Tài liệu Báo cáo khoa học: Regulators of G-protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide pptx

Regulators of G-protein signalling are modulated by
bacterial lipopeptides and lipopolysaccharide
Sabine Riekenberg
1
, Katja Farhat
1
, Jennifer Debarry
2
, Holger Heine
2
,Gu
¨
nther Jung
3
, Karl-Heinz
Wiesmu
¨
ller
4
and Artur J. Ulmer
1
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:
(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 G
a
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.
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 649

to 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 Pam
3
C-SK
4
and 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 Ga
i
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
G
a
,5G
b
and 6 G
c
isoforms [13]. Furthermore the
a subunits are divided into four main categories: Ga
i
,
Ga
s
,Ga
q
and Ga
12 ⁄ 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
Aspergillus and 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 Ga
i
and Ga
q
subunits as much as 100- to 300-fold [15,19].
They can also modulate the lifetime and kinetics of
slow-acting signalling responses like Ca
2+
oscillations
[20]. Different studies have shown that RGS1 stimu-

lates the GTPase activity of several members of the
Ga
i
subfamily but is ineffective against Ga
s
[21],
whereas RGS2 does not interact with Ga
i
,Ga
o
,Ga
s
and Ga
12 ⁄ 13
at all; RGS2 acts selectively as a GAP for
Ga
q
subunits [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 stimulation
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
RGS are modulated by lipopeptides and LPS S. Riekenberg et al.
650 FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS
(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 Pam

2
C-SK
4
(TLR2 ⁄ 1 and TLR2 ⁄ 6 ligand) and PamOct
2
C-
(VPGVG)
4
VPGKG (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 Pam
3
C-SK
4
) 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
)1
LPS (A) or 100 nM FSL-1 (B) and
mRNA was determined by microarray analysis. The results are expressed as relative fluorescence and fold induction compared with control.
A Stimulation 3 h B Stimulation 2 h Stimulation 6 h

Gene
Control
relative
fluorescence
LPS relative
fluorescence
(fold) Gene
Control relative
fluorescence
FSL-1 relative
fluorescence
(fold)
Control
relative
fluorescence
FSL-1 relative
fluorescence
(fold)
RGS1 168 1366 (8.13) RGS1 492 420 ()1.2) 574 190 ()3.0)
RGS2 1532 773 ()2.0) RGS2 1872 149 ()12.6) 1540 22 ()70.0)
RGS3 153 149 ()1.0) RGS3 172 152 ()1.1) 206 188 ()1.1)
RGS4
a
8 8 (1.0) RGS4
a
13 11 ()1.2) 14 14 (1.0)
RGS5 6 6 (1.0) RGS5
a
8 8 (1.0) 7 9 (1.3)
RGS6

a
87()1.0) RGS6
a
87()1.1) 9 9 (1.0)
RGS7
a
8 8 (1.0) RGS7
a
11 9 ()1.2) 9 8 ()1.1)
RGS9
a
63 68 (1.1) RGS8
a
10 9 ()1.1) 10 9 ()1.1)
RGS10 2161 1485 ()1.5) RGS9 105 107 (1.0) 108 114 (1.1)
RGS11
a
20 15 ()1.4) RGS10 1535 1191 ()1.3) 1790 605 ()3.0)
RGS14 247 323 (1.3) RGS11 14 17 (1.2) 23 27 (1.2)
RGS16 10 16 (1.3) RGS12 39 33 ()1.2) 25 19 ()1.3)
RGS17
a
6 7 (1.1) RGS13
a
5 6 (1.2) 5 5 (1.0)
RGS18 407 249 ()1.9) RGS14 224 158 ()1.4) 311 240 ()1.3)
RGS19 382 212 ()1.8) RGS16 26 53 (2.0) 20 23 (1.2)
RGS20
a
76()1.0) RGS18 292 176 ()1.7) 742 316 ()2.3)

RGS19 691 412 ()1.7) 990 832 ()1.2)
RGS20
a
8 9 (1.1) 10 9 ()1.1)
a
RGS mRNA not expressed in BMDM.
S. Riekenberg et al. RGS are modulated by lipopeptides and LPS
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 651
and 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
TNF-α
Ctr.
0.25
0.5
1
2
4

6
12
24
TNF-α (pg·mL
–1
)
0
5000
10 000
15 000
20 000
25 000
Pam
3
C-SK
4
Ctr.
0.25
0.5
1
2
4
6
12
24
0
5000
10 000
15 000
20 000

25 000
30 000
LPS
Ctr.
0.25
0.5
1
2
4
6
12
24
0
500
1000
1500
2000
2500
3000
3500
(h)
100 nM FSL-1
0
0.25
0.5
1
2
4
6
12

24
Relative expression of RGS1 mRNA
0
2
4
6
8
10
A

B
100 nM Pam
3
C-SK
4
RGS1
0
0.25
0.5
1
2
4
6
12
24
100 ng·mL
–1
LPS
0
0.25

0.5
1
2
4
6
12
24
(h)
100 nM FSL-1
0
0.25
0.5
1
2
4
6
12
24
Relative expression of RGS2 mRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
100 nM Pam
3
C-SK

4
RGS2
0
0.25
0.5
1
2
4
6
12
24
100 ng·mL
–1
LPS
0
0.25
0.5
1
2
4
6
12
24
(h)
Fig. 1. Modulation of RGS1 and RGS2 mRNA. Expression was measured after stimulation of BMDM with 100 ngÆmL
)1
LPS, 100 nM 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.
RGS are modulated by lipopeptides and LPS S. Riekenberg et al.
652 FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS
24 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 stimulation with a TLR2,
TLR3 or TLR4 ligand (data not shown). Also, the
inhibition of the Ga
i
subunit by pertussis toxin [24]
100 nM FSL-1
Relative expression of RGS1 mRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
100 nM Pam
3
C-SK
4
R
GS
1
100 ng·mL
–1
LPS
(h)

100 nM FSL-1
Relative expression of RGS2 mRNA
0.0
0.2
0.4
0.6
0.8
1.0
1.2
100 nM Pam
3
C-SK
4
RGS2
100 ng·mL
–1
LPS
(h)
100 nM FSL-1
0 2 14
0 2 14 0 2 14 0 2 14
0 2 14 0 2 14 0 2 14
0 2 14 0 2 14
Relative expression of TNF-α
α
mRNA
0
2
4
6

8
100 nM Pam
3
C-SK
4
TNF-α
100 ng·mL
–1
LPS
(
h
)

Fig. 2. Expression of RGS1, RGS2 and
TNFa mRNA in J774. After stimulation with
100 ngÆmL
)1
LPS, 100 nM 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.
S. Riekenberg et al. RGS are modulated by lipopeptides and LPS
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 653
has 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 stimulation 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
Rel. express
i
on o
f
mRNA
0
20

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
R
GS
1

Ctr. 0.5 12 24
Rel. express
i
on o
f
mRNA
0.0
0.5
1.0
1.5
2.0
2.5
3.0
ODN
R
GS
2
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
)1
poly(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
Rel. express
i
on o
f
mRNA

0
50
100
150
200
Wild-type
Trif
–/–
RGS1
RGS2
(
h
)

(
h
)

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.
RGS are modulated by lipopeptides and LPS S. Riekenberg et al.
654 FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS
upregulates 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 Ga
i
subunits, but selectively regulates
the function of Ga
q
[23]. These findings are supported
by unique structural features of its G-protein-binding
interface [26]. RGS2 inhibits Ga
q
-induced activation of
phospholipase C in cell membranes [23]. After downre-
gulation of RGS2 the Ga
q

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, Ca
2+
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 stimulation 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 stimulation 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 Ga
i
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 Ga
i
subunits and thereby inhibits the Ga
i
subunit signal-
ling, which presumably results in compensation of the
inhibition of the adenylyl cyclases and Ca

2+
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 activation 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
S. Riekenberg et al. RGS are modulated by lipopeptides and LPS
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 655
resembles 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 upregulation 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
Rel. expresss
i
on o
f
mRNA
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
R
el. expresss
i
on o
f
mRNA
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.
RGS are modulated by lipopeptides and LPS S. Riekenberg et al.
656 FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS
lipopeptides were synthesized and characterized by EMC
microcollections (Tuebingen, Germany).
Cell culture
J774 macrophages were cultured at 37 °C, 5% CO
2
in 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 · 10
5
cells were seeded in 48-well cell
culture dishes for 2 h and stimulated with 100 ngÆmL
)1
LPS, 100 nm LP, 50 lgÆmL
)1
poly(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
)1
LPS 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·
LightCycler
Ò
Fast Start DNA Master
Plus
SYBR Green I mix
(Roche Diagnostics). The following primers were used:
muRGS1 5¢-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
RGS1RGS2RGS1RGS2RGS1Response
Early
RGS2
CpG-DNA
LPS
Poly(I:C)
TLR4
TLR3
CpG-DNA
Pam
3

C-SK
4
TLR9 TLR2/X
MyD88 TRIF
Fig. 6. Schematic of modulation of RGS1 and RGS2 mRNA due to
the MyD88 and ⁄ or TRIF pathway after activation of different TLR.
S. Riekenberg et al. RGS are modulated by lipopeptides and LPS
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 657
of 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 cell-activat-
RGS are modulated by lipopeptides and LPS S. Riekenberg et al.
658 FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS
ing chemokine, CXC chemokine ligand 13. J Immunol
172, 7031–7042.
31 Lattin J, Zidar DA, Schroder K, Kellie S, Hume DA &
Sweet MJ (2007) G-protein-coupled receptor expression,
function, and signaling in macrophages. J Leukoc Biol
82, 16–32.
32 Xu X, Zeng W, Popov S, Berman DM, Davignon I, Yu
K, Yowe D, Offermanns S, Muallem S & Wilkie TM
(1999) RGS proteins determine signaling specificity of
Gq-coupled receptors. J Biol Chem 274, 3549–3556.
33 Munir I, Fukunaga K, Miyazaki K, Okamura H
& Miyamoto E (1999) Mitogen-activated protein kinase
activation and regulation of cyclooxygenase 2
expression by platelet-activating factor and hCG in
human endometrial adenocarcinoma cell line HEC-1B.
J Reprod Fertil 117, 49–59.
34 Kang YJ, Wingerd BA, Arakawa T & Smith WL (2006)
Cyclooxygenase-2 gene transcription in a macrophage
model of inflammation. J Immunol 177, 8111–8122.
35 Heximer SP, Knutsen RH, Sun X, Kaltenbronn KM,
Rhee MH, Peng N, Oliveira-dos-Santos A, Penninger
JM, Muslin AJ, Steinberg TH et al. (2003) Hyperten-

sion and prolonged vasoconstrictor signaling in RGS2-
deficient mice. J Clin Invest 111, 445–452.
36 Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi
H, Kikuchi H, Sasaki S, Imai K, Shibue T, Honda K
et al. (2003) Integration of interferon-alpha ⁄ beta
signalling to p53 responses in tumour suppression and
antiviral defence. Nature 424, 516–523.
37 Kehrl JH & Sinnarajah S (2002) RGS2: a multifunc-
tional regulator of G-protein signaling. Int J Biochem
Cell Biol 34, 432–438.
38 Muzio M, Polentarutti N, Bosisio D, Manoj Kumar PP
& Mantovani A (2000) Toll-like receptor family and
signalling pathway. Biochem Soc Trans 28, 563–566.
39 Metzger J, Wiesmu
¨
ller KH, Schaude R, Bessler WG &
Jung G (1991) Synthesis of novel immunologically
active tripalmitoyl-S-glycerylcysteinyl lipopeptides as
useful intermediates for immunogen preparations. Int J
Pept Protein Res 37, 46–57.
40 Bolstad BM, Collin F, Simpson KM, Irizarry RA &
Speed TP (2004) Experimental design and low-level
analysis of microarray data. Int Rev Neurobiol 60,
25–58.
S. Riekenberg et al. RGS are modulated by lipopeptides and LPS
FEBS Journal 276 (2009) 649–659 ª 2008 Research Center Borstel. Journal compilation ª 2008 FEBS 659