The Tyrosine Kinase Pyk2 Mediates Lipopolysaccharide-Induced
IL-8 Expression in Human Endothelial Cells1
Appakkudal R. Anand,* Magali Cucchiarini,†Ernest F. Terwilliger,†and Ramesh K. Ganju2*
Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis.
However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a
nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed
a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the
catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9
significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity
blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the
inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates
Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step
downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in
LPS-induced IL-8 production in endothelial cells. The Journal of Immunology, 2008, 180: 5636–5644.
inflammatory agents during local and systemic inflammation (1,
2). Endothelial cells express chemokines that initiate the activation
and recruitment of circulating leukocytes at sites of tissue inflam-
mation (3, 4). The bacterial endotoxin, LPS, an essential compo-
nent of the surface of Gram-negative bacteria (5), has potent proin-
flammatory properties by acting on many cell types including
endothelial cells (3, 6). High levels of LPS are a major cause of
Gram-negative septic shock, in which LPS induces numerous
changes, including up-regulation of adhesion molecules as well as
procoagulant activity, enhanced endothelial permeability, and se-
cretion of proinflammatory mediators by the endothelium (3, 4, 7).
Among these activities, secretion of IL-8 (CXCL8) by LPS-acti-
vated endothelial cells contributes substantially to inflammatory
responses (8). IL-8 displays a chemotactic activity for neutrophils,
which are the first line of immune cells to be recruited to infected
areas (9, 10). IL-8 also activates neutrophils to generate several
toxic products, such as arachidonic acid metabolites. Thus, during
septicemia, IL-8 participates in a series of cellular events that se-
verely damage the endothelium and surrounding tissues. However,
the precise mechanism of LPS-induced signaling that leads to IL-8
secretion in endothelial cells is poorly understood. Elucidation of
this pathway may be of immense significance in the design of
he endothelium lines the inner surface of blood vessels
and functions as an interactive barrier between blood and
tissue. Exposed to blood flow, it is the primary target for
anti-inflammatory therapies that act by regulating chemokine re-
sponses leading to septic shock-related events.
The proline-rich kinase 2, Pyk2, also known as RAFTK or
CAK?, is a cytoplasmic tyrosine kinase related to focal adhesion
kinase (FAK)3(11). However, unlike FAK, Pyk2 exhibits a more
restricted tissue expression pattern primarily in epithelial cells,
neuronal cells, fibroblasts, hematopoietic cells, and endothelial
cells (11–13). Pyk2 has been shown to be activated in response to
a broad range of stimuli, including extracellular signals that elevate
intracellular Ca2?concentration, agonists of G protein-coupled re-
ceptors, and engagement of Ag receptors on T cells, B cells, and
mast cells (12, 14–16). Pyk2 is also activated in response to in-
flammatory cytokines, stress signals, and integrin-mediated cell
adhesion (12, 17, 18). It is fast emerging as a critical “platform”
kinase that couples several receptors (including integrin and che-
mokine receptors) with a variety of downstream effectors, thus
regulating various functions such as cell adhesion, migration, pro-
liferation, and survival (11). Although the expression of Pyk2 has
been identified in endothelial cells, very few reports have focused
on its role in these cells. In view of recent studies that link the
nonreceptor tyrosine kinase Pyk2 with inflammation (19, 20), our
present study evaluated its role in mediating IL-8 secretion in LPS-
stimulated endothelial cells.
Materials and Methods
Reagents, cells, and culture conditions
LPS and cyclohexamide were obtained from Sigma-Aldrich. The Pyk2
inhibitor (Tyrphostin A9), p38 MAPK inhibitor (SB203580), ERK kinase
inhibitor (PD98059), Pam3Cys (TLR2 agonist), and polymyxin B were
obtained from Calbiochem. Py99, Pyk2, phospho-Pyk2, and phospho-FAK
Abs were obtained from BioSource International, whereas TLR2, TLR4,
IL-8, phospho-ERK, ERK, phospho-p38, and p38 Abs were obtained from
Santa Cruz Biotechnology. Isotype controls were purchased from BD
Transduction Laboratories. HUVEC were purchased from Clonetics. Cells
were grown at 37°C in 5% CO2in endothelial growth medium EGM2-MV
*Department of Pathology, Ohio State University Medical Center, Columbus, OH
43210; and†Division of Experimental Medicine, Beth Israel Deaconess Medical Cen-
ter, Harvard Medical School, Boston, MA 02115
Received for publication February 12, 2008. Accepted for publication February
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 is supported in part by Grants AI49140 and CA109527 from the National
Institutes of Health (to R.K.G.).
2Address correspondence and reprint requests to Dr. Ramesh K. Ganju, Department
of Pathology, Ohio State University Medical Center, 1645 Neil Avenue, 166 Ham-
ilton Hall, Columbus, OH 43210. E-mail address: Ramesh.Ganju@osumc.edu
3Abbreviations used in this paper: FAK, focal adhesion kinase; siRNA, small inter-
fering RNA; AAV, adeno-associated virus; EBM, endothelial basal medium; TEM,
transendothelial migration; ?-Gal, ?-galactosidase.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
containing 2% FBS, 12 ?g/ml bovine brain extract, 10 ng/ml human re-
combinant epidermal growth factor, 1 ?g/ml hydrocortisone, and GA-1000
(1 ?g/ml gentamicin and amphotericin B), according to the recommenda-
tions of the supplier. The human monocytic THP-1 cell line obtained from
the American Type Culture Collection was cultured in RPMI 1640 medium
supplemented with 10% FBS at 37°C in 5% CO2atmosphere.
In all experiments, HUVEC were grown to 80% confluency in 6-well assay
plates. The cells were stimulated with LPS in the presence of 0.5% FBS.
In the case of inhibitor treatments (Tyrphostin A9, SB203580, PD98059),
HUVEC were pretreated with inhibitor for 1 h after which they were stim-
ulated with LPS for various time periods. The supernatant was used for the
IL-8 or transendothelial migration (TEM) assays, and the cell lysates were
used for the Western blotting and immunoprecipitation analyses. THP-1
cells were differentiated into macrophage-like cells by treating with 100
nM 1?,25-dihydroxy-vitamin D3(Biomol) for 3 days. Vitamin D3-differ-
entiated THP-1 cells were incubated with either Pam3Cys or LPS in the
presence or absence of TLR2 and TLR4 blocking Abs, respectively, for
24 h. After stimulation, the culture supernatants were collected and the
levels of IL-8 determined by ELISA.
Recombinant adeno-associated virus (AAV) transduction
High-efficiency gene delivery of the dominant negative Pyk2 mutant,
Pyk2K457A (Pyk2MT), or a control gene, ?-galactosidase (?-Gal), was
accomplished using an recombinant AAV-based method. The AAV vectors
were prepared as previously described (21). Briefly, the kinase-inactive
Pyk2 mutant or the same vector encoding ?-Gal cDNA (as a control for
nonspecific effects of viral infection) were inserted between a CMV-im-
mediate early promoter and an SV40 fragment (providing an intron as well
as a polyadenylation function) in one of our lab’s standard AAV vector
plasmids, pACP. Each gene cassette was framed between AAV2 inverted
terminal repeats. The vectors were then packaged in AAV2 virions using
a three-plasmid transient cotransfection system consisting of the vector
plasmids pXX2 and pXX6. Cell lysates were collected 2 days after trans-
fection, and the virions were isolated. The vector preps were then dialyzed
before use. Genomic titers of the preparations, as determined by real-time
PCR were ?1011copies/ml. Before being exposed to the virus, the HUVEC
were cultured overnight in complete medium. HUVEC were transduced by
at 37°C in a cell culture incubator. Equal volumes of complete endothelial
basal medium (EBM) containing 4% serum were added to the cells to
achieve a final serum concentration of 2%. The cells were finally cultured
for 36 h before being used for the experiments described later. After trans-
duction, LPS was added to the medium and the cells were incubated for an
additional 24 h. The culture supernatant was removed and evaluated for
IL-8 content. Alternatively, the cells were lysed and subjected to Western
blot analysis by using rabbit anti-human Pyk2 Ab or ?-Gal staining in the
case of the control. In each well, at least 80% of the HUVEC appeared to
be ?-Gal-positive, confirming the high efficiency of our recombinant AAV-
based approach (data not shown).
HUVEC were either unstimulated or stimulated for 24 h with LPS (100
ng/ml) in the presence or absence of 10 ?g/ml cycloheximide. Supernatants
were harvested and the IL-8 production measured by ELISA.
After stimulation, the culture supernatants were collected, centrifuged, and
processed for IL-8 quantification by commercially available ELISA kits
(Endogen), per the manufacturer’s instructions.
for the indicated periods of time (A) or with various concentrations of LPS for 15 min (B). The lysates were then immunoprecipitated with Abs to Pyk2.
The immunoprecipitates were analyzed by Western blotting with Abs to phosphotyrosine (Py99). The same blot was then probed with anti-Pyk2 Ab. C and
D, Lysates obtained from HUVEC stimulated with LPS (100 ng/ml) for various periods of time were also analyzed by Western blotting with Abs to
phospho-FAK (p-FAK) (C) or with Abs to phospho-Pyk2 (Tyr 402) and phospho-Pyk2 (Tyr 580) (D). E, The kinase activity in the immunoprecipitates
was assessed as the ability of Pyk2 to phosphorylate the synthetic substrate poly(Glu-Tyr)4:1. A typical autoradiograph is shown (117 kDa). (top). NRS,
Normal rabbit serum; PC, positive control. The total abundance of Pyk2 protein was analyzed by Western blot analysis of the lysates, as indicated (bottom).
Data show one representative experiment of three independent experiments performed.
LPS induces tyrosine phosphorylation and enzymatic activity of Pyk2 in endothelial cells. HUVEC were stimulated with LPS (100 ng/ml)
5637 The Journal of Immunology
Isolation of neutrophils
Human neutrophils were purified from normal donors by dextran sedimen-
tation and Ficoll gradient centrifugation followed by hypotonic lysis of
erythrocytes. The purity of the prepared neutrophils was ?95% (as judged
by the morphology of stained cytocentrifuged preparations) and the via-
bility was ?98% (as judged by the trypan blue dye exclusion method).
TEM assay of neutrophils
Briefly, ?100,000 HUVEC were added to fibronectin-coated 24-well Tran-
sculture chambers with a pore size of 3 ?m (Costar; Corning) and grown
for 3 days in 5% CO2at 37°C. A total of 0.6 ml of medium from the
untreated or LPS/Tyrphostin A9 or AAV/Pyk2MT-treated HUVEC was
added to the lower compartment. In the upper compartment, 1 ? 106neu-
trophils in 0.1 ml of the EBM containing 0.5% FBS were added onto the
HUVEC monolayer. Supernatants pretreated with 1000 U/ml polymyxin B
and 20 ?g/ml IL-8 Ab served as controls. The chambers were incubated for
4 h at 37°C in 5% CO2. The cells in the lower compartment were counted
on a hemocytometer. The results are presented as mean ? SD of three
separate experiments and are expressed as the increase in the number of
cells migrating toward the lower compartment.
Western blotting and immunoprecipitation
Total cellular extracts from the LPS-treated cells were prepared by lysing
the cells in radioimmunoprecipitation assay buffer (50 mM Tris-HCl (pH
7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM
PMSF, 10 ?g/ml aprotinin, leupeptin, and pepstatin, 10 mM sodium van-
adate, 10 mM sodium fluoride, and 10 mM sodium pyrophosphate). Pro-
teins (50 ?g) were size-fractionated by 8% SDS-PAGE and transferred
onto nitrocellulose membranes. The membranes were blocked for 2–3 h
with 5% nonfat milk and then incubated with the respective primary and
secondary Abs for 2–3 h each. The membranes were washed three to four
times for 15 min each with TBS and 0.05% Tween 20, and later developed
by chemiluminescence (ECL System; GE Healthcare).
For the immunoprecipitation, equal amounts of protein from the stim-
ulated time points were clarified by incubation with protein A-Sepharose
CL-4B or GammaBind Sepharose beads (Amersham Biosciences) for 1 h
AAV-expressing Pyk2 mutant (Pyk2MT) or AAV ?-Gal vector control (VC). Overexpression of the mutant was demonstrated by Western blot analysis
with anti-Pyk2 Abs 48 h after transduction (top left). Anti-actin Ab was used as an internal control (bottom left). Transduced cells were tested for their ability
to produce IL-8 upon LPS stimulation (100 ng/ml) using a commercial ELISA kit (Endogen) (right). ?, p ? 0.05 vs the AAV vector control (AAV-VC).
Data represent the mean ? SD of three independent experiments. B, HUVEC were pretreated with vehicle or Tyrphostin A9 (5 ?M) for 1 h at 37°C. The
cells were then stimulated with LPS for various time periods in EBM with 0.5% FBS. The cells were lysed and analyzed by Western blotting with
anti-phospho-Pyk2 (Tyr 402) Abs (left). The same blots were probed with anti-Pyk2 Abs. HUVEC preincubated with vehicle or various concentrations of
Tyrphostin A9 for 1 h were cultured without or with LPS (100 ng/ml) and the concentration of IL-8 in the culture supernatants was determined 24 h after
stimulation (right). ?, p ? 0.05 compared with the vehicle control. Data represent the mean ? SD of three independent experiments. C, HUVEC were
transfected with Pyk2-specific siRNA and nontargeting siRNA (NT siRNA) using Lipofectamine in the absence or presence of LPS (100 ng/ml). The knock
down of Pyk2 expression was analyzed by Western blotting with anti-Pyk2 Abs (left). Anti-actin Ab was used as an internal control. The supernatants from
the siRNA transfected cells were analyzed for IL-8 expression after stimulation with LPS (100 ng/ml) (right). ?, p ? 0.05 compared with the nontargeting
siRNA control. D, HUVEC were treated with LPS (100 ng/ml) in the absence or presence of 10 ?g/ml cyclohexamide (CX). After 24 h, the supernatants
were collected and analyzed for IL-8 expression. ?, p ? 0.05 compared with the LPS control. Data represent the mean ? SD of three independent
Inhibition of Pyk2 activation blocks LPS-induced IL-8 production in endothelial cells. A, HUVEC were transduced with recombinant
5638Pyk2 REGULATES IL-8 PRODUCTION IN ENDOTHELIUM
at 4°C. The Sepharose beads were removed by brief centrifugation, and the
supernatants were incubated with different primary Abs for 2 h at 4°C.
Immunoprecipitation of the antigen-antibody complexes was performed by
incubation at 4°C overnight with 50 ?l of protein A-Sepharose or Gam-
mabind Sepharose (50% suspension). Nonspecific interacting proteins were
removed by washing the beads thrice with radioimmunoprecipitation RIPA
buffer and once with PBS. Immunoprecipitated complexes were solubilized
in 50 ?l of 2X Laemmli buffer, and further analyzed by Western blotting,
Immune complex kinase assay
HUVEC were lysed in modified RIPA buffer and equal amounts of cell
lysates were immunoprecipitated with anti-Pyk2 Ab. The immunoprecipi-
tates were incubated for 12 min at 23°C with 50 ?l of Tris buffer containing
5 ?Ci [32P]ATP and 30 ?g/ml poly(Glu-Tyr)4:1. The reaction was stopped
by adding 2X SDS sample buffer and the32P-labeled immune complexes
were resolved on 7.5% SDS-PAGE. The gel was dried and the32P-labeled
proteins were made visible by autoradiography.
Cell surface staining and flow cytometry analysis
To detect TLR2 and TLR4 expression, HUVEC and THP-1 cells were
stained with the monoclonal primary Abs, respectively, followed by stain-
ing with FITC- or PE-conjugated secondary Abs. Flow cytometry was
conducted using a FACSCalibur cytometer and these data were analyzed
by CellQuest software (BD Biosciences).
Small interfering RNA (siRNA)-mediated knockdown of Pyk2
RNA interference-mediated knockdown of Pyk2 was performed using
SMARTpool Pyk2 duplex RNA oligonucleotides obtained from Dharma-
con. A nontargeting siRNA (Qiagen) was used as the control. HUVEC
were transfected with the siRNA using Lipofectamine 2000 reagent
(Invitrogen Life Technologies), according to the manufacturer’s in-
structions. Pyk2 siRNA-mediated knockdown was estimated by detec-
tion of Pyk2 expression by Western blot analysis, 48 h after the initial
Reported data are mean ? SD of at least three independent experiments
performed in duplicate or triplicate. The statistical significance was deter-
mined by the Student’s t test.
LPS induces the tyrosine phosphorylation of Pyk2, but not
FAK, in endothelial cells
To investigate the signaling processes involved in LPS-induced
cytokine production, we examined the ability of LPS to activate
Pyk2. Pyk2 is a nonreceptor tyrosine kinase that is expressed abun-
dantly in endothelial cells (22). Stimulation of endothelial cells
with LPS (100 ng/ml) was accompanied by increased tyrosine
phosphorylation of Pyk2, as demonstrated by immunoprecipitation
experiments. We observed that LPS induces both a concentration
and time-dependent tyrosine phosphorylation of Pyk2 (Fig. 1, A
and B). The increase in Pyk2 phosphorylation was observed with
as low as 10 ng/ml LPS and was most evident at 100 ng/ml LPS,
5–30 min after stimulation. Similar amounts of Pyk2 were immu-
noprecipitated in the samples, as demonstrated by blotting with
anti-Pyk2 Ab (Fig. 1, A and B, bottom). In contrast, LPS did not
induce an increase in FAK phosphorylation (Fig. 1C). We, there-
fore, focused the rest of our studies on Pyk2.
Pyk2 has a structure similar to FAK as it contains an autophos-
phorylation site (Tyr402), sites involved in kinase activation
(Tyr579/580), and a site homologous to the Grb2-binding site
(Tyr881) in FAK (11). Endothelial cells treated with LPS were
analyzed for the phosphorylation and activation of Pyk2 using Abs
specific for these sites of tyrosine phosphorylation. We observed
that LPS enhanced the phosphorylation of residues 402 and 580
(Fig. 1D), indicating an important function for these residues in
LPS-induced Pyk2 activation. However, LPS did not markedly
induce phosphorylation of residue 881 (data not shown).
LPS induces Pyk2 tyrosine kinase activity
To assess whether increased Pyk2 tyrosine phosphorylation leads
to the induction of Pyk2 enzyme activity, Pyk2 immunoprecipi-
tates from control or LPS-stimulated HUVEC were assayed for
(Tyr A9) and Pyk2 mutant-overexpressing (AAV-Pyk2MT) (A) or Pyk2-specific siRNA-treated (Pyk2 siRNA) and nontargeting siRNA (NT siRNA) (B)
HUVEC cells were stimulated with LPS (100 ng/ml) and tested for their ability to induce neutrophil migration using a TEM assay. ?, p ? 0.05 vs the vehicle
control. ??, p ? 0.05 compared with the AAV vector control (AAV-VC). ???, p ? 0.05 vs the nontargeting siRNA control. C, HUVEC pretreated with
various concentrations of polymyxin B were stimulated with 100 ng/ml LPS. The IL-8 content in the supernatants was measured after 24 h of stimulation.
?, p ? 0.05 vs the LPS control. D, Supernatants from untreated or LPS-treated HUVEC pretreated with IL-8 blocking Ab or an isotype control were also
tested for their ability to induce neutrophil migration across the endothelium using the TEM assay. ?, p ? 0.05 vs the isotype control. Data represent mean ?
SD of three independent experiments.
Inhibition of the LPS-induced production of IL-8 decreased the TEM of human neutrophils. Supernatants from the Tyrphostin A9-treated
5639The Journal of Immunology
Pyk2 activity based on its ability to phosphorylate a synthetic sub-
strate poly(Glu-Tyr)4:1in an in vitro kinase assay (Fig. 1E). As a
positive control, Pyk2 activity was stimulated with 100 ng/ml vas-
cular endothelial growth factor for 2 min. LPS significantly in-
creased the activation of Pyk2 and its tyrosine kinase activity
peaked at 15 min.
Inhibition of Pyk2 activation attenuates LPS-induced IL-8
Because Pyk2 is rapidly being recognized as a tyrosine kinase of
central importance in diverse cell types, we hypothesized that Pyk2
could be involved in the LPS signaling that mediates acute inflam-
matory responses in endothelium. Thus, we tested the possible role
of Pyk2 in LPS-induced cytokine production. Tyrphostin A9
(AG17) is the most selective of tyrosine kinase inhibitors that
block the Pyk2 signaling pathway (23), thus providing an effective
tool for investigating the role of this tyrosine kinase in cellular
signaling. Our initial studies testing the possible involvement of
Pyk2 in LPS-induced cytokine expression using Ab arrays dem-
onstrated that certain cytokines that are known to be up-regulated
on stimulation with LPS, such as GRO?, IL-1ra, IL-6, IL-8, IP-10,
and migration inhibitory factor, were inhibited by the specific Pyk2
inhibitor, Tyrphostin A9 (data not shown). Because IL-8 is the
prototypic CXC chemokine (9) and it contributes most signifi-
cantly to the inflammatory process in endothelial cells (7), we fur-
ther investigated the molecular mechanisms of Pyk2 in LPS-in-
duced IL-8 production.
We used three approaches to study the role of Pyk2 in regulating
IL-8 production: 1) expression of a dominant negative form of
Pyk2 that removes a key lysine involved in ATP binding, 2) use of
a specific chemical inhibitor, and 3) knockdown of Pyk2 protein
expression using siRNA.
As a first approach, we constructed AAV vectors expressing a
kinase-inactive Pyk2 mutant (Pyk2K457A) and then HUVEC were
transduced with this dominant negative Pyk2. In this mutant, mu-
tation of Lys457to alanine in the tyrosine kinase domain of Pyk2
creates an inactive Pyk2 that inhibits wild-type Pyk2 activity. We
observed that endothelial cells transduced with the catalytically
inactive Pyk2 mutant (AAV-Pyk2MT) exhibited significantly
attenuated LPS-induced IL-8 production as compared with the
HUVEC and THP-1 were stained us-
ing Abs against TLR4 (left) or TLR2
(right) and analyzed by flow cytom-
etry. Cells stained with control IgG
represent the Ab control (AbC). Ab
control (filled histogram) and TLR
expression (open histogram) are indi-
cated. B, HUVEC were pretreated
with anti-TLR4 or anti-TLR2 block-
ing Ab (10 ?g/ml) or isotype control
for 1 h at 37°C. The cells were then
stimulated with LPS (100 ng/ml) for
15 min in EBM with 0.5% FBS. The
cells were lysed and analyzed by
Western blotting with anti-phospho-
Pyk2 (Tyr 402) Abs (top). The same
blots were probed with anti-Pyk2 Abs
(bottom). C, HUVEC preincubated
with anti-TLR4 Ab, anti-TLR2 Ab, or
isotype control for 1 h were cultured
with or without LPS (100 ng/ml). The
concentration of IL-8 in the culture
supernatants was determined 24 h af-
ter stimulation. ?, p ? 0.05 compared
with the vehicle control. Data repre-
sent the mean ? SD of three indepen-
dent experiments. D, Vitamin D3-dif-
ferentiated THP-1 cells preincubated
with isotype control (Iso. Ab) or anti-
TLR2 Ab for 1 h were cultured with
or without Pam3Cys (10 pg/ml). Sim-
ilarly, THP-1 cells, preincubated with
isotype control (Iso. Ab) or anti-
TLR4 Ab for 1 h were cultured with
or without LPS (100 ng/ml). The con-
centration of IL-8 in the culture su-
pernatants was determined 24 h after
stimulation. ?, p ? 0.05 compared
with the isotype control. ??, p ? 0.05
compared with the isotype control.
5640Pyk2 REGULATES IL-8 PRODUCTION IN ENDOTHELIUM
vector control (?-Gal) transduced cells (Fig. 2A, right). The inhib-
itory effect was present over a range of LPS stimulations (10–100
ng/ml) (data not shown) with maximal inhibition occurring at 100
ng/ml LPS. It should be noted that a basal production of IL-8 was
always present in the supernatant. Overexpression of Pyk2MT in
HUVEC was confirmed by Western blot analysis of the cell lysates
(Fig. 2A, left). This result provides direct evidence for the role of
Pyk2 in LPS-induced IL-8 production.
We also demonstrated that Tyrphostin A9, a specific inhibitor of
Pyk2, inhibited LPS-induced Pyk2 phosphorylation in a time-de-
pendent manner (Fig. 2B, left). Furthermore, the inhibitor blocked
LPS-induced IL-8 expression in the endothelial cells in a concen-
tration-dependent manner (Fig. 2B, right).
As a third approach, we decreased Pyk2 expression in HUVEC
using siRNA Pyk2. Our optimization studies indicated that the
expression of Pyk2 protein was specifically decreased by transfec-
tion with the siRNA Pyk2 duplex (100 nM), but not by transfection
with a nontargeting siRNA (Fig. 2C, left). At this siRNA concen-
tration (100 nM), there was minimal loss of cell viability (?10%)
(data not shown). There was no difference in the Pyk2 knock down
in the absence or presence of LPS (100 ng/ml). The Pyk2 siRNA-
treated cells showed reduced IL-8 expression compared with cells
treated with nontargeting siRNA control (Fig. 2C, right). Taken
together, these data suggest that Pyk2-dependent activation is an
important part of the LPS-IL-8 signaling pathway.
Cycloheximide treatment at 10 ?g/ml completely blocked LPS-
induced IL-8 production, confirming that the up-regulation of IL-8
expression was due to de novo synthesis (Fig. 2D). To exclude a
possible toxic effect of cyclohexamide, cells that had been cultured
in its presence were washed and restimulated with LPS in the
absence of cyclohexamide, and IL-8 production was measured af-
ter 24 h. HUVEC were still able to respond to LPS (data not
Inhibition of Pyk2 activation reduces the LPS-induced TEM
IL-8 has been shown to be a potent mediator of the extravasation
and accumulation of neutrophils at the sites of injury by their ad-
hesion to and migration through the endothelial lining (24). Hence,
the TEM of neutrophils toward the supernatants of LPS-stimulated
endothelial cells transduced with AAV-Pyk2MT or pretreated with
Tyrphostin A9 was assessed to determine whether the IL-8 pro-
duced was functionally active. As shown, transduction of LPS-
stimulated endothelial cells with Pyk2MT and pretreatment with
Tyrphostin A9 significantly inhibited neutrophil migration across a
HUVEC monolayer compared with the supernatants of cells
treated with LPS only (Fig. 3A). To confirm that the carryover of
Tyrphostin A9 did not influence IL-8-mediated neutrophil migra-
tion, rIL-8 (100 ng/ml) was added to the supernatants with or with-
out Tyrphostin A9. There was no difference in IL-8-mediated
transmigration of neutrophils toward these supernatants with or
without Tyrphostin A9 (data not shown). In addition, we also ob-
served that supernatants from the Pyk2-specific siRNA-treated
cells showed attenuated neutrophil transmigration, in comparison
(5 ?M) for 1 h at 37°C. The cells were then stimulated with LPS for various periods of time in EBM with 0.5% FBS. The cells were lysed and analyzed
by Western blotting with anti-phospho-ERK1/2 (p-ERK1/2) Abs (upper left blots) or anti-phospho-p38 (p-38) Abs (lower left blots). The same blots were
probed with anti-ERK1/2 or anti-p38 Abs, respectively. Data show one representative experiment of three independent experiments. For quantitative
analysis of protein phosphorylation, the ratio of phosphorylation vs total protein in each lane was obtained by densitometry. The phosphorylation index of
ERK and p38 was determined by calculating the value of this ratio in each lane and presenting the ratio as the fold increase over the control value
(unstimulated sample; 0), which was designated as 1 (right). B, HUVEC pretreated with vehicle, the p38 MAPK inhibitor (SB203580, SB) (left) or the ERK
kinase inhibitor (PD98059, PD) (right) for 1 h were stimulated with 100 ng/ml LPS and then the concentration of IL-8 in the supernatants was measured
24 h after stimulation. ?, p ? 0.05 compared with the vehicle control. Data represent mean ? SD of three independent experiments.
Pyk2 regulates LPS-induced IL-8 expression through the p38 MAPK pathway. A, HUVEC was pretreated with vehicle or Tyrphostin A9
5641 The Journal of Immunology
to the supernatants of cells treated with nontargeting siRNA (Fig.
3B). The TEM of neutrophils toward any residual LPS present in
the supernatant was eliminated by pretreatment with polymyxin B.
Polymyxin B was used at a concentration of 1000 U because al-
most complete inhibition of LPS-induced IL-8 expression was
seen at this concentration (Fig. 3C).
To specifically test the role of IL-8 in migration, 20 ?g/ml hu-
man IL-8-specific goat Ab was added to the supernatants for 1 h
before the assay was initiated. The results demonstrated that anti-
IL-8 Ab blocked the neutrophil migration across the HUVEC
monolayer, which was induced by the LPS-treated supernatants
(Fig. 3D). These results confirmed that the secreted IL-8 in the
supernatants was functionally active and hence indicated a phys-
iological function of Pyk2 activity in regulating IL-8 production.
LPS-induced Pyk2 activation leading to IL-8 expression is
mediated by TLR4
Previous studies have indicated that TLR4, and to a certain extent
TLR2, are the main receptors for LPS in endothelial cells (25–27).
To investigate the involvement of TLR2 and TLR4 in LPS-respon-
sive HUVEC, we first analyzed the expression pattern of TLRs in
HUVEC by FACS analysis. TLR4 was detected at moderate levels
on the surface of HUVEC (Fig. 4A, top left). However, TLR2
expression was minimal (Fig. 4A, bottom left). THP-1, a mono-
cytic cell line was used as a positive control for expression of
TLR2 and TLR4 (Fig. 4A, right).
To further determine the functional role of TLR4 and TLR2 in
LPS-induced Pyk2 activation and IL-8 expression, the ability of
anti-TLR4- and anti-TLR2-neutralizing Abs to block these LPS-
induced effects was examined. At concentrations of 10 ?g/ml, anti-
TLR4 Ab significantly inhibited both the Pyk2 activation (Fig. 4B)
and IL-8 production (Fig. 4C) induced by LPS. However, anti-
TLR2 Ab did not have any effect. The functional ability of the
TLR2 and TLR4 blocking Ab was confirmed by inhibition of
Pam3Cys-induced IL-8 production and LPS-induced IL-8 produc-
tion, respectively, in vitamin D3-differentiated THP-1 cells (Fig.
4D). In THP-1 cells, Pam3Cys, an analog of bacterial lipoprotein
has been shown to induce IL-8 production via TLR2 (28), whereas
LPS has been shown to induce IL-8 production through TLR4.
(29). Taken together, these findings suggest that LPS requires cell
surface TLR4 to induce Pyk2 activation leading to IL-8 production
in endothelial cells.
Pyk2 regulates IL-8 production through the p38 MAPK pathway
Pyk2 has been shown to act as an essential intermediate providing
a link between extracellular stimuli and MAPK signaling pathways
in various cell lines (30). Of the signaling pathways activated by
LPS in endothelial cells, the p44/42 MAPK and p38 MAPK path-
ways have been shown to be directly involved in the production
of cytokines (31–34). Hence, we determined whether inhibition of
Pyk2 with Tyrphostin A9 blocked MAPK activation. Treatment of
HUVEC with LPS resulted in the phosphorylation of both ERK
and p38 MAPK, as reported previously (31–34). However, pre-
treatment with Tyrphostin A9 significantly inhibited the LPS-in-
duced p38 phosphorylation (Fig. 5A, bottom left), but did not sig-
nificantly influence ERK phosphorylation (Fig. 5A, top left),
suggesting that p38 MAPK may be an important downstream mol-
ecule of Pyk2 in LPS-induced IL-8 production. The phosphoryla-
tion indices of ERK and p38 MAPK are shown in Fig. 5A (right).
The involvement of the p38 MAPK pathway was confirmed by the
dose-dependent blocking of LPS-induced IL-8 with a specific in-
hibitor of p38 MAPK, SB203580 (Fig. 5B, left). Though the use of
a specific ERK kinase inhibitor, PD98059, reduced the LPS-in-
duced expression of IL-8 slightly, the difference was not statisti-
cally significant (Fig. 5B, right).
Pyk2 is rapidly becoming recognized as a tyrosine kinase of cen-
tral importance in diverse cell types such as neuronal cells, hema-
topoietic cells, liver epithelial cells, and vascular smooth muscle
cells (11, 12, 14, 35). However, little is known about Pyk2 regu-
lation in vascular endothelial cells. In this study, we present data
indicating that activation of Pyk2 plays a key role in modulating
LPS-induced IL-8 production in endothelial cells. We have dem-
onstrated Pyk2 activation by an increase in both tyrosine phos-
phorylation and enzyme activity of Pyk2. Though previous studies
on endothelial cells have shown that Pyk2 is tyrosine-phosphory-
lated in response to stimulation with G protein-coupled receptor of
agonists, vascular endothelial growth factor, and the cytokine
IL-1? (13, 36), this study is the first to our knowledge to demon-
strate the activation of Pyk2 in response to LPS in endothelial
The proline-rich kinase-2 Pyk2 is a cytoplasmic tyrosine kinase
showing considerable sequence homology and structural similarity
to FAK, including consensus motifs in the catalytic domain. De-
spite the high sequence homology, structural similarities, and com-
mon downstream effector molecules between FAK and Pyk2, re-
cent studies provide evidence of their different subcellular
localization and regulatory mechanisms (37, 38). In the present
study, LPS did not induce the tyrosine phosphorylation of FAK,
and therefore the activities of Pyk2 and FAK are likely to be reg-
ulated by different upstream molecules in LPS-stimulated endo-
Several lines of recent evidence suggest that Pyk2 tyrosine
phosphorylation could be closely linked to inflammatory processes
in multiple cell types (12, 13, 20). In endothelial cells, the LPS-
induced production of inflammatory cytokines has been reported to
contribute significantly to the inflammatory process (7). Prelimi-
nary cytokine array studies in our laboratory indicated IL-8 to be
among the most prominent chemokines attenuated in LPS-stimu-
lated endothelial cells treated with Tyrphostin A9 (data not
shown). Hence, we further investigated the molecular mechanism
by which Pyk2 regulates LPS-induced IL-8 production using
AAV-expressing a Pyk2 kinase-inactive mutant, Tyrphostin A9,
and Pyk2-specific siRNA. Using all three approaches, we were
able to demonstrate decreased LPS-induced IL-8 production, in
comparison to the controls. Overexpression of a catalytically in-
active form of Pyk2, Pyk2-K457A in pulmonary endothelial cells
has been demonstrated to result in defects in cell adhesion, spread-
ing and migration (39, 40). Although Pyk2-specific siRNA
blocked IL-8 expression compared with the nontargeted siRNA
control, the basal levels of IL-8 in the controls treated only with
siRNA were marginally higher than the levels in untreated control.
The higher basal levels of IL-8 produced on treatment with only
siRNA are in agreement with a recent study that shows that
siRNA may nonspecifically trigger the production of IL-8 (41).
The physiological relevance of these experiments was con-
firmed by the inhibition of human neutrophil transmigration to-
ward the supernatants of the LPS-treated HUVEC that were
transduced with AAV-Pyk2MT, pretreated with Tyrphostin A9
or transfected with Pyk2-specific siRNA, in comparison to the
LPS-treated controls, suggesting that Pyk2 modulates neutro-
phil infiltration across the endothelial cells via regulation of
Increasing evidence suggests that central to the recognition of
LPS expression in endothelial cells is a family of transmembrane
proteins known as TLRs that have leucine-rich repeats in their
5642Pyk2 REGULATES IL-8 PRODUCTION IN ENDOTHELIUM
extracellular domains (42, 43). Most effector cells of the innate
immune system, such as monocytes and endothelial cells express
TLR2 and TLR4 (26). Our studies on the expression of these two
receptors in HUVEC confirmed a previous report (26) that indi-
cated that HUVEC predominantly express TLR4 and weakly ex-
press TLR2. Using anti-TLR4 blocking Abs, we were able to dem-
onstrate the inhibition of both LPS-induced Pyk2 activation and
IL-8 production. From this demonstration, it seems reasonable to
suggest that TLR4 is an important part of the Pyk2 signaling path-
way, acting to transmit LPS-dependent signals that lead to IL-8
Pyk2 has been shown to function as an essential intermediate
providing a link between extracellular stimuli and signaling path-
ways involving MAPKs (30, 44). The central role of p38 MAPK-
dependent signaling for LPS-induced endothelial cell activation
has been highlighted in several recent studies (32, 33, 45). We
were able to demonstrate that p38 MAPK is an important part of
the signaling cascade downstream of Pyk2 in LPS-IL-8 pathway in
endothelial cells. Based on our data, we therefore propose a model
that suggests that, as a result of the interaction of LPS with TLR4
on the cell surface, a signaling route is initiated via Pyk2 and the
p38 MAPK pathway leading to the production of IL-8 (Fig. 6),
possibly through NF-?B activation. Activation of NF-?B, down-
stream to p38 MAPK, is known to be an essential prerequisite for
LPS-induced IL-8 expression in endothelial cells (33, 46).
In summary, we show that LPS induces Pyk2 activation in
endothelial cells. The specific inhibition of Pyk2 activity was
paralleled by a strong reduction in both LPS-induced IL-8 pro-
duction and neutrophil chemotaxis, pinpointing a key role of
Pyk2 in the LPS-induced production of the inflammatory cyto-
kine. Because LPS-induced IL-8 production is known to be in-
volved in the pathogenesis of sepsis and several inflammatory
diseases, these results indicate that Pyk2 may represent a novel
target for the development of innovative therapeutic strategies
against inflammatory conditions.
We thank Janet Delahanty for editing the manuscript.
The authors have no financial conflict of interest.
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5644 Pyk2 REGULATES IL-8 PRODUCTION IN ENDOTHELIUM