (PI3K) and the downstream serine/
threonine kinases AKT and FRAP (Fukuda et al., 2002; Laughner et al.,
2001). In addition, acetylcholine or prostaglandin E2 also activates
HIF-1 by increase of neosynthesis of HIF-1α protein (Fukuda et al.,
2003; Hirota et al., 2004). Notably, cellular receptors for these agents
are heterotrimeric guanine nucleotide binding (G) protein-coupled
receptors.
We previously reported that opioid receptor stimulation does not
affect cellular hypoxia-induced gene responses mediated by HIF-1 in
cultured cell lines (Takabuchi et al., 2005). However, in that study we
only investigated the effects of opioids on HIF-1 activation over a 4-h
period. Opioids have recently been used over a prolonged perioper-
ative period; therefore, we examined the effect of opioid receptor
stimulation over a prolonged treatment time. In this study, we aimed
to investigate whether opioid receptor stimulation by fentanyl,
morphine and remifentanil induces HIF-1α protein expression and
HIF-1-dependent gene expression. Furthermore, we investigated
whether intraperitoneal administration of fentanyl induces HIF-1
downstream gene expression in the brain in vivo.
2. Materials and methods
2.1. Cell culture and reagents
Human SH-SY5Y neuroblastoma cells obtained from ATCC,
Manassas, VA, were maintained in RPMI 1640 medium containing
10% fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml
streptomycin (Takabuchi et al., 2005; Tanaka et al., 2010a; Wakamatsu
et al., 2009). Human hepatoma Hep3B and lung adenocarcinoma A549
cells were maintained in Dulbecco's modified Eagle's medium supple-
mented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin
(Tanaka et al., 2010a,b). Naloxone, desferrioxamine (DFX) and kinase
inhibitors LY294002, PD98059 and GF109203X were obtained from
Sigma (St. Louis, MO). Cycloheximide (CHX) and dithiothreitol (DTT)
was obtained from Calbiochem (San Diego, CA). The selective opioid
receptor agonists [D-Ala2,N-Me-Phe4,Gly5-ol]-ENKEPHALIN (DAMGO),
[D-Pen2,5]-ENKEPHALIN (DPDPE) and (±)-trans-U-50488 methanesul-
fonate salt (U-50488) were also obtained from Sigma. Anti-HIF-1α
antibody was purchased from BD Biosciences (San Jose, CA) and anti-
HIF-1β antibody was purchased from Novus Biologicals (Littleton, CO).
Anti-β-actin antibody was purchased from Sigma.
2.2. Hypoxic treatment
Cells were maintained in a multigas incubator (APMW-36; Astec,
Fukuoka, Japan) and exposed to hypoxia (1% O2:5% CO2:94% N2)
(Hirota and Semenza, 2001; Tanaka et al., 2010b; Wakamatsu et al.,
2009).
2.3. Immunoblot Assays
Whole cell lysates were prepared using ice-cold lysis buffer [0.1%
SDS, 1% Nonidet P-40, 5 mM EDTA, 150 mM NaCl, 50 mM Tris–Cl
(pH 8.0), 2 mM DTT, 1 mM sodium orthovanadate and the Complete
Protease Inhibitor™ (Roche Diagnostic, Tokyo, Japan)] as described
previously (Kasuno et al., 2004). Samples were centrifuged at
10,000×g to pellet cell debris. For HIF-1α and -β, 100 μg of protein
was fractionated by SDS-PAGE (7.5% gel) and subjected to an
immunoblot assay using primary antibodies at a dilution of 1:1000.
Anti-β-actin mouse monoclonal antibody (Sigma) was used as the
control at a dilution of 1:5000. Horseradish peroxidase-conjugated
sheep anti-mouse IgG (GE Healthcare, Piscataway, NJ) was used as the
secondary antibody at a dilution of 1:1000. The signal was developed
using enhanced chemiluminescence reagent (GE Healthcare). The
intensity of each band was quantified using Image J software (Tanaka
et al., 2010b).
2.4. Gene silencing by siRNA
SH-SY5Y cells grown until 30%–50% confluence were plated on a
24-well plate using RPMI 1640 without antibiotics, and transfected
with the Validated Stealth RNAi (100 pmol/mL) for HIF-1α gene
(Invitrogen Corp.) using Lipofectamine RNAiMAX (Invitrogen Corp.),
according to the manufacturer's instructions (Harada et al., 2009).
Transfected cells were incubated in a normoxic incubator for 24 h
following fentanyl treatment. The Stealth RNAi Negative Control Kit
(Invitrogen Corp.) was used as the negative control. The human HIF-1α
siRNA sequence used was 5′-GGAUGCUGGUGAUUUGGAUAUUGAA′.
2.5. Reverse transcriptase (RT)-PCR analysis for HIF-1α
The RT-PCR protocol is described elsewhere (Nishi et al., 2008).
Cells were harvested, and RNA was isolated with TRIzol (Invitrogen).
1 μg of total RNA was subjected to first strand cDNA synthesis using
random hexamers (SuperScript II RT kit, Invitrogen). cDNAs were
amplified with TaqGold polymerase (Roche, Manheim, Germany) in a
thermal cycler with the specific primers (sequences of the primers can
be pro- vided by request). PCR was optimized for cycle number to
obtain linearity between the amount of input RT product and output
PCR product. Thermocycling conditions were 30 s at 94 °C, 60 s at
57 °C, and 30 s at 72 °C for 25 cycles preceded by 10 min at 94 °C. PCR
products were fractionated by 1.5% SeaKem GTG Agarose gel
electrophoresis, stained with ethidium bromide, and visualized with
UV. Primers for HIF-1α used are as follows: upper; 5′-GAAAGCG-
CAAGTCCTCAAA-3′, lower; 5′-CTATATGGTGATGATGTGGCACTA-3′.
2.6. Quantitative real-time reverse transcriptase (RT)-PCR analysis
(qRT-PCR)
RNAwas purified using RNeasy™ (Qiagen, Valencia, CA) and treated
withDNase (Odaet al., 2008; Tanakaet al., 2010b). First-strand synthesis
and real-timePCRwereperformedusing theQuantiTect SYBRGreenPCR
Kit (Qiagen) following the manufacturer's protocol. Amplification and
detectionwere performed using the Applied Biosystems 7300 Real-time
PCR System (Applied Biosystems, Foster City, CA). PCR primers were
purchased from Qiagen (human Glut-1; QT00068957, human VEGF;
QT01682072, human LDHA; QT00001687, humanHIF-1α; QT00083664,
human μ-opioid receptor; QT00001512, human δ-opioid receptor;
QT00000210, human κ-opioid receptor; QT00015316, mouse Glut-1;
QT01044953 andmouse VEGF; QT00160769). The change in expression
of each target mRNA relative to 18S rRNA was calculated (Oda et al.,
2008; Tanaka et al., 2010b).
2.7. Reporter gene assay
The reporter plasmid p2.1, harbouring a 68-bp hypoxia response
element (HRE) from the human enolase 1 gene inserted upstream of
an SV40 promoter and Photinus pyralis (firefly) luciferase coding
sequences, has been described previously (Jiang et al., 1997; Kasuno
et al., 2004; Semenza et al., 1996). Cells were plated at a density of
5×104 cells per well a day before transfection. During each
transfection, 200 ng of reporter gene plasmid p2.1 or 50 ng of the
control plasmid pRL-SV40 (Promega, Madison, WI), containing the
SV40 promoter upstream of Renilla reniformis (sea pansy) luciferase
145H. Daijo et al. / European Journal of Pharmacology 667 (2011) 144–152