RhoA/Rho-Associated Kinase Pathway Selectively Regulates
Thrombin-Induced Intercellular Adhesion Molecule-1
Expression in Endothelial Cells via Activation of I?B Kinase ?
and Phosphorylation of RelA/p651
Khandaker N. Anwar,†Fabeha Fazal,* Asrar B. Malik,†and Arshad Rahman2*
We investigated the involvement of the RhoA/Rho-associated kinase (ROCK) pathway in regulating ICAM-1 expression in en-
dothelial cells by the procoagulant, thrombin. Exposure of HUVECs to C3 exoenzyme, a selective inhibitor of Rho, markedly
reduced thrombin-induced ICAM-1 expression. Inhibition of ROCK, the downstream effector of Rho, also prevented thrombin-
induced ICAM-1 expression. Blockade of thrombin-induced ICAM-1 expression was secondary to inhibition of NF-?B activity, the
key regulator of ICAM-1 expression in endothelial cells. In parallel studies we observed that inhibition of the RhoA/ROCK
pathway by the same pharmacological and genetic approaches failed to inhibit TNF-?-induced NF-?B activation and ICAM-1
expression. The effect of RhoA/ROCK inhibition on thrombin-induced NF-?B activation was secondary to inhibition of I?B kinase
activation and subsequent I?B? degradation and nuclear uptake and the DNA binding of NF-?B. Inhibition of the RhoA/ROCK
pathway also prevented phosphorylation of Ser536within the transactivation domain 1 of NF-?B p65/RelA, a critical event
conferring transcriptional competency to the bound NF-?B. Thus, the RhoA/ROCK pathway signals thrombin-induced ICAM-1
expression through the activation of I?B kinase, which promotes NF-?B binding to ICAM-1 promoter and phosphorylation of
RelA/p65, thus mediating the transcriptional activation of bound NF-?B. The Journal of Immunology, 2004, 173: 6965–6972.
endothelium involving the expression of ICAM-1 (CD54) on the
endothelial cell surface and activation of its counter-receptor ?2
integrins (CD11/CD18) on the PMN surface (1). The interaction of
ICAM-1 with ?2integrins enables PMN to adhere firmly and sta-
bly to the vascular endothelium and migrate across the endothelial
barrier (2, 3). Although ICAM-1 is constitutively expressed in low
levels in endothelial cells, its expression can be induced by proin-
flammatory mediators such as the procoagulant thrombin (4–6),
which is released during intravascular coagulation initiated by tis-
sue injury or sepsis (7–9). We have shown that the transcription
factor NF-?B p65 (RelA) is the key regulator of thrombin-induced
ICAM-1 gene transcription in endothelial cells and that this re-
sponse is mediated through activation of the GTP-binding protein
(G protein)-coupled receptor, protease-activated receptor-1 (4, 10).
NF-?B is a ubiquitously expressed family of transcription fac-
tors controlling the expression of numerous genes involved in im-
he recruitment of polymorphonuclear leukocytes (PMN)3
from blood to the site of infection is a highly ordered
process. A key step is the stable adhesion of PMN to the
munity and inflammation (11). The prototypical NF-?B complex,
a heterodimer of 50-kDa (p50) and 65-kDa (RelA) subunits, is
sequestered in the cytoplasm in an inactive form by its association
with I?B?, the prototype of a family of inhibitory proteins termed
I?B proteins (12). Activation of NF-?B requires the degradation
of I?B? achieved through serine phosphorylation (Ser32and Ser36)
of I?B? by a macromolecular I?B kinase (IKK) complex (13, 14).
Phosphorylation targets I?B? for polyubiquitination by the E3-
SCF?-TrCP ubiquitin ligase and subsequently its degradation by
the 26S proteasome (15). The released NF-?B rapidly translocates
to the nucleus to activate the transcription of target genes such as
ICAM-1. Another important mechanism regulating NF-?B activity
is through modulation of its transcriptional function by phosphor-
ylation of RelA/p65 (16–20). Studies have shown that phosphor-
ylation of RelA/p65 at serine 276, 311, 529, or 536 increases the
transcriptional capacity of NF-?B in the nucleus (16–20). How-
ever, unlike I?B? phosphorylation, the RelA/p65 phosphorylation
site and the kinase involved vary in a stimulus- and cell type-
specific manner (16–20).
The Rho family of monomeric GTPases, Rho, Rac, and Cdc42,
serve as molecular switches by cycling between the inactive GDP-
bound state and the active GTP-bound state (21). The active con-
formation facilitates the interaction of Rho GTPases with their
downstream targets to activate intracellular signaling. Cycling be-
tween the two conformations is regulated by guanine nucleotide
exchange factors, which promote the release of GDP and allow
GTP to bind, and GTPase-activating proteins, which stimulate the
hydrolysis of GTP (21, 22). In addition to the role of Rho GTPases
in the regulation of cytoskeletal dynamics, actin stress fiber for-
mation, and myosin L chain phosphorylation, RhoA can affect
gene expression through activation of transcription factors such as
serum response factor and NF-?B (23–25). Rho-associated kinase
(p160ROCK/Rho kinase), a downstream effector of RhoA, is im-
plicated in a variety of RhoA-mediated responses (26, 27). We
*Department of Pediatrics, University of Rochester School of Medicine, Rochester,
NY 14642; and†Department of Pharmacology, University of Illinois College of Med-
icine, Chicago, IL 60612
Received for publication February 19, 2004. Accepted for publication October
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 Heart Lung and Blood Institute Grants
HL67424, HL46350, and HL64573.
2Address correspondence and reprint requests to Dr. Arshad Rahman, Department of
Pediatrics, Box 850, University of Rochester School of Medicine, 601 Elmwood
Avenue, Rochester, NY 14642. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; EBM2, en-
dothelial basal medium 2; IKK, I?B kinase; LUC, luciferase; ROCK, Rho-associated
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc.0022-1767/04/$02.00
have demonstrated that thrombin activation of RhoA is critical in
regulating thrombin-induced endothelial barrier function (28). In
the present study we addressed the possible role of the RhoA/Rho-
associated kinase (ROCK) pathway in signaling ICAM-1 expres-
sion in endothelial cells. Our data demonstrate that the RhoA/
ROCK pathway signals thrombin-induced ICAM-1 expression in
endothelial cells by the activation of IKK?, which, in turn, medi-
ates NF-?B binding to ICAM-1 promoter and phosphorylation of
RelA/p65, thereby conferring transcriptional competency to the
Materials and Methods
Human thrombin was purchased from Enzyme Research Laboratories
(South Bend, IN). Polyclonal Abs to IKK?, I?B?, p65/RelA, and ?-actin;
an mAb to ICAM-1; and GST-I?B? fusion protein were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA). Abs that detect RelA/p65
when phosphorylated at Ser536or that detect I?B? when phosphorylated at
Ser32and Ser36were obtained from Cell Signaling Technology (Beverly,
MA). The polyvinylidene difluoride membrane was obtained from Milli-
pore (Bedford, MA); Y27632 was purchased from Calbiochem-Novabio-
chem (La Jolla, CA); the protein assay kit and nitrocellulose membrane
were obtained from Bio-Rad (Hercules, CA); the plasmid Maxi Kit was
purchased from Qiagen (Valencia, CA). All other materials were obtained
from Fisher Scientific (Pittsburgh, PA) or VWR Scientific Products
HUVECs (Cambrex, La Jolla, CA) were cultured as previously described
(10) in gelatin-coated flasks using endothelial basal medium 2 (EBM2)
with Bullet Kit additives (Cambrex). Confluent cells were incubated in
EBM2-containing heat-inactivated 0.5% FBS for 2 h or in 1% FBS for 12 h
before thrombin challenge. All experiments were performed with cells be-
tween the third and eighth passages, except for the transfection experi-
ments, in which cells were between the third and fifth passages.
Cell lysis and immunoblotting
After thrombin challenge of HUVEC transfected with C3 transferase or
pretreated with Y27632, cells were lysed in radioimmune precipitation
buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.25 mM
EDTA (pH 8.0), 1% deoxycholic acid, 1% Triton X-100, 5 mM NaF, and
1 mM sodium orthovanadate supplemented with complete protease inhib-
itors (Sigma-Aldrich, St. Louis, MO). Cell lysates were analyzed by SDS-
PAGE and transferred onto nitrocellulose (Bio-Rad) or polyvinylidene di-
fluoride membranes, and the residual binding sites on the filters were
blocked by incubation with 5% (w/v) nonfat dry milk in TBST (10 mM
Tris (pH 8.0), 150 mM NaCl, and 0.05% Tween 20) for 1 h at room
temperature or overnight at 4°C. The membranes were subsequently incu-
bated with the indicated Abs and developed using an ECL method as pre-
viously described (5).
In vitro IKK assay
Cells were starved by 12-h incubation in 1% FBS containing EBM2. The
cells were subsequently challenged with thrombin (2.5–5 U/ml) for 1 h in
the absence or the presence of Y-27632 (10 ?M), which was added 1 h
before thrombin treatment. The cells were then lysed with kinase lysis
buffer (20 mM Tris-HCl (pH 7.5), 125 mM NaCl, 100 ?M sodium or-
thovanadate, 25 mM ?-glycerophosphate, 50 mM sodium fluoride, 1 mM
EDTA, 1 mM magnesium chloride, 1% Triton X-100, 1 mM PMSF, and
protease inhibitor mixture (Sigma-Aldrich)). Cell lysates were immuno-
precipitated with an Ab against IKK? using protein A/G-agarose (Santa
Cruz Biotechnology) as previously described (5). The immunocomplexes
were washed three times with kinase lysis buffer and twice with kinase
assay buffer (20 mM HEPES (pH 7.5), 20 mM MgCl2, 0.5 mM EGTA, and
20 mM DTT) and were resuspended in 30 ?l of kinase assay buffer con-
taining 1 ?g of GST-I?B?, 20 ?M ATP, and 10–15 ?Ci of [?-32P]ATP.
The reaction was incubated for 30 min at 30°C and was terminated by the
addition of SDS sample buffer. Proteins were analyzed by SDS-PAGE, and
the phosphorylated form of GST-I?B? was detected by autoradiography.
HUVEC transfected with C3 transferase or pretreated with Y27632 were
challenged with thrombin, and total RNA was isolated using an RNeasy kit
(Qiagen) according to the manufacturer’s recommendations. Quantification
and purity of RNA were assessed by A260/A280absorption, and an aliquot
of RNA (20 ?g) from samples with a ratio ?1.6 was fractionated using a
1% agarose formaldehyde gel. The RNA was transferred to Duralose-UV
nitrocellulose membrane (Stratagene, La Jolla, CA) and covalently linked
by UV irradiation using a Stratalinker UV cross-linker (Stratagene). Hu-
man ICAM-1 (0.96-kb SalI to PstI fragment) (29) and rat GAPDH (1.1-kb
PstI fragment) were labeled with [?-32P]dCTP using the random primer kit
(Stratagene), and hybridization was conducted as previously described (5).
Briefly, the blots were prehybridized for 30 min at 68°C in QuikHyb so-
lution (Stratagene), then hybridized for 2 h at 68°C with random-primed,
32P-labeled probes. After hybridization, the blots were washed twice for 30
min each time at room temperature in 2? SSC with 0.1% SDS, followed
by two washes for 15 min each time at 60°C in 0.1? SSC with 0.1% SDS.
Autoradiography was performed with an intensifying screen at ?70°C for
12–24 h. The nitrocellulose membrane was soaked for stripping the probe
with boiled water or with 0.1? SSC /0.1% SDS.
Reporter gene constructs, endothelial cell transfection, and
luciferase (LUC) assay
The plasmid pNF-?B-LUC containing five copies of consensus NF-?B
sequences linked to a minimal E1B promoter-LUC gene was purchased
from Stratagene. The ICAM-1 LUC reporter plasmid containing approxi-
mately 1393 bp of ICAM-1 5?-flanking DNA linked to the firefly LUC
gene has been described previously (30). The constructs encoding the dom-
inant negative (N19RhoA) and constitutively active (V14RhoA) mutants of
RhoA were gifts from Dr. A. Hall (University College London, London,
U.K.). The construct encoding the kinase-defective mutant of IKK? was
described previously (5). Transfections were performed using the DEAE-
dextran method (30, 31) with slight modifications (5). Briefly, 5 ?g of
DNA was mixed with 50 ?g/ml DEAE-dextran in serum-free EBM2, and
the mixture was added to cells that were 70–80% confluent. In some ex-
periments HUVEC were also transfected with C3 transferase. We used
0.125 ?g of pTKRLUC plasmid (Promega, Madison, WI) containing Re-
nilla LUC gene driven by the constitutively active thymidine kinase pro-
moter to normalize the transfection efficiencies. After 1 h, cells were in-
cubated for 4 min with 10% DMSO in serum-free EBM2. The cells were
then washed twice with EBM2/10% FBS and grown to confluence. We
achieved a transfection efficiency of 16 ? 3% (mean ? SD; n ? 3) in these
In some experiments we used Superfect (Qiagen) to transfect the cells as
previously described (5). Briefly, reporter DNA (1 ?g) was mixed with 5
?l of Superfect in 100 ?l of serum-free EBM2 (Cambrex). We used 0.1 ?g
of pTKRLUC to normalize the transfection efficiencies. After a 5- to 10-
min incubation at room temperature, 0.6 ml of EBM2/10% FBS was added,
and the mixture was applied to the cells that had been washed once with
PBS. Three hours later, the medium was changed to EBM2/10% FBS, and
the cells were grown to confluence. This protocol resulted in a transfection
efficiency of 20 ? 2% (mean ? SD; n ? 3). Cell extracts were prepared
and assayed for firefly and Renilla LUC activities using a Biotech Dual
Luciferase Reporter Assay System (Promega). The data were expressed as
the ratio of firefly to Renilla luciferase activity.
Cytoplasmic and nuclear extract preparation
After thrombin challenge of HUVEC transfected with C3 transferase or
pretreated with Y27632, cells were washed twice with ice-cold TBS and
resuspended in 400 ?l of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl,
0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). After 15
min, Nonidet P-40 was added to a final concentration of 0.6%. Samples
were centrifuged to collect the supernatants containing cytosolic proteins
for determining I?B? degradation by Western blot analysis. The pelleted
nuclei were resuspended in 50 ?l of buffer B (20 mM HEPES (pH 7.9), 0.4
M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). After
30 min at 4°C, lysates were centrifuged, and supernatants containing the
nuclear proteins were transferred to new vials. The protein concentration of
the extract was measured using a protein determination kit (Bio-Rad).
EMSAs were performed as previously described (5). Briefly, 10 ?g of
nuclear extract was incubated with 1 ?g of poly(dI-dC) in a binding buffer
(10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, and 10% glycerol
(20 ?l final volume)) for 15 min at room temperature. Then end-labeled,
double-stranded oligonucleotides containing an NF-?B site (30,000 cpm
each) were added, and the reaction mixtures were incubated for 15 min at
room temperature. The DNA-protein complexes were resolved in 5% na-
tive PAGE in low ionic strength buffer (0.25? Tris-borate-EDTA). The
6966 STIMULUS-SPECIFIC ENDOTHELIAL ICAM-1 REGULATION BY RhoA/ROCK
oligonucleotide used for the gel-shift analysis was NF-?B (5?-AGTT
GAGGGGACTTTCCCAGGC-3?) or ICAM-1 NF-?B (5?-AGCTTG
GAAATTCCGGAGC-TG-3?). The Ig-?B oligonucleotide contains the
consensus NF-?B binding site sequence (underlined) present in pNF-?B-
LUC. The ICAM-1 NF-?B oligonucleotide represents a 21-bp sequence of
ICAM-1 promoter encompassing the NF-?B binding site located 183 bp
upstream of transcription initiation site (32). The sequence motifs within
the oligonucleotides are underlined.
Results are expressed as the mean ? SE. Data were analyzed by Student’s
Inhibition of Rho impairs thrombin-induced ICAM-1 expression
in endothelial cells
We addressed the role of Rho in signaling thrombin-induced
ICAM-1 mRNA expression in endothelial cells. Northern blot
analysis showed that transfection of HUVEC with Clostridium
botulinum C3 exoenzyme, an inhibitor of Rho (33, 34), markedly
reduced ICAM-1 mRNA expression (Fig. 1A). We also determined
the effect of inhibition of Rho on thrombin-induced ICAM-1 pro-
tein expression. Western blot analysis showed that thrombin chal-
lenge of HUVEC resulted in increased ICAM-1 protein expression
dothelial cells. A, HUVEC were transfected with C3 transferase (0.75 ?g/
ml) using Superfect as described in Materials and Methods. After 12–16 h,
cells were challenged with thrombin (5 U/ml) for 3 h or with TNF-? (100
U/ml) for 2 h. Total RNA was isolated and analyzed by Northern hybrid-
ization with a human ICAM-1 cDNA, which hybridizes to a 3.3-kb tran-
script. Blots were stripped and reprobed to determine GAPDH mRNA
expression as a measure of RNA loading. The autoradiogram and the cor-
responding bar graph showing the relative ICAM-1 mRNA expression are
representative of two separate experiments. B, HUVEC were transfected
with C3 transferase (0.75 ?g/ml) using Superfect as described in Materials
and Methods. After 12–16 h, cells were challenged with thrombin (5 U/ml)
or TNF-? (100 U/ml) for 6 h. Total cell lysates (10 ?g/lane) were separated
by SDS-PAGE and immunoblotted with an Ab to ICAM-1. The blots were
subsequently stripped and reprobed with an Ab to actin to verify equal
loading of the gel. The blot is a representative of three separate experi-
ments. The bar graph showing the relative ICAM-1 protein expression
represents the average of two experiments.
Rho signals thrombin-induced ICAM-1 expression in en-
ICAM-1 expression in endothelial cells. A, Confluent HUVEC monolayers
were pretreated for 1 h with the indicated concentrations of Y-27632 before
challenge with thrombin (5 U/ml) for 3 h or with TNF-? (100 U/ml) for
2 h. Total RNA was isolated and analyzed by Northern hybridization for
ICAM-1 mRNA expression as described in Fig. 1A. The autoradiogram
and the corresponding bar graph showing the relative ICAM-1 mRNA
expression are representative of two separate experiments. B, Confluent
HUVEC monolayers were pretreated for 1 h with Y-27632 (10 ?M) before
challenge with thrombin (5 U/ml) or TNF-? (100 U/ml) for 6 h. Total cell
lysates (10 ?g/lane) were analyzed by Western blotting for ICAM-1 pro-
tein expression as described in Fig. 1B. The blot and the corresponding bar
graph showing the relative ICAM-1 protein expression are representative
of two separate experiments.
Involvement of ROCK in signaling thrombin-induced
6967 The Journal of Immunology
and that this response was inhibited with C3 exoenzyme (Fig. 1B).
Because Rho is implicated in a number of TNF-? responses (35,
36), we evaluated the function of Rho in the mechanism of TNF-
?-induced ICAM-1 expression in endothelial cells. We found that
inhibition of Rho by C3 exoenzyme failed to prevent ICAM-1
expression induced by TNF-? (Fig. 1).
Rho signals thrombin-induced ICAM-1 expression through ROCK
We investigated the possibility that ROCK, a downstream effector
of Rho (26, 27, 37), participates in Rho signaling of thrombin-
induced ICAM-1 expression. We found that pretreatment of cells
with Y-27632, an inhibitor of ROCK (38), prevented mRNA as
well as protein expression of ICAM-1 in response to thrombin
challenge of endothelial cells (Fig. 2). In related experiments, in-
hibition of ROCK failed to prevent ICAM-1 expression by TNF-?
(Fig. 2) in contrast to the effect of Rho inhibition on thrombin-
induced ICAM-1 expression (Fig. 1).
Inhibition of RhoA/ROCK prevents thrombin-induced NF-?B
Because of the essential role of NF-?B in mediating thrombin-
induced ICAM-1 transcription, we addressed whether the RhoA/
ROCK pathway signals ICAM-1 expression by regulating NF-?B
activity. We determined the effects of inhibition of RhoA on NF-
?B-dependent reporter activity. Cotransfection of cells with C3
exoenzyme inhibited thrombin-induced NF-?B activity (Fig. 3A).
Also, the expression of the dominant negative mutant of RhoA
(RhoAmut) inhibited NF-?B activity induced by thrombin (Fig.
3C). However, inhibition of RhoA by C3 exoenzyme or RhoAmut
had no significant effect on TNF-?-induced NF-?B activity (Fig. 3,
B and D).
We next evaluated whether inhibition of ROCK mimicked the
effects of inhibition of RhoA on NF-?B activity. We found that
inhibition of ROCK by exposing the cells to Y-27632 impaired
thrombin-induced NF-?B activity (Fig. 3A). In parallel experi-
ments, inhibition of ROCK showed no significant effect on TNF-
?-induced NF-?B activity (Fig. 3B).
RhoA/ROCK signals thrombin-induced NF-?B activation
We addressed the possibility that RhoA activation is sufficient to
induce NF-?B activity. We observed that the expression of con-
stitutively active RhoA mutant (RhoAcat) induced NF-?B activity
in the absence of thrombin challenge (Fig. 4A). In another exper-
iment, cotransfection of RhoAcatincreased ICAM-1 promoter-de-
pendent reporter activity (Fig. 4B), consistent with its ability in
inducing NF-?B activation (Fig. 4A). We next determined the
function of RhoA in mediating thrombin-induced I?B? degrada-
tion, a requirement for NF-?B activation (39, 40). Because I?B?
degradation is contingent on its phosphorylation by IKK? (41–
bin-induced NF-?B activity in endothelial
cells. A and B, HUVEC were transfected with
NF-?B-LUC construct in combination with
C3 transferase (0.75 ?g/ml) using DEAE-dex-
tran as described in Materials and Methods.
After 12–16 h, cells were challenged with
thrombin (5 U/ml) or TNF-? (100 U/ml) for
6 h. In some experiments HUVEC transfected
with NF-?B-LUC construct were pretreated
with Y-27632 (10 ?M) for 1 h before chal-
lenge with thrombin (5 U/ml) or TNF-? (100
U/ml) for 6 h. Cell extracts were prepared and
assayed for firefly and Renilla luciferase ac-
tivities. Firefly luciferase activity normalized
to Renilla luciferase activity is expressed as
the fold increase relative to the untreated con-
trol value. Values shown are the mean ? SE
of three experiments performed in triplicate.
?, p ? 0.01 vs control; #, p ? 0.01 vs throm-
bin-stimulated control. C and D, HUVEC
were cotransfected with NF-?B-LUC and the
constructs encoding dominant negative mu-
tant of RhoA (RhoAmut) using DEAE-dextran
as described in Materials and Methods.
pcDNA3 was used as the vector alone control.
Cells were stimulated for 8 h with thrombin (5
U/ml) or for 6 h with TNF-? (100 U/ml) be-
fore harvesting the cells. Cell extracts were
prepared and assayed for firefly and Renilla
luciferase activities. Firefly luciferase activity
normalized to Renilla luciferase activity is ex-
pressed as the fold increase relative to the un-
treated control value. Values shown are the
mean ? SE of three experiments performed in
triplicate. ?, p ? 0.005 vs control; #, p ? 0.05
vs thrombin-stimulated control.
RhoA/ROCK regulates throm-
6968STIMULUS-SPECIFIC ENDOTHELIAL ICAM-1 REGULATION BY RhoA/ROCK
43), we evaluated the function of IKK? in RhoA-mediated NF-?B
activation. Coexpression of IKK?mutprevented RhoAcat-induced
NF-?B activity (Fig. 4A), indicating that RhoA mediates thrombin-
induced NF-?B activity through the activation of IKK?. To deter-
mine the role of ROCK in signaling RhoA activation of IKK?, we
assessed the effects of ROCK inhibition on thrombin-induced
IKK? activity using an in vitro kinase assay in which GST-I?B?
was used as an exogenous substrate. We observed that IKK? immu-
noprecipitated from thrombin-stimulated cells showed increased
phosphorylation of GST-I?B? compared with IKK? from control
cells (Fig. 4C), indicative of activation of IKK?. Inhibition of ROCK
by Y-27632 prevented thrombin-induced IKK? activation (Fig. 4C).
The above data led us to investigate whether IKK? activated by
RhoA/ROCK catalyzes I?B? phosphorylation (Ser32and Ser36),
and its degradation after thrombin challenge of endothelial cells.
The results showed that inhibition of the RhoA/ROCK pathway
interfered with the ability of thrombin to induce I?B? phosphor-
ylation, and consequently its degradation (Fig. 4D and Fig. 5, A
and B). Because I?B? degradation results in nuclear localization
and DNA binding of NF-?B, we determined whether inhibition of
RhoA/ROCK would lead to inhibition of these events. We found
that inhibition of the RhoA/ROCK pathway abrogated thrombin-
induced nuclear translocation and the DNA binding function of
nuclear NF-?B (Fig. 5, C and D). In contrast, RhoA/ROCK inhi-
bition had no effect on TNF-?-induced I?B? degradation and
DNA binding activity of nuclear NF-?B (Fig. 5, B–D).
Inhibition of ROCK prevents thrombin-induced RelA/p65
Because phosphorylation of Ser536in the transactivation domain 1
of RelA/p65 regulates transcriptional activity of NF-?B in the nu-
cleus (20, 44, 45), we addressed the possibility that RhoA/ROCK
combination with the constructs encoding the kinase-defective mutant of IKK? and the constitutively active RhoA mutant (RhoAcat) using Superfect as
described in Materials and Methods. pcDNA3 was used as the vector alone control. Twenty-four hours later, cell extracts were prepared and assayed for
firefly and Renilla luciferase activities. Firefly luciferase activity normalized to Renilla luciferase activity is expressed as the fold increase relative to the
pcDNA3 alone control value. Values shown are the mean ? SE of three experiments performed in triplicate. ?, p ? 0.01 vs pcDNA3 alone control; #, p ?
0.01 vs RhoAcatcontrol. B, HUVEC were cotransfected with 1393-bp ICAM-1LUC construct in combination with the construct encoding RhoAcatusing
DEAE-dextran as described in Materials and Methods. pcDNA3 was used as the vector alone control. At 24 h, cell extracts were prepared and assayed
for firefly and Renilla luciferase activities. The data are expressed as firefly/Renilla luciferase activity. Results are representative of two experiments
performed in triplicate. Bars indicate the mean ? SD. ?, p ? 0.05 vs vector control. C, Confluent HUVEC monolayers were pretreated with Y-27632 (10
?M) for 1 h before challenge with thrombin (5 U/ml) for 1 h. Cell lysates were immunoprecipitated with an Ab to IKK?, and in vitro kinase assays were
conducted on immunoprecipitates using GST-I?B? as an exogenous substrate. Proteins were analyzed by SDS-PAGE and transferred to the membrane, and the
phosphorylated form of GST-I?B? was detected by autoradiography. The blot was subsequently immunoblotted with an Ab to IKK?. The autoradiogram and the
corresponding bar graph showing the IKK? activity are representative of two separate experiments. D, Confluent HUVEC monolayers were pretreated with
Y-27632 (10 ?M) for 1 h before challenge with thrombin (5 U/ml) for 1 h. Total cell lysates (10 ?g/lane) were separated by SDS-PAGE and immunoblotted with
an Ab to the phosphorylated (Ser32and Ser36) form of I?B?. The blots were subsequently stripped and reprobed with an Ab to RelA/p65 to verify equal loading
of the gel. The blot and the corresponding bar graph showing the relative I?B? phosphorylation are representative of two separate experiments.
RhoA/ROCK signals thrombin-induced NF-?B activity by activating IKK?. A, HUVEC were cotransfected with NF-?B-LUC construct in
6969The Journal of Immunology
contributes to NF-?B activity by controlling thrombin-induced
phosphorylation of RelA/p65. Western blot analysis showed that
thrombin challenge of HUVEC resulted in Ser536phosphorylation
of RelA/p65, and inhibition of RhoA/ROCK after pretreatment of
cells with Y-27632 prevented this response (Fig. 6).
We have previously shown that thrombin promotes endothelial
adhesiveness toward PMN by a mechanism involving the expres-
sion of ICAM-1 via a NF-?B-dependent pathway (4). In this study
we provide evidence that activation of the RhoA/ROCK pathway
plays an essential role in signaling the thrombin-induced ICAM-1
expression in endothelial cells. Our data demonstrate that the
RhoA/ROCK pathway mediates ICAM-1 expression by promoting
NF-?B binding to the ICAM-1 promoter by phosphorylation and
degradation of I?B? as well as enhancing the transactivation ca-
pacity of the bound NF-?B through Ser536phosphorylation of
RelA/p65. Interestingly, RhoA/ROCK mediated NF-?B activation
and ICAM-1 expression in response to thrombin, but not TNF-?,
the prototypic inducer of NF-?B activation and ICAM-1 expres-
sion. These data show that the RhoA/ROCK pathway activates
NF-?B-dependent ICAM-1 expression in endothelial cells in an
agonist-specific manner, possibly secondary to activation of the G
protein-coupled receptor, protease-activated receptor-1, that is li-
gated by thrombin in endothelial cells (4, 10).
We used pharmacological and genetic approaches to address the
role of the RhoA/ROCK pathway in mediating NF-?B activation
and ICAM-1 expression after thrombin challenge. We used Clos-
tridium botulinum C3 exoenzyme, specifically inactivates Rho by
ADP-ribosylating Asn41in its effector domain (33, 34), to inves-
tigate whether inhibition of RhoA influences ICAM-1 expression.
Inhibiting Rho activity markedly reduced thrombin-induced
ICAM-1 expression. These data led us to investigate whether
RhoA signals thrombin-induced ICAM-1 expression through the
activation of the RhoA kinase ROCK, the downstream effector of
RhoA (26, 27, 37). Pretreatment of cells with Y-27632, the pyri-
dine-derived smooth muscle relaxant that selectively inhibits
ROCK (38), reproduced the effect of RhoA inhibition on ICAM-1
expression induced by thrombin. We have previously shown that
thrombin induces ICAM-1 expression via a G?q/PKC?-dependent
mechanism (5, 10). A recent study has also reported the role of
PKC? in mediating thrombin-induced RhoA activation in endo-
thelial cells (46). These and other studies (47, 48) suggest that a
linkage between the G?q/PKC? and RhoA/ROCK pathways sig-
nals ICAM-1 expression in response to thrombin challenge of en-
dothelial cells. That the G?q/PKC? and RhoA/ROCK signaling
pathway is important in mediating the thrombin response finds
further support from our observation that inhibition of ROCK
mimicked the effects PKC? inhibition on ICAM-1 expression. We
also observed that ICAM-1 protein expression was inhibited to a
greater extent than NF-?B activity or ICAM-1 mRNA, raising the
possibility that ROCK may regulate ICAM-1 expression at post-
transcriptional and translational levels.
and NF-?B DNA binding activity. HUVEC transfected with C3 transferase
(A and C) or pretreated with Y-27632 (10 ?M; B and D) were stimulated
with thrombin (5 U/ml) for 1 h or with TNF-? (100 U/ml) for 20 min where
indicated. Cytoplasmic (A and B) and nuclear extracts (C and D) were
prepared and assayed for I?B? degradation by Western blot analysis (A
and B) and for NF-?B DNA binding activity by EMSA (C and D) as
described in Materials and Methods. The blot and the corresponding bar
graph are representative of two separate experiments.
RhoA/ROCK signals thrombin-induced I?B? degradation
6970STIMULUS-SPECIFIC ENDOTHELIAL ICAM-1 REGULATION BY RhoA/ROCK
We evaluated in parallel studies the effects of RhoA/ROCK in-
hibition on ICAM-1 expression after TNF-? challenge of endo-
thelial cells. These results showed that inhibition of the RhoA/
ROCK pathway failed to prevent TNF-?-induced ICAM-1
expression in these cells. Studies have shown that RhoA/ROCK is
required for TNF-?-induced reorganization of the actin cytoskel-
eton and endothelial cell apoptosis (35, 36, 49). Thus, the lack of
involvement of the RhoA/ROCK pathway in mediating TNF-?-
induced ICAM-1 expression cannot be ascribed to the absence of
RhoA/ROCK activation by TNF-? in endothelial cells. The rea-
sons for the different effects of RhoA/ROCK inhibition on throm-
bin- vs TNF-?-induced ICAM-1 expression are not clear. A pos-
sible explanation is that there may be distinct mechanisms of
thrombin and TNF-? activation of RhoA/ROCK as well as a spe-
cialized set of downstream effectors activated by each agonist. An
indication of this difference is that although TNF-? induces
ICAM-1 by a PKC?-dependent mechanism, thrombin, as described
above, mediates this response via a PKC?-dependent pathway (5,
50). Although PKC? has been implicated in thrombin-induced
RhoA activation (46), the role of PKC? in signaling TNF-?-in-
duced RhoA activation in endothelial cells is not known. It is pos-
sible that PKC?-dependent activation of RhoA/ROCK by throm-
bin targets it to an intermediate protein, which, in turn, links it to
the NF-?B signaling pathway, whereas RhoA/ROCK activated by
TNF-? interacts with other downstream targets mediating re-
sponses such as endothelial cell apoptosis (49).
In the present study we addressed the mechanism by which the
RhoA/ROCK pathway regulates ICAM-1 expression in response
to thrombin challenge of endothelial cells. We observed that
thrombin-induced NF-?B-dependent reporter activity was signifi-
cantly reduced in cells pretreated with either C3 exoenzyme or
Y-27632. The expression of dominant negative RhoA mutant also
prevented NF-?B-dependent reporter activity induced by throm-
bin. However, inhibition of the RhoA/ROCK pathway using these
approaches failed to inhibit the TNF-?-induced, NF-?B-dependent
reporter activity. We also used the constitutively active RhoA mu-
tant (RhoAcat) to examine whether activation of RhoA is sufficient
to induce NF-?B activity in endothelial cells. The expression of
RhoAcatinduced NF-?B activity as well as ICAM-1 promoter ac-
tivation in the absence of thrombin challenge. In other experiments
the expression of kinase-defective IKK? mutant (IKK?mut) pre-
vented the RhoAcat-induced NF-?B activity. These data indicate
that the RhoA/ROCK pathway acts upstream of IKK? in signaling
thrombin-induced NF-?B activation in endothelial cells. Consis-
tent with the crucial involvement of IKK?, we also showed that
RhoA/ROCK-mediated I?B? phosphorylation and degradation re-
sulted in the migration of NF-?B to the nucleus, where its binding
to the promoter activated ICAM-1 gene transcription. Cammarano
and Minden (24) have reported that RhoA activates NF-?B in
NIH-3T3 cells in the absence of IKK stimulation. It is not clear
what determines IKK-dependent vs -independent activation of
NF-?B by RhoA in endothelial compared with NIH-3T3 cells. It is
also not known whether ROCK is involved in RhoA activation of
NF-?B in NIH-3T3 cells. Recently, Kato et al. (51) showed that
IKK-independent activation of NF-?B by UV radiation is medi-
ated by CK2 (formerly casein kinase II) through C-terminal phos-
phorylation of I?B?. Thus, it is possible that RhoA interacts with
different downstream targets depending upon the cell type and
stimulus, which, in turn, can dictate whether NF-?B activation
occurs by an IKK-dependent or -independent mechanism.
Recent studies have established that signal-induced phosphory-
lation of RelA/p65 is an additional regulatory pathway activated in
parallel with I?B? degradation, and that it plays an important role
in conferring transcriptional competency to DNA-bound NF-?B
(16–20). Our results show that thrombin induced the phosphory-
lation of p65/RelA at Ser536in transactivation domain 1 mediated
by the RhoA/ROCK-dependent pathway. Because IKK? is impli-
cated in phosphorylating RelA/p65 at Ser536(20, 44, 45), it is
possible that the RhoA/ROCK-mediated activation of IKK? cat-
alyzes Ser536phosphorylation of RelA/p65 and thus renders bound
NF-?B transcriptionally competent. This is in addition to the other
function of Rho/ROCK discussed above to induce I?B? phosphor-
ylation and promote NF-?? binding to DNA. However, given that
the same serine residue of RelA/p65 can be phosphorylated by
more than one kinase (16, 17, 44, 45), the role of other kinases in
this response cannot be excluded. We have shown that p38 MAPK
also contributes to thrombin-induced ICAM-1 expression by in-
ducing the transactivation capacity of RelA/p65 bound to ICAM-1
promoter (5). Others have shown that p38 MAPK signals down-
stream of IKK to induce transcriptional activation of RelA/p65
(52). It remains to be determined whether RhoA/ROCK-dependent
Ser536phosphorylation of RelA/p65 by thrombin is directly me-
diated by IKK? or it requires the participation of p38 MAPK.
In summary, the present study has identified the important role
of the RhoA/ROCK pathway in regulating thrombin-induced
NF-?B activation and ICAM-1 expression in endothelial cells. The
role of RhoA/ROCK in the thrombin response is distinct because
TNF-?-mediated NF-?B activation and ICAM-1 occur indepen-
dently of this mechanism. Thus, the specific targeting of the RhoA/
ROCK pathway may be a useful strategy for preventing the throm-
bin-activated inflammatory response associated with intravascular
We are grateful to Alan Hall for kindly providing the RhoA constructs
used, and to Anser Azim for useful discussions.
1. Springer, T. A. 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell 76:301.
2. Smith, C. W., S. D. Marlin, R. Rothlein, C. Toman, and D. C. Anderson. 1989.
Cooperative interactions of LFA-1 and Mac-1 with intercellular adhesion mole-
cule-1 in facilitating adherence and transendothelial migration of human neutro-
phils in vitro. J. Clin. Invest. 83:2008.
tion of RelA/p65. Confluent HUVEC monolayers were pretreated with
Y-27632 (10 ?M) for 1 h before challenge with thrombin (5 U/ml) for 1 h.
Total cell lysates (10 ?g/lane) were separated by SDS-PAGE and immu-
noblotted with an Ab against the phosphorylated (Ser536) form of RelA/
p65. The blots were subsequently stripped and reprobed with an Ab RelA/
p65 to verify equal loading of the gel. The blot and the corresponding bar
graph showing the relative p65 phosphorylation are representative of two
ROCK signals thrombin-induced serine 536 phosphoryla-
6971The Journal of Immunology
3. Doerschuk, C. M., S. Tasaka, and Q. Wang. 2000. CD11/CD18-dependent and Download full-text
-independent neutrophil emigration in the lungs: how do neutrophils know which
route to take? Am. J. Respir. Cell Mol. Biol. 23:133.
4. Rahman, A., K. N. Anwar, A. L. True, and A. B. Malik. 1999. Thrombin-induced
p65 homodimer binding to downstream NF-?B site of the promoter mediates
endothelial ICAM-expression and neutrophil adhesion. J. Immunol. 162:5466.
5. Rahman, A., K. N. Anwar, S. Uddin, N. Xu, R. D. Ye, L. C. Platanias, and
A. B. Malik. 2001. Protein kinase C-? regulates thrombin-induced ICAM-1 gene
expression in endothelial cells via activation of p38 mitogen-activated protein
kinase. Mol. Cell. Biol. 21:5554.
6. Minami, T., A. Sugiyama, S. Q. Wu, R. Abid, T. Kodama, and W. C. Aird. 2004.
Thrombin and phenotypic modulation of the endothelium. Arterioscler. Thromb.
Vasc. Biol. 24:41.
7. Fenton, J. W. 1981. Thrombin specificity. Ann. NY Acad. Sci. 370:468.
8. Dickneite, G., and E. P. Paques. 1993. Reduction of mortality with antithrombin
III in septicemic rats: a study of Klebsiella pneumoniae induced sepsis. Thromb.
9. Aird, W. C. 2003. The role of the endothelium in severe sepsis and multiple organ
dysfunction syndrome. Blood 101:3765.
10. Rahman, A., A. L. True, K. N. Anwar, R. D. Ye, T. A. Voyno-Yasenetskaya, and
A. B. Malik. 2002. G?q and G?? regulate PAR-1-induced NF-?B activation and
ICAM-1 transcription in endothelial cells. Circ. Res. 91:398.
11. Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-?B puzzle. Cell
12. Baldwin, A. S. 1996. The NF-?B and I-?B proteins: new discoveries and insights.
Annu. Rev. Immunol. 14:649.
13. Israel, A. 2000. The IKK complex: an integrator of all signals that activate NF-
?B? Trends Cell Biol. 10:129.
14. Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the
control of NF-?B activity. Annu. Rev. Immunol. 18:621.
15. Ben-Neriah, Y. 2002. Regulatory functions of ubiquitination in the immune sys-
tem. Nat. Immunol. 3:20.
16. Zhong, H., M. J. May, E. Jimi, and S. Ghosh. 2002. The phosphorylation status
of NF-?B determines its association with CBP/p300 or HDAC-1. Cell. 9:625.
17. Vermeulen, L., G. De Wilde, P. Van Damme, W. Vanden Berghe, and
G. Haegeman. 2003. Transcriptional activation of the NF-?B p65 subunit by
mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J. 22:1313.
18. Duran, A., M. T. Diaz-Meco, and J. Moscat. 2003. Essential role of RelA Ser311
phosphorylation by ?PKC in NF-?B transcriptional activation. EMBO J. 22:3910.
19. Wang, D., S. D. Westerheide, J. L. Hanson, and A. S. Baldwin. 2000. Tumor
necrosis factor ?-induced phosphorylation of RelA/p65 on Ser529is controlled by
casein kinase II. J. Biol. Chem. 275:32592.
20. Sakurai, H., H. Chiba, H. Miyoshi, T. Sugita, and W. Toriumi. 1999. I?B kinases
phosphorylate NF-?B p65 subunit on serine 536 in the transactivation domain.
J. Biol. Chem. 274:30353.
21. Etienne-Manneville, S., and A. Hall. 2002. Rho GTPases in cell biology. Nature
22. Scita, G., P. Tenca, E. Frittoli, A. Tocchetti, M. Innocenti, G. Giardina, and
P. P. Di Fiore. 2000. Signaling from Ras to Rac and beyond: not just a matter of
GEFs. EMBO J. 19:2393.
23. Hill, C. S., J. Wynne, and R. Treisman. 1995. The Rho family GTPases RhoA,
Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81:1159.
24. Cammarano, M. S., and A. Minden. 2001. Dbl and the Rho GTPases activate
NF?B by I?B kinase (IKK)-dependent and IKK-independent pathways. J. Biol.
25. Montaner, S., R. Perona, L. Saniger, and J. C. Lacal. 1999. Activation of serum
response factor by RhoA is mediated by the nuclear factor-?B and C/EBP tran-
scription factors. J. Biol. Chem. 274:8506.
26. Kaibuchi, K., S. Kuroda, and M. Amano. 1999. Regulation of the cytoskeleton
and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev.
27. Riento, K., and A. J. Ridley. 2003. Rocks: multifunctional kinases in cell behav-
iour. Nat. Rev. Mol. Cell Biol. 4:446.
28. Mehta, D., A. Rahman, and A. B. Malik. 2001. Protein kinase C-? signals Rho-
guanine nucleotide dissociation inhibitor phosphorylation and Rho activation and
regulates the endothelial cell barrier function. J. Biol. Chem. 276:22614.
29. Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, and T. A. Springer.
1988. Primary structure of ICAM-1 demonstrates interaction between members
of immunoglobulin and integrin supergene families. Cell 54:925.
30. Voraberger, G., R. Schafer, and C. Stratowa. 1991. Cloning of the human gene
for intercellular adhesion molecule-1 and analysis of its 5?-regulatory region.
J. Immunol. 147:2777.
31. Lopata, M. A., D. W. Cleveland, and B. Sollner-Webb. 1984. High level of
transient expression of a chloramphenicol acetyl transferase gene by DEAE-dex-
tran mediated DNA transfection coupled with a dimethyl sufoxide or glycerol
shock treatment. Nucleic Acids Res. 12:5707.
32. Hou, J., V. Baichwal, and Z. Cao. 1994. Regulatory elements and transcription
factors controlling basal and cytokine-induced expression of the gene encoding
ICAM-1. Proc. Natl. Acad. Sci. USA 91:11641.
33. Sekine, A., M. Fujiwara, and S. Narumiya. 1989. Asparagine residue in the rho
gene product is the modification site for botulinum ADP-ribosyltransferase.
J. Biol. Chem. 264:8602.
34. Yamamoto, M., N. Marui, T. Sakai, N. Morii, S. Kozaki, K. Ikai, S. Imamura,
S. Narumiya. 1993. ADP-ribosylation of the rhoA gene product by botulinum C3
exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell
cycle. Oncogene 8:1449.
35. Wojciak-Stothard, B., A., Entwistle, R. Garg, and A. J. Ridley. 1998. Regulation
of TNF-?-induced reorganization of the actin cytoskeleton and cell-cell junctions
by Rho, Rac, and Cdc42 in human endothelial cells. J. Cell Physiol. 176:150.
36. Wojciak-Stothard, B., L. Williams,and A. J. Ridley. 1999. Monocyte adhesion
and spreading on human endothelial cells is dependent on Rho-regulated receptor
clustering. J. Cell Biol. 145:1293.
37. Van Aelst, L., and C. D’Souza-Schorey. 1997. Rho GTPases and signaling net-
works. Genes Dev. 11:2295.
38. Uehata, M., T. Ishizaki, H. Satoh, T. Ono, T. Kawahara, T. Morishita,
H. Tamakawa, K. Yamagami, J. Inui, M. Maekawa, et al. 1997. Calcium sensi-
tization of smooth muscle mediated by a Rho-associated protein kinase in hy-
pertension. Nature 389:990.
39. Brown, K. S., S. Gerstberger, L. Carlson, G. Franzoso, and U. Siebenlist. 1995.
Control of I??? proteolysis by site-specific, signal-induced phosphorylation. Sci-
40. Traenckner, EB-M., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, and
P. A. Baeuerle. 1995. Phosphorylation of human I?B? on serines 32 and 36
controls I?B? proteolysis and NF-?B activation in response to diverse stimuli.
EMBO J. 14:2876.
41. Zandi, E., D. M. Rothwarf, M. Delhase, M. Hayakawa, and M. Karin. 1997. The
I?B kinase complex (IKK) contains two kinase subunits, IKK? and IKK?, nec-
essary for I?B phosphorylation and NF-?B activation. Cell 91:243.
42. Delhase, M., M. Hayakawa, Y. Chen, and M. Karin. 1999. Positive and negative
regulation of I?B kinase activity through IKK? subunit phosphorylation. Science
43. Li, Q., D. Van Antwerp, F. Mercurio, K. F. Lee, and I. M. Verma. 1999. Severe
liver degeneration in mice lacking the I?B kinase 2 gene. Science 284:321.
44. Jiang, X., N. Takahashi, N. Matsui, T. Tetsuka, and T. Okamoto. 2003. The
NF-?B activation in lymphotoxin ? receptor signaling depends on the phosphor-
ylation of p65 at serine 536. J. Biol. Chem. 278:919.
45. Yang, F., E. Tang, K. Guan, and C. Y. Wang. 2003. IKK? plays an essential role
in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide.
J. Immunol. 170:5630.
46. Harrington, E. O., J. L. Brunelle, C. J. Shannon, E. S. Kim, K. Mennella, and
S. Rounds. 2003. Role of protein kinase C isoforms in rat epididymal microvas-
cular endothelial barrier function. Am. J. Respir. Cell Mol. Biol. 28:626.
47. Chikumi, H., J. Vazquez-Prado, J. M. Servitja, H. Miyazaki, and J. S. Gutkind.
2002. Potent activation of RhoA by G?q and Gq-coupled receptors. J. Biol.
48. Vogt, S., R. Grosse, G. Schultz, and S. Offermanns. 2003. Receptor-dependent
RhoA activation in G12/G13-deficient cells: genetic evidence for an involvement
of Gq/G11. J. Biol. Chem. 278:28743.
49. Petrache, I., M. T. Crow, M. Neuss, and J. G. Garcia. 2003. Central involvement
of Rho family GTPases in TNF-?-mediated bovine pulmonary endothelial cell
apoptosis. Biochem. Biophys. Res. Commun. 306:244.
50. Rahman, A., K. N. Anwar, and A. B. Malik. 2000. Protein kinase C-zeta mediates
TNF-?-induced ICAM-1 gene transcription in endothelial cells. Am. J. Physiol.
51. Kato, T., Jr., M. Delhase, A. Hoffmann, and M. Karin. 2003. CK2 Is a C-terminal
I?B kinase responsible for NF-?B activation during the UV response. Mol. Cell
52. Madrid, L. V., M. W. Mayo, J. Y. Reuther, and A.S. Baldwin, Jr. 2001. Akt
stimulates the transactivation potential of the RelA/p65 subunit of NF-?B through
utilization of the I?B kinase and activation of the mitogen-activated protein ki-
nase p38. J. Biol. Chem. 276:18934.
6972 STIMULUS-SPECIFIC ENDOTHELIAL ICAM-1 REGULATION BY RhoA/ROCK