However, the TLR3 ligand polyI:C permanently upregu-lates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway.. These observations led us
Trang 1Regulators of G-protein signalling are modulated by
bacterial lipopeptides and lipopolysaccharide
Sabine Riekenberg1, Katja Farhat1, Jennifer Debarry2, Holger Heine2, Gu¨nther Jung3, Karl-Heinz Wiesmu¨ller4and Artur J Ulmer1
1 Cellular Immunology, Department of Immunology and Cell Biology, Research Center Borstel, Germany
2 Innate Immunity, Department of Immunology and Cell Biology, Research Center Borstel, Germany
3 Institute of Organic Chemistry, University of Tuebingen, Germany
4 EMC microcollections GmbH, Tuebingen, Germany
The innate immune system is the first barrier against
pathogens and is initiated rapidly after recognition of
microbial products by receptors such as the Toll-like
receptors (TLR) TLR recognize a broad range of
ligands like lipopolysaccharides (LPS) and lipopeptides
(LP) representing pathogen-associated molecular
patterns [1,2] TLR contain two major domains: the
extracellular ligand-binding domain, characterized by
leucine-rich repeats and the intracellular Toll⁄ IL-1
receptor domain (TIR domain) [3] In mammals, 13
TLR homologues recognizing specific bacterial or viral
ligands have been identified [4] Bacterial LP and LPS are recognized by the membrane receptors TLR2 and TLR4, respectively Intracellular TLR3 is a receptor for poly(I:C) [5], and CpG oligo-nucleotides are ligands for the intracellular TLR9 [6,7] TLR2 is unique among all TLR, developing heteromers with TLR1 and TLR6 In previous studies we investigated the ligand specificity of different TLR2 dimers in spleen cells from TLR2-, TLR6- and TLR1-deficient mice [8,9] LP have strong TLR2-dependency but differ
in their requirement for TLR6 and TLR1, according
Keywords
gene expression; lipopeptides;
macrophages; regulator of G-protein
signalling; Toll-like receptors
Correspondence
A J Ulmer, Cellular Immunology and Cell
Biology, Research Center Borstel, Parkallee
22, 23845 Borstel, Germany
Fax: +49 4537 188435
Tel: +49 4537 188448
E-mail: ajulmer@fz-borstel.de
(Received 26 August 2008, revised 12
November 2008, accepted 20 November
2008)
doi:10.1111/j.1742-4658.2008.06813.x
Regulators of G-protein signalling accelerate the GTPase activity of Ga subunits, driving G proteins in their inactive GDP-bound form This property defines them as GTPase activating proteins Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed After stimulation with TLR2⁄ 1 or TLR2 ⁄ 6 lipopeptide ligands and the TLR4⁄ MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein sig-nalling tested Real-time PCR confirmed modulation of RGS1 and RGS2
In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downre-gulation Similar results were also found in the murine macrophage cell line J774 The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner However, the TLR3 ligand poly(I:C) permanently upregu-lates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway This was confirmed using MyD88) ⁄ ) and TRIF) ⁄ ) bone marrow-derived macrophages Modulation
of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection
Abbreviations
BMDM, bone marrow-derived macrophages; FSL-1, fibroblast-stimulating lipopeptide-1; GAP, GTPase activating protein; GPCR, G-protein coupled receptor; LP, lipopeptide; LPS, lipopolysaccharide; RGS, regulator of G-protein signalling; TLR, Toll-like receptor.
Trang 2to the number and length of their fatty acids and the
amino acid sequence of their peptide tail To address
TLR2⁄ 1- and TLR2 ⁄ 6-mediated signalling we used the
lipopeptide Pam3C-SK4and fibroblast-stimulating
lipo-peptide-1 (FSL-1), respectively
Activation of macrophages by microbes or their
cel-lular components induces the release of different
inflammatory mediators Stimulation of TLR leads to
activation of a series of signalling proteins, and to the
expression of pro- and inflammatory cytokines There
is evidence that the heteromeric guanine
nucleotide-binding regulatory protein (G protein) is also involved
in TLR4 activation It has been shown that LPS
induced TNFa production which can be blocked by
pertussis toxin [10] Also, TLR4-induced ERK1⁄ 2
phosphorylation is inhibited by dominant-negative Gai
protein constructs [11] G proteins are located
down-stream of G-protein-coupled receptors (GPCR) [12]
GPCR represent a large family of cell-surface proteins
mediating the effects of a broad spectrum of biological
signals After ligand binding, the receptor undergoes a
conformational change Ligands include hormones,
biogenic amines, histamine, serotonin and lipid
deri-vates, but also immunological and inflammatory
medi-ators such as chemokines Heterotrimeric G proteins
are localized on the inner surface of cell membranes
They comprise a superfamily of at least 17 distinct
Ga, 5 Gb and 6 Gc isoforms [13] Furthermore the
a subunits are divided into four main categories: Gai,
Gas, Gaqand Ga12⁄ 13 [14] In their inactive
conforma-tion G proteins consist of a-, b- and c subunits,
whereas only the a subunit is bound to GDP GPCR
are transmembrane receptor proteins, containing seven
membrane-spanning segments After binding of the
relevant ligand and activation of the GPCR, the
receptor acts as a guanine nucleotide-exchange factor
that exchanges GTP for GDP on the a subunit In the
active GTP-bound form, the a subunit–GTP complex
dissociates from the bc dimer Each of the separated
subunits can regulate downstream effectors Signalling
is terminated when the a subunit hydrolyses GTP,
returns to the GDP-bound state and again associates
with bc subunits to give the inactive heterotrimeric
form [15]
Regulators of G-protein signalling (RGS) interact
directly with the G protein a subunit in order to
inhi-bit G-protein signalling [16] RGS proteins belong to a
large gene family, whose members are widespread from
yeast to mammals [13] RGS proteins differ widely in
their size and amino acid identity They were first
dis-covered genetically as negative regulators of G-protein
signalling in lower eukaryotic organisms including
Aspergillusand Caenorhabditis elegans
Currently, more than 25 mammalian RGS proteins have been identified by molecular cloning [17] Each RGS protein contains a conserved sequence of 120 amino acids which is responsible for binding to the Ga subunit [18] The functional effect of most of RGS proteins is unclear Biochemical studies have shown that RGS proteins have GTPase activity and act as a GTPase activating protein (GAP) As a result, RGS proteins enhance GTP hydrolysis rates for purified Gai and Gaq subunits as much as 100- to 300-fold [15,19] They can also modulate the lifetime and kinetics of slow-acting signalling responses like Ca2+ oscillations [20] Different studies have shown that RGS1 stimu-lates the GTPase activity of several members of the
Gai subfamily but is ineffective against Gas [21], whereas RGS2 does not interact with Gai, Gao, Gas and Ga12⁄ 13at all; RGS2 acts selectively as a GAP for
Gaqsubunits [22,23]
In this study, we show using microarray analyses that RGS2 belongs to the most downregulated mRNA after stimulation of murine bone marrow-derived mac-rophages (BMDM) with LP, whereas RGS1 was upregulated after stimulation with LPS Similar results were found in dendritic cells after activation with LPS These observations led us to investigate the modula-tion of RGS1 and RGS2 in BMDM after stimulamodula-tion with LP and LPS in more detail, because regulation of RGS1 and RGS2 after activation of different TLR may modify the effects of G-protein signalling after posterior activation of GPCR Our results indicate that RGS1 and RGS2 have important immunomodulating functions in murine macrophages because these two RGS proteins demonstrate strong modulation of expression after stimulation with LPS and LP LP and LPS mediate immunomodulating functions, at least
in part, through regulation of RGS1 and RGS2 expression
Results RGS1 and RGS2 mRNA expression is regulated
by LP and LPS BMDM were stimulated with FSL-1, a ligand for the TLR2⁄ 6 heteromer, and LPS, a ligand for TLR4⁄ MD2 After various culture times, mRNA was isolated and the expression of multiple probe sets was analysed by microarray analysis According to the microarray data, only 11 of 18 tested RGS mRNAs are expressed in LP-stimulated BMDM, and 9 of 16 tested RGS mRNAs were expressed in LPS-stimulated BMDM We did not detect the mRNA of several of the tested RGS genes in control or stimulated cells
Trang 3(Table 1) We gave special regard to RGS2, because
we observed that the mRNA of RGS2 was the
strongest downregulated mRNA of 45 101 probe
sets after 6 h of stimulation with FSL-1 Interleukin-6,
by contrast, was the strongest upregulated gene
(data not given) In addition, RGS1 and RGS10, but
none of the other listed RGS mRNAs, were also
found to be modulated Stimulation with Pam2C-SK4
(TLR2⁄ 1 and TLR2⁄ 6 ligand) and PamOct2
C-(VPGVG)4VPGKG (TLR2⁄ 1 ligand) showed similar
results (data not shown) Interestingly, after
micro-array analysis with LPS-stimulated BMDM strong
upregulation of RGS1 was found, but no modulation
(more than twofold) of other RGS mRNAs
(Table 1A) These finding led us to investigate the
modulation of RGS1 and RGS2 after stimulation with
LP and LPS in more detail
To confirm modulation of RGS1 and RGS2 mRNA
determined by microarray analysis, real-time PCR was
performed with BMDM as described in Materials and
methods To control stimulation of BMDM, the TNFa
release in the supernatant by ELISA was measured
(Fig 1B) Expression levels of RGS1 and RGS2 after
real-time PCR were referred to the housekeeping gene
HPRT After activation of BMDM with LPS or LP
(FSL-1 and Pam3C-SK4) there was an increase in
RGS1 mRNA at a very early period (15 min) of
stimu-lation After 1–2 h, the expression decreased, was found to be at control levels 12 h after stimulation (Fig 1A), and was further decreased at 24 h of culture For RGS2, no upregulation but rather a decrease in mRNA expression in BMDM after stimulation with LPS and LP could be detected, which was seen after 30–60 min of stimulation Expression of RGS2 mRNA was further reduced after stimulation with LP up to a culture period of 24 h Similar expression of RGS1 and RGS2 mRNA was also found after stimulation of the macrophages cell line J774 with LPS and LP at 2 and
14 h (Fig 2) The control of this stimulation is given
by the relative expression of TNFa (Fig 2) Cytokine mRNA expression can be increased by LP and LPS Thus, these results demonstrate that both BMDM and J774 cells express RGS1 and RGS2 mRNA and modu-late expression after stimulation with LP and LPS
Expression patterns of RGS1 and RGS2 after activation of TLR3 and TLR9
To investigate the expression levels of RGS1 and RGS2 after activation of other TLR, BMDM were stimulated with ODN1826 for 0–24 h to activate TLR9 signalling After real-time PCR we found a slight increase in RGS1 mRNA ( 2.6-fold) 30 min after stimulation After 12–24 h expression decreased
Table 1 Gene regulation of different RGS in BMDM Macrophages were stimulated with 10 ngÆmL)1LPS (A) or 100 n M FSL-1 (B) and mRNA was determined by microarray analysis The results are expressed as relative fluorescence and fold induction compared with control.
Gene
Control
relative
fluorescence
LPS relative fluorescence
Control relative fluorescence
FSL-1 relative fluorescence (fold)
Control relative fluorescence
FSL-1 relative fluorescence (fold)
a
RGS mRNA not expressed in BMDM.
Trang 4and was found at control level (Fig 3), similar to the
modulation after activation of TLR2 and TLR4 by LP
and LPS In contrast to RGS1, RGS2 showed only a
decrease in mRNA expression in BMDM after
stimu-lation with ODN1826
We measured mRNA expression in BMDM
trea-ted with poly(I:C) to activate TLR3 signalling in a
kinetic manner Strong upregulation of RGS1 mRNA was found only after 12 and 24 h (Fig 3) Treatment with poly(I:C) increased the mRNA level
of RGS1 150-fold compared with the control after
24 h Surprisingly, in contrast to the other TLR ligands, we detected an upregulation for RGS2 mRNA (approximately fivefold changes) after 12 and
FSL-1
Ctr 0.25 0.5
1 2 4 6
12 24
0
5000
10 000
15 000
20 000
25 000
Pam3C-SK4
0
5000
10 000
15 000
20 000
25 000
30 000
LPS
0
500
1000
1500
2000
2500
3000
3500
(h)
2
4
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10
A
B
RGS1
Relative expression of RGS2 mRNA 0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
RGS2
Fig 1 Modulation of RGS1 and RGS2 mRNA Expression was measured after stimulation of BMDM with 100 ngÆmL)1LPS, 100 n M FSL-1 and 100 n M Pam 3 C-SK 4 for 0–24 h (A) Specific mRNA expression was determined by real-time PCR The release of TNFa into the culture supernatants was determined by ELISA (B) For real-time PCR, similar data were obtained in three independent experiments Data for ELISA are the mean ± SE from two experiments.
Trang 524 h Therefore poly(I:C) was a strong stimulator of
RGS1 mRNA production, and of RGS2 mRNA,
suggesting that regulation of RGS1 and RGS2 after
stimulation with poly(I:C) is due to the
TRIF-depen-dent pathway
Poly(I:C) induced upregulation of RGS1 and RGS2
mRNA expression via a TRIF-dependent pathway
To further analyse the regulation of RGS1 and RGS2
mRNA after activation of TLR3 signalling we
mea-sured mRNA expression in wild-type and TRIF) ⁄ )
BMDM after stimulation with poly(I:C) Fig 4 shows
that poly(I:C) induced a 180-fold increase in RGS1
mRNA in cells from wild-type mice A slight increase
in expression occurred as early as 0.5 h and reached a peak after 24 h (Fig 4) As shown in Fig 3, there was also strong upregulation of RGS2 mRNA after stimu-lation with poly(I:C) We detected a 17-fold increase in RNA expression after 24 h As expected in BMDM of TRIF) ⁄ ) mice, we found no regulation of RGS1 and RGS2 mRNA, indicating that poly(I:C) can only acti-vate genes via a TRIF-dependent pathway Looking at downstream signalling events after stimulation, the involvement of different MAP kinases was determined Use of PD98059, an inhibitor of Erk, or SB203580, an inhibitor of p38, had no effect on RGS1 or RGS2 reg-ulation after 0.5 and 6 h of stimreg-ulation with a TLR2, TLR3 or TLR4 ligand (data not shown) Also, the inhibition of the Gai subunit by pertussis toxin [24]
100 n M FSL-1
Relative expression of RGS1 mRNA 0.0
0.2 0.4 0.6 0.8 1.0
1.2
100 n M Pam 3 C-SK4 RGS1
100 ng·mL –1 LPS
(h)
100 n M FSL-1
Relative expression of RGS2 mRNA 0.0
0.2 0.4 0.6 0.8 1.0
1.2
100 n M Pam 3 C-SK4
RGS2
100 ng·mL –1 LPS
(h)
100 n M FSL-1
0 2 14
0
2
4
6
8
100 n M Pam 3 C-SK4
TNF-α
100 ng·mL –1 LPS
(h)
Fig 2 Expression of RGS1, RGS2 and
TNFa mRNA in J774 After stimulation with
100 ngÆmL)1LPS, 100 n M FSL-1 and
100 n M Pam 3 C-SK 4 for 0–14 h, specific
mRNA expression was determined by
real-time PCR Similar data were obtained in
three independent experiments.
Trang 6has no influence on RGS1 modulation after
stimula-tion with LPS and FSL-1
Involvement of TRIF in the upregulation of RGS1
and RGS2 mRNA
The findings obtained from activation of TLR3 by
stimulation with poly(I:C) indicate a different
modula-tion of RGS1 and RGS2 mRNA by the MyD88- or
TRIF-dependent signalling pathway To confirm this
we stimulated wild-type, TRIF) ⁄ ) and MyD88) ⁄ )
BMDM with LP, which induce only the
MyD88-dependent signalling pathway, or with LPS, which
induced the MyD88- and TRIF-dependent signalling
pathways Kinetic studies showed that RGS1 mRNA
was found to be first upregulated and then
downregu-lated to the same degree after stimulation of wild-type
and TRIF) ⁄ ) mice with LP, indicating that the TRIF
signalling pathway is not involved The same kinetics
of RGS1 modulation was found after stimulation of
the cells with LPS in the absence of the TRIF path-way, indicating that LP and LPS regulate RGS1 in the same manner via the MyD88 pathway In the absence
of the MyD88-dependent signalling pathway in cells of MyD88) ⁄ ) mice, there is no modulation of RGS1 mRNA expression after stimulation with LP but a strong upregulation after stimulation of the cells with LPS This indicates that activation of the TRIF path-way resulted in a different modulation of RGS1 mRNA than after activation of the MyD88 pathway following stimulation with LPS This differential response of the BMDM resulted in prolonged upregu-lation of RGS1 mRNA after stimuupregu-lation with LPS, depending on whether the MyD88- or TRIF pathway was activated
Downregulation of RGS2 mRNA by FSL-1 was seen only in wild-type and TRIF) ⁄ ) BMDM, whereas
in MyD88) ⁄ ) BMDM no modulation of RGS2 was found By contrast, LPS downregulates RGS2 mRNA expression in wild-type and TRIF) ⁄ )cells but strongly
Poly(I:C)
RGS1
Ctr 0.5 12 24
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40
60
80
100
120
140
160
Poly(I:C)
RGS2
Ctr 0.5 12 24 Rel expression of mRNA 0
1
2
3
4
5
6
(h) (h)
ODN
RGS1
Ctr 0.5 12 24
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ODN
RGS2
Ctr 0.5 12 24 Rel expression of mRNA 0.0
0.2 0.4 0.6 0.8 1.0 1.2
(h) (h)
Fig 3 Verification of RGS1 and RGS2 mRNA after activation of TLR9 and TLR3 BMDM were stimulated with 1 l M
ODN1826 or 50 lgÆmL)1poly(I:C) RNA level was detected by real-time PCR Data were representative for three independent experiments.
Poly (I:C)
Ctr 0.5 3 6 12 24 Rel expression of mRNA 0
2
4
6
8
10
12
14
16
18
Wild-type Trif –/–
Poly (I:C)
Ctr 0.5 3 6 12 24
0
50
100
150
200
Wild-type
Trif –/–
Fig 4 Modulation of RGS1 and RGS2 mRNA in wild-type or TRIF) ⁄ )BMDM after stimulation with poly(I:C) Specific mRNA expression was determined by real-time PCR Similar data were obtained in three independent experiments.
Trang 7upregulates this RGS mRNA in MyD88) ⁄ )
macro-phages This indicates a different modulation of RGS
mRNA via the MyD88 and TRIF pathways
Discussion
RGS1 and RGS2 are proven to be the main RGS
mRNA modulated in murine macrophages after
stimu-lation with LP, LPS, poly(I:C) and ODN1826
Micro-array analysis identified RGS2 mRNA as the most
downregulated gene after 6 h of stimulation (Table 1),
whereas interleukin-6 was the strongest upregulated
gene within 45 101 probe sets [25] These findings
sug-gest that RGS2 plays an important role in the
biolo-gical consequences after activation of TLR by different
ligands However, little is known about the
involve-ment of RGS2 proteins in the context of inflammation
Under all RGS proteins, RGS2 contains a unique
function, because it is the only RGS protein that does
not interact with Gaisubunits, but selectively regulates
the function of Gaq [23] These findings are supported
by unique structural features of its G-protein-binding
interface [26] RGS2 inhibits Gaq-induced activation of
phospholipase C in cell membranes [23] After
downre-gulation of RGS2 the Gaq subunit stays active, with
the consequence that phospholipase C can cleave
phos-phatidylinositol 4,5-bisphoshate into two second
mes-sengers, inositoltriphosphate and diacylglycerol [27]
These secondary messengers can themselves mediate,
for example, Ca2+ flux and activate protein kinase C
[28] This activation leads then to further downstream
effects like changes in gene transcription or
morpho-logical and cytoskeletal changes Another function of
RGS2 proteins is to bind directly to certain subtypes
of adenylyl cyclases [29] This interaction between the
cyclases and RGS2 leads to an inhibition of the cAMP
production [20] After downregulation of RGS2 it is
likely that the inhibition of the adenylyl cyclase is
com-pensated Taken together, downregulation of RGS2
mRNA prohibits the deactivation of phospholipase C
and adenylyl cyclases, followed by different signal
cascades to counteract against microbial lipids
It is interesting to see the strong upregulation of
RGS1 mRNA between 30 and 60 min after stimulation
with LPS and also with LP (Fig 1) and ODN1826
(Fig 3) in BMDM Similar results were obtained in
J774 cells (Fig 2) The fast kinetics of RGS regulation
indicates a primary effect due to the TLR activation
and not a secondary effect due to G-protein signalling
Using microarray analysis we found a relevant
modu-lation of RGS1 after 3 h of stimumodu-lation with LPS
(Table 1) The gene was upregulated eightfold and
rep-resents the only upregulated RGS gene tested using
this microarray approach Confirming the data by real-time PCR, strong upregulation of RGS1 mRNA after 30 min of stimulation was observed However, the real-time PCR assay does not show strong regula-tion at 2 or 4 h of stimularegula-tion in several experiments This effect may be due to the peculiarity of this single gene array experiment indicating that such an experi-ment should be confirmed by real-time PCR Never-theless such early RGS1 modulation is likely to participate in appropriate cellular responses like RGS2 Comparable results were found in dendritic cells after stimulation with LPS RGS16, a RGS pro-tein similar to RGS1 and RGS2, was strongly upregu-lated [30] and the regulation of different RGS proteins
in murine macrophages are discussed, but no function
is known to date [31] RGS1 proteins stimulate the intrinsic GTPase activity of Gai subunits These subunits are responsible for the activation of different ion channels, several phospholipases and for the inhi-bition of the cAMP production Upregulation of RGS1 accelerates the GTP hydrolyse of the Gai subunits and thereby inhibits the Gai subunit signal-ling, which presumably results in compensation of the inhibition of the adenylyl cyclases and Ca2+ channels
as well as the activation of K+ channels or phospho-diesterases [32] Upregulation of RGS1 leads to a higher cAMP level and this second messenger activates protein kinase A Protein kinase A phosphorylation leads to an increased expression of cyclo-oxygenase-2, also known as prostaglandin synthase-2 in HEC-1B cells [33] We found also strong upregulation of Cox-2
in BMDM after stimulation with different lipopeptides
in our microarray analysis [25] That means that upregulation of RGS1 mRNA may lead to modulation
of cyclo-oxygenase-2 transcription involved in inflam-mation [34]
Another surprising point was the strong upregula-tion of RGS1 and RGS2 mRNA after activaupregula-tion of the TLR3 signalling pathway with poly(I:C) (Fig 4) Upregulation in this dimension ( 180-fold of RGS1 mRNA) has an enormous effect in BMDM, because the modulation of RGS1 and therefore the regulation
of different proteins is enforced by RGS2 mRNA after
12 h of stimulation The upregulation of both RGS mRNA was found only after activation of the TLR3 signalling pathway This upregulation of RGS1 and RGS2 mRNA is due to the TRIF pathway We veri-fied the data by stimulation experiments with LPS in wild-type versus TRIF) ⁄ ) and MyD88) ⁄ ) BMDM (Fig 5) It is known that LPS can signal via TLR4 in
a MyD88- and TRIF-dependent manner Stimulation
of MyD88) ⁄ ) mice with LPS activates the TRIF-dependent pathway The effect on RGS modulation
Trang 8resembles the results we obtained after poly(I:C)
stimu-lation, thus proving the responsibility of the TRIF
activation for the upregulation of both RGS mRNA
Stimulation of TLR3 and activation of the TRIF
path-way leads to interferon-b production [35] Takaoka
et al [36] demonstrated that interferon-b can induce
the transcription of p53 and this is critical for an
antiviral defence of the host In addition, T cells with
a lack of RGS2 impair antiviral immunity [37] In
con-clusion, after activation of TLR3 by poly(I:C) RGS2 is
necessary for an adequate antiviral immune response
After stimulation of distinct TLR pathways different
MAP kinases and several transcription factors like
Nf-jB are activated and the induction of
proinflamma-tory cytokines are found [38] The participation of these
signal transduction molecules in RGS1 and RGS2
modulation is not obvious, because usage of different
inhibitors (PD98059 an inhibitor of Erk, SB203580 an
inhibitor of p38) had no influence on modulating
RGS1 and RGS2 mRNA This indicates that
modula-tion of both RGS transcripts is regulated by a pathway
independent of these two MAP kinases It is possible
that the modulation is due to the activation of JNK
Other MAP kinase inhibitors as well as G-protein
inhibitors should be investigated to find out the
partici-pating proteins in RGS1 and RGS2 modulation
In conclusion, our results show strong modulation
of RGS1 and RGS2 mRNA induced by different TLR
ligands After stimulation with bacterial LP, LPS and ODN we detected strong upregulation and afterwards downregulation of RGS1 and a decrease in RGS2 because of the MyD88-dependent pathway Stimula-tion with poly(I:C) only leads to upregulaStimula-tion of both RGS1 and RGS2 mRNA, as a result of the TRIF-dependent pathway, without involvement of MyD88 (Fig 6) We suggest that the inflammatory and the adjuvant activities of TLR-ligands are at least partially mediated through modulation of RGS1 and RGS2 The molecular mechanisms, leading to this modulation and the consequences of the modulation of RGS1 and RGS2 remain to be investigated
Materials and methods Reagents
Dulbecco’s modified Eagles medium, RPMI-1640, penicil-lin-streptomycin, l-glutamine, sodium pyruvate and Hepes buffer were obtained from Invitrogen (Karlsruhe, Germany) Fetal calf serum (Linaris, Wertheim-Bettingen, Germany) was heat-inactivated before use LPS from Salmonella enterica serovar Friedenau was a gift from
H Brade (Research Center Borstel, Germany) Poly(I:C) and ODN1826 was received from InvivoGen (San Diego,
CA, USA) Pertussis toxin, SB203580 and PD98059 were obtained from Calbiochem (San Diego, CA, USA) All
FSL- 1
Ctr 0.5 3 6 12 24
0
2
4
6
8
10
12
14
Wild-type MyD88 –/–
TRIF –/–
LPS
Ctr 0.5 3 6 12 24
Wild-type MyD88 –/–
TRIF –/–
FSL-1
Ctr 0.5 3 6 12 24
0
1
2
3
4
5
6
Wild-type
MyD88 –/–
TRIF –/–
LPS
Ctr 0.5 3 6 12 24
Wild-type MyD88 –/–
TRIF –/–
RGS1
RGS2
(h)
(h)
Fig 5 Modulation of RGS1 and RGS2 mRNA in wild-type, TRIF) ⁄ )and MyD88) ⁄ ) BMDM Expression after stimulation was measured by real-time PCR Data were obtained in three independent experiments.
Trang 9lipopeptides were synthesized and characterized by EMC
microcollections (Tuebingen, Germany)
Cell culture
J774 macrophages were cultured at 37C, 5% CO2in
Dul-becco’s modified Eagles medium supplemented with 10%
fetal calf serum and 100 UÆmL)1 penicillin–streptomycin
Bone marrow-derived macrophages of C57N BL⁄ 6 mice
were differentiated by incubation with macrophage
colony-stimulating factor as described elsewhere [39] All animal
experiments were approved by the Ministerium fu¨r Umwelt,
Naturschutz und Landwirtschaft, Schleswig-Holstein
(Germany)
For stimulation, 2.5· 105cells were seeded in 48-well cell
culture dishes for 2 h and stimulated with 100 ngÆmL)1
LPS, 100 nm LP, 50 lgÆmL)1poly(I:C) or 2 lm ODN1826
Affymetrix gene chip analysis
BMDM were stimulated with 100 nm LP for 2 and 6 h or
10 ngÆmL)1LPS for 3 h Control samples were treated only
with medium and gene chip analyses were performed for
each experiment Total RNA (3 lg) was processed and
hybridized to mouse expression array MOE430 2.0
accord-ing to manufacturer’s protocol (Affymetrix, Santa Clara,
CA, USA) Arrays were scanned and fluorescence
intensi-ties were analyzed using affymetrix gcos software CEL
files were processed for global normalization and generation
of expression values using the robust multi-array analysis
algorithm implemented in the R-affy package (http://
www.bioconductor.org/) [40] Data from 11 oligis for each
probe set were statistically analysed by s-score test
ELISA After stimulation, cell-free supernatants were collected and assayed for TNFa measurement using commercial ELISA (Biosource, Solingen, Germany) according to the manufac-turer’s protocol
RNA isolation Total RNA was isolated using Absolutely RNA Miniprep kit (Stratagene, Amsterdam, the Netherlands), including DNase treatment, in accordance with the manufacture’s instructions The integrity of RNA was examined by gel electrophoresis before real-time PCR analysis
cDNA synthesis and real-time PCR First-strand cDNA were synthesized from 1 lg RNA by using SuperScript III reverse transcriptase (Invitrogen) Amplification was performed in a fluorescence temperature cycler (Light Cycler 2.0 system, Roche Diagnostics, Mann-heim, Germany) cDNA (20 ng) was used as template in a
10 lL reaction volume containing 0.5 lm of each primer, 1· LightCyclerFast Start DNA MasterPlusSYBR Green I mix (Roche Diagnostics) The following primers were used: muRGS1 TCTGCTAGCCCAAAGGATTC-3¢ (sense), 5¢-TTCACGTCCATTCCAAAAGTC-3¢ (anti-sense); muRGS2 5¢-GAGAAAATGAAGCGGACACTCT-3¢ (sense), 5¢-TTG CCAGTTTTGGGCTTC-3¢ (antisense); muHPRT as house-keeping gene 5¢-ACTTTGCTTTCCCTGGTTA-3¢ (sense), 5¢-CAAAGTCTGGCCTGTATCC-3¢ (antisense); muTNF-a 5¢-GACCCTCACACTCAGATCATCTTC-3¢ (sense), 5¢-CC ACTTGGTTTGCTACGA-3¢ (antisense)
Acknowledgements
We appreciate the excellent technical assistance of Suhad Al-Badri and Franziska Daduna We thank Roland Lang and Jo¨rg Mages (Technical University Munich, Institute of Medical Microbiology) for micro-array analysis This work was supported by the Deutsche Forschungsgemeinschaft UL68⁄ 3-2
References
1 Janeway CA & Medzhitov R (2002) Innate immune recognition Annu Rev Immunol 20, 197–216
2 Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, Carroll JD, Espevik T, Ingalls RR, Radolf JD et al (1999) Toll-like receptor 2 functions as
a pattern recognition receptor for diverse bacterial products J Biol Chem 274, 33419–33425
3 Shi GX, Harrison K, Han SB, Moratz C & Kehrl JH (2004) Toll-like receptor signaling alters the expression
Late
RGS1 RGS2 RGS1 RGS2 RGS1
Response
Early
RGS2
CpG-DNA Pam3C-SK4
Fig 6 Schematic of modulation of RGS1 and RGS2 mRNA due to
the MyD88 and ⁄ or TRIF pathway after activation of different TLR.
Trang 10of regulator of G protein signaling proteins in dendritic
cells: implications for G protein-coupled receptor
signal-ing J Immunol 172, 5175–5184
4 Hoebe K, Janssen E & Beutler B (2004) The interface
between innate and adaptive immunity Nat Immunol 5,
971–974
5 Yamamoto M, Sato S, Hemmi H, Hoshino K, Kaisho
T, Sanjo H, Takeuchi O, Sugiyama M, Okabe M,
Takeda K et al (2003) Role of adaptor TRIF in the
MyD88-independent toll-like receptor signaling
pathway Science 301, 640–643
6 Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S,
Sanjo H, Matsumoto M, Hoshino K, Wagner H,
Takeda K et al (2000) A Toll-like receptor recognizes
bacterial DNA Nature 408, 740–745
7 Latz E, Verma A, Visintin A, Gong M, Sirois CM,
Klein DC, Monks BG, McKnight CJ, Lamphier MS,
Duprex WP et al (2007) Ligand-induced
conforma-tional changes allosterically activate Toll-like receptor 9
Nat Immunol 8, 772–779
8 Buwitt-Beckmann U, Heine H, Wiesmu¨ller KH, Jung
G, Brock R & Ulmer AJ (2005) Lipopeptide structure
determines TLR2 dependent cell activation level FEBS
J 272, 6354–6364
9 Buwitt-Beckmann U, Heine H, Wiesmu¨ller KH, Jung
G, Brock R, Akira S & Ulmer AJ (2006) TLR1- and
TLR6-independent recognition of bacterial lipopeptides
J Biol Chem 281, 9049–9057
10 Fan H, Williams DL, Zingarelli B, Breuel KF, Teti G,
Tempel GE, Spicher K, Boulay G, Birnbaumer L,
Halushka PV et al (2007) Differential regulation of
lipopolysaccharide and Gram-positive bacteria induced
cytokine and chemokine production in macrophages by
Galpha(i) proteins Immunology 122, 116–123
11 Fan H, Peck OM, Tempel GE, Halushka PV &
Cook JA (2004) Toll-like receptor 4 coupled GI
pro-tein signaling pathways regulate extracellular
signal-regulated kinase phosphorylation and AP-1 activation
independent of NFkappaB activation Shock 22,
57–62
12 Krauss G (2006) G Protein-Coupled Signal Transmission
Pathways Biochemistry of Signal Transduction and
Reg-ulation Wiley-VCH, Weinheim
13 Dohlman HG & Thorner J (1997) RGS proteins and
signaling by heterotrimeric G proteins J Biol Chem
272, 3871–3874
14 Gilman AG (1987) G proteins: transducers of
receptor-generated signals Annu Rev Biochem 56, 615–649
15 Koelle MR (1997) A new family of G-protein regulators
– the RGS proteins Curr Opin Cell Biol 9, 143–147
16 Neer EJ (1997) Intracellular signalling: turning down
G-protein signals Curr Biol 7, R31–R33
17 Heximer SP, Cristillo AD & Forsdyke DR (1997)
Comparison of mRNA expression of two regulators of
G-protein signaling, RGS1⁄ BL34 ⁄ 1R20 and
RGS2⁄ G0S8, in cultured human blood mononuclear cells DNA Cell Biol 16, 589–598
18 De Vries L, Zheng B, Fischer T, Elenko E & Farquhar
MG (2000) The regulator of G protein signaling family Annu Rev Pharmacol Toxicol 40, 235–271
19 Sato M, Blumer JB, Simon V & Lanier SM (2006) Accessory proteins for G proteins: partners in signaling Annu Rev Pharmacol Toxicol 46, 151–187
20 Hollinger S & Hepler JR (2002) Cellular regulation of RGS proteins: modulators and integrators of G protein signaling Pharmacol Rev 54, 527–559
21 Watson N, Linder ME, Druey KM, Kehrl JH & Blu-mer KJ (1996) RGS family members: GTPase-activating proteins for heterotrimeric G-protein alpha-subunits Nature 383, 172–175
22 Zerangue N & Jan LY (1998) G-protein signaling: fine-tuning signaling kinetics Curr Biol 8, R313–R316
23 Heximer SP, Watson N, Linder ME, Blumer KJ & Hepler JR (1997) RGS2⁄ G0S8 is a selective inhibitor
of Gqalpha function Proc Natl Acad Sci USA 94, 14389–14393
24 Denecke B, Meyerdierks A & Bottger EC (1999) RGS1
is expressed in monocytes and acts as a GTPase-activat-ing protein for G-protein-coupled chemoattractant receptors J Biol Chem 274, 26860–26868
25 Farhat K, Riekenberg S, Heine H, Debarry J, Lang R, Mages J, Buwitt-Beckmann U, Ro¨schmann K, Jung G, Wiesmu¨ller KH et al (2008) Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling
J Leukoc Biol 83, 692–701
26 Heximer SP, Srinivasa SP, Bernstein LS, Bernard JL, Linder ME, Hepler JR & Blumer KJ (1999) G protein selectivity is a determinant of RGS2 function J Biol Chem 274, 34253–34259
27 Cunningham ML, Waldo GL, Hollinger S, Hepler JR
& Harden TK (2001) Protein kinase C phosphorylates RGS2 and modulates its capacity for negative regula-tion of Galpha 11 signaling J Biol Chem 276, 5438– 5444
28 Rey O, Reeve JR Jr, Zhukova E, Sinnett-Smith J & Rozengurt E (2004) G protein-coupled receptor-mediated phosphorylation of the activation loop of protein kinase D: dependence on plasma membrane translocation and protein kinase Cepsilon J Biol Chem
279, 34361–34372
29 Roy AA, Lemberg KE & Chidiac P (2003) Recruitment
of RGS2 and RGS4 to the plasma membrane by G pro-teins and receptors reflects functional interactions Mol Pharmacol 64, 587–593
30 Perrier P, Martinez FO, Locati M, Bianchi G, Nebuloni
M, Vago G, Bazzoni F, Sozzani S, Allavena P & Mantovani A (2004) Distinct transcriptional programs activated by interleukin-10 with or without lipopolysac-charide in dendritic cells: induction of the B