In summary, TGF-b1 opposes LPS-induced stabilization of TNF-a mRNA and reduces the amount of TNF-a protein, through induction of expression of the mRNA-binding protein FXR1.. TGF-b1 indu
Trang 1transcriptional suppression of lipopolysaccharide-induced tumour necrosis factor-a production by transforming
growth factor-b1
Tarnjit K Khera1, Andrew D Dick1,2and Lindsay B Nicholson1,2
1 Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, UK
2 Department of Clinical Sciences South Bristol, Academic Unit of Ophthalmology, University of Bristol, UK
Introduction
Tumour necrosis factor-a (TNF-a) is a key mediator of
inflammation, during which it plays a crucial role in the
early phase of a host’s defence against infection [1,2] It
is also produced during autoimmune inflammatory
diseases, where it contributes to tissue damage [3,4]
Septic shock is an extreme example of dysregulated
inflammation, in which TNF-a is expressed rapidly and
at high levels [5–8]
To limit the potentially devastating effects that can follow the release of TNF-a, its expression is under strict control It is regulated at the level of transcription, pre-mRNA processing, mRNA stability, translation,
Keywords
FXR1; macrophages; RNA-binding proteins;
TGF-b1; TNF-a
Correspondence
T K Khera, Department of Cellular and
Molecular Medicine, School of Medical
Sciences, University Walk,
Bristol, BS8 1TD, UK
Fax: +44 117 3312091
Tel: +44 117 3312012
E-mail: t.khera@bristol.ac.uk
Website: http://www.bris.ac.uk/cellmolmed/
air/
(Received 26 February 2010, revised 11
April 2010, accepted 20 April 2010)
doi:10.1111/j.1742-4658.2010.07692.x
Tumour necrosis factor-a (TNF-a) is a key mediator of inflammation in host defence against infection and in autoimmune disease Its production is controlled post-transcriptionally by multiple RNA-binding proteins that interact with the TNF-a AU-rich element and regulate its expression; one
of these is Fragile X mental retardation-related protein 1 (FXR1) The anti-inflammatory cytokine transforming growth factor-b1 (TGF-b1), which is involved in the homeostatic regulation of TNF-a, causes post-transcriptional suppression of lipopolysaccharide (LPS)-induced TNF-a production We report here that this depends on FXR1 Using RAW 264.7 cells and bone marrow-derived macrophages (BMDMu) stimulated with LPS and TGF-b1, we show that TGF-b1 inhibits TNF-a protein secretion, whereas TNF-a mRNA expression remains unchanged This response is recapitulated by the 3¢-UTR of TNF-a, which is known to bind FXR1 TGF-b1 induces FXR1 with a pattern of expression distinct from that of tristetraprolin, T-cell intracellular antigen 1, or human antigen R When FXR1 is knocked down, TGF-b1 is no longer able to inhibit LPS-induced TNF-a protein production, and overexpression of FXR1 suppresses LPS-induced TNF-a protein production Targeting the p38 mitogen-activated protein kinase pathway of LPS-treated cells with small molecule inhibitors can induce FXR1 protein and mRNA expression In summary, TGF-b1 opposes LPS-induced stabilization of TNF-a mRNA and reduces the amount of TNF-a protein, through induction of expression of the mRNA-binding protein FXR1
Abbreviations
ARE, AU-rich element; BMDMu, bone marrow-derived macrophages; CMV, cytomegalovirus; FXR1, Fragile X mental retardation-related protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HuA, human antigen R; IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; Q-PCR, quantitative PCR; RFP, red fluorescent protein; siRNA, small interfering RNA; TGF-b1, transforming growth factor-b1; TIA-1, T-cell intracellular antigen 1; TNF-a, tumour necrosis factor-a; TTP, tristetraprolin.
Trang 2and retention at the plasma membrane [9–13] The
TNF-a mRNA 3¢-UTR contains AU-rich elements
(AREs) AREs, which are found in many cytokine,
inflammatory gene and oncogene mRNAs, are targets
for binding proteins that regulate mRNA stability [14]
Mice with targeted deletion of the TNF-a ARE show
spontaneous production of TNF-a and develop
chronic inflammatory arthritis and inflammatory bowel
disease [15] Multiple RNA-binding proteins that
inter-act with the TNF-a ARE and regulate its expression
have been identified These include tristetraprolin
(TTP), T-cell intracellular antigen 1 (TIA-1),
TIA-1-related protein, human antigen R (HuA), AU-rich
element binding factor 1, and Fragile X mental
retar-dation-related protein 1 (FXR1) [16–22] FXR1 is a
homologue of the Fragile X mental retardation
syn-drome protein, and, together with Fragile X mental
retardation-related protein 2P, forms the Fragile X
mental retardation-related family of RNA-binding
pro-teins [23] Targeted deletion of FXR1 produced a mouse
that died soon after birth, but macrophage cell lines
generated from these animals had enhanced TNF-a
pro-tein production as compared with wild-type
macrophag-es [21] The exprmacrophag-ession of several other proinflammatory
proteins was also affected by FXR1 deficiency, but the
cytokines involved in the induction of FXR1 remain
un-characterized The regulation by the anti-inflammatory
cytokine transforming growth factor-b1 (TGF-b1) of
the proinflammatory cytokine TNF-a via the induction
of FXR1 is the focus of this article
TGF-b1, a member of the large transforming growth
factor-b superfamily [24,25], is an anti-inflammatory
cytokine that can regulate TNF-a Loss of TGF-b1
is associated with chronic inflammation, indicating that
a failure to produce anti-inflammatory factors (i.e
TGF-b1) or defective signalling by anti-inflammatory
cytokines can contribute to the pathogenesis of
inflam-matory autoimmune diseases [26,27] Both recombinant
TGF-b1 protein and antibodies against TNF-a have
been shown to be protective against collagen type II
arthritis in mice, whereas recombinant TNF-a protein
or antibodies against TFG-b1 increased the severity
of this disease, emphasizing the opposing effects of
these cytokines in vivo [28] Other studies have shown
that TGF-b1 can suppress TNF-a production during
infection, increasing the severity of disease [29–32]
In the present study, we show for the first time that
TGF-b1 regulates TNF-a post-transcriptionally via the
induction of FXR1 expression TGF-b1 induces the
expression of this RNA-binding protein, which can
downregulate lipopolysaccharide (LPS)-induced TNF-a
protein production Furthermore, inhibition of FXR1
production can abolish the suppression of TNF-a
protein production induced by TGF-b1 FXR1 there-fore plays an important role in the negative regulation
of TNF-a
Results
TGF-b1 inhibits LPS-induced TNF-a protein production by a TNF-a mRNA expression-independent mechanism
TGF-b1 is known to destabilize the mRNA of LPS-induced chemokines and regulate the mRNA stability
of various other genes [33] It was reported to inhibit TNF-a protein production without concomitant altera-tions in the levels of mRNA, although the mechanism was unknown [34,35]
Bone marrow-derived macrophages (BMDMu) and RAW 264.7 cells treated with LPS (100 pgÆmL)1 to
1 lgÆmL)1) for 4 h produced TNF-a protein in a dose-dependent fashion, maximum production being reached at 100 ngÆmL)1 (Fig S 1A,B) When TGF-b1 (10 ngÆmL)1) [35] was added to either BMDMu or RAW 264.7 cells treated with LPS (100 ngÆmL)1) for
4 h, the level of TNF-a protein induced showed a decrease (Fig 1A,B) The response of the BMDMu was comparable to that of the RAW 64.7 cells Assess-ment by intracytoplasmic staining and flow cytometry gave results comparable to those obtained by measure-ment of the TNF-a protein concentration by ELISA (Fig S1A–C)
To determine whether TGF-b1-dependent inhibition
of LPS-induced TNF-a protein production occurred at the level of transcription, RAW 264.7 cells were stimu-lated as above (Fig 1C) LPS treatment for 4 h increased TNF-a mRNA levels, as compared with the nontreated or TGF-b1-treated controls, and the addi-tion of TGF-b1 with LPS had no effect on the level of TNF-a mRNA Therefore, changes in TNF-a protein production do not correlate with changes in TNF-a mRNA expression, and, as expected, the control of TNF-a induction is not solely transcriptional
Addition of TGF-b1 induces the expression of factors that target the TNF-a 3¢-UTR
Most cytokines contain an ARE in the 3¢-UTR of their mRNA, which modulates stability [36] To deter-mine whether TGF-b1 induced the expression of factors that targeted the TNF-a 3¢-UTR, the TNF-a 3¢-UTR was cloned into the pGL3 control vector after the luciferase ORF (SV40–Luc–TNF-3¢-UTR; Fig 2A) RAW 264.7 cells were cotransfected with SV40–Luc–TNF-3¢-UTR and a Renilla control, and
Trang 3treated with LPS and TGF-b1; untreated cells acted as
controls In unstimulated cells, no luciferase protein
expression was seen LPS treatment induced luciferase
expression (Fig 2B), whereas the simultaneous
addi-tion of TGF-b1 with LPS led to a reducaddi-tion in
lucifer-ase activity from 100% to 43.5 ± 14.1% (P = 0.01)
In agreement with published data, these experiments
show that the TNF-a 3¢-UTR is sufficient to give LPS
the ability to stabilize mRNA [12], but they also
dem-onstrate that this process is regulated by TGF-b1
Induction of FXR1 expression by TGF-b1 leads to
post-transcriptional downregulation of TNF-a
protein
Many RNA-binding proteins, such as TTP, TIA-1,
HuA, TIA-1-related protein, and FXR1, are known to
bind to the ARE in the 3¢-UTR of cytokines, including
TNF-a, and regulate translation We therefore studied the effect of TGF-b1 on RNA-binding proteins, including the mRNA expression of HuA (Fig 3A), TIA-1 (Fig 3B), and TTP (Fig 3C) LPS induced an increase in HuA expression and a decrease in TIA-1 expression, but TGF-b1 did not have an effect
on these mRNA levels As the production of these proteins was not induced by TGF-b1, they were not likely candidates for mediating its effects As expected [37], LPS induced TTP mRNA expression, although, unexpectedly, TGF-b1 decreased the LPS-induced increase in TTP levels TTP is a negative regulator
of TNF-a [38], so the reduction in its level in the pre-sence of TGF-b1 is not consistent with a role in con-trolling TNF-a protein production following TGF-b1 stimulation
The patterns of FXR1 mRNA expression in BMDMu (Fig 3D) and RAW 264.7 cells (Fig 3E)
Fig 1 TGF-b1 can suppress LPS-induced TNF-a protein production, without significant changes in mRNA expression BMDMu (A) and RAW 264.7 cells (B) were treated with
100 ngÆmL)1LPS for 4 h To some samples,
10 ngÆmL)1TGF-b1 was added at the same time as LPS (TGF-b1 ⁄ LPS) TNF-a production was quantified by flow cytometry and ELISA RAW 264.7 cells were treated
as above, and relative TNF-a mRNA expression was quantified using Q-PCR and normalized to GAPDH expression (C) Nontreated cells were used as a control;
n = 3–4, *P < 0.05.
Trang 4were different LPS treatment did not alter the level of
FXR1 mRNA, but this was increased by TGF-b1
(P = 0.009), and this induction was augmented when
TGF-b1 and LPS were present together (P = 0.009) in
RAW 264.7 cells TGF-b1 alone and TGF-b1 with
LPS also induced FXR1 protein production in this
sys-tem (Fig 3F) As expected, two different isoforms of
FXR1 were visualized by western blotting with
anti-body against FXR1 following the addition of TGF-b1
and TGF-b1⁄ LPS [39,40]
The pattern of FXR1 induction suggests that it
could play a role in regulating TNF-a protein
expres-sion in cells treated with TGF-b1 To test this directly,
FXR1 was inhibited with small interfering RNA
(siRNA) (Fig 4A) FXR1 siRNA inhibited FXR1
mRNA expression by 74% as compared with a control
siRNA Inhibition of FXR1 protein production
was assessed by using RAW 264.7 cells treated with
TGF-b1 for 1 h (Fig 4Ba) and RAW 264.7 cells
stably transfected with FXR1 under a cytomegalovirus
(CMV) promoter (FXR1-OE cells; Fig 4Bb) In both
the RAW 264.7 cells treated with TGF-b1 and the
FXR1-OE cells, siRNA 1 led to the greatest inhibition
of FXR1 protein production, so siRNA 1 was used for
all experiments In the control siRNA-transfected cells,
LPS induced TNF-a protein production, and the
addi-tion of TGF-b1 suppressed TNF-a protein producaddi-tion
by 63% When FXR1 was inhibited, TGF-b1 was no
longer able to suppress LPS-induced TNF-a protein
production (Fig 4C) These findings show that TGF-b1-induced inhibition of TNF-a protein production is reversed when FXR1 is inhibited
Overexpression of FXR1 can suppress LPS-induced TNF-a protein production
To determine whether FXR1 overexpression is suffi-cient to oppose the effects of LPS on TNF-a secretion from RAW 264.7 cells, the FXR1-OE cells were com-pared with control cells transfected with red fluores-cent protein (RFP) under a CMV promoter (RFP-OE cells) Increased expression of FXR1 protein in these cell lines could be detected by western blot (Fig 5A) and by quantitative PCR (Q-PCR) (Fig 5B), and FXR1 mRNA expression was 5.5-fold higher in FXR1-OE cells than in RFP-OE cells TNF-a protein from these cells treated with LPS for 4 h was quanti-fied At all concentrations of LPS, TNF-a protein production was suppressed in FXR1-OE cells as com-pared with controls (Fig 5C) This effect was rela-tively greater at lower LPS concentrations, and shows directly that overexpression of FXR1 can suppress TNF-a protein production However, the effects of consistent inhibition were partially reversed by increas-ing the stimulus drivincreas-ing TNF-a protein production This could be because higher amounts of LPS lead to increased TNF-a protein production as measured by ELISA and intracellular staining (Fig S1A–C)
Inhibition of p38 mitogen-activated protein kinase (MAPK) can induce FXR1 production in LPS-treated cells
LPS is known to activate the p38 MAPK pathway, which is important, for example, in the stabilization of chemokine mRNA TGF-b1 opposes LPS-induced chemokine stabilization by inhibiting p38 MAPK [33]
To determine whether this signalling pathway was involved in FXR1 induction, SB203580, a cell-perme-able p38 MAPK inhibitor, was used RAW 264.7 cells were treated with LPS (100 ngÆmL)1) and 0–10 lm SB203580 for 4 h, and FXR1 protein (Fig 6A) and mRNA (Fig 6B) were quantified This treatment led
to substantial upregulation of FXR1 protein produc-tion as well as an increase in mRNA levels Further experiments showed that the MAPKAP kinase 2 inhib-itor also induced FXR1 mRNA expression (Fig 2C) These inhibitors were also tested with BMDMu trea-ted with LPS A negative control inhibitor (SB202474) did not lead to expression of FXR1 mRNA, whereas the MAPKAP kinase 2 inhibitor and SB203580 both induced FXR1 mRNA expression (Fig 6D) These
A
B
Fig 2 TGF-b1 can inhibit LPS-induced protein production via the
3¢-UTR of TNF-a (A) Schematic representation of the SV40–Luc–
TNF-3¢-UTR plasmid used (B) The SV40–Luc–TNF-3¢-UTR plasmid
was cotransfected into RAW 264.7 cells with Renilla, also on a
con-stitutive promoter After 24 h, the cells were treated with
100 ngÆmL)1LPS, with addition of 10 ngÆmL)1TGF-b1 alone or at
the same time as LPS Luciferase expression was normalized using
Renilla Cells treated with LPS alone were set at 100% luciferase
expression and the nontreated cells at 0% luciferase expression;
n = 3, *P < 0.05.
Trang 5results show that, in LPS-treated cells, inhibition of
p38 MAPK leads to FXR1 induction at the protein
and mRNA levels, a pattern consistent with the known
signalling properties of TGF-b1
Discussion
Regulation of TNF-a plays a central role in
autoim-mune disease [41–44], and therapy targeting TNF-a is
effective in patients with inflammatory disorders such
as uveitis and rheumatoid arthritis [45–49] However,
this treatment has significant side effects, and better
understanding of its control may lead to more selective
therapies Here, we investigated the homeostatic
regu-lation of TNF-a by the anti-inflammatory cytokine
TGF-b1 and demonstrated that FXR1, an
mRNA-binding protein, plays an essential role in this process
Building on previous work showing that TGF-b1 can
inhibit LPS-induced TNF-a and chemokine production
[33,35], we confirmed that this occurs
post-transcrip-tionally We then showed that the TNF 3¢-UTR is a
target for factors induced by TGF-b1 that counteract
the LPS-induced increased stability of TNF-a mRNA
One of these factors is FXR1, which, unlike other mRNA-binding proteins known to modulate TNF-a,
is induced by TGF-b1 but not by LPS The specific role of FXR1 in this process was shown by inhibition
by siRNA on the one hand, and stable overexpression
of FXR1 on the other These results are consistent with the phenotype of macrophages derived from FXR1) ⁄ ) mice [21] The reporter assay showed more suppression than quantification of TNF-a protein by ELISA or intracellular staining The most likely reason for this is that only the effects of the TNF-a mRNA 3¢-UTR are being taken into account Although the luciferase data show that TGF-b1 can suppress LPS-induced TNF-a production post-transcriptionally,
it does not provide information about whether this occurs via mRNA instability and a decrease in the half-life of TNF-a mRNA or via translational suppression
TGF-b1 inhibits the action of LPS, in part, by inter-fering with p38 MAPK-dependent stabilization of mul-tiple mRNAs [33] This has downstream effects on a number of genes, including those for TNF-a [50], interleukin (IL)-3 [51], and IL-8 [52] Inhibiting p38
A
B
Fig 3 TGF-b1 can induce FXR1 mRNA and protein expression RAW 264.7 cells were treated with 100 ngÆmL)1LPS and
10 ngÆmL)1, TGF-b1 alone or in combination, for 4 h The relative expression of mRNA was then quantified by Q-PCR, and represented as a fold increase as compared with nontreated control cells GAPDH was used to normalize the results; *P < 0.05; (A) HuA, n = 3; (B) TIA-1, n = 3; and (C) TTP,
n = 4 FXR1 mRNA expression was quantified in BMDMu (D), n = 3, and RAW 264.7 cells (E), n = 5 FXR1 protein expression was detected by western blot using RAW 264.7 cells (F).
Trang 6MAPK signalling with specific inhibitors in both a macrophage cell line and primary macrophages treated with LPS led to an increase in FXR1 expression simi-lar to that seen in cells treated with TGF-b1, indicat-ing that this pathway plays a role in the control of FXR1
It is also notable that TGF-b1 did not change the expression of HuA and TIA-1, although it did lead to a significant reduction in TTP mRNA expression, either
on its own or in combination with LPS (Fig 3C), in contrast to previously published data showing that TGF-b1 can increase TTP expression in a T-cell line and a human monocytic cell line [53] The reduction of TTP expression by TGF-b1 is difficult to explain, but
A
C B
Fig 5 Overexpression of FXR1 suppresses LPS-induced TNF-a protein production RAW 264.7 cells were transfected with the RFP-OE or FXR1-OE plasmid and cultured in the presence of G418 for 4 weeks FXR1 protein was detected by western blot (A), and FXR1 mRNA expression was quantified using Q-PCR and normal-ized using GAPDH; n = 3, *P < 0.05 (B) RFP-OE and FXR1-OE cells were cultured with or without 100–1 ngÆmL)1LPS for 4 h in the presence of GolgiPlug Intracellular analysis of TNF-a was carried out by flow cytometry (C); n = 3–4, *P < 0.05.
a) RAW 264.7 cells
b) FXR1-OE cells
None Control siRN
FXR1 Actin
FXR1 Actin
B
1
0.8
0.6
0.4
0.2
0
125
100
75
50
25
0
Control
siRNA
*
*
TGF- β1/LPS LPS
C
Fig 4 FXR1 is necessary for TGF-b1-mediated suppression of
LPS-induced TNF-a production RAW 264.7 cells were transfected
with 50 n M control siRNA or siRNA targeting FXR1 for 24 h (A).
TGF-b1, 10 ngÆmL)1, was added 1 h prior to quantification of FXR1
mRNA expression by Q-PCR to induce FXR1 expression The
results were normalized using GAPDH; n = 3, *P < 0.05 FXR1
pro-tein was also detected by western blot (Ba), using three siRNAs
that target FXR1 FXR1-OE cells were transfected with siRNA as
described, and, after 24 h, FXR1 protein was detected by western
blot (Bb) RAW 264.7 cells were transfected with siRNA, and, after
24 h, 100 ngÆmL)1LPS was added plus 10 ngÆmL)1TGF-b1 for 4 h
in the presence of GolgiPlug (C) Intracellular TNF-a was quantified
by flow cytometry; n = 3, *P > 0.05.
Trang 7is worthy of further investigation, as TTP is a negative
regulator of TNF-a On the other hand, TGF-b1, but
not LPS alone, significantly increased FXR1
expres-sion, whereas LPS in combination with TGF-b1
fur-ther increased FXR1 expression FXR1 is known to
bind to the TNF-a mRNA ARE and suppress
transla-tion [21] Other mRNA-binding proteins, such as TTP,
are known to be controlled by phosphorylation by p38
MAPK⁄ MK2 There is evidence suggesting that
phos-phorylation of another member of the FXR1 family,
Fragile X mental retardation protein, on Ser 144 may
be important in translational repression [54], but the
effects of phosphorylation of FXR1 are unknown In
this article, we have shown that inhibition of the p38
MAPK pathway can upregulate FXR1, but the
mecha-nism for this remains unknown and is under
investiga-tion It is also possible that other anti-inflammatory
cytokines may also upregulate FXR1, leading to
post-transcriptional regulation of TNF-a and possibly other
proinflammatory cytokines This remains an area for
further investigation FXR1 is intimately involved in
mRNA regulation, but there are other mechanisms in
which it may play a role It will be important to
deter-mine whether the reported association of FXR1 with
the RNA-induced silencing complex protein AGO2 is
critical to its action [39,55,56] This work shows the
induction of FXR1 by the regulatory cytokine
TGF-b1 We have focused on the downstream effects of
FXR1 on TNF-a, but it is likely that other cytokines
will also be regulated by the same mechanisms In
other experiments, IL-6 has been reported to be a
tar-get for FXR1 [21], and investigation of further
poten-tial targets is ongoing
Little is known about FXR1 in human disease, in which it has not been extensively investigated, although it has been identified as an autoantigen in systemic sclerosis [57] We also have scant information
on the significance that the different isoforms of FXR1 have in terms of function Although these have been characterized carefully at the molecular level [40], their patterns of expression in inflammatory disease remain
to be investigated TNF-a overproduction has been shown to be an important driving force in many auto-immune diseases, including rheumatoid arthritis [58], uveitis [59], multiple sclerosis [60,61], and inflamma-tory bowel disease [62] Blockade of TNF-a in autoim-mune disease has been successfully introduced into the clinic for some of these conditions In many instances, however, these treatments have been associated with impaired host defence against infections [63–65] Understanding the role that FXR1 plays in the control
of TNF-a by TGF-b1 could allow the development of therapies that complement the blockade of TNF-a, to give full efficacy while reducing unwanted side effects The study of RNA-binding proteins is therefore essen-tial for the understanding of intracellular regulatory pathways and molecular mechanisms of pathology
Experimental procedures
BMDMu
C57BL⁄ 6 Ly.5.2 congenic mice were obtained from Charles River Laboratories (Margate, UK), and were reared under specific pathogen-free conditions The mice were maintained
in accordance with the Home Office Regulations for
A
C
B
D
Fig 6 Inhibition of the p38 MAPK pathway can induce FXR1 protein production in LPS-treated macrophages RAW 264.7 cells were cultured with 100 ngÆmL)1LPS for 4 h plus SB203580 FXR1 protein was detected
by western blot (A), and relative FXR1 mRNA expression was quantified by Q-PCR (B) The results were normalized using GAPDH; n = 3, *P < 0.05 RAW 264.7 cells were also cultured with 100 ngÆmL)1LPS for 4 h plus either the negative control inhibitor SB202474 or the MAPKAP kinase 2 inhibitor, and this was followed by FXR1 mRNA quantification by Q-PCR; n = 3 (C) BMDMu were cultured with 100 ngÆmL)1 LPS for 4 h plus either the negative control inhibitor SB202474, SB203580, or the MAPKAP kinase 2 inhibitor, and this was followed by FXR1 mRNA quantification by Q-PCR; n = 3 (D).
Trang 8Animal Experimentation, UK Bone marrow cells were
obtained by flushing the femurs of mice, and the cells were
cultured as previously described [66] in hydrophobic Teflon
bags in DMEM containing 10% heat-inactivated fetal
bovine serum, 5% normal horse serum, and the
superna-tant of macrophage colony-stimulating factor-secreting
L929 fibroblasts at a final concentration of 15% (v⁄ v) for
8 days at 37C in the presence of 5% CO2
RAW 264.7 cell culture
The RAW 264.7 murine cell line was cultured in DMEM
supplemented with 10% fetal bovine serum, 10 UÆmL)1
penicillin, 10 lgÆmL)1streptomycin, and 2 mm l-glutamine
(all from Invitrogen, Paisley, UK) Cells were maintained at
37C in the presence of 5% CO2
Inhibitors
SB203580 was purchased from Promega The negative
con-trol inhibitor SB2025880 and MAPKAP kinase 2 (Hsp25)
inhibitor were purchased from Calbiochem (Nottingham,
UK)
Stable cell lines
RAW 264.7 cells were electroporated at 300 V and 960 lF
with plasmids containing RFP or FXR1 on the CMV
pro-moter (Cambridge Biosciences, Cambridge, UK) After
24 h, 500 lgÆmL)1 G418 (Sigma Aldrich, Dorset, UK) was
added to the medium [67], and cells were used after 4 weeks
of culture
Flow cytometry
Cells were seeded in macrophage serum-free medium
(Invi-trogen, Paisley, UK) The cells were treated with TGF-b1
(R&D Systems, Abingdon, UK) or LPS (Sigma Aldrich,
Dorset, UK) at the concentrations stated for 4 h in the presence of 1 lgÆmL)1 GolgiPlug (BD Bioscience, Oxford, UK) The cells were washed in buffer containing a balanced salt solution with 0.1% BSA and 0.08% azide (Media Ser-vices, University of Bristol, UK), and fixed using
Cyto-fix⁄ Cytoperm according to the manufacturer’s instructions (BD Pharmingen, Oxford, UK) The cells were stained with rat anti-mouse TNF-a-APC Ig, IgG1 isotype (clone MP6-XT22; BD Pharmingen, Oxford, UK) Analysis was carried out using a FACS Canto II and FACS diva 5.0.2 software (BD Biosciences, San Jose, CA, USA) The data are shown
as percentage change, using the geometric mean values, with control, nontreated cells set at 0%, and cells treated with only LPS set at 100%
ELISA
Cells were seeded in macrophage serum-free medium and treated with TGF-b1 or LPS at the concentrations stated for 4 h TNF-a in the supernatant was measured by ELISA according to the manufacturer’s protocol (R&D Systems, Abingdon, UK)
Q-PCR
Alterations in mRNA expression were examined by Q-PCR, using Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK), performed using specific oligonucleotide primers (Table 1, from Sigma Genosys, Dorset, UK) and StepOnePlus (Applied Biosystems, War-rington, UK) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control to normalize the results
Luciferase assay
The 3¢-UTR of TNF-a was cloned using the following prim-ers: forward, 5¢-CCCGACTACGTGCTCCTCAC-3¢; and reverse, 5¢-TTTATTTCTCTCAATGACCCGT-3¢ (Sigma
Table 1 Primers: names and sequences of primers used for Q-PCR The sequences from the database RT (http://medgen.ugent.be/ rtprimerdb/) were used.
Reverse: GGCATGGACTGTGGTCATGA
RTPrimerDB 2920
Reverse: CCACATGGCTCTTGGTCATTTGCT
–
Reverse: TGGGAGTAGACAAGGTACAACCC
RTPrimerDB 147
Reverse: TAGGAACGGATCCACCCAAACACT
–
Reverse: AGGCTGCTTTGATGTCTTCGGTTG
–
Reverse: AAGCTTTGCAGATTCAACCTCGCC
–
Trang 9Genosys, Dorset, UK) The PCR product was purified using
the QIAquick PCR purification kit (Qiagen, Crawley, UK)
and inserted into the XbaI site in the pGL3 control vector
(Promega, Southampton, UK) The plasmid was amplified
in Top10 Escherichia coli (Invitrogen, Paisley, UK) and
purified using the HiSpeed Plasmid Midikit (Qiagen) A
Renillaplasmid (pRL–TK–renilla; Promega) was used as a
control RAW 264.7 cells were seeded to a density of
1.5· 106cells per well in a six-well plate on the day before
transfection, in DMEM containing l-glutamine only On the
day of transfection, the cells were washed in Optimem
med-ium (Invitrogen) The plasmids were prepared for
transfec-tion [per well, in a 1.5 mL tube: 20 lL of Optimem, 5 lg of
each plasmid, and 6 lL of Lipofectamine LTX (Invitrogen)
and incubated at room temperature for 30 min This mixture
was then added slowly to cells in 1 mL of Optimem The
cytokines were added on the next day, and luciferase activity
was measured
Western blot
FXR1 protein was examined by western blot analysis, using
standard methodologies with a polyclonal antibody against
FXR1 raised in goats (ab51970; Abcam, Cambridge, UK)
An polyclonal antibody against actin, also raised in goats,
was used as a control (Santa Cruz Biotechnology,
Heidel-berg, Germany) Briefly, cells were scraped off into NaCl⁄ Pi
before resuspension of the pellet in cell lysis buffer (Cell
Signaling Technology, Hitchin, UK) The protein was
pre-pared in SDS sample buffer, and boiled for 5 min prior to
loading onto 10% SDS⁄ PAGE gels After electrophoresis,
the separated proteins were transferred to a nitrocellulose
membrane (Amersham Pharmacia, Biotech UK Ltd, Little
Chalfont, UK) The membrane was blocked with NaCl⁄
Tris containing 5% BSA for 1 h, and then incubated with
the primary antibody overnight at 4C The blots were
subsequently washed in NaCl⁄ Tris-Tween, and then
incu-bated with an appropriate horseradish
peroxidase-conju-gated secondary antibody (Santa Cruz Biotechnology)
Proteins were visualized by enhanced chemiluminescence
(Amersham, Little Chalfont, UK), according to the
manu-facturer’s instructions
siRNA
Cells were plated in a six-well plate at a density of
1· 106
cells per well overnight in DMEM containing 2 mm
l-glutamine only On the following day, the cells were
washed with Optimem and transfected using Lipofectamine
RNAiMax (Invitrogen) with 50 nm control siRNA
(BLOCK-iT Alexa Fluor Red Fluorescent Control;
Invitro-gen) or FXR1 siRNA (InvitroInvitro-gen) for 24 h The following
FXR1 siRNA sequences were used: siRNA 1, 5¢-GGG
CCC UAA UUA CAC CUC CGG UUA U-3¢; siRNA 2,
5¢-GCA AUC CAU ACA GCU UAC UUG AUA A-3¢; and
siRNA 3, 5¢-GAA GUU GAU GCU UAU GUC CAG AAA U-3¢
Statistical analysis
Results are expressed as mean ± standard error of the mean The Mann–Whitney U-test, two-tailed, was used to determine significance
Acknowledgements
The flow cytometric analysis was carried out with assistance from Dr A Herman and Mr T Curry, Flow Cytometry Facility, Cellular and Molecular Medicine, Bristol University The authors would like to thank Professor N Perkins, University of Bristol, for review-ing the manuscript Mr O Whitton assisted with the preparation for publication of this manuscript This work was supported by grants from the National Eye Research Centre (NERC) and the James Tudor Foun-dation, and a Royal College of Pathologists⁄ Jean Shanks Foundation Pilot Award
References
1 Vassalli P (1992) The pathophysiology of tumor necro-sis factors Annu Rev Immunol 10, 411–452
2 Beutler B, Greenwald D, Hulmes JD, Chang M, Pan
YC, Mathison J, Ulevitch R & Cerami A (1985) Identity of tumour necrosis factor and the macrophage-secreted factor cachectin Nature 316, 552–554
3 Tincani A, Andreoli L, Bazzani C, Bosiso D & Sozzani S (2007) Inflammatory molecules: a target for treatment of systemic autoimmune diseases Autoimmun Rev 7, 1–7
4 Clark IA (2007) How TNF was recognized as a key mechanism of disease Cytokine Growth Factor Rev 18, 335–343
5 Waage A, Halstensen A & Espevik T (1987) Associa-tion between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease Lancet
1, 355–357
6 Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S, Milsark IW, Hariri RJ, Fahey TJ 3rd, Zentella A, Albert JD et al (1986) Shock and tissue injury induced by recombinant human cachectin Science 234, 470–474
7 Parrillo JE (1993) Pathogenetic mechanisms of septic shock N Engl J Med 328, 1471–1477
8 Cohen J (2002) The immunopathogenesis of sepsis Nature 420, 885–891
9 Weil D, Brosset S & Dautry F (1990) RNA processing
is a limiting step for murine tumor necrosis factor beta expression in response to interleukin-2 Mol Cell Biol
10, 5865–5875
Trang 1010 Moreira AL, Sampaio EP, Zmuidzinas A, Frindt P,
Smith KA & Kaplan G (1993) Thalidomide exerts its
inhibitory action on tumor necrosis factor alpha by
enhancing mRNA degradation J Exp Med 177, 1675–
1680
11 Han J, Huez G & Beutler B (1991) Interactive effects of
the tumor necrosis factor promoter and 3¢-untranslated
regions J Immunol 146, 1843–1848
12 Han J, Brown T & Beutler B (1990)
Endotoxin-respon-sive sequences control cachectin⁄ tumor necrosis factor
biosynthesis at the translational level J Exp Med 171,
465–475
13 Biragyn A & Nedospasov SA (1995)
Lipopolysaccha-ride-induced expression of TNF-alpha gene in
the macrophage cell line ANA-1 is regulated at the
level of transcription processivity J Immunol 155,
674–683
14 Asirvatham AJ, Gregorie CJ, Hu Z, Magner WJ &
Tomasi TB (2008) MicroRNA targets in immune genes
and the Dicer⁄ Argonaute and ARE machinery
compo-nents Mol Immunol 45, 1995–2006
15 Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli
F & Kollias G (1999) Impaired on⁄ off regulation of
TNF biosynthesis in mice lacking TNF AU-rich
ele-ments: implications for joint and gut-associated
immun-opathologies Immunity 10, 387–398
16 Zhang W, Wagner BJ, Ehrenman K, Schaefer AW,
DeMaria CT, Crater D, DeHaven K, Long L &
Brewer G (1993) Purification, characterization, and
cDNA cloning of an AU-rich element RNA-binding
protein, AUF1 Mol Cell Biol 13, 7652–7665
17 Vasudevan S, Tong Y & Steitz JA (2008) Cell-cycle
control of microRNA-mediated translation regulation
Cell Cycle 7, 1545–1549
18 Piecyk M, Wax S, Beck AR, Kedersha N, Gupta M,
Maritim B, Chen S, Gueydan C, Kruys V, Streuli M,
et al.(2000) TIA-1 is a translational silencer that
selec-tively regulates the expression of TNF-alpha EMBO J
19, 4154–4163
19 Lai WS, Carballo E, Strum JR, Kennington EA,
Phil-lips RS & Blackshear PJ (1999) Evidence that
triste-traprolin binds to AU-rich elements and promotes the
deadenylation and destabilization of tumor necrosis
fac-tor alpha mRNA Mol Cell Biol 19, 4311–4323
20 Gueydan C, Droogmans L, Chalon P, Huez G, Caput
D & Kruys V (1999) Identification of TIAR as a
pro-tein binding to the translational regulatory AU-rich
element of tumor necrosis factor alpha mRNA J Biol
Chem 274, 2322–2326
21 Garnon J, Lachance C, Di Marco S, Hel Z, Marion D,
Ruiz MC, Newkirk MM, Khandjian EW & Radzioch
D (2005) Fragile X-related protein FXR1P regulates
proinflammatory cytokine tumor necrosis factor
expres-sion at the post-transcriptional level J Biol Chem 280,
5750–5763
22 Dean JL, Wait R, Mahtani KR, Sully G, Clark AR & Saklatvala J (2001) The 3¢ untranslated region of tumor necrosis factor alpha mRNA is a target of the mRNA-stabilizing factor HuR Mol Cell Biol 21, 721–730
23 Bardoni B, Schenck A & Mandel JL (2001) The Frag-ile X mental retardation protein Brain Res Bull 56, 375–382
24 Miyazono K (2000) Positive and negative regulation of TGF-beta signaling J Cell Sci 113, 1101–1109
25 Dijke PT & Hill CS (2004) New insights into TGF-b-Smad signalling Trends Biochem Sci 29, 265–273
26 Monteleone G, Kumberova A, Croft NM, McKenzie
C, Steer HW & MacDonald TT (2001) Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease J Clin Invest 108, 601–609
27 MacDonald TT (1999) Effector and regulatory lym-phoid cells and cytokines in mucosal sites Curr Top Microbiol Immunol 236, 113–135
28 Thorbecke GJ, Shah R, Leu CH, Kuruvilla AP, Hardi-son AM & Palladino MA (1992) Involvement of endog-enous tumor necrosis factor alpha and transforming growth factor beta during induction of collagen type II arthritis in mice Proc Natl Acad Sci USA 89, 7375– 7379
29 Mendez-Samperio P, Hernandez-Garay M & Nunez Vazquez A (1998) Inhibition of Mycobacterium bovis BCG-induced tumor necrosis factor alpha secretion in human cells by transforming growth factor beta Clin Diagn Lab Immunol 5, 588–591
30 Dlugovitzky D, Bay ML, Rateni L, Urizar L, Rondelli
CF, Largacha C, Farroni MA, Molteni O & Bottaso
OA (1999) In vitro synthesis of interferon-gamma, inter-leukin-4, transforming growth factor-beta and interleu-kin-1 beta by peripheral blood mononuclear cells from tuberculosis patients: relationship with the severity of pulmonary involvement Scand J Immunol 49, 210–217
31 D’Andrea A, Ma X, Aste-Amezaga M, Paganin C & Trinchieri G (1995) Stimulatory and inhibitory effects
of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: priming for IL-12 and tumor necrosis factor alpha production J Exp Med 181, 537–546
32 Chantry D, Turner M, Abney E & Feldmann M (1989) Modulation of cytokine production by transforming growth factor-beta J Immunol 142, 4295–4300
33 Dai Y, Datta S, Novotny M & Hamilton TA (2003) TGFbeta inhibits LPS-induced chemokine mRNA stabilization Blood 102, 1178–1185
34 Hausmann EH, Hao SY, Pace JL & Parmely MJ (1994) Transforming growth factor beta 1 and gamma interferon provide opposing signals to lipopolysaccha-ride-activated mouse macrophages Infect Immun 62, 3625–3632