The role of sphingosine 1-phosphate in the TNF-α induction of
IL-8 gene expression in lung epithelial cells
Hemakumar Chandru, Vijayakumar Boggaram⁎
Department of Molecular Biology, University of Texas Health Center at Tyler, 11937 US Highway 271 Tyler, TX 75708-3154, United States
Received 24 August 2006; received in revised form 13 November 2006; accepted 11 December 2006
Available online 22 December 2006
Received by A.J. van Wijnen
Tumor necrosis factor-α (TNF-α) is an important cytokine involved in the pathogenesis of inflammatory diseases of the lung. Interleukin-8
(IL-8), a C-X-C chemokine, is induced by TNF-α and initiates injury by acting as a chemoattractant for neutrophils and other immune cells.
Although sphingolipids such as ceramide and sphingosine 1-phosphate (S1-P) have been shown to serve as signaling molecules in the TNF-α
inflammatory response, their role in the TNF-α induction of IL-8 gene expression in lung epithelial cells is not known. We investigated the role of
sphingolipids in the TNF-α induction of IL-8 gene expression in H441 lung epithelial cells. We found that TNF-α induced IL-8 mRNA levels by
increasing gene transcription, and the stability of IL-8 mRNA was not affected. Exogenous S1-P but not ceramide or sphingosine increased IL-8
mRNA levels and IL-8 secretion. Dimethylsphingosine, an inhibitor of sphingosine kinase, partially inhibited TNF-α induction of IL-8 mRNA
levels indicating the importance of intracellular increases in S1-P in the IL-8 induction. S1-P induction of IL-8 mRNA was due to an increase in
gene transcription, and the stability of IL-8 mRNAwas not affected. S1-P induction of IL-8 mRNAwas associated with an increase in the binding
activity of AP-1 but the activities of NF-κB and NF IL-6 were unchanged. S1-P induced the phosphorylation of ERK, p38 and JNK MAPKs.
Pharmacological inhibitors of ERK and p38 but not JNK partly inhibited S1-P induction of IL-8 mRNA levels. These data show that increases in
the intracellular S1-P partly mediate TNF-α induction of IL-8 gene expression in H441 lung epithelial cells via ERK and p38 MAPK signaling
pathways and increased AP-1 DNA binding.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Sphingolipids; Transcription; Lung injury; Inflammation
by maintaining alveolar stability through secretion of surfactant,
clearance of inhaled particulates by mucociliary action, and
facilitates phagocytosis of pathogens by secretory immunoglob-
ulin and surfactant production. Additionally the respiratory
epithelium plays important roles in the control of various aspects
of inflammation such as initiation, amplification and down
regulation as well as tissue repair through the elaboration of
various mediators including cytokines (Standiford et al., 1990;
necrosis factor-α (TNF-α), a cytokine expressed predominantly
by the alveolar macrophages has been implicated in the
pathophysiology of inflammatory diseases of the lung resulting
Gene 391 (2007) 150–160
Abbreviations: IL-8, interleukin-8; TNF-α, tumor necrosis factor-alpha;
ARDS, acute respiratory distress syndrome; PAF, platelet activating factor; SSC,
0.15 M NaCl/0.015 M Na3·citrate pH7.6; ELISA, enzyme-linked immunosorbent
assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; EMSA, electropho-
retic mobility shift assay; DMS, N,N-dimethyl-D-erythro-sphingosine; DTT,
dithiothreitol; MAP kinase, mitogen activated protein kinase; MAPP, D-erythro-
2-(N-myristoylamino)-1-phenol-1-propanol; MOPS, [3-(N-morpholino) propane-
sulfonic acid]; NOE, N-oleoyl-ethanolamine; S1-P, sphingosine 1-phosphate;
SiRNA, small inhibitor RNA; SP, surfactant protein; NF-κB, nuclear factor
kappaB; AP-1, activator protein 1; NF IL-6, nuclear factor IL-6; CCT-α, CTP:
⁎Corresponding author. Tel.: +1 903 877 7780.
E-mail address: firstname.lastname@example.org (V. Boggaram).
0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
(Tracey et al., 1988; Piguet, 1990; Suter et al., 1992). TNF-α
levels are markedly elevated in the bronchoalveolar lavage fluid
from patients with acute respiratory distress syndrome (ARDS)
(Suter et al., 1992). Elevated TNF-α levels are associated
with increased IL-8 levels, and TNF-α is a major inducer of
IL-8 expression in lung epithelial cells (Standiford et al., 1990).
IL-8, a member of the C-X-C family of chemokines is a potent
immune cells (Kunkel et al., 1995). Neutralizing IL-8 antibodies
prevented lung injury in animal models of lung disease,
indicating that IL-8 is an important mediator of lung injury
(Broaddus et al., 1994; Modelska et al., 1999).
The sphingolipid ceramide has long been recognized as a
signaling molecule in the inflammatory response. It acts as a
a host of stress agents including TNF-α (Kim et al., 1991),
ionizing radiation (Haimovitz-Friedman et al., 1994) and platelet
activating factor (PAF) (Goggel et al., 2004). Ceramide can be
produced via de novo synthesis or hydrolysis of membrane
sphingomyelin by sphingomyelinase enzymes. TNF-α increases
intracellular levels of ceramide by activating lysosomal and
plasma membrane-associated sphingomyelinases that display
different pH and metal requirements. There is accumulating
pathophysiology of acute lung injury. Lung cells express high
sphingolipid levels are elevated in acute lung injury. Intratracheal
administration of TNF-α and ceramide into rats increases lung
permeability and decreases surface tension lowering effects of
surfactant (Ryan et al., 2003). In other studies, platelet activating
2004). PAF induced edema was suppressed in acid sphingomye-
linase deficient animals and was reduced by the non-specific acid
acid sphingomyelinase-mediated production of ceramide is
responsible for the edema (Goggel et al., 2004). Ceramide once
produced can be metabolized further into sphingosine and S1-P
by the sequential actions of ceramidase and sphingosine kinase
in diverse cellular activities such as cell growth and survival, cell
motility and immunity (Spiegel and Milstien, 2003).
Although TNF-α induction of IL-8 gene expression in lung
epithelial cells has been studied previously, information is
lacking on the role of sphingolipid metabolites in the induction
process. Considering that the respiratory epithelium plays key
roles in the initiation and amplification of inflammatory
responses and that sphingolipid metabolites are recognized as
important signaling molecules in the inflammatory response, we
sought to understand the roles of sphingomyelin metabolites in
the TNF-α induction of IL-8 mRNA. In this investigation, we
studied the effects of TNF-α and sphingolipids on IL-8 gene
expression in lung H441 cells, a cell line with characteristics of
bronchiolar (Clara) epithelial cells. We found that TNF-α
induced IL-8 mRNA levels by increasing gene transcription
without altering IL-8 mRNA stability. Of the sphingolipid
metabolites tested, S1-P but not ceramide or sphingosine in-
duced IL-8 mRNA and protein levels. TNF-α induction of
IL-8 mRNA levels was partly reduced by dimethylsphingo-
sine, an inhibitor of sphingosine kinase, indicating the impor-
tance of elevated levels of intracellular S1-P in the induction.
Consistent with the effects of dimethylsphingosine, SiRNA-
mediated inhibition of sphingosine kinase partially blocked
TNF-α induction of IL-8 mRNA. S1-P induction of IL-8mRNA
was associated with an increase in AP-1 DNA binding activity,
however NF-κB binding activity was unchanged. S1-P in-
inhibitors, SB203580 and PD98059 indicating the involve-
ment of ERK and p38 MAPKs in the S1-P regulation of
2.1. Cell culture
NCI-H441 cells [American Type Culture Collection (ATCC)
HTB-174], a human lung adenocarcinoma cell line of
bronchiolar (Clara) cell lineage were grown on plastic tissue
culture dishes in RPMI 1640 medium containing 10% fetal
bovine serum, penicillin (100 unit/ml), streptomycin (100 μg/
ml) and amphotericin B (0.25 μg/ml). In all experiments the
medium was changed to RPMI 1640 without serum for 24 h
before the start of the experiment. In experiments to test the
effects of S1-P on IL-8 expression, BSA (0.4% w/v) was added
to the culture medium.
TNF-α was purchased from R & D Sytems (Minneapolis,
MN). TRI Reagent was from Molecular Research Center (Cin-
cinnati, OH). Actinomycin D, bovine serum albumin [low endo-
toxin (≤0.1 ng/ml) and fatty acid-free], Staphylococcus aureus
sphigomyelinase and sphingosine were from Sigma (St. Louis,
MO). 5,6-dichloro-1-b-D-ribofuranozyl-benzimidazole (DRB)
was from Calbiochem. C2-ceramide, sphingosine 1-phosphate
and N,N-Dimethyl-D-erythro-sphingosine (DMS) were obtained
from Avanti (Alabaster, AL). S1-P was dissolved in a mixture
of methanol–water (95:5) at 0.5 mg/ml by heating at 45 °C for
10–15 min followed by sonication for 10 s each time for three
times. Solubilized S1-P was dried under nitrogen and recon-
stituted in cell culture medium containing 0.4% bovine serum
2.3. RNA isolation and Northern blot analysis
Experimental procedures for total RNA isolation and
Northern blotting analysis are as described previously (Bog-
garam and Margana, 1994). Cytosolic RNA was isolated
according to published protocol (Greenberg and Bender,
1997). IL-8 and GAPDH RNA bands were quantified with a
PhosphorImager using Quantity One Image Acquisition and
Analysis Software (Bio-Rad) and IL-8 mRNA levels were
normalized to 18S rRNA levels to correct for variations in the
quantification, loading and transfer of RNA. The expression of
151H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
GAPDH mRNA was assessed as an internal control. Plasmids
encoding human IL-8 cDNA were kindly provided by Drs.
Edmund Miller and Usha Pendurthi, University of Texas Health
Center at Tyler, Tyler, TX.
2.4. Determination of IL-8
IL-8 levels in cell medium were determined by enzyme-
linked immunosorbent assay (ELISA) using a matched antibody
pair according to the manufacturer's protocol (R & D Systems,
2.5. Isolation of nuclei and nuclear run-on transcription assay
Methods for the isolation of nuclei and run-on transcription
assay are as described previously (Greenberg and Bender,
1997; Boggaram and Margana, 1994). Total RNA from labeled
nuclei was isolated and equal amounts of radioactive RNAs
(10–30×106counts/min) were hybridized to nitrocellulose
membranes containing immobilized plasmid DNAs containing
human IL-8 and GAPDH cDNAs and pBluescript. After wash-
ing, radioactivity bound to the filters was quantified with a
PhosphorImager. Radioactivity bound to pBluescript was con-
sidered as background.
2.6. Plasmid DNA isolation
Luciferase reporter plasmids containing −546/+44 and
−133/+44 bp sequences of human IL-8 gene were kindly pro-
vided by Dr. Naofumi Mukaida, Cancer Research Institute,
Kanazawa University, Kanazawa, Japan. Plasmid DNAs were
amplified in Escherichia coli top 10 strain (Invitrogen,
Carlsbad, CA) and purified by anion exchange chromatography
using QIA filter plasmid purification kit (Qiagen, Valencia,
2.7. Transient transfection and reporter gene assay
Plasmid DNAs were transiently transfected into cells by lipo-
some-mediated DNA transfer with Lipofectamine 2000 (Invitro-
gen) according to the manufacturer's instructions. β-galactosidase
and luciferase reporter activities in cell extracts were measured by
chemiluminescence assays (Tropix, Bedford, MA and Promega,
2.8. Transfection of SiRNA oligonucleotides
SiRNA duplex oligonucleotides (siGENOME SMARTpool
reagent) targeting human sphingosine kinase (Human SPHK1)
and non-targeting SiRNA duplex oligonucleotides were pur-
SMARTpool SiRNA oligonucleotides comprise of four SiRNAs
negative control having no perfect matches to known human or
mouse genes. H441 cells (30–50% confluent) plated on T25
oligonucleotide and 20 μl Oligofectamine (Invitrogen) per dish
according to the manufacturer's protocol. Transfected cells were
cultured for 72 h in order to achieve maximum silencing effects
and then subjected to treatments.
2.9. Preparation of nuclear extracts and electrophoretic
mobility shift analysis (EMSA)
Nuclear extracts were prepared according to the methods
described previously (Schreiber et al., 1989; Singh and
Aggarwal, 1995). Protein concentrations of nuclear extracts
were determined by Bradford's method using Bio-Rad protein
Synthetic oligonucleotides were annealed by heating equi-
molar concentrations of sense and antisense oligonucleotides in
10 mM Tris–HCl, pH 7.5 containing 10 mM MgCl2and 50 mM
NaCl at 95 °C for 5 min and then allowed to cool to room
temperature over a period of 1 h. The sense strand sequences of
the human IL-8 promoter oligonucleotides (binding sites are
underlined) used in EMSA are as follows:
NF IL-6: 5′-TCCATCAGTTGCAAATCGTGGA-3′
Double stranded oligonucleotides were 5′end labeled using
[γ32P] and T4 polynucleotide kinase. EMSAs were performed
as described previously (Berhane and Boggaram, 2001) by
incubating 0.5–1.0 ng (100,000 cpm) of the labeled oligonu-
cleotide with 5 μg of nuclear protein in 20 μl of binding buffer
[13 mM Hepes, pH 7.9 containing 13% glycerol, 80 mM KCl,
as non-specific competitor DNA] at 30 °C for 20 min. After
electrophoresis, the gel was dried and exposed to an X-ray film.
2.10. Immunoblotting analysis
Cells were rinsed twice with cold phosphate buffered saline
and incubated in lysis buffer (50 mM Tris–Cl, pH 7.4 containing
150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM sodium
vanadate, 2.5 mM sodium pyrophosphate, 2 μg/ml leupeptin and
aprotinin, 1 mM PMSF and 15% glycerol) for 15 min on ice. The
and the supernatant was used for Western immunoblotting
analysis. SDS-PAGE separation and transfer of proteins to
membrane were carried out with an XCell II Mini-Cell apparatus
(Novex, San Diego, CA) according to the manufacturer's
instructions. Equal amounts of cellular protein (10 μg) were
separated by SDS-PAGE on 10% Bis-Tris gels with MOPS
running buffer and electrophoretically transferred to PVDF
membranes. Membranes were successively incubated with rabbit
polyclonal antibodies against phosphorylated and total p38, p44/
42 and JNK MAPKs (Cell Signaling, Beverly, MA) at 1:1000
dilution overnight at 4 °C followed by goat anti-rabbit alkaline
phosphatase conjugated-secondary antibody (Cell Signaling) at
152 H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
1:2000 dilution for 1 h at room temperature. Protein bands were
visualized by the Enhanced Chemifluorescence (ECF) detection
method (Amersham Pharmacia Biotech, Piscataway, NJ) accord-
ing to the manufacturer's instructions. Membranes were scanned
witha fluorescencescanner for visualizationofproteinbandsand
the intensity of the bands was quantified using Quantity One
Image Acquisition and Analysis Software (Bio-Rad).
Data are shown as means±SD/SE. In experiments where IL-
8 mRNA levels in control cells were arbitrarily set at 100%,
statistical significance was analyzed by one-sample t-test. For
other samples, unpaired t-test was used to analyze statistical
significance. One-tailed P values of b0.05 were considered
3.1. TNF-α increases IL-8 mRNA in H441 lung epithelial cells
in a time-dependent manner
H441 cells are human lung epithelial cells that possess many
of the characteristics of Clara cells such as morphologic and
ultrastructural features as well as expression of lung-specific
a cell line representingthe distal lung epithelium.We studied the
We found that IL-8 mRNA was barely detectable in untreated
of incubation (Fig. 1A and B). Elevated IL-8 mRNA levels in
TNF-α-treated cells were sustained for extended periods of
time. The levels of GAPDH mRNAwere unaffected by TNF-α
3.2. TNF-α increases IL-8 gene transcription rate in H441 cells
TNF-α induction of IL-8 gene expression is subject to
regulation at the transcriptional and mRNA stabilization levels.
To determine the roles of transcriptional and posttranscriptional
mechanisms, we analyzed the effects of TNF-α on IL-8 gene
transcription rate and IL-8 mRNA levels by transcription run-on
assay and Northern blotting, respectively. We found that in cells
treated with TNF-α IL-8 transcription increased after 0.5 h and
declined thereafter (Fig. 2A and C, dot # 1). Northern
blot analysis of cytosolic RNA isolated from the same cells
showed that, in agreement with our earlier data, the steady state
IL-8 mRNA levels increased to maximum levels after 1 h of
treatment (Fig. 2B). TNF-α induction of IL-8 gene transcription
(∼10-fold relative to control at 1 h) was similar to the increase
of cytosolic IL-8 mRNA levels (∼10-fold relative to control at
1 h) indicating that the inductive effects of TNF-α are exerted
Fig. 1. TNF-α induces IL-8 mRNA levels in H441 lung epithelial cells. IL-8 and
GAPDH mRNA levels in H441 cells incubated in control (C) or TNF-α
containing medium (25 ng/ml) for the indicated periods of time were determined
by Northern blotting. A. A representative autoradiogram showing the effects of
TNF-α on IL-8, GAPDH mRNA levels. 18S rRNA levels visualized by
ethidium bromide staining are also shown. B. IL-8 mRNA levels in control cells
were arbitrarily set at 100, and the levels in treated cells relative to control cells
are shown. Data are means±SD of 2–3 independent experiments.⁎Pb0.05 and
⁎⁎Pb0.01 for TNF-α-treated samples compared to control.
Fig. 2. TNF-α increases IL-8 gene transcription rate. IL-8 and GAPDH
transcription rates and cytosolic IL-8 mRNA levels in H441 cells incubated in
control (C) or TNF-α (25 ng/ml) containing medium were determined by
transcription run-on assay and Northern blotting, respectively. A. A represen-
tative autoradiogram showing the effects of TNF-α on IL-8 and GAPDH
transcription rates. 1, IL-8; 2, GAPDH; 3, pBluescript; 4, blank. B. A
representative autoradiogram of the effects of TNF-α on cytosolic IL-8 mRNA
levels. C. Radioactivity bound to pBluescript was considered as background and
IL-8 gene transcription rate was normalized to GAPDH transcription rate. Data
shown are means±SD of 2 independent experiments.
153H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
primarily at the transcriptional level. GAPDH transcription was
unaffected by TNF-α (Fig. 2A, dot # 2). We further evaluated the
effect of TNF-α on the posttranscriptional regulation of IL-8
mRNA by determining its effect on the stability of IL-8 mRNA.
control cells and was unaffected by TNF-α indicating that TNF-α
obtained when 5,6-dichlororibofuranosylbenzimidazole (DRB)
was used as a transcriptional inhibitor (data not shown). The
stability of GAPDH mRNA was not affected by TNF-α. Taken
together the data of transcription run-on assays and the RNA half-
life experiments showed that TNF-α increases IL-8 mRNA levels
in H441 cells primarily by increasing gene transcription.
In agreement with transcription run-on experiments, TNF-α
increased luciferase reportergene expression from IL-8 minimal
promoter (−133/+44 bp) in transient transfection assays (data
not shown). TNF-α induction of IL-8 mRNA was associated
with an induction of NF-κB DNA binding activity, however
AP-1 binding activity was not changed (data not shown).
3.3. Sphingomyelin metabolite(s) mediate TNF-α induction of
IL-8 mRNA levels
Sphingomyelin metabolites have been shown to play
important roles in mediating the inflammatory responses in
the lung. To determine the role of sphingomyelin metabolites in
the regulation of IL-8 gene expression, we investigated the
effects of exogenously added sphingomyelinase, ceramide,
sphingosine and S1-P on IL-8 mRNA and IL-8 secretion in
H441 cells. As S1-P was reconstituted in serum-free medium
containing 0.4% BSA, we determined the effects of 0.4% BSA
on IL-8 mRNA and IL-8 secretion and found that BSA did not
alter IL-8 expression (data not shown). Results (Fig. 4A)
showed that at 1 h incubation, sphingomyelinase (1 unit/ml) and
S1-P (1 μM) increased IL-8 mRNA levels by ∼3.5- and 4.8-
fold, respectively, compared to control whereas ceramide
(10 μM) and sphingosine (10 μM) did not cause any increase.
Preliminary experiments to test the effects of different
concentrations of ceramide (1–100 μM) did not show any
inductive effects on IL-8 mRNA levels after 1 h incubation. As
expected,TNF-α was highly effective inincreasing IL-8 mRNA
levels. Measurement of IL-8 levels by ELISA in the medium
cells treated with ceramide was also found (control=4.64±
0.5 pg/mg protein, ceramide=8.15±1 pg/mg protein) (Fig. 4B).
A time point of 6 h was chosen to allow sufficient time for
changes in protein synthesis and secretion to occur as S1-P
maximally stimulates IL-8 mRNA levels at 1 h of incubation.
The inductive effect of S1-P on IL-8 secretion was significantly
greater than its effect on IL-8 mRNA levels. H441 cells were
Fig. 3. TNF-α does not alter the stability of IL-8 mRNA. H441 cells were
incubated in control (♦) or TNF-α (25 ng/ml) (▪) containing medium for 1 h
and then incubation continued in the presence of actinomycin D (5 μM) for the
indicated times. The levels of IL-8 and GAPDH mRNAs were determined by
Northern blotting. A. A representative autoradiogram showing IL-8, GAPDH
and 18 S rRNA bands. B. IL-8 mRNA level at 0 h was arbitrarily set at 100 and
levels at other times are shown relative to 0 h. Data are means±SD of 2
Fig. 4. Effects of sphingomyelinase and sphingolipids on IL-8 mRNA and
proteinlevels. IL-8 mRNA and IL-8 levels in mediumin H441 cells incubatedin
control medium (C) or medium containing TNF-α (25 ng/ml), sphingomyeli-
nase (Smase) (1 unit/ml), C2-ceramide (10 μM), sphingosine (10 μM) or S1-P
(1 μM) were determined by Northern blotting and ELISA, respectively. Cells
were incubated for 1 and 6 h for Northern blotting (A) and ELISA (B),
respectively. Data are means±SEM of 3–4 independent experiments. Effects on
IL-8 mRNA:⁎⁎⁎Pb0.001 for TNF-α-treated cells vs control;⁎Pb0.05 for
Smase and S1-P-treated cells vs control. Effects on IL-8 protein:⁎Pb0.05 for
ceramide and S1-P-treated cells vs control;⁎⁎⁎Pb0.001 for TNF-α-treated cells
154H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
treated with ceramidase inhibitors, D-erythro-2-(N-myristoyla-
mino)-1-phenol-1-prop (MAPP) and N-oleoyl-ethanolamine
(NOE), for 1–24 h to increase the intracellular accumulation
of ceramide, and their effects on IL-8 mRNA levels were
determined. Results (Fig. 5) showed that treatment of H441
cells with ceramidase inhibitors for 1–24 h did not increase
IL-8 mRNA levels consistent with the lack of inductive effects
of exogenously added ceramide.
The inductive effect of S1-P on IL-8 mRNA levels was time-
dependent with a maximum effect at 1 h of incubation (Fig. 6).
The inductive effects of S1-P were concentration-dependent with
the compound at 0.5 and 1 μM causing maximum induction
after 1 h of treatment (data not shown). S1-P similarly increased
type II epithelial cells (Sparkman, L. and Boggaram, V., unpub-
of IL-8 gene expression is due to the generation of intracellular
S1-P or not, the effects of inhibition of sphingosine kinase by N,
N-dimethylsphingosine (DMS) on the sphingomyelinase induc-
tion of IL-8 mRNAwere determined. Results showed that DMS
of IL-8 mRNA (control=1, sphingomyelinase=9.78±0.80,
DMS (10 μM)+sphingomyelinase=7.49±0.55, DMS (20 μM)
+sphingomyelinase=4.47±0.46, mean±SEM, n=3) indicating
the involvement of S1-P in the induction.
We further assessed the role of S1-P in the TNF-α induction
of IL-8 mRNA expression using DMS, an inhibitor of
sphingosine kinase. DMS reduced TNF-α induction of IL-
8 mRNA levels in a dose-dependent manner indicating that
intracellular increase in S1-P levels contributes to the TNF-α
induction of IL-8 mRNA levels (Fig. 7A and B). To further
prove that TNF-α induced increase in the intracellular S1-P is
involved in the induction of IL-8 mRNA, we used RNA-
mediated interference to reduce sphingosine kinase expression
and studied its effects on TNF-α induction of IL-8 mRNA
expression. RNA-mediated interference results in gene-specific
silencing providing superior advantage over chemical inhibi-
tors. SiRNA duplex oligonucleotides targeting sphingosine
kinase were transfected into H441 cells and the effects of TNF-
α on IL-8 mRNA induction was determined. Results (Fig. 7C
and D) showed that in agreement with the results of DMS,
SiRNA inhibition of sphingosine kinase partially reduced TNF-
α induction of IL-8 mRNA. The effects of silencing were
apparent in cells exposed to TNF-α for 1 h (data not shown) and
more pronounced after 24 h of treatment. We were unable to
detect sphingosine kinase levels by Western blotting in control
or SiRNA transfected cells.
3.4. S1-P induction of IL-8 gene expression is regulated at the
Our data showed that S1-P induced IL-8 mRNA levels and
IL-8 secretion in H441 cells. To understand molecular mecha-
nisms that mediate S1-P induction of IL-8 mRNA levels, we
determined its effects on IL-8 gene transcription and mRNA
transcriptionrate andmRNA stabilityshowedthatS1-Pincreased
IL-8 gene transcription rate (Fig. 8A, dot # 1) and IL-8 promoter
(Fig. 8B) indicating that the inductive effects are mediated
primarily at the transcriptional level. GAPDH transcription was
not significantly increased by S1-P (Fig. 8A, dot # 2).
3.5. Effects of S1-P on NF-κB and AP-1 DNA binding activities
Our data showed that TNF-α-induced intracellular S1-P
levels play important roles in the induction of IL-8 mRNA. Our
data also showed that S1-P increased IL-8 mRNA levels by
increasing gene transcription. As NF-κB and AP-1 transcription
Fig. 5. Effects of ceramidase inhibitors on IL-8 mRNA levels. IL-8 mRNA
levels in H441 cells incubated in control medium (C) or medium containing
ceramidase inhibitors D-erythro-2-(N-myristoylamino)-1-phenol-1-propanol
(MAPP) (5 μM) or N-oleoyl-ethanolamine (NOE) (5 μM) for the indicated
times were determined by Northern blotting. Data are means±SD of 2
Fig. 6. S1-P induces IL-8 mRNA levels. A. IL-8 mRNA levels in H441 cells
incubated in control medium (C) or medium containing S1-P for the indicated
times were determined by Northern blotting. B. Data are means±SEM of 3
independent experiments.⁎⁎Pb0.01 for the 30 min sample comparedto control,
⁎Pb0.05 for the 1 h sample compared to control,⁎⁎Pb0.01 for the 2 h sample
compared to control.
155H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
factors play key roles in the induction of IL-8 expression, we
were interested to determine the effects of S1-P on NF-κB
and AP-1 DNA binding activities. Results (Fig. 9) showed that
NF-κB DNA binding activity was undetectable in control cells
(lane 2) and exposure of cells to S1-P (lane 3) did not increase
NF-κB DNA binding activity, however AP-1 DNA binding
activity was modestly increased (lanes 4 and 5) and NF IL-6
binding activity remained unchanged (lanes 6 and 7). Similar
results were obtained when cells were incubated with S1-P for
different periods of time ranging from 10 min to 6 h (data not
3.6. Involvement of MAPKs in the S1-P induction of IL-8
Sphingolipids are known to activate MAPK signaling path-
ways in a variety of cells. We studied the involvement of MAPK
signaling pathways in the S1-P induction of IL-8 mRNA
expression. S1-P stimulated the phosphorylation of p38, ERK
and JNK MAPKs in a time-dependent manner in H441 cells;
enhanced phosphorylation was observed as early as 10 min after
exposure to S1-P and leveled off to control levels after 2 h
(Fig. 10A). The stimulatory effect of S1-P on p46 form of JNK
that MAPK signaling pathways may be involved in the S1-P
induction of IL-8 mRNA expression. We ascertained the
involvement of MAPKs in the S1-P induction of IL-8 mRNA
by determining the effects of pharmacological inhibitors of
MAPKs on S1-P induction of IL-8 mRNA. ERK, p38 and JNK
MAPKs were inhibited with PD98059, SB203580 and JNK II
inhibitor respectively and the effects of S1-P on IL-8 mRNA
levels by approximately 50 and 40%, respectively indicating the
involvement of p38 and P44/42 MAPK signaling pathways. In
contrast JNK II inhibitor alone modestly increased IL-8 mRNA
levels and additively enhanced S1-P induction of IL-8 mRNA.
IL-8 gene expression is induced by a wide variety of agents
including cytokines, growth factors, bacterial and viral products,
oxidants and others (Roebuck, 1999). Induction of IL-8 gene
expression is subject to both transcriptional and posttranscrip-
tional regulation in a cell/tissue- and stimulus-specific manner
(Roebuck, 1999; Hoffmann et al., 2002). We found that in H441
cells TNF-α induced IL-8 mRNA levels primarily by increasing
to activate IL-8 promoter activity via recruitment of NF-κB
to a TNF-α response element consistent with a role for tran-
scriptional mechanisms (Brasier et al., 1998) in the induction of
IL-8 gene expression in lung epithelial cells. A relatively short
sequence of DNA spanning −133/ +41 bp is necessary and
sufficient for the basal and TNF-α induction of IL-8 promoter
Fig. 7. Inhibition of sphingosine kinase reduces TNF-α induction of IL-8 mRNA levels. A. The effects of dimethylsphingosine (DMS), an inhibitor of sphingosine
DMS (40 μM)+TNF-α compared to TNF-α alone,⁎⁎Pb0.01 for DMS (30 μM)+TNF-α compared to TNF-α alone,⁎⁎⁎Pb0.001 for DMS (50 μM)+TNF-α
compared to TNF-α alone. C. SiRNA duplex oligonucleotides for sphingosine kinase were transfected into H441 cells by liposome-mediated transfer with
Oligofectamine. After 72 h, cells were treated with medium±TNF-α (25 ng/ml) for 24 h and IL-8 and GAPDH mRNAs were analyzed by Northern blotting. 1 and 2,
non-targeting SiRNA; 3, sphingosine kinase SiRNA. D. Data are means±SD of 3 independent experiments.⁎Pb0.05 for TNF-α compared to control;⁎Pb0.05 for
sphingosine kinase SiRNA transfected cells compared to non-targeting SiRNA transfected cells.
156H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
activity (Yasumoto et al., 1992; Brasier et al., 1998). The
NF-κB and AP-1 that act independently and synergistically
to activate IL-8 promoter in response to stimulatory agents in a
cell type-specific manner [reviewed in Roebuck, 1999]. In this
study wefound that TNF-α and S1-P induced IL-8 expression by
increasing gene transcription and without altering the stability of
IL-8 mRNA. However, regulation of IL-8 mRNA stability has
different cells. Nitric oxide, lipopolysaccharide, adenovirus and
Shiga toxin increase IL-8 mRNA expression in lung epithelial
cells, THP cells, fibroblasts and A549 cells, respectively, by
increasing the stability of IL-8 mRNA (Leland Booth and
Boggaram, 2004). The half-life of IL-8 mRNA in untreated cells
was in the range of 0.5–2 h and increased by several fold
depending on the cell type and the stimulus.
Sphingomyelin metabolites are increasingly recognized as
important mediators of inflammation in the lung, and ceramide
has emerged as a putative lipid mediator in TNF-α signaling.
Despite the important roles that sphingomyelin metabolites play
in TNF-α signaling, little is known about their involvement in
the TNF-α induction of IL-8 gene expression in lung cells. Our
data showed that among the sphingomyelin metabolites, S1-P
but not ceramide or sphingosine induced IL-8 mRNA levels and
IL-8 secretion. Consistent with the lack of significant effects of
ceramide, inhibition of ceramidase to increase intracellular
ceramide levels did not increase IL-8 mRNA levels. We found
that the inductive effect of S1-P on IL-8 level in the medium
was significantly greater than its effect on IL-8 mRNA level.
Similarly we found that although ceramide did not increase
IL-8 mRNA level it caused a small increase in IL-8 level. The
observed discrepancy between IL-8 mRNA and IL-8 levels in
S1-P- and ceramide-treated cells point to possible translational
and/or posttranslational regulation of IL-8 expression. Togeth-
er our data indicated that elevated intracellular S1-P generated
as a result of activation of sphingosine kinase partly mediates
TNF-α induction of IL-8 gene expression in H441 cells.
sphingosine-phosphate lyase that catalyzes the irreversible
cleavage of S1-P (Reiss, 2004). Whether TNF-α regulates
sphingosine-phosphate lyase expression and/or activity to
modulate intracellular S1-P levels in H441 cells is not
known. In human umbilical vein endothelial cells (HUVEC)
TNF-α was found to induce the expression of E-selectin and
vascular adhesion molecule-1 (VCAM-1) via increased
generation of S1-P by the activation of sphingosine kinase
(Xia et al., 1998). Although TNF-α induced sphingomyelin
breakdown and ceramide generation, ceramide failed to mimic
the effects of TNF-α to induce E-selectin and VCAM-1
Changes in the levels of sphingolipid metabolites can occur
via coordinate activation of the entire cascade of sphingolipid
metabolizing enzymes as in the case of oxidized low density
Fig. 8. S1-P increases IL-8 gene transcription and promoter activity. A. Effect on
IL-8 gene transcription. IL-8 and GAPDH transcription rates in H441 cells
incubated in control medium (C) or medium containing S1-P (1 μM) for 1 h
were determined by transcription run-on assay. The data is representative of 2
independent experiments. B. Effect on IL-8 mRNA stability. H441 cells were
first incubated in control medium (C) or medium containing S1-P (1 μM)
(♦, control;▪, S1-P) for 1 h and then incubation continued in the presence of
actinomycinD (2.5μM) for the indicatedtimes. IL-8 and GAPDHmRNA levels
were determined by Northern blotting. Data are means±SD of 2 independent
experiments. C. Effect on IL-8 promoter activity. H441 cells were transiently
2 independent experiments.
Fig. 9. Effects of S1-P on NF-κB, AP-1 and NF IL-6 DNA binding activities.
H441 cells were incubated in control medium (C) or medium containing S1-P
(1 μM) for 1 h and nuclear extracts prepared. Binding activities of NF-κB, AP-1
and NF IL-6 to IL-8 promoter elements were analyzed by EMSA. The arrows
indicate the mobility of protein–DNA complexes. Ns, non-specific band.
157H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
lipoprotein induced mitogenesis of smooth muscle cells (Auge
et al., 1999) or via selective activation of one of the enzymes of
the pathway as in the case of TNF-α activation of sphingosine
kinase to inhibit apoptosis in HUVEC cells (Xia et al., 1999). In
some cells the activation of ceramidase may be so robust that
the levels of sphingosine and S1-P are vastly increased in the
absence of substantial increases in ceramide levels (Kolesnick,
2002). Our experiments showed that exogenous ceramide and
inhibition of ceramidase to increase intracellular ceramide levels
failed to increase IL-8 mRNA and protein levels suggesting that
the inability of ceramide to increase IL-8 may not be due to low
intracellular ceramide levels.
TNF-α activates sphingomyelin hydrolysis to increase
intracellular ceramide levels in lung (Ryan et al., 2003) and
lung cells (Vivekananda et al., 2001). Sphingolipids generated in
cytidyltransferase (CCTα) (Vivekananda et al., 2001) the rate-
an important component of lung surfactant, leading to the
perturbation of surfactant lipid composition. These findings
suggest that perturbations in surfactant lipid synthesis contribute
to lung injury associated with inflammation and that sphingoli-
pids play important roles in mediating these effects. Our findings
of the inductive effects of S1-P on IL-8 mRNA levels reveal yet
another pathway that potentially contributes to lung injury in
inflammation. Increases in IL-8 levels can lead to increased
recruitment of neutrophils into the lung contributing to lung
The molecular mechanisms and signal transduction pathways
by which S1-P induces the expression of IL-8 in H441 lung
epithelial cells remains to be investigated. Our studies showed
that S1-P increased AP-1 DNA binding activity in H441 cells
increase of IL-8 gene transcription. S1-P is known to enhance the
DNA binding activity of AP-1 (Su et al., 1994).
MAPKs regulate IL-8 expression and secretion in a variety
of cells including lung epithelial cells (Hashimoto et al., 1999;
Matsumoto et al., 1998) and MAPK regulation of IL-8 expres-
sion occurs via transcriptional and mRNA stabilization mechan-
isms (Holtmann et al., 1999). Transcription factors NF-κB and
AP-1 play central roles in the transcriptional regulation of IL-
in the regulation of IL-8 expression appears to be dependent
on the cell type and the nature of the stimulus. Induction of
IL-8 expression in BEAS-2B bronchial epithelial cells by
S1-P (Wang et al., 2002) and lysophosphatidic acid (Saatian
et al., 2006) required activation of p44/p42 whereas induction
by Streptococcus pneumoniae (Schmeck et al., 2006) and zinc
(Kim et al., 2006) involved activation of JNK and ERK
plus JNK respectively. Our studies showed that S1-P activated
p44/42, p38 and JNK phosphorylation in H441 cells, however,
pharmacological inhibitors of ERK and p38 but not JNK
MAPKs inhibited S1-P induction of IL-8 mRNA levels in-
dicating that ERK and p38 signaling pathways are required for
S1-P induction. S1-P increased AP-1 DNA binding activity
suggesting a role for AP-1 in the induction of IL-8 mRNA
expression. It remains to be determined if ERK and p38
MAPK pathways control AP-1 DNA binding activity to in-
crease IL-8 expression. It is known that p38, p44/p42 and JNK
MAPKs regulate AP-1 activity (Whitmarsh and Davis, 1996).
In summary, our studies have shown that TNF-α induces
IL-8 gene expression in H441 lung epithelial cells by increa-
sing gene transcription and that intracellular increases in S1-P
levels play important roles in mediating TNF-α induction.
S1-P induced IL-8 mRNA expression via activation of p38
and p44/42 MAPK signaling pathways and increase in AP-1
Fig. 10. A. Effects of S1-P on MAPK activation. H441 cells were serum-starved
for 24 h and then treated with S1-P (1 μM) for the indicated periods of time. The
levels of phosphorylated and total p38, p44/42, and JNK MAP kinases were
analyzed by Western immunoblotting. Similar results were obtained in 2 other
independent experiments. B and C. Effects of pharmacological inhibitors of
MAPK activation on S1-P induction of IL-8 mRNA levels. H441 cells were first
incubated in medium±inhibitor (SB 203850, 10 μM; PD98059, 50 μM; JNK
inhibitor II, 10 μM) for 1 h and then treated with S1-P (1 μM) for an additional
1 h. IL-8, and GAPDH mRNA levels were determined by Northern blotting
analysis. A representative Northern blot is shown. Similar results were obtained
in 2 other independent experiments.
158H. Chandru, V. Boggaram / Gene 391 (2007) 150–160
DNA binding activity. Thus TNF-α induces IL-8 gene ex-
pression in H441 lung epithelial cells via two pathways —
one involving the activation of NF-κB and the other via
intracellular elevation of S1-P that results in an increase in
AP-1 but not NF-κB binding.
This work was supported partly by the National Heart, Lung,
and Blood Institute Grant HL48048. We thank Dr. Anna
Kurdowska for the measurement of IL-8 levels by ELISA.
Human IL-8 promoter plasmids were kindly provided by Dr.
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