Báo cáo khoa học: Rapamycin inhibits lipopolysaccharide induction of granulocyte-colony stimulating factor and inducible nitric oxide synthase expression in macrophages by reducing the levels of octamer-binding factor-2 doc

Rapamycin inhibits lipopolysaccharide induction of
granulocyte-colony stimulating factor and inducible nitric
oxide synthase expression in macrophages by reducing the
levels of octamer-binding factor-2
Yuan-Yi Chou1, Jhen-I Gao1, Shwu-Fen Chang2, Po-Yuan Chang3 and Shao-Chun Lu1
1 Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
2 Graduate Institute of Medical Sciences, Taipei Medical University, Taiwan
3 Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University, Taipei, Taiwan

Keywords
granulocyte-colony stimulating factor
(G-CSF); inducible nitric oxide synthase
(iNOS); lipopolysaccharide (LPS);
macrophage; mammalian target of
rapamycin (mTOR); octamer-binding factor-2
(Oct-2); rapamycin
Correspondence
S.-C. Lu, Room 810, No.1, Jen Ai Road
Section 1, Department of Biochemistry and
Molecular Biology, College of Medicine,
National Taiwan University, Taipei 10051,
Taiwan
Tel: +886 2 2312 3456, ext. 88224
Fax: +886 2 2391 5295
E-mail:
Website: />department/ibmb/
(Received 22 July 2010, revised 5 October
2010, accepted 21 October 2010)
doi:10.1111/j.1742-4658.2010.07929.x

This article reports an inhibitory effect of rapamycin on the lipopolysaccharide (LPS)-induced expression of both inducible nitric oxide synthase

(iNOS) and granulocyte-colony stimulating factor (G-CSF) in macrophages
and its underlying mechanism. The study arose from an observation that
rapamycin inhibited the LPS-induced increase in octamer-binding factor-2
(Oct-2) protein levels through a mammalian target of rapamycin (mTOR)dependent pathway in mouse RAW264.7 macrophages. As both iNOS and
G-CSF are potential Oct-2 target genes, we tested the effect of rapamycin
on their expression and found that it reduced the LPS-induced increase in
iNOS and G-CSF mRNA levels and iNOS and G-CSF protein levels.
Blocking of mTOR-signaling using a dominant-negative mTOR expression
plasmid resulted in inhibition of the LPS-induced increase in iNOS and
G-CSF protein levels, supporting the essential role of mTOR. Forced
expression of Oct-2 using the pCG–Oct-2 plasmid overcame the inhibitory
effect of rapamycin on the LPS-induced increase in iNOS and G-CSF
mRNA levels. Chromatin immunoprecipitation assays showed that LPS
enhanced the binding of Oct-2 to the iNOS and G-CSF promoters and that
this effect was inhibited by pretreatment with rapamycin. Moreover, RNA
interference knockdown of Oct-2 reduced iNOS and G-CSF expression in
LPS-treated cells. The inhibitory effect of rapamycin on the LPS-induced
increase in Oct-2 protein levels and on the iNOS and G-CSF mRNA levels
was also detected in human THP-1 monocyte-derived macrophages. This
study demonstrates that rapamycin reduces iNOS and G-CSF expression at
the transcription level in LPS-treated macrophages by inhibiting Oct-2
expression.

Introduction
Macrophages play a critical role in the host defense
against bacterial pathogens. The toll-like receptor 4
(TLR4) on the surface of macrophages recognizes

lipopolysaccharide (LPS), a Gram-negative bacterial
endotoxin, and induces the production of proinflammatory cytokines [1,2]. In LPS-stimulated macrophages,

Abbreviations
Akt-in, Akt inhibitor; ChIP, chromatin immunoprecipitation; DN, dominant-negative; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
G-CSF, granulocyte-colony stimulating factor; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; mTOR, mammalian target of
rapamycin; NO, nitric oxide; Oct-1, octamer-binding factor-1; Oct-2, octamer-binding factor-2; PI3K, phosphoinositide 3-kinase; PMA,
4b-phorbol 12-myristate 13-acetate; RR, rapamycin-resistant; TLR4, toll-like receptor 4; TSA, trichostatin A.

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

Y.-Y. Chou et al.

gene expression is controlled by the activation of
various protein kinases, such as protein kinase A, protein kinase C, Src-related kinases, mitogen-activated
protein kinases and phosphoinositide 3-kinase (PI3K),
downstream of the TLR4-signaling pathway [3–5]. Of
the LPS-induced genes, those coding for inducible
nitric oxide synthase (iNOS) and granulocyte-colony
stimulating factor (G-CSF) attracted our interest,
because both proteins are important in the host’s
defense against microbial infection. iNOS catalyzes the
production of nitric oxide (NO) to combat invading
pathogens [6] and G-CSF stimulates the production,
growth and function of neutrophils [7,8]. Both iNOS
and G-CSF genes are expressed in macrophages and
their expression is strongly induced by LPS at the

transcriptional level [9–11]. In addition, a nuclear factorkappa B-binding element, a nuclear factor-interleukin-6
(also named C ⁄ EBPb)-binding element and an octamer element in the promoter of the iNOS and G-CSF
genes have been reported to be essential for full promoter activities of these two genes following stimulation with LPS [9,10,12,13]. However, there is clinical
evidence that excessive concentrations of NO or
G-CSF exacerbates inflammatory responses and causes
tissue damage [14,15]. It is therefore necessary to maintain appropriate levels of both iNOS and G-CSF
during inflammation, and the expression of these two
genes should be tightly controlled and regulated by
similar mechanisms.
Activation of PI3K leads to the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [16,17], which subsequently
activate downstream signaling molecules, such as Akt
[18] and mammalian target of rapamycin (mTOR), regulating various biological processes, including cell
cycling, cell survival and protein synthesis [19]. There
is growing evidence that mTOR (activated by TLR4
via PI3K ⁄ Akt) is crucial in monocytes and macrophages for coordinating the innate immune response, but
how mTOR exerts its effect is poorly understood
[20,21]. A well-known function of mTOR is to regulate
protein synthesis by activating p70 S6 kinase and by
inhibiting eukaryotic translation initiation factor
4E-binding protein (eIF4E-BP1) [19]. Rapamycin, a
potent immunosuppressor, exerts its function by binding
to the FK506-binding protein (FKBP12) and inhibits
the activity of mTOR complex 1 (mTORC1) and subsequently inhibits the translation of target mRNA, but
has no effect on mTOR complex 2 (mTORC2) [22]. In
contrast, in LPS-stimulated innate immune cells, rapamycin alters the expression of cytokine genes at the
transcriptional level [21], and LPS-induced expression of
iNOS in macrophages has been reported to be partially
86

inhibited by rapamycin at the mRNA level [23].

These findings suggest that mTOR may be involved in
gene expression through mechanisms other than translational control. Octamer-binding factor-2 (Oct-2) is a
transcription factor that binds to the octamer element
(ATGCAAAT) in the promoter of its target gene. We
have previously demonstrated that expression of Oct-2
can be induced by LPS in macrophages and that it is
involved in the LPS-induced upregulation of resistin
and iNOS expression [24,25]. In addition, we reported
that treatment of macrophages with LY294002 decreased LPS-induced Oct-2 expression at the protein level,
subsequently reducing the expression of resistin [24].
As LY294002 inhibits the activity of both PI3K and
mTOR [26], we speculated that the induction of Oct-2
expression by LPS might occur through an mTORdependent pathway and be inhibited by the mTOR
inhibitor, rapamycin. In addition, Oct-2 is involved in
the expression of LPS-inducible genes that contain
octamer in their promoters [24,25]. Thus, rapamycin
may inhibit the expressions of iNOS and G-CSF at the
transcriptional level in LPS-treated macrophages. In
this study, the effects of rapamycin on the LPSinduced increase in Oct-2 protein and on the expression of iNOS and G-CSF protein and mRNA were
evaluated in macrophages, and the involvement of
Oct-2 in LPS ⁄ mTOR-induced iNOS and G-CSF
expression was investigated further.

Results
Rapamycin inhibits the LPS-induced increase in
Oct-2 protein levels in RAW264.7 macrophages
Oct-2 mRNA and Oct-2 protein levels increased in a
time-dependent manner when RAW264.7 cells were
exposed to LPS for 1 to 24 h. Oct-2 mRNA levels
increased by 60% at 1 h and reached a maximum, of

about threefold higher than the basal level, after 4–8 h
of stimulation with LPS, then showed a subsequent
decrease at 24 h (Fig. 1A, upper panel). Oct-2 protein
levels showed an 80% increase at 1 h, reached a maximum of about 10-fold higher than the basal level at
8 h and were maintained at this level for at least 24 h
(Fig. 1A, bottom panel). To confirm that the LPSinduced increase in Oct-2 protein was inhibited by the
PI3K inhibitor LY294002 [24], the cells were treated
with 12.5, 25 or 50 lm LY294002 for 30 min before,
and during, treatment with LPS for 4 h. Fig. 1B shows
that LY294002 inhibited the LPS-induced increase in
Oct-2 protein levels in a dose-dependent manner,
reaching  70% inhibition at 50 lm LY294002. A similar, but lower, inhibitory effect on the LPS-induced

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

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Fig. 1. LPS induces the expression of Oct-2 mRNA and Oct-2 protein in RAW264.7 macrophages, and the induction of Oct-2 protein by LPS
is blocked by a PI3K or an Akt inhibitor. (A) RAW264.7 cells were treated with 100 ngỈmL)1 of LPS for 0, 1, 2, 4, 8 or 24 h, then the levels
of Oct-2 mRNA (upper panels) or Oct-2 protein (lower panels) were determined by RT-PCR or western blot analysis, respectively. GAPDH or
b-actin was used as the respective internal control. The asterisk indicates a nonspecific signal. (B and C) RAW264.7 cells were untreated
(lane 1), were treated with LPS for 4 h (lane 2), or were pretreated for 30 min with increasing concentrations of LY294002 (B) or Akt-in (C)
before treatment with LPS (100 ngỈmL)1) for 4 h (lanes 3–5); the levels of Oct-2 and b-actin were then measured by western blotting. (D)
RAW264.7 cells were untreated (lanes 1 and 2) or were pretreated with 50 lM LY294002 (upper panels) or 50 lM Akt-in (lower panels) for
30 min (lanes 3 and 4), then incubated with (lanes 2 and 4) or without (lanes 1 and 3) LPS for 4 h. The Oct-2 and b-actin levels were
measured by western blotting. (E) Cells were transiently transfected with 7.5 lg of empty vector or DN-Akt expression plasmid, then, after
24 h, were incubated for 4 h in the presence or absence of LPS and the levels of Oct-2 and b-actin were analyzed by western blotting. (F)
Cells were untreated (lane 1) or were treated for 4 h with LPS in the absence (lane 2) or presence of 25 lM LY294002 (lane 3) or 50 lM
Akt-in (lane 4), then the Oct-2 and GAPDH mRNA levels were analyzed by RT-PCR. The results are representative of three independent
experiments. LY, LY294002.

Oct-2 protein increase was also seen in cells treated
with 0.5 lm wortmannin, another PI3K inhibitor (data
not shown). As Akt is a well-characterized downstream
effecter of the PI3K signaling pathway, we examined
whether it is involved in the LPS-induced increase in
Oct-2 protein. Pretreatment with 25, 50 or 75 lm Akt
inhibitor (Akt-in) before treatment with LPS for 4 h
resulted in a dose-dependent decrease in Oct-2 protein,
with maximal inhibition (approximately 70%) being
achieved using 50 or 75 lm Akt-in (Fig. 1C). Inhibitors (LY294002 and Akt-in) had no effect on Oct-2
expression in cells not stimulated with LPS (Fig. 1D).
To further evaluate the involvement of Akt in the
LPS-induced increase of Oct-2 protein, RAW264.7
cells were transfected with the dominant-negative

(DN)-Akt expression plasmid for 24 h before 4 h of
stimulation with LPS, and this resulted in a reduction
of about 60% in Oct-2 protein expression compared
with cells not treated with the expression plasmid
(Fig. 1E). However, the levels of Oct-2 mRNA were

not altered by either LY294002 or Akt-in in LPS-treated cells (Fig. 1F). These results show that LPS upregulates Oct-2 protein in macrophages through a
PI3K ⁄ Akt-dependent pathway. As mTOR is one of
several downstream targets of PI3K and because
LY294002 is also an mTOR inhibitor [26], it is possible that mTOR is also involved in LPS-induced Oct-2
expression. To test this possibility, cells were pretreated
with increasing amounts of rapamycin before treatment with LPS and then the expression of Oct-2 was
analyzed. Fig. 2A shows that rapamycin inhibited the
increase in Oct-2 protein levels in LPS-treated cells in
a dose-dependent manner, whereas the Oct-2 mRNA
levels in LPS-treated cells were not affected (Fig. 2C).
Rapamycin had no effect on Oct-2 expression in cells
not stimulated with LPS (Fig. 2B). To verify the
involvement of mTOR in LPS-induced Oct-2 expression, RAW264.7 cells were transfected with either the
DN-mTOR expression plasmid or the rapamycinresistant (RR)-mTOR expression plasmid 24 h before

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Fig. 2. Rapamycin reduces the LPS-induced increase in Oct-2 protein levels, but not in Oct-2 mRNA levels, in RAW264.7 macrophages
through an mTOR-dependent pathway. (A) RAW264.7 cells were untreated (lane 1), were treated with 100 ngỈmL)1 of LPS for 4 h (lane 2)
or were pretreated for 30 min with increasing concentrations of rapamycin before LPS treatment for 4 h (lanes 3–5); the concentrations of
Oct-2 and b-actin were then measured after western blotting. (B) RAW264.7 cells were untreated (lanes 1 and 2), or were pretreated with
200 ngỈmL)1 of rapamycin for 30 min (lanes 3 and 4), followed by incubation with (lanes 2 and 4) or without (lanes 1 and 3) LPS for 4 h. The
levels of Oct-2 and b-actin were measured after western blotting. (C) RAW264.7 cells were untreated (lane 1), were treated with LPS for
4 h (lane 2), or were pretreated for 30 min with 200 ngỈmL)1 of rapamycin before LPS treatment for 4 h, then the Oct-2 and GAPDH mRNA
levels were measured by RT-PCR. The levels of Oct-2 mRNA were normalized to those for GAPDH and the results were expressed relative
to those in the untreated control (relative value = 1). The values are expressed as the mean ± sd of three independent experiments. (D and
E) RAW264.7 cells were transiently transfected with 7.5 lg of empty vector or plasmid encoding DN-mTOR (D) or RR-mTOR (E), then, 24 h
later, were incubated for 4 h with or without LPS in the absence or presence of 200 ngỈmL)1 of rapamycin; the Oct-2 and b-actin levels
were analyzed after western blotting. (F) RAW264.7 cells were untreated (lanes 1 and 2) or were preincubated with 200 ngỈmL)1 of rapamycin for 30 min (lane 3) or transiently transfected for 24 h with 7.5 lg of empty vector (lanes 4 and 5) or plasmid encoding DN-mTOR (lane
6), then incubated in the presence or absence of LPS for 30 min and the levels of total and phosphorylated p70 S6 kinase were determined
by western blot analysis. Similar results were obtained in three separate experiments. Rapa, rapamycin.

treatment with LPS for 4 h. Transfection with the
DN-mTOR expression plasmid inhibited the increase
in Oct-2 protein in LPS-treated RAW264.7 macrophages by  60% (Fig. 2D), while transfection with
the RR-mTOR expression plasmid overcame the
inhibitory effect of rapamycin on the increase in Oct-2
protein levels in LPS-treated cells (Fig. 2E). The phosphorylation levels of p70 S6 kinase were assayed to
verify the activation of mTOR. Fig. 2F shows that

phospho-p70 S6 kinase was detectable after 30 min of
LPS treatment and that this effect was blocked by
rapamycin treatment or by transfection with the
DN-mTOR expression plasmid. None of these treatments affected the total amount of p70 S6 kinase protein.
These results strongly suggest that mTOR activation is
essential for LPS-induced Oct-2 protein expression.
Rapamycin decreases LPS-induced iNOS
expression in RAW264.7 macrophages
The octamer element plays an essential role in the
LPS-induced activation of the promoters of the iNOS,
88

G-CSF and resistin [12,13,24,25] and we previously
demonstrated that inhibition of Oct-2 expression by
trichostatin A (TSA), a potent histone deacetylase
inhibitor, or by LY294002 leads to reduced iNOS
or resistin expression, respectively, in LPS-treated macrophages [24,25]. It was possible that rapamycin might
also reduce the expression of these genes. We therefore
tested the effect of rapamycin on the LPS-induced
expression of iNOS and G-CSF. Fig. 3A shows that
detectable levels of iNOS and G-CSF mRNAs were
induced in RAW264.7 cells by 4 h of incubation with
LPS and that both effects were inhibited by pretreatment with rapamycin. LPS-induced iNOS protein
expression (4 h of incubation, Fig. 3B) and nitrite production (24 h of incubation, Fig. 3C) were also inhibited in cells pretreated with rapamycin. Furthermore,
DN-mTOR-transfected cells expressed only 46% as
much iNOS protein as control cells in response to LPS
(Fig. 3D) and produced only 43% as much nitrite
(Fig. 3E). These results show that rapamycin inhibited
LPS-induced iNOS expression in RAW264.7 macrophages through an mTOR-dependent pathway.

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

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Fig. 3. Rapamycin reduces LPS-induced NO production and the expression of iNOS mRNA and iNOS protein in RAW264.7 macrophages.
(A and B) RAW264.7 cells were either untreated or pretreated with LY294002 (50 lM) or rapamycin (200 ngỈmL)1) for 30 min and then
treated with LPS (100 ngỈmL)1) for 4 h. The levels of iNOS and G-CSF mRNAs (A) and the level of iNOS protein (B) were analyzed by
RT-PCR and western blotting, respectively. GAPDH mRNA or b-actin was used as the internal control. Similar results were obtained in at
least three independent experiments. (C) Cells were treated with LPS in the absence or presence of 200 ngỈmL)1 of rapamycin for 0, 16 or
24 h, then the NO levels in the culture medium were determined using a Griess reagent system kit. (D and E) RAW264.7 cells were
transiently transfected with 7.5 lg of empty vector or DN-mTOR expression plasmid, then, after 24 h, were incubated for 4 h (D) or 24 h (E)
in the presence or absence of LPS; the levels of iNOS and b-actin were analyzed by western blotting (D) and the NO levels in the medium
were determined using a Griess reagent system kit (E). The values for the LPS-treated cells were divided by those for the non-LPS-treated
cells and are shown as a percentage of the values for the cells transfected with control vector (relative value = 100). All results are
expressed as the mean ± SD of three independent experiments. *P < 0.01 compared with the untreated control or the LPS-treated control,
as appropriate. LY, LY294002; Rapa, rapamycin.

Rapamycin inhibits G-CSF expression in
LPS-treated RAW264.7 macrophages
To examine whether LPS-induced G-CSF expression
was also sensitive to rapamycin treatment, RAW264.7
cells were pretreated with rapamycin for 30 min before
treatment with LPS for different periods of time. The
levels of G-CSF mRNA (Fig. 4A) and of G-CSF protein (Fig. 4B) were below the detection limit in
untreated cells and increased in a time-dependent manner in response to LPS stimulation. Pretreatment with
rapamycin resulted in approximately 50% less G-CSF

protein in the medium (Fig. 4B), which was probably
caused by attenuation of G-CSF promoter activity
(Fig. 4C) and mRNA levels (Fig. 3A) by rapamycin.
Transfection of RAW264.7 cells with the DN-mTOR
expression plasmid also resulted in a reduction of
about 50% in the LPS-induced increase in G-CSF protein in the medium (6 h of LPS treatment, Fig. 4D),
suggesting that activation of mTOR is essential for
induction of G-CSF expression by LPS. In a previous
study, we showed that forced expression of Oct-2

restores iNOS protein levels in TSA- and LPS-treated
cells [25]. In the present study, ectopic expression of
Oct-2 by transfection with increasing amounts of
pCG-Oct-2 overcame the rapamycin-mediated inhibition of iNOS and G-CSF mRNA expression in LPStreated RAW264.7 cells in a dose-dependent manner
(4 h of incubation; Fig. 4E). These results confirm that
Oct-2 plays a critical role in the upregulation of iNOS
and G-CSF expression by LPS in RAW264.7 macrophages; moreover, these results also show that the
LPS-induced increase in G-CSF expression is sensitive
to rapamycin and suggest that mTOR activation is
required for G-CSF expression in response to LPS.
Oct-2 is directly involved in LPS-induced iNOS
and G-CSF expression in RAW264.7 macrophages
To further examine the involvement of Oct-2 in LPSinduced iNOS and G-CSF gene expression in
RAW264.7 cells, a chromatin immunoprecipitation
(ChIP) assay was performed to examine the binding of
Oct-2 to the iNOS and G-CSF promoters in vivo. In

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

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Fig. 4. Rapamycin reduces LPS-induced G-CSF expression in RAW264.7 macrophages. (A and B) RAW264.7 cells were untreated or were
pretreated with rapamycin (200 ngỈmL)1) for 30 min, followed by treatment with 100 ngỈmL)1 of LPS for 0, 2, 4, 6, 8, 12 or 24 h. (A) Total
RNA was then isolated and the levels of G-CSF and GAPDH mRNAs were determined by RT-PCR. The result is representative of those

obtained in three similar experiments. (B) The levels of G-CSF protein in the medium were determined by ELISA. The values are the
mean ± sd of an experiment performed in triplicate, and similar results were observed in three separate experiments. (C) RAW264.7 cells
were transfected with pG-CSF()283 ⁄ +35)-Luc and phRLTK, then, 24 h later, were pretreated with rapamycin and with LPS for 6 h and the
Photinus and Renilla luciferase activities were measured. The levels of Photinus luciferase activity were normalized to Renilla luciferase
activity and expressed relative to those in the LPS-treated controls (relative value = 100). (D) RAW264.7 cells were transfected and treated
as in Fig. 3E, except that LPS treatment was for 6 h, then the G-CSF protein levels in the medium were determined by ELISA. The values
for the LPS-treated cells were divided by those for the non-LPS-treated cells and are shown as a percentage of the values for the cells transfected with control vector (relative value = 100). The values are the mean ± SD of three independent experiments. *P < 0.01 compared with
the untreated control or with the LPS-treated control, as appropriate. (E) RAW264.7 cells were transfected with 2 lg of control pCG vector
or with increasing amounts of pCG–Oct-2 (made up to 2 lg with pCG) and cultured for 24 h, then untreated (lane 1), treated with LPS for
4 h (lane 2) or pretreated for 30 min with 200 ngỈmL)1 of rapamycin, then treated with LPS for 4 h (lanes 3–6); iNOS and G-CSF mRNA were
detected by RT-PCR (upper panels) and Oct-2 protein in cell lysates was measured by western blotting (lower panels). GAPDH or b-actin
was used as the respective internal control. The results are representative of three independent experiments. Rapa, rapamycin.

cells treated with LPS for 6 h, but not in control cells,
an iNOS promoter region from nucleotides )90 to 154
and a G-CSF promoter region from nucleotides )70 to
)248, encompassing the octamer, were pulled down by
anti-Oct-2 IgG (Fig. 5), but not by a control IgG, and
this effect was blocked by pretreatment with rapamycin.
This shows that Oct-2 binds to the iNOS and G-CSF
promoters and that the binding is sensitive to
rapamycin. Moreover, specific knockdown of Oct-2 by
transfection with an Oct-2 RNA interference (RNAi)
plasmid (pLL3.7–Oct-2) resulted in decreased levels of
iNOS protein in the cell lysate (4 h of incubation,
Fig. 6A) and G-CSF protein in the culture medium
(6 h of incubation, Fig. 6B) of LPS-induced cells.
These results support the critical role of Oct-2 in LPSinduced iNOS and G-CSF expression.
90

LPS-induced expression of Oct-2, G-CSF and
iNOS in the THP-1 human monocyte/macrophage
cell line is sensitive to rapamycin treatment
To determine whether rapamycin also inhibited Oct-2,
iNOS, and G-CSF expression in human macrophages,
THP-1, a human monocyte ⁄ macrophage cell line, was
induced to differentiate by incubation with 160 nm
4b-phorbol 12-myristate 13-acetate (PMA) for 24 h,
then the cells were treated with rapamycin and LPS
(100 ngỈmL)1, 6 h). As in mouse macrophages, Oct-2
protein expression was induced by LPS, and rapamycin inhibited this effect by 34% (Fig. 7A). Moreover,
rapamycin also inhibited the LPS-induced expression
of iNOS and G-CSF mRNAs by 37% and 45%,
respectively (Fig. 7B).

FEBS Journal 278 (2011) 85–96 ª 2010 The Authors Journal compilation ª 2010 FEBS


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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

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LPS
LY
Akt-in
Rapa

LPS
Rapa
Fig. 5. Rapamycin inhibits the binding of Oct-2 to the iNOS and
G-CSF promoters in LPS-treated RAW264.7 macrophages. RAW264.7
cells were either untreated or pretreated with rapamycin for 30 min
and treated with or without LPS for 6 h, then ChIP assays were
performed using anti-Oct-2 IgG (top row) or control IgG (center
row), and a 179 bp G-CSF promoter fragment ()248 to )70 bp) and
a 244 bp iNOS promoter fragment ()90 to 154 bp) were amplified
by PCR. Ten per cent of the chromatin DNA used for immunoprecipitation was subjected to PCR and is indicated as ‘input’ (bottom
row). The results are representative of three independent experiments. Rapa, rapamycin.

A

1

2

3

Oct -2
iNOS
β-actin

LPS
Control
RNAi
LPS-induced G-CSF
production (% of control)

B

Oct -2
RNAi

B
iNOS
G-CSF
GAPDH
LPS
Rapa

Fig. 7. LPS induces Oct-2 protein and iNOS and G-CSF mRNA
expression in PMA-differentiated human THP-1 monocyte ⁄ macrophages, and this effect is blocked by a PI3K, Akt or mTOR inhibitor.
(A) THP-1 cells were induced to differentiate by exposure to
160 nM PMA for 24 h and then incubated with or without LPS for
6 h in the absence or presence of LY294002 (50 lM), Akt-in (50 lM)
or rapamycin (200 ngỈmL)1); the levels of Oct-2 and b-actin protein
were then determined by western blotting. (B) Differentiated THP-1
cells were untreated (lane 1), treated with 200 ngỈmL)1 of rapamycin for 30 min (lane 3), or treated for 6 h with LPS in the absence
(lane 2) or presence (lane 4) of 200 ngỈmL)1 of rapamycin; iNOS,
G-CSF and GAPDH mRNA levels were then determined by RT-PCR.
The results are representative of three independent experiments.
LY, LY294002; Rapa, rapamycin.

100

Discussion

80
*

60
40
20
0

Control

Oct -2

RNAi
Fig. 6. Knockdown of Oct-2 reduces the LPS-induced increase in
iNOS and G-CSF expression in RAW264.7 macrophages.
RAW264.7 cells were transiently transfected with either pLL 3.7scrambled (Sc) or pLL 3.7-Oct-2 RNA interference (RNAi), then,
24 h later, were untreated or were treated with LPS for 4 h (A) or
6 h (B); the amounts of Oct-2, iNOS and b-actin proteins were then
detected by western blotting (A) and the concentration of G-CSF
protein in the medium was measured by ELISA (B). The values for
the LPS-treated cells were divided by those for the non-LPS-treated
cells and are shown as a percentage of the values for the cells
transfected with control vector (relative value = 100). The values
are expressed as the mean ± sd of three independent experiments. *P < 0.01 compared with the LPS-treated control.

We evaluated the effects of rapamycin on the expression of Oct-2 and its potential target genes, iNOS and
G-CSF, in RAW264.7 cells and in THP-1 cells. The
results demonstrated that rapamycin reduced Oct-2
protein levels and attenuated iNOS and G-CSF expression at the level of transcription in LPS-stimulated
macrophages. Similar results were obtained by transfection of cells with DN-mTOR, indicating that the
LPS-induced increase in Oct-2, iNOS and G-CSF
expression occurs through an mTOR-dependent pathway. It is very likely that the increase in Oct-2, iNOS,
or G-CSF expression occurs through the PI3K ⁄ Akt ⁄ mTOR signaling pathway, as mTOR is downstream of
PI3K-Akt, and treatment of cells with LY294002 or an
Akt inhibitor, or transfection of cells with DN-Akt,
also resulted in a decrease in the LPS-induced increase
in Oct-2, iNOS and G-CSF expression (Figs. 1B,C,E,
and 3B and 7A, and data not shown).
Rapamycin has been reported to downregulate NO
production by inhibiting phosphorylation [27] or

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

Y.-Y. Chou et al.

inducing proteasomal degradation [28] of iNOS protein, or by decreasing the secretion of interferon-b [29].
However, Attur et al. [23] showed that rapamycin
inhibits the LPS-induced accumulation of iNOS
mRNA in macrophages. Our data showed that rapamycin inhibited the accumulation of iNOS mRNA at
the transcriptional level and that this effect was caused

by a lower amount of Oct-2. These results are in agreement with our previous results showing that downregulation of Oct-2 is responsible for the decrease in LPSinduced iNOS expression in TSA-pretreated macrophages [25]. Moreover, we observed that LPS-induced
expression of G-CSF was decreased by pretreating
macrophages with rapamycin (this study) or TSA (data
not shown), which may also be attributed to the
decrease in Oct-2 expression caused by these reagents.
The involvement of Oct-2 in iNOS and G-CSF expression was further supported by the finding that knockdown of Oct-2 expression also attenuated iNOS and
G-CSF protein expression (Fig. 6). Mutation of the
octamer in the iNOS and G-CSF promoters results in
a reduction of more than 90% in the LPS-induced promoter activities of these genes [13,30], suggesting the
essential role of the octamer and the factors binding to
it in LPS-induced gene expression. Although involvement of octamer-binding factor-1 (Oct-1), another octamer-binding protein, cannot be excluded in the LPSinduced expression of iNOS and G-CSF, it is very
likely that Oct-2 plays a more important role, as the
level of expression of iNOS and G-CSF changed in
parallel with that of Oct-2, whereas Oct-1 is constitutively expressed and its expression is not changed by
treatment with LPS (data not shown). Interestingly, we
observed that LPS induced an increase of about 1.6fold in Oct-2 mRNA levels, which was not changed by
treatment with rapamycin. This result suggests that
rapamycin may regulate the production of Oct-2
protein at the post-transcriptional level. Treatment
with lactacystin, a specific inhibitor of the 26S proteasome, did not change the levels of Oct-2 protein in the
presence or absence of LY294002, Akt-in or rapamycin
in LPS-treated cells (data not shown). As mTOR regulates cell growth and protein synthesis by increasing
the phosphorylation of two major downstream targets
– 4E-BP and ribosomal p70 S6 kinase – it is very possible that rapamycin downregulates the Oct-2 protein
level by inhibiting Oct-2 protein synthesis.
Although recombinant G-CSF is routinely used to
treat neutropenia and to mobilize hematopoietic stem
cells from the bone marrow into the peripheral blood
before transplantation [31], it is also expressed endogenously in a variety of cell types in response to treatment with LPS, tumor necrosis factor-a, interleukin-1b,
92

PMA or interferon-c [32]. Expression of G-CSF can
be regulated at both the transcriptional and posttranscriptional levels [33]; however, the regulatory
mechanisms are poorly understood. To our knowledge,
this is the first study showing that rapamycin reduces
the LPS-induced expression of G-CSF at the transcriptional level in macrophages by blocking mTOR activity. G-CSF functions as an anti-inflammatory cytokine
by activating antibacterial defense by neutrophils and
by reducing the release of proinflammatory mediators,
thereby preventing the overactivation of monocytes
and lymphocytes [34]. Thus, a decrease in G-CSF
might decrease the host-defense response during infection. This could explain, at least in part, the higher
mortality rate in LPS-induced shock in rapamycintreated mice compared with control mice [21]. In contrast, G-CSF has been demonstrated to induce chronic
inflammation and autoimmunity and to exacerbate
underlying inflammatory diseases in humans and mice.
G-CSF deficiency protects mice from collagen-induced
arthritis [35], and this effect might be a result of
G-CSF not only inducing neutrophil production, but
also promoting neutrophil trafficking into inflamed
joints [36]. Further investigations are required to
confirm the clinical usefulness of rapamycin in the
treatment of inflammatory diseases.
In summary, we demonstrated that rapamycin inhibits the LPS-induced increase in iNOS and G-CSF
expression through an Oct-2-dependent pathway. Our
results provide evidence for an interesting role of
mTOR in the transcriptional control of LPS-induced
gene expression. Because of its potent immunosuppressive and antiproliferative properties, there is considerable interest in the use of rapamycin for the treatment
of inflammatory diseases. As G-CSF can function as
either an anti-inflammatory or a proinflammatory
cytokine, understanding the mechanism of the effect of
rapamycin on G-CSF expression is important.

Whether rapamycin also inhibits G-CSF expression in
other cells, such as rheumatoid synovial fibroblasts,
deserves further investigation.

Materials and Methods
Materials
LPS from Escherichia coli (serotype 0111:B4) and PMA
were purchased from Sigma-Aldrich (St Louis, MO, USA).
LY294002, a PI3K inhibitor, 1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate, an
Akt inhibitor (Akt-in), and rapamycin, an mTOR inhibitor, were purchased from Calbiochem (San Diego, CA,
USA) and were dissolved in dimethylsulfoxide. Dulbecco’s

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Y.-Y. Chou et al.

modified Eagle’s medium (DMEM), penicillin ⁄ streptomycin
and fetal bovine serum were obtained from GibcoBrl ⁄ LifeTechnologies (Rockville, MD, USA). Rabbit polyclonal
anti-Oct-2 and anti-iNOS IgGs, and horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit
polyclonal anti-p70 S6 kinase and phospho-p70 S6 kinase
(Thr389) IgGs and horseradish peroxidase-conjugated antimouse IgG were purchased from Cell Signaling Technology
(Beverly, MA, USA). Mouse monoclonal anti-b-actin IgG
was purchased from Chemicon (Temecula, CA, USA).
Restriction endonucleases were obtained from New England Biolabs (Beverly, MA, USA). The pGL3-Basic and
phRLTK reporter plasmids, the Dual-LuciferaseÒ Reporter
Assay System and the Griess reagent system were from Promega (Madison, WI, USA). The SuperFect Transfection
Reagent was purchased from Qiagen (Hilden, Germany),
the mouse G-CSF Quantikine ELISA kit from R&D Systems (Minneapolis, MN, USA), the ChIP assay kit from

Upstate Biotechnology (Lake Placid, NY, USA) and protein A–Sepharose beads from Amersham Biosciences
(Chandler, AZ, USA). Plasmids pCG-Oct-1 and pCG-Oct2, and their parent plasmid, pCG, were gifts from Dr W.
Herr (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) [37]. The expression plasmid for DN-Akt
(hemagglutinin-tagged Akt K179M) was kindly provided
by Dr Mien-Chie Hung (University of Texas M. D.
Anderson Cancer Center) [38], and those for DN-mTOR
(hemagglutinin-tagged mTOR N2343K) and RR-mTOR
(hemagglutinin-tagged mTOR S2035T) were kindly provided by Dr Kazuyoshi Yonezawa (Biosignal Research
Center, Kobe University) [39].

Rapamycin inhibits LPS-induced G-CSF and iNOS expression

Cell culture and LPS treatment
RAW264.7, a murine macrophage cell line, was cultured in
DMEM supplemented with 10% fetal bovine serum, 4 mm
glutamine, 100 mL)1 of penicillin and 100 lgỈmL)1 of
streptomycin at 37 °C in 5% CO2, as described previously
[24,25]. The human acute monocytic leukemia cell line THP1 was obtained from the American Type Culture Collection,
and was maintained and induced to differentiate using
160 nm PMA, as described previously [40]. In these experiments, Oct-2, iNOS and G-CSF mRNA and protein levels,
NO production, the phosphorylation levels of p70 S6 kinase
and the transactivation and activity of the G-CSF promoter
were compared between untreated cells and cells treated with
100 ngỈmL)1 of LPS (RAW264.7 and THP-1 cells). When
inhibitors were used, they were added 30 min before the LPS.

Plasmid construction
A DNA fragment containing nucleotides )283 to +35 of
the mouse G-CSF promoter was PCR-amplified from genomic DNA using the primers listed in Table 1; the underlined sequences are MluI and BglII sites created to facilitate
cloning. The DNA fragment was cloned into the MluI ⁄

BglII sites of the pGL3-basic luciferase reporter vector to
obtain the pG-CSF()283 ⁄ +35)-Luc reporter plasmid. The
transcription start site (+1) was assigned according to Nagata et al. [12,41]. The Oct-2 short hairpin RNA plasmid
(pLL 3.7-Oct-2) was constructed as described previously [24].
All constructs were verified by restriction mapping and
sequencing.

Table 1. Primers used in this study.
Sequence (5¢ fi 3¢)
Oligonucleotides used for plasmid construction
G-CSF promoter
Oligonucleotides used for RT-PCR
Oct-2 a
iNOS a
Mouse G-CSF
Human G-CSF
GAPDH a
Oligonucleotides used for the ChIP assay
G-CSF promoter
iNOS promoter

a

Forward ACGCGTAGATCCAACACCCTGCAGCGAT
Reverse AGATCTGATTCTGGGTGATCTGGGCTGCA
Forward
Reverse
Forward
Reverse
Forward

Reverse
Forward
Reverse
Forward
Reverse

AATGGACCCGACATTAACCA
AAATGGTCGTTTGGCTGAAG
AGGAACATCTGGCCAGGCTG
ACTTGGGATGCTCCATGGTC
CTCAACTTTCTGCCCAGAGG
CTGGAAGGCAGAAGTGAAGG
CACTCTGGACAGTGCAGGAAG
CGACACCTCCAGGAAGCTCTG
AAAGGATCCACTGGCGTCTTCACCACC
GAATTCGTCATGGATGACCTTGGCCAG

Forward
Reverse
Forward
Reverse

TGGCTGGAAGAGAGGAAGAG
CTGGGGCAACTCAGGCTTA
AACTGGGGACTCTCCCTTTG
CTACTCCGTGAAGTGAACAA

Primers that can be used to amplify both human and mouse Oct-2, iNOS and GAPDH cDNA.

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Rapamycin inhibits LPS-induced G-CSF and iNOS expression

Y.-Y. Chou et al.

Transient transfection

Reporter gene activity assay

Transient transfection was carried out using the SuperFect
Transfection Reagent, as described previously [24]. Briefly,
RAW264.7 cells were plated and cultured overnight before
transfection. Twenty-four hours after transfection, the cells
were either untreated or were pretreated with vehicle or
specific inhibitors for 30 min before incubation with
100 ngỈmL)1 of LPS.

To measure G-CSF promoter activity, 0.9 lg of the G-CSF
promoter-luciferase reporter plasmid pG-CSF ()283 ⁄ +35)Luc was mixed with 0.1 lg of phRLTK plasmid and the
mixture was used to transiently transfect RAW264.7 cells,
as described above. Photinus and Renilla luciferase activities
in the cell lysates were assayed using the Dual-Luciferase
Reporter Assay System, as described previously [24], and
the light intensity produced by Photinus luciferase (test
plasmid) was normalized to that produced by Renilla luciferase (control plasmid).

RNA isolation and RT-PCR

Following treatment, total RNA in RAW264.7 and THP-1
cells was isolated by acid guanidinium thiocyanate ⁄ phenol ⁄ chloroform extraction, according to the method of
Chomczynski and Sacchi [42]. The concentration and purity
of the RNA were measured by reading the absorbances at
260 nm and 280 nm, respectively, on a spectrophotometer.
The levels of Oct-2, G-CSF, iNOS or glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNAs were determined by semiquantitative RT-PCR using the primers listed
in Table 1. The amplified DNA fragments were separated
by electrophoresis through a 1% agarose gel and sequenced
to confirm their identity.

Western blot analysis
Samples of cell lysates (40 lg of protein per lane) from
RAW264.7 and THP-1 cells were separated by SDS ⁄ PAGE
on 8% gels and transferred onto a poly(vinylidene difluoride) membrane, which was then blocked for 1 h at room
temperature with blocking buffer [TBST (150 mm NaCl,
10 mm Tris ⁄ HCl and 0.05% Tween 20, pH 7.4) containing
5% (w ⁄ v) nonfat dried milk]. The blots were then incubated overnight at 4 °C with primary antibodies in blocking
buffer, washed with TBST, and incubated for 40 min at
room temperature with horseradish peroxidase-conjugated
secondary antibodies in blocking buffer. Immunoreactive
bands were detected using Western LightningÒ Western
Blot Chemiluminescence Reagent Plus (Perkin-Elmer, Boston, MA, USA), following the manufacturer’s instructions,
and by autoradiography using Kodak BioMax MR films
(Eastman Kodak, Rochester, NY, USA).

Quantification of G-CSF and nitrite in culture
medium
The concentration of G-CSF in the culture medium of
RAW264.7 cells was measured by an ELISA using a mouse
G-CSF Quantikine ELISA kit (R&D Systems) according to

the manufacturer’s instructions. The levels of NO in the
culture medium of RAW264.7 cells were measured using a
Griess reagent system kit, according to the manufacturer’s
instructions [25]. The limit of detection for G-CSF and
nitrite was 5 pgỈmL)1 and 2.5 lm, respectively.

94

ChIP assay
The ChIP assay was performed as described previously [24].
Briefly, after various treatments, the cells were fixed with
1% (w ⁄ w) formaldehyde for 10 min at 37 °C to crosslink
proteins to DNA, collected by scraping and then sonicated
on ice by pulsing eight times for 10 s at a power setting of
30% using a Microson Ultrasonic Cell Disrupter KL.
A portion of the sample was kept as input material and the
rest of the fragmented chromatin was immunoprecipitated
with anti-Oct-2 IgG or control rabbit IgG, then the crosslinks were reversed by incubation at 65 °C for 4 h, the proteins were digested with proteinase K for 1 h at 45 °C and
the DNA was recovered by phenol ⁄ chloroform extraction
and ethanol precipitation and used as the template for PCR
with the primers listed in Table 1. The input material was
similarly subjected to de-crosslinking, DNA recovery and
PCR analysis. Thirty PCR cycles were used for all ChIP
experiments and 28 PCR cycles were used for the input
samples.

Statistical analysis
The results are shown as the mean ± SD. Differences
between means were evaluated using the Student’s t-test
and were considered significant at P < 0.01.

Acknowledgements
This study was supported by the National Science
Council of Taiwan grant NSC97-2320-B-002-057-MY3
and NSC99-2320-B-038-009-MY3.

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