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

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

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,

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

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-

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

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

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

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

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-

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

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

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

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

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

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

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

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

(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
¨

(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

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
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