RhoA GTPase Activation by TLR2 and TLR3 Ligands:
Connecting via Src to NF-?B1
Maria Manukyan,* Perihan Nalbant,†Sylvia Luxen,* Klaus M. Hahn,‡and Ulla G. Knaus2*
Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive
immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as
NF-?B, C/EBP, and serum response factor. The multifaceted host cell activation triggered by TLRs in response to soluble and
particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and
monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA
activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered re-
cruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated
stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-?B activation, whereas RhoA was
dispensable for type I IFN generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and
-independent TLR signaling and acts as a molecular switch downstream of TLR-Src-initiated pathways. The Journal of Immu-
nology, 2009, 182: 3522–3529.
these pathogens. In the lung, the pulmonary innate immune system
serves as the first line of defense against aerosolized pathogens. A
major component of this lung immune response is the airway ep-
ithelium, which provides not only the mechanical barrier and in-
terface between the environment and the host but also is uniquely
equipped for sensing and responding to inhaled bacterial and viral
organisms. Lung epithelial cells respond to many TLR ligands,
although the participation of TLR4 under noninflammatory con-
ditions seems to be limited due to the intracellular localization and
decreased LPS sensitivity of this receptor in airway epithelial cells
(1–4). In contrast, TLR2 represents an important functional recep-
tor for bacterial recognition at the lung epithelial cell surface. Key
bacterial lung pathogens such as Staphylococcus aureus, Strepto-
coccus pneumonia, and Pseudomonas aeruginosa initiate TLR2-
mediated signaling responses (2, 5). Recognition of viral lung
pathogens is accomplished by TLR3, located in vesicular endoso-
mal compartments, or by cytosolic receptors such as RIG-I and
MDA5 (6, 7). TLR2- and TLR3-initiated signaling differs in its use
of adapter molecules, where TLR2 connects to TIRAP/MyD88,
while dsRNA-stimulated TLR3 recruits TIR-domain-containing
adapter-inducing IFN-? (TRIF).3Further downstream, both TLR-
oll-like receptors recognize conserved microbial and viral
ligands and trigger signaling pathways required for
mounting a host immune response against infection by
adapter complexes cause activation of NF-?B-dependent gene tran-
scription and of MAPK/JNK/p38 pathways, although TLR3 mediates
additionally type I IFN generation. Thus, receptor-proximal events
differ between TLR2 and TLR3, connecting TLR-adapter complexes
to separate signaling cascades.
Certain signaling molecules have been tentatively connected to
MyD88-dependent and -independent TLR signaling. For example,
a PI3K-Akt pathway is triggered by several TLRs, independently
of their adapter usage or cellular localization (8–10). The associ-
ation between TLR2 and the p85 regulatory subunit of PI3K re-
quires the tyrosine-phosphorylated intracellular TIR domain of
TLR2 (8). Inducible TLR tyrosine phosphorylation has been linked
to activation of Src family kinases, which seem to be an integral
part of the TLR2 and TLR3 signaling complex (11–14). Another
component of TLR complexes is the tyrosine kinase Syk, which
relays signals downstream of integrin engagement and takes part in
ITAM-containing immunoreceptor signal transduction (9, 15, 16).
Syk activation seems to be the result of receptor cooperation, in
case of TLR2 and TLR4 with the ?-glucan receptor dectin-1 (17),
or with CD36 for TLR2/TLR6 (18, 19). Signaling events down-
stream of TLRs have also been connected to Rho family GTPases.
We and others (8, 20, 21) reported a rapid and transient increase in
Rac1 and RhoA activity when TLR2 and TLR4 stimulation by
soluble pathogen-associated molecular patterns occurred. Rho GT-
Pases are molecular switches that regulate essential cellular pro-
cesses including actin dynamics, gene transcription and motility.
The activity of Rho GTPases is controlled by guanine nucleotide
exchange factors (GEFs), which are commonly stimulated by ty-
rosine phosphorylation and/or phospholipid binding. In the context
of TLR signaling, the RacGEF Vav-1 is activated via a TLR9-Src
pathway, while the RhoGEF AKAP13 is involved in TLR2 re-
sponses (22, 23). Previous studies in TLR2-expressing HEK293
cells indicated that RhoA was not an integral part of the TLR2/
IL-1R-associated kinase/TNFR-associated factor 6 complex but
was required for PKC?-dependent NF-?B transactivation (20).
*Department of Immunology and Microbial Science, The Scripps Research Institute,
La Jolla, CA 92037;†Center for Medical Biotechnology, University of Duisburg-
Essen, Essen, Germany; and‡Department of Pharmacology, University of North
Carolina, Chapel Hill, NC 27599
Received for publication July 14, 2008. Accepted for publication January 9, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grants AI35947 and
GM37696 (to U.G.K.) and GM57464 (to K.M.H.).
2Address correspondence and reprint requests to Dr. Ulla G. Knaus, Department of
Immunology and Microbial Science, The Scripps Research Institute, 10550 North
Torrey Pines Road, IMM28, La Jolla, CA 92037. E-mail address: uknaus@scripps.
3Abbreviations used in this paper: TRIF, TIR-domain-containing adapter-inducing
IFN-?; EGF, epidermal growth factor; FRET, fluorescence resonance energy transfer;
GEF, guanine nucleotide exchange factor; MEF, murine embryonic fibroblast; SALE,
small airway lung epithelial; SI, Src kinase inhibitor I; siRNA, small interfering RNA.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
Since RhoA, Src, and Syk play a role in transmitting signals to
NF-?B in several cellular systems, we hypothesized that these pro-
teins could be connected in a TLR-proximal signaling cascade,
regulating NF-?B-dependent gene transcription. Thus, biochemi-
cal and spatiotemporal detection of active, GTP-bound RhoA in
pathogen-associated molecular pattern-stimulated lung epithelial
cells was conducted. RhoA activation was observed in distinct
cellular compartments and required Src activity. TLR2 and TLR3
stimulation triggered the Src-RhoA pathway to NF-?B-dependent
gene transcription, whereas RhoA was not involved in TLR3/Src-
initiated type I IFN generation.
Materials and Methods
Cells and reagents
The immortalized primary human lung epithelial cells (small airway lung
epithelial (SALE)) were a gift from Dr. W. C. Hahn (Harvard Medical
School, Boston, MA) and were described previously (24). SABM medium,
SAGM SingleQuots, and trypsin/EDTA used for SALE cells were pur-
chased from Lonza. L929-ISRE cell line (ISRE-luciferase reporter) was
provided by Dr. B. Beutler (The Scripps Research Institute, La Jolla, CA).
The synthetic lipopeptide MALP-2 was obtained from Alexis, Pam3CSK4
from InvivoGen, and poly(I:C) was purchased from Amersham Bio-
sciences and dissolved in pyrogen-free distilled water. dsRNA (a 34-bp
sequence from human rhinovirus 16) labeled with Alexa 633 at the 3?-end
was synthesized by Invitrogen. RhoA mAb (26C4), I?B? polyclonal Ab
(C-21), and TLR2 Ab (H-175) were from Santa Cruz Biotechnology; phos-
pho-IRF3 (Ser386) was from IBL; IRF3, phospho-IRF3 (Ser396), phospho-
Src (Tyr416), phospho-Syk (Tyr525–526), phospho-p38, p38, Fyn, Hck, Src,
Yes, and Lyn Abs were from Cell Signaling Technology; Myc (9E10) mAb
was from Covance Research Products; and GM130 mAb was from BD
Pharmingen. AKAP-13 Ab was from Bethyl Laboratories. Secondary
Ab labeled with Alexa 647 was purchased from Invitrogen. PP2, PP3,
Src kinase inhibitor I (SI), and piceatannol were obtained from
All plasmids were prepared using endotoxin-free plasmid DNA purifica-
tion (Qiagen). RhoA biosensor (RhoA-CFP-RBD-YFP) was described pre-
viously (25). An expression plasmid containing the RhoA biosensor, len-
tiviral packaging plasmids, and pVSV-G were provided by Dr. K. Wong
(University of California, San Francisco, CA (26)). Myc-RhoA wt and
Myc-RhoA T19N were described previously (20). The NF-?B-responsive
luciferase reporter (5? NF-?B-Luc) was from Promega. c-Src K295M was
provided by Dr. D. Schlaepfer (University of California, San Diego, CA).
RhoA biosensor expressing SALE
Cotransfection of HEK293T cells with RhoA biosensor, packaging plas-
mid, and VSV-G was performed by calcium phosphate method. After 2–3
days, virus-containing supernatants were collected and used for transduc-
tion of SALE cells. Cells were incubated with viral supernatant for 6–8 h,
and medium was then replaced with fresh SABM medium with SAGM
SingleQuots. The procedure was repeated 24 h later. RhoA biosensor-ex-
pressing SALE cells were positively selected by FACS (98% positive) for
low to medium expressers.
Transfection and reporter assays
Transfection of SALE cells was done using Lipofectamine Plus (Invitro-
gen). SALE cells were plated in 6-well plates at a density of 0.2 ? 106/
well. Twenty-four hours later, cells were transiently transfected with 100
ng of NF-?B luciferase reporter plasmid alone or cotransfected with RhoA
wt (200 ng), RhoA T19N (400 ng), c-Src K295M (400 ng), or empty
vector. Eighteen hours later (36 h for cotransfection), cells were starved for
3 h in SABM medium with 0.5% FBS. Stimulation with MALP-2 (10
ng/ml), Pam3CSK4 (100 ng/ml), or poly(I:C) (10–30 ?g/ml) was for 4 h.
Cells were lysed in luciferase assay buffer (Promega), and luminescence of
lysates was measured in triplicates for each independent assay (n ? 3). For
small interfering RNA (siRNA) treatment, Stealth Select Syk RNA inter-
ference (HSS110401, HSS110402, and HSS110403) (Invitrogen) was
tested using Dharmafect 4 (Dharmacon), and HSS110402 RNA interfer-
ence (20 nM) was used for Syk knockdown. Forty-eight hours later, 5?
NF-?B luc was transfected, followed 24 h later by starvation and cell
stimulation as described earlier.
IFN type I production
L929 cells stably expressing an ISRE luciferase reporter construct were
provided by Dr. B. Beutler. For measurements of IFN type I production,
L929-ISRE cells were plated in 96-well plate at a density 5 ? 104/well.
Twenty-four hours later, medium was removed before adding superna-
tants of SALE cells stimulated with poly(I:C) (100 ?l/well). After 4 h,
L292-ISRE cells were washed with PBS, lysed in luciferase assay buffer
(Promega), and luminescence was measured with a LB 960 Centro Mi-
croplate Luminometer in triplicates (n ? 3 independent experiments).
Rho activation assay
SALE cells were plated in 10-cm dishes (Fisher Scientific) at a concen-
tration of 0.8 ? 106/ml in SABM medium with supplements. Forty-eight
hours later, cells were starved in SABM/0.5% FBS overnight, followed by
RBD pull-down assay as described previously (20). Stimulation with
I?B? degradation and p38 phosphorylation are induced by MALP-2.
SALE cells were stimulated for indicated time points with MALP-2, and
lysates were analyzed by immunoblotting. Phospho-p38 and I?B? blots
were stripped and reprobed for p38 and actin as loading controls. One
representative experiment is shown (n ? 3). B, I?B? degradation and p38
phosphorylation are induced by poly(I:C). SALE cells were stimulated for
indicated time points with poly(I:C), and lysates were analyzed by immu-
noblotting. Phospho-p38 and I?B? blots were stripped and reprobed for
p38 and actin as loading controls. One representative experiment is shown
(n ? 3). C, IRF3 phosphorylation is induced by poly(I:C). SALE cells were
stimulated for indicated time points with poly(I:C), and lysates were ana-
lyzed by immunoblotting. Phospho-IRF3 (S396) and total IRF3 are shown
in upper two panels. Phospho-IRF3 (Ser386) was analyzed by native PAGE,
followed by immunoblotting (panel 3). Blots were stripped and probed for
total IRF3 as loading control (panel 4). One representative experiment is
shown (n ? 2).
SALE airway cells contain functional TLR2 and TLR3. A,
3523The Journal of Immunology
MALP-2 (10 ng/ml) or poly(I:C) (30 ?g/ml) was performed for the indi-
cated times. For some experiments, cells were preincubated with inhibitors
30 min before stimulation. Protein samples were analyzed by SDS-PAGE
and immunoblotting with anti-RhoA Ab.
For the detection of phospho-IRF3 (Ser386), the procedure was as described
previously (27). All other immunoblots were performed as described else-
where (28). Densitometry of immunoblots was performed by using the
GS-800 Calibrated Densitometer (BioRad) and Quantity One software
(version 4.5.2). Average density of the protein bands was normalized to the
corresponding bands of the loading controls.
SALE cells stably expressing Myc-RhoA were plated in 10-cm dishes
(Fisher Scientific) at a concentration of 1 ? 106/ml in SABM medium with
supplements. Forty-eight hours later, cells were starved in SABM/0.5%
FBS overnight. After stimulation with MALP-2 (10 ng/ml) for 10 min,
cells were washed with PBS and lysed (10 mM Tris-Cl, 100 mM NaCl, 1
mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na2P2O7, 1% Triton X-100,
10% glycerol, 0.1% SDS, 0.5% deoxycholate, 2 mM orthovanadate, 1 ?M
microcystein, 2 ?g/ml leupeptin, 2 ?g/ml aprotinin, 1 ?g/ml pepstatin, and
2 mM PMSF). Lysates were cleared by centrifugation and incubated with
AKAP13, TLR2, or rabbit IgG Abs for 1.5 h at 4°C. Incubation with
Sepharose G beads was performed for 45 min at 4°C. After four washes,
samples were resuspended in sample buffer, boiled for 5 min, and analyzed
by SDS-PAGE and immunoblotting.
Sample preparation for confocal microscopy
For fluorescence resonance energy transfer (FRET) experiments SALE/
RhoA cells (0.2 ? 105) were seeded on glass coverslips in 35-mm tissue
culture dishes (Fisher Scientific). Stimulation with MALP-2 (10 ng/ml) for
0–10 min or poly(I:C) (30 ?g/ml) for 0–30 min in SABM with 0.5% FBS
was performed after overnight starvation in the same media. When indi-
cated, SI (1 ?M) was added to cells 30 min before stimulation. Cells were
fixed with 4% paraformaldehyde for 15 min at room temperature and
mounted in Pro Long Gold mounting medium (Invitrogen). For colocal-
ization experiments, cells were incubated with dsRNA-Alexa 633 (3 ?M)
for 30 min and then fixed with 4% paraformaldehyde for 15 min at room
temperature and mounted in Pro Long Gold mounting medium (Invitro-
gen). An average of 50 cells was analyzed per condition at each time point.
cells. Upper panel shows RhoA-GTP, and lower panel shows total RhoA in lysates (10% of pull-down lysate). One representative experiment is shown
(n ? 3). B, RhoA biosensor activation by MALP-2 and poly(I:C). Representative FRET ratio images and RhoA-YFP images of SALE/RhoA cells
stimulated with MALP-2 for 5 and 10 min or poly(I:C) for 30 and 60 min (scale bar, 10 ?m). Quantification of whole-cell emission ratios is shown; number
of cells first panel: n (?) ? 59; n (MALP-2, 5 min) ? 78; second panel: n (?) ? 70; n (MALP-2, 10 min) ? 70; third panel: n (?) ? 29; n (poly(I:C)
30, min) ? 19; fourth panel: n (?) ? 29; n (poly(I:C), 60 min) ? 34; error bars represent SEs; values of p ? 0.001 (???), ? 0.01 (??), and ? 0.05 (?).
C, Colocalization of the RhoA biosensor with dsRNA. Representative FRET ratio images and RhoA-YFP images (upper panel) as well as dsRNA
colocalization with FRET and RhoA-YFP (lower panel) in SALE/RhoA cells stimulated with dsRNA/A633 (3 ?M) for 30 min. Colocalization is in white;
the colocalization coefficient for red to green was 0.83 (FRET) and 0.75 (Rho/YFP) analyzed by Image J (version 1.33). FRET ratio images in B and C
are scaled so that regions of highest RhoA activity are shown in red. Scale bar, 10 ?m.
TLR2- and TLR3-mediated activation of RhoA. A, Time-dependent activation of endogenous RhoA by MALP-2 and poly(I:C) in SALE
3524RhoA IN TLR SIGNALING
Confocal microscopy and image processing
Imaging was performed with a confocal microscope (2100 Radiance; Bio-
Rad) using a ?60 oil immersion objective. Intramolecular FRET, as pre-
viously described (29), was measured by exciting CFP with a 405-nm laser
line and using the sequential scan acquisition mode for the CFP and FRET
(YFP) channels; additionally, a YFP image was acquired by excitation with
a 488-nm laser line. Three images were acquired in the same order: CFP,
FRET, and YFP in all experiments. Ratiometric image analysis of FRET
was performed using Image Pro 3DS software (version 6.0; Media Cyber-
netics) and LSM examiner software. The YFP image with high signal-to-
noise ratio served to create a binary mask with a value of zero outside and
value of one inside the cell using a threshold-based procedure. Subse-
quently, CFP and FRET images were multiplied by the mask. Dividing a
FRET-masked image by a CFP-masked image and multiplying this value
by a factor of 1000, a ratio image was obtained. Colocalization analysis
was performed using NIH image analysis software packages Image J (ver-
sion 1.33) and LSM examiner software (version 6; BioRad-Zeiss Laser-
Indicated experimental data were analyzed for statistical significance using
the Student t test for paired samples. Significance is indicated by asterisks
and described in figure legends.
Responsiveness of SALE cells to TLR ligands
Human airway epithelial (SALE) cells express a variety of TLRs,
although they are not very responsive to LPS stimulation (30). Cell
surface expression of TLR2 and CD14 as well as intracellular ex-
pression of TLR3 was confirmed by flow cytometry (data not
shown). TLR2 and TLR3 reside in different cellular compartments
and use distinct adapters such as TIRAP/MyD88 and TRIF. SALE
cells were analyzed for their responsiveness to TLR2 and TLR3
ligands by using diacylated lipopeptide (MALP-2), Pam3CSK4,
and dsRNA (poly(I:C)). The specificity of TLR2 and TLR3 ligands
was confirmed using TLR2 blocking Ab or chloroquine pretreat-
ment, followed by NF-?B-luciferase reporter assays (data not
shown). For initial screening of the NF-?B pathway and of MAPK
activation, I?B? degradation and p38 phosphorylation by these
ligands were assessed. Airway cells did not respond well to the
TLR2 ligand Pam3CSK4 (see NF-?B activation; supplemental
Fig. 1A).4In contrast, MALP-2-induced degradation of I?B? was
detected after 15 min, and I?B? was completely resynthesized in
90 min (Fig. 1A). Phosphorylation of p38 by MALP-2 occurred at
30 min. A similar pattern was observed with poly(I:C) (Fig. 1B),
although the overall kinetic was delayed due to internalization of
the TLR ligand and the TLR3 response from the endosomal com-
partment. It has been previously shown that TLR3 stimulation gen-
erates IRF3-dependent type I IFN. Accordingly, poly(I:C) stimu-
lation of SALE cells induced IRF3 phosphorylation at Ser396and
at Ser386. Dimer formation of phospho-IRF3 (S386), a prerequisite
for IRF3 transcriptional activity, was observed after 60 min (Fig.
1C). Thus, major TLR2 and TLR3 signaling pathways are func-
tional in SALE cells.
TLR2 and TLR3 signaling leads to RhoA activity at distinct
TLR2 stimulation triggers activation of the Rho GTPase RhoA in
TLR2-expressing model cell lines and monocytic cells (20). Thus,
RhoA activation was determined in SALE cells stimulated either
with MALP-2 or for comparison with poly(I:C). Pull-down assays
showed a rapid increase in RhoA activity after 5 min, whereas
poly(I:C)-induced RhoA activation was delayed, as expected for
signaling by intracellular TLR3 (Fig. 2A). SALE cells are charac-
terized by large, spreading cell bodies, which is advantageous for
visualization of signaling molecule recruitment. To visualize local
RhoA activity changes caused by TLR-mediated signals, a single-
chain FRET sensor was introduced into SALE cells. A RhoA bi-
osensor, which permits detection of RhoA activation in real time,
was lentivirally transduced into SALE cells. These cells, termed
SALE/RhoA, were characterized for their RhoA content and
screened for their TLR ligand responsiveness. SALE/RhoA cells
expressed similar levels of endogenous RhoA and the RhoA bio-
sensor (supplemental Fig. 1B). Expression of the RhoA biosensor
did not disturb TLR signaling, because SALE/RhoA cells re-
sponded equally well to TLR ligands as nontransduced SALE cells
(supplemental Fig. 1, C and D). To study the spatiotemporal dy-
namics of TLR-triggered RhoA activation, SALE/RhoA cells were
either left unstimulated or stimulated with MALP-2 or poly(I:C)
for various time periods. Cells were fixed and analyzed by confo-
cal microscopy for increased FRET. RhoA activation is presented
as CFP/FRET emission ratio and is visualized by color-coding the
images with scaling from low (blue) to high (red) FRET, whereas
biosensor localization is shown in RhoA-YFP images. Short stim-
ulation of SALE/RhoA cells with MALP-2 (5 min) increased the
emission ratios with areas of the highest intensity close to cell
edges. At the same time, enrichment of RhoA was detected at
membrane ruffles (Fig. 2B, panel 1). Active RhoA moved from the
cell edges to the cytoplasm at later time points (10 min shown).
Performance of the RhoA biosensor was validated by coexpression
4The online version of this article contains supplemental material.
and TLR3 stimulation. SALE cells were transiently transfected with NF-
?B-luc and plasmids as indicated before stimulation with MALP-2 (A) or
poly(I:C) (B and C) for 4 h. Lysates of A and B were analyzed in chemi-
luminescence and by immunoblot for protein expression (data not shown),
and supernatants (C) were collected and used to determine type I IFN
production. Error bars represent SDs; p values are ?0.001 (???) to stim-
ulated vector control. Data of one representative experiment are shown
(n ? 3).
Inhibition of RhoA blocks NF-?B transactivation by TLR2
3525The Journal of Immunology
of dominant-negative RhoA, which reduced the emission ratio of
MALP-2-stimulated, Myc-RhoA DN-positive cells (supplemental
Fig. 1E). An increase in FRET was also observed after 15–30 min
of poly(I:C) stimulation (Fig. 2B, panel 3), although highest RhoA
activity was in this case concentrated in perinuclear, vesicular
structures. After 60 min of poly(I:C) stimulation, RhoA activity
was still maintained. Thus, the imaging time course of RhoA ac-
tivity correlated well with the biochemical assay. To confirm co-
localization of active RhoA with dsRNA-containing vesicles,
which are TLR3 signaling compartments (11), SALE/RhoA cells
were incubated with labeled dsRNA. After 30 min of stimulation,
the labeled TLR3 ligand colocalized with areas of high FRET sig-
nal (active RhoA) (Fig. 2C, upper panel) as well as with RhoA-
YFP (Fig. 2C, lower panel).
RhoA is required for NF-?B activation, but dispensable for type
I IFN generation
Previous studies in HEK293-TLR2 cells demonstrated a regulatory
role for RhoA in TLR2-initiated NF-?B transactivation. These data
were verified in SALE cells, which present a more physiologically
relevant cell type. As shown in Fig. 3, expression of dominant-
negative RhoA or Clostridium botulinum C3 toxin in SALE cells
harboring a NF-?B responsive, luciferase-coupled promoter in-
hibited TLR2 NF-?B activation. Overexpression of RhoA wt
(?3- to 6-fold) in these cells augmented NF-?B activity. RhoA
signaling was also required for TLR3-mediated NF-?B activa-
tion (Fig. 3B), although production and release of IFN-?? was
independent of RhoA (Fig. 3C).
Src family kinase activity is required for TLR2 and TLR3
signaling to NF-?B
Recent reports (11, 14) placed Src family kinases as receptor-prox-
imal regulators of TLR signaling. Similarly, the tyrosine kinase
Syk was implicated in TLR pathways (9, 15, 16). When analyzing
MALP-2-induced Src kinase activation, several immunoreactive
bands were detected using pan-phospho-Src Tyr416Ab (Fig. 4A).
Tyrosine phosphorylation of these proteins was blocked by the Src
kinase inhibitor PP2. Immunoblotting revealed that SALE cells
express multiple Src family kinases. The tyrosine-phosphorylated
band at 68 kDa may represent c-Src or Yes kinases; the 130-kDa
band remains unidentified (supplemental Fig. 2). In addition, rapid
activation of Syk by the TLR ligand MALP-2 was detected, using
epidermal growth factor (EGF) stimulation of SALE cells as pos-
itive control (Fig. 4B).
TLR2- and TLR3-induced NF-?B-de-
pendent gene transcription. A, SALE
cells were stimulated with MALP-2
for 10 min with or without preincuba-
tion with PP2. Lysates were analyzed
Tyr416). Actin served as control. B,
SALE cells were stimulated with
MALP-2 or EGF (1 ng/ml), and ly-
sates were analyzed for Syk activation
using phospho-Syk Tyr525/526Ab. To-
tal Syk served as control. C and F,
SALE cells were transiently trans-
fected with NF-?B-luc and preincu-
bated with SI (10 ?M), PP2 (10 ?M),
PP3 (10 ?M), or piceatannol (25 ?M)
before incubation with MALP-2 or
poly(I:C) for 4 h. D and E, SALE cells
were either cotransfected with c-Src
K295M (24 h) and 5? NF-?B luc (D)
or treated with Syk siRNA (no. 2, 20
nM, 48 h) before 5? NF-?B luc trans-
fection (E). Lysates of C–F were an-
alyzed for chemiluminescence. Super-
natants (G) from F were collected to
determine type I IFN production. Er-
ror bars represent SDs; p values are
?0.01 (??) and ?0.05 (?) to stimu-
lated control. Data of one representa-
tive experiment are shown (n ? 3).
Src and Syk mediate
3526RhoA IN TLR SIGNALING
Next, the involvement of Src family kinases and Syk in TLR2-
and TLR3-initiated pathways was probed. Inhibition of Src kinases
with PP2 or SI, and of Syk with piceatannol, significantly reduced
MALP-2-stimulated NF-?B activation (Fig. 4C). These data were
confirmed using transfection of dominant-negative c-Src or by
siRNA knockdown of Syk (Fig. 4, D and E). Similarly, poly(I:C)-
stimulated NF-?B activation was abolished by Src kinase inhibi-
tion (Fig. 4F), whereas PP3, an inactive analog of PP2, did not
alter NF-?B-dependent gene transcription. Piceatannol treatment
of SALE cells had a modest effect on TLR3 ligand-stimulated
NF-?B activation. Both, Src kinase and Syk inhibitors abolished
poly(I:C)-induced IFN-?? production (Fig. 4G). Supernatants de-
rived from unstimulated, piceatannol-treated SALE cells reduced
the baseline transcriptional activity in L929-ISRE cells, which
could indicate some unspecific effect of this compound.
Src family kinases are upstream of TLR2- and TLR3-induced
Previous studies indicated that the TLR2-RhoA pathway may di-
verge from the canonical TLR2-MyD88 pathway (20). Src family
kinases have been implicated in TLR-TIR domain phosphoryla-
tion, thus providing initial binding sites for association of signaling
complexes. Src-mediated phosphorylations are also often prereq-
uisite for GEF and thus GTPase activation, placing Src upstream of
GTPases such as RhoA. Preincubation of SALE cells with the Src
kinase inhibitors SI and PP2 abolished RhoA activation by
MALP-2 and by poly(I:C) when pull-down assays were analyzed
(Fig. 5A). The effect of Src kinase inhibitors on RhoA activation
was also studied by FRET using biosensor-expressing SALE/
RhoA cells. As shown in Fig. 5B, MALP-2-stimulated RhoA ac-
tivity was reduced in the presence of SI. Analysis of the whole-cell
emission ratios revealed a significant FRET reduction upon Src
inhibition (Fig. 5B). Overall, these data indicate that Src signaling
is essential for transmitting TLR2 and TLR3 signals to RhoA.
Syk kinase is upstream of TLR2-induced RhoA activation
Syk seems to be involved in transmitting TLR2 and TLR3 signals
to NF-?B in SALE cells. Analysis of MALP-2-stimulated SALE
cells showed that Syk kinase activity was necessary for RhoA
activation (Fig. 5C). Although piceatannol treatment of SALE
cells increased RhoA activity in unstimulated cells, MALP-2-trig-
gered RhoA activity was decreased. Interestingly, poly(I:C)-medi-
ated RhoA activation was not altered by Syk inhibition (data not
shown). The Syk inhibitor piceatannol showed strong autofluores-
cence, thus precluding imaging studies and confirmation of
changes in biosensor FRET in the presence or absence of this
Rho GTPases are master regulators of many immunoreceptors,
ranging from receptors required for differentiation and maturation
of immune cells and for pathogen recognition to receptors in-
volved in pathogen uptake and the subsequent host signaling re-
sponses. Several TLRs induce the activation of the Rho family
GTPases Rac, Rho, and Cdc42. Almost every cell type, including
professional innate immune cells such as macrophages, neutro-
phils, and dendritic cells, responds to TLR stimulation by activat-
ing Rho GTPases rapidly (8, 20, 31). Similarly, lung epithelial
cells induce Rho GTPase activation when they encounter TLR
ligands. It seems apparent that RhoA activation depends on the
interaction of a specific TLR ligand with its cognate TLR, inde-
pendently of the connecting adapters or the cellular compartment
where TLR signaling occurs. A RhoA FRET probe was used to
inhibitors. SALE cells were stimulated with MALP-2 for 10 min with or without preincubation with SI or PP2. Lysates were analyzed for RhoA activity
by pull-down assay (RhoA-GTP, total RhoA 10% of lysate) and quantitated by densitometry (see Materials and Methods). B, RhoA activity was analyzed
by FRET using RhoA biosensor (representative FRET ratio images and RhoA-YFP images of SALE/RhoA cells are shown; scale bar is 10 ?m). FRET
ratio images are scaled so that regions of highest RhoA activity are shown in red. Quantification of whole-cell emission ratios are shown; number of cells
analyzed was n (?) ? 32; n (MALP-2) ? 52; n (SI/MALP-2) ? 45; error bars represent SEs; values of p ? 0,001 (???) and ? 0.01 (??). C,
MALP-2-stimulated RhoA activity is inhibited by Syk kinase inhibitor. SALE cells were stimulated with MALP-2 for 10 min with or without piceatannol
preincubation. Lysates were used for RBD pull-down assay and total RhoA immunoblotting. Data in A–C are representative of three independent
TLR2- and TLR3-mediated RhoA activation requires Src. A, MALP-2 and poly(I:C)-stimulated RhoA activity is inhibited by Src kinase
3527 The Journal of Immunology
examine localized RhoA activity at early time points after initia-
tion of TLR2 or TLR3 signaling. After 3–5 min of treatment with
the TLR2 ligand MALP-2, RhoA was rapidly activated at ruffling
areas at the cell surface. Later on, RhoA activity was detected in
vesicular structures, which moved to the perinuclear region and
may contain internalized TLR2. To visualize colocalization of
TLR3 ligands with RhoA-YFP and with areas of high FRET,
which indicate high RhoA activity, a labeled dsRNA ligand was
used. Areas of colocalization and FRET were predominantly de-
tected in perinuclear, speckle-like structures. These structures are
reminiscent of TRIF speckles, which formed in HeLa cells in re-
sponse to poly(I:C) stimulation (32). These speckles contained the
receptor-interacting protein 1, a signaling molecule connecting
TRIF to NF-?B activation, and NF-?B-activating kinase-associ-
ated protein 1, which links TRIF to TANK-binding kinase 1 and
the IRF3-IFN-?? pathway. Energy transfer from TRIF to NAP1
was not efficient, whereas RIP1 was tightly associated with TRIF
(32). Our data suggest that RhoA is part of this putative speckle
signalosome, and that incorporation of RhoA into the TRIF sig-
naling complex is essential for NF-?B activation. On the other
hand, RhoA activation occurs also rapidly when TLR2-connected
adapter signaling is triggered at the plasma membrane.
Recent reports (5, 11–14, 22, 33, 34) connect the Src family
kinases c-Src, Hck, Lyn, Fyn, Yes, and Fgr to TLR3, TLR2, TLR4,
and TLR9 signaling in various cell types. These kinases have been
implicated in the tyrosine phosphorylation of the intracellular
TLR-TIR domain to provide docking sites for signaling molecules
and may initiate activation of downstream targets. Quite likely,
some of these targets are GEFs, which control positively the GT-
Pase activation cycle. So far, only the GEFs Vav and AKAP13/
Lbc have been implicated in TLR signaling (22, 23). Probing
RhoA activation by rhothekin RBD binding assay or by biosensor
FRET clearly shows that Src family kinases provide an essential
upstream signal for RhoA activity when TLR2 or TLR3 ligands
are used. The contribution of Syk to the TLR-RhoA pathway was
investigated as several reports (9, 15, 16) linked Syk kinase di-
rectly to the TLR4 signaling complex. TLR2 and TLR3 ligands
stimulated Syk activity in lung epithelial cells (see MALP-2;
poly(I:C) not shown). Using a Syk inhibitor or Syk siRNA, a sub-
stantial decrease in MALP-2-stimulated NF-?B activation was ob-
served. Where to place Syk in TLR signaling to RhoA needs to be
explored in more detail, as the specificity of the widely used in-
hibitor piceatannol has been questioned (35).
Src family kinases and Syk kinase were required for NF-?B
activation and type I IFN production in lung epithelial cells,
whereas inhibition of RhoA activity reduced only NF-?B activa-
tion. The IFN-? promoter contains the positive regulatory ele-
ments PRD-I, -II, -III, and -IV that bind to the transcription factors
AP-1, IRF3, and NF-?B. Several studies reported a critical role of
NF-?B in poly(I:C)- or virus-induced type I IFN expression (36–
38). Our data implicate RhoA as regulator of the NF-?B pathway
and dispensable for IFN-?? production. This observation is con-
sistent with studies by Wang and coworkers, who assigned only a
minor role to NF-?B transcription factors when analyzing IFN-??
expression in virus-infected MEFs lacking individual members of
the NF-?B family (39).
The question still remains how TLR-initiated cellular responses
connect via Src/Syk to the Rho GTPase-regulated network. A re-
cent report (40) connects the TLR2-TNFR-associated factor 6
pathway to c-Src. Attempts to link RhoA to this pathway were not
successful. We also explored the recently described TLR2-
AKAP13-NF-?B pathway, because the RhoA-specific GEF
AKAP13 may convey TLR2 signals to RhoA. Although constitu-
tive and MALP-2-triggered complex formation of endogenous
TLR2 and AKAP13 was observed (supplemental Fig. 3), the pres-
ence of RhoA in this complex could not be confirmed. It seems
likely that rapid association and dissociation of RhoA from its
GEF takes place. In general, the well-characterized role of Rho
GTPases in controlling cytoskeletal remodeling may provide a
platform for constant assembly and disassembly of the TLR sig-
nalosome. Receptor dimerization, coreceptor usage, and involve-
ment of cooperating receptors and integrins may be needed for
association of recruited signaling components. During these
events, Rho GTPases may participate in the dynamic assembly of
signaling platforms at specific cellular locations.
We thank Dr. W. Kiosses for advice, Katrina Schreiber for graphic assis-
tance, and Monica Ruse for critical reading of the manuscript.
The authors have no financial conflict of interest.
1. Guillot, L., S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and
M. Si-Tahar. 2004. Response of human pulmonary epithelial cells to lipopoly-
saccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways:
evidence for an intracellular compartmentalization of TLR4. J. Biol. Chem. 279:
2. Hippenstiel, S., B. Opitz, B. Schmeck, and N. Suttorp. 2006. Lung epithelium as
a sentinel and effector system in pneumonia—molecular mechanisms of pathogen
recognition and signal transduction. Respir. Res. 7: 97.
3. Mayer, A. K., M. Muehmer, J. Mages, K. Gueinzius, C. Hess, K. Heeg, R. Bals,
R. Lang, and A. H. Dalpke. 2007. Differential recognition of TLR-dependent
microbial ligands in human bronchial epithelial cells. J. Immunol. 178:
4. Jia, H. P., J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, and
P. B. McCray, Jr. 2004. Endotoxin responsiveness of human airway epithelia is
limited by low expression of MD-2. Am. J. Physiol. 287: L428–L437.
5. Chun, J., and A. Prince. 2006. Activation of Ca2?-dependent signaling by TLR2.
J. Immunol. 177: 1330–1337.
6. Guillot, L., R. Le Goffic, S. Bloch, N. Escriou, S. Akira, M. Chignard, and
M. Si-Tahar. 2005. Involvement of Toll-like receptor 3 in the immune response
of lung epithelial cells to double-stranded RNA and influenza A virus. J. Biol.
Chem. 280: 5571–5580.
7. Matsumoto, M., and T. Seya. 2008. TLR3: interferon induction by double-
stranded RNA including poly(I:C). Adv. Drug Deliv. Rev. 60: 805–812.
8. Arbibe, L., J. P. Mira, N. Teusch, L. Kline, M. Guha, N. Mackman,
P. J. Godowski, R. J. Ulevitch, and U. G. Knaus. 2000. Toll-like receptor 2-me-
diated NF-?B activation requires a Rac1-dependent pathway. Nat. Immunol. 1:
9. Arndt, P. G., N. Suzuki, N. J. Avdi, K. C. Malcolm, and G. S. Worthen. 2004.
Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neu-
trophils: role of phosphatidylinositol 3-kinase and Syk-mediated pathways.
J. Biol. Chem. 279: 10883–10891.
10. Schmeck, B., S. Huber, K. Moog, J. Zahlten, A. C. Hocke, B. Opitz,
S. Hammerschmidt, T. J. Mitchell, M. Kracht, S. Rosseau, et al. 2006. Pneumo-
cocci induced TLR- and Rac1-dependent NF-?B recruitment to the IL-8 promoter
in lung epithelial cells. Am. J. Physiol. 290: L730–L737.
11. Johnsen, I. B., T. T. Nguyen, M. Ringdal, A. M. Tryggestad, O. Bakke, E. Lien,
T. Espevik, and M. W. Anthonsen. 2006. Toll-like receptor 3 associates with
c-Src tyrosine kinase on endosomes to initiate antiviral signaling. EMBO J. 25:
12. Gong, P., D. J. Angelini, S. Yang, G. Xia, A. S. Cross, D. Mann,
D. D. Bannerman, S. N. Vogel, and S. E. Goldblum. 2008. TLR4 signaling is
coupled to SRC family kinase activation, tyrosine phosphorylation of zonula
adherens proteins, and opening of the paracellular pathway in human lung mi-
crovascular endothelia. J. Biol. Chem. 283: 13437–13449.
13. Aki, D., R. Mashima, K. Saeki, Y. Minoda, M. Yamauchi, and A. Yoshimura.
2005. Modulation of TLR signalling by the C-terminal Src kinase (Csk) in mac-
rophages. Genes Cells 10: 357–368.
14. Medvedev, A. E., W. Piao, J. Shoenfelt, S. H. Rhee, H. Chen, S. Basu,
L. M. Wahl, M. J. Fenton, and S. N. Vogel. 2007. Role of TLR4 tyrosine phos-
phorylation in signal transduction and endotoxin tolerance. J. Biol. Chem. 282:
15. Ulanova, M., S. Asfaha, G. Stenton, A. Lint, D. Gilbertson, A. Schreiber, and
D. Befus. 2007. Involvement of Syk protein tyrosine kinase in LPS-induced re-
sponses in macrophages. J. Endotoxin Res. 13: 117–125.
16. Chaudhary, A., T. M. Fresquez, and M. J. Naranjo. 2007. Tyrosine kinase Syk
associates with Toll-like receptor 4 and regulates signaling in human monocytic
cells. Immunol. Cell Biol. 85: 249–256.
17. Dennehy, K. M., G. Ferwerda, I. Faro-Trindade, E. Pyz, J. A. Willment,
P. R. Taylor, A. Kerrigan, S. V. Tsoni, S. Gordon, F. Meyer-Wentrup, et al. 2008.
Syk kinase is required for collaborative cytokine production induced through
Dectin-1 and Toll-like receptors. Eur. J. Immunol. 38: 500–506.
3528RhoA IN TLR SIGNALING
18. Hoebe, K., P. Georgel, S. Rutschmann, X. Du, S. Mudd, K. Crozat, S. Sovath, Download full-text
L. Shamel, T. Hartung, U. Zahringer, and B. Beutler. 2005. CD36 is a sensor of
diacylglycerides. Nature 433: 523–527.
19. Stuart, L. M., S. A. Bell, C. R. Stewart, J. M. Silver, J. Richard, J. L. Goss,
A. A. Tseng, A. Zhang, J. B. El Khoury, and K. J. Moore. 2007. CD36 signals
to the actin cytoskeleton and regulates microglial migration via a p130Cas com-
plex. J. Biol. Chem. 282: 27392–27401.
20. Teusch, N., E. Lombardo, J. Eddleston, and U. G. Knaus. 2004. The low mo-
lecular weight GTPase RhoA and atypical protein kinase C? are required for
TLR2-mediated gene transcription. J. Immunol. 173: 507–514.
21. Chen, L. Y., B. L. Zuraw, F. T. Liu, S. Huang, and Z. K. Pan. 2002. IL-1
receptor-associated kinase and low molecular weight GTPase RhoA signal mol-
ecules are required for bacterial lipopolysaccharide-induced cytokine gene tran-
scription. J. Immunol. 169: 3934–3939.
22. Stovall, S. H., A. K. Yi, E. A. Meals, A. J. Talati, S. A. Godambe, and
B. K. English. 2004. Role of vav1- and src-related tyrosine kinases in macro-
phage activation by CpG DNA. J. Biol. Chem. 279: 13809–13816.
23. Shibolet, O., C. Giallourakis, I. Rosenberg, T. Mueller, R. J. Xavier, and
D. K. Podolsky. 2007. AKAP13, a RhoA GTPase-specific guanine exchange
factor, is a novel regulator of TLR2 signaling. J. Biol. Chem. 282: 35308–35317.
24. Lundberg, A. S., S. H. Randell, S. A. Stewart, B. Elenbaas, K. A. Hartwell,
M. W. Brooks, M. D. Fleming, J. C. Olsen, S. W. Miller, R. A. Weinberg, and
W. C. Hahn. 2002. Immortalization and transformation of primary human airway
epithelial cells by gene transfer. Oncogene 21: 4577–4586.
25. Pertz, O., L. Hodgson, R. L. Klemke, and K. M. Hahn. 2006. Spatiotemporal
dynamics of RhoA activity in migrating cells. Nature 440: 1069–1072.
26. Wong, K., O. Pertz, K. Hahn, and H. Bourne. 2006. Neutrophil polarization:
spatiotemporal dynamics of RhoA activity support a self-organizing mechanism.
Proc. Natl. Acad. Sci. USA 103: 3639–3644.
27. Mori, M., M. Yoneyama, T. Ito, K. Takahashi, F. Inagaki, and T. Fujita. 2004.
Identification of Ser-386 of interferon regulatory factor 3 as critical target for
inducible phosphorylation that determines activation. J. Biol. Chem. 279:
28. Lombardo, E., A. Alvarez-Barrientos, B. Maroto, L. Bosca, and U. G. Knaus.
2007. TLR4-mediated survival of macrophages is MyD88 dependent and requires
TNF-? autocrine signalling. J. Immunol. 178: 3731–3739.
29. Hodgson, L., O. Pertz, and K. M. Hahn. 2008. Design and optimization of ge-
netically encoded fluorescent biosensors: GTPase biosensors. Methods Cell Biol.
30. Ritter, M., D. Mennerich, A. Weith, and P. Seither. 2005. Characterization of
Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3
ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and
inflammatory response. J. Inflamm. 2: 16.
31. Knaus, U. G., A. Bamberg, and G. M. Bokoch. 2007. Rac and Rap GTPase
activation assays. Methods Mol. Biol. 412: 59–67.
32. Funami, K., M. Sasai, Y. Ohba, H. Oshiumi, T. Seya, and M. Matsumoto. 2007.
Spatiotemporal mobilization of Toll/IL-1 receptor domain-containing adaptor
molecule-1 in response to dsRNA. J. Immunol. 179: 6867–6872.
33. Achuthan, A., C. Elsegood, P. Masendycz, J. A. Hamilton, and G. M. Scholz.
2006. CpG DNA enhances macrophage cell spreading by promoting the Src
family kinase-mediated phosphorylation of paxillin. Cell. Signal. 18: 2252–2261.
34. Kannan, S., A. Audet, J. Knittel, S. Mullegama, G. F. Gao, and M. Wu. 2006. Src
kinase Lyn is crucial for Pseudomonas aeruginosa internalization into lung cells.
Eur. J. Immunol. 36: 1739–1752.
35. Mocsai, A., H. Zhang, Z. Jakus, J. Kitaura, T. Kawakami, and C. A. Lowell.
2003. G protein-coupled receptor signaling in Syk-deficient neutrophils and mast
cells. Blood 101: 4155–4163.
36. Garoufalis, E., I. Kwan, R. Lin, A. Mustafa, N. Pepin, A. Roulston, J. Lacoste,
and J. Hiscott. 1994. Viral induction of the human beta interferon promoter:
modulation of transcription by NF-?B/rel proteins and interferon regulatory fac-
tors. J. Virol. 68: 4707–4715.
37. Thanos, D., and T. Maniatis. 1995. Identification of the rel family members
required for virus induction of the human ? interferon gene. Mol. Cell. Biol. 15:
38. Merika, M., A. J. Williams, G. Chen, T. Collins, and D. Thanos. 1998. Recruit-
ment of CBP/p300 by the IFN-? enhanceosome is required for synergistic acti-
vation of transcription. Mol. Cell 1: 277–287.
39. Wang, X., S. Hussain, E. J. Wang, M. O. Li, A. Garcia-Sastre, and A. A. Beg.
2007. Lack of essential role of NF-?B p50, RelA, and cRel subunits in virus-
induced type 1 IFN expression. J. Immunol. 178: 6770–6776.
40. Lee, I. T., S. W. Wang, C. W. Lee, C. C. Chang, C. C. Lin, S. F. Luo, and
C. M. Yang. 2008. Lipoteichoic acid induces HO-1 expression via the TLR2/
MyD88/c-Src/NADPH oxidase pathway and Nrf2 in human tracheal smooth
muscle cells. J. Immunol. 181: 5098–5110.
3529 The Journal of Immunology