ORIGINAL RESEARCH PAPER
The p38 mitogen-activated protein kinases modulate endothelialcell survival and tissue repair
Nobuhiro Kanaji • Amy Nelson • Diane S. Allen-Gipson • Tadashi Sato •
Masanori Nakanishi • Xingqi Wang • YingJi Li • Hesham Basma •
Joel Michalski • Maha Farid • Stephen I. Rennard • Xiangde Liu
Received: 21 April 2011 / Revised: 17 October 2011 / Accepted: 11 November 2011 / Published online: 3 December 2011
� Springer Basel AG 2011
Abstract
Objective and design This study is designed to investi-
gate the role of p38 MAPK in modulating human
pulmonary artery endothelial cells (HPAECs) survival and
tissue repair functions.
Methods HPAECs (passage 8–12) were used for all
experiments. Cells were treated with IL-1b (0.5 or 2 ng/ml)
or p38 inhibitor (SB203580 or SB220025, 5 lM each).
Cells were also transfected with 50 nM siRNAs. Cell
length was measured using ImageJ software. Collagen gel
contraction and wound close assay were performed to
evaluate tissue repair functions.
Results IL-1b activated p38 MAPK and induced
morphologic change of HPAECs. The p38 inhibitors further
augmented IL-1b-induced cell morphologic change, pre-
vented cell death, and augmented collagen gel contraction.
Suppression of p38a, c, or d, but not p38b resulted in cell
morphologic alteration, and suppressing any one of p38
isoforms by siRNAs increased cell survival. Suppression of
p38a or d augmented gel contraction. While p38a sup-
pression stimulated cell migration, suppressing the rest of
three isoforms inhibit cell migration. Nuclear factor p65-
siRNA blocked IL-1b-induced cell morphologic change,
but did not affect p38 inhibitor-induced change.
Conclusion These findings suggest that p38 MAPK may
negatively modulate tissue repair functions of endothelial
cells via p65 independent pathway.
Keywords p38 � Interleukin-1 � Endothelial cells �Repair � Apoptosis
Introduction
Airway inflammation is believed to play an important role
in the development of a variety of lung diseases. Endo-
thelial cells are essential in normal development and
angiogenesis and also play an important role in inflam-
matory responses. In this regard, up-regulation of adhesion
molecules on endothelial cells causes leucocytes to attach
to the endothelium at sites of injuries and infection [1]. The
inflammatory cytokine interleukin (IL)-1b can induce these
endothelial cell responses [1]. IL-1b can also contribute to
development of tissue remodeling and fibrosis by modu-
lating endothelial cell functions [2]. IL-1b induces
endothelial cells to undergo a fibroblast-like morphologic
change and increases resistance to apoptosis [3].
Responsible Editor: Liwu Li.
N. Kanaji
Division of Endocrinology and Metabolism, Hematology,
Rheumatology and Respiratory Medicine, Kagawa University,
Kagawa, Japan
N. Kanaji � A. Nelson � D. S. Allen-Gipson � X. Wang �H. Basma � J. Michalski � M. Farid � S. I. Rennard � X. Liu (&)
Pulmonary, Critical Care, Sleep and Allergy Division,
University of Nebraska Medical Center, 985910 Nebraska
Medical Center, Omaha, NE 68198-5910, USA
e-mail: [email protected]
T. Sato
Department of Respiratory Medicine, Juntendo University
School of Medicine, Tokyo, Japan
M. Nakanishi
Third Department of Internal Medicine, Wakayama Medical
University School of Medicine, Wakayama, Japan
Y. Li
Department of Hygiene and Public Health, Nippon Medical
School, Tokyo, Japan
Inflamm. Res. (2012) 61:233–244
DOI 10.1007/s00011-011-0405-7 Inflammation Research
123
Mitogen-activated protein kinases (MAPKs) regulate
diverse cellular programs including embryogenesis, pro-
liferation, differentiation, migration and apoptosis [4].
Among more than a dozen MAPKs in mammals, p38 is
known to respond to a wide range of extracellular cues,
particularly cellular stressors, including growth factors and
inflammatory cytokines such as IL-1b [4, 5]. Recent studies
indicate that p38 MAPK plays a role in the development,
maintenance and/or exacerbation of a number of pulmon-
ary diseases, such as asthma, cystic fibrosis, idiopathic
pulmonary fibrosis, and chronic obstructive pulmonary
disease [6–10]. In addition, p38 MAPK has been reported
to be involved in regulating several critical functions of
endothelial cells including cell migration, vasodilatation,
angiogenesis and chemoattraction [11, 12].
The p38 MAPK family consists of four distinct isoforms,
i.e., p38a (MAPK14), p38b (MAPK11), p38c (MAPK12),
and p38d (MAPK13) [4]. The p38a and b isoforms are
expressed in most tissues, whereas p38c and d appear to
be more tissue restricted [4, 13]. Although several studies
have reported that several isoforms of p38 are expressed
in vascular endothelial cells [14, 15], expression of p38
isoforms in pulmonary artery endothelial cells and their
role in regulating cellular functions have not been
reported.
The current study was, therefore, designed to investigate
the role of p38 MAPK isoforms in mediating inflammatory
cytokine-induced HPAEC morphologic change and effects
on cell survival and tissue repair functions. We here show
that inhibition of p38 MAPK per se causes endothelial cells
to undergo morphologic change from a polygonal-shape
into spindle-shaped fibroblast-like cells, and also modu-
lates viability and repair functions. These effects are
differentially mediated by different p38 isoforms and are
independent of the nuclear factor (NF)-jB pathway.
Materials and methods
Reagents
Recombinant human IL-1b was purchased from R&D
systems (Minneapolis, MN). Anti-p38a, anti-p38b, anti-
p38c, anti-p38d and anti-phosphorylated p38 antibodies
were purchased from Cell Signaling Technology, Inc
(Danvers, MA). Anti-b-actin antibody and FITC–phalloi-
din were purchased from Sigma-Aldrich (St. Louis, MO).
HRP-conjugated anti-mouse and rabbit IgG antibodies
were purchased from Rockland (Gilbertsville, PA). Selec-
tive p38 MAPK inhibitors SB203580 and SB220025
were purchased from Calbiochem (San Diego, CA) and
dissolved in dimethyl sulfoxide (DMSO). The small inter-
ference RNAs (siRNAs) targeting p38 MAPK isoforms
(a, b, c, and d), NF-jB p65 and non-targeting control siRNA
were purchased from Dharmacon (Lafayette, CO).
Cell culture and treatment
Human pulmonary artery endothelial cells (HPAEC) were
purchased from Clonetics, Lonza (Walkersville, MD).
Cells were maintained in endothelial basal medium (EBM-2,
Lonza) supplemented with growth factors and 2% fetal
bovine serum according to the manufacturer’s recommen-
dations (complete EBM). Cells passaged between 8 and 12
were used for all experiments. Cells were treated with
IL-1b (0.5 or 2 ng/ml) or p38 inhibitor (SB203580 or
SB220025 (5 lM)) alone or in combination in a 1:1 mix-
ture complete EBM to EBM-2 resulting in a final
concentration of 1% serum (1:1 EBM) for 2 days. Lower
concentration of IL-1b, that is, 0.5 ng/ml, was used when it
was combined with p38 inhibitors in order to maximize the
p38 inhibitors’ effect. DMSO was used as a solvent control
for the p38 inhibitors. To induce apoptosis, cells were
incubated in EBM-2, which is without any growth factors
or serum, for 4 days in the presence or absence of p38
inhibitors (5 lM) and/or IL-1b (0.5 ng/ml). Cells were
then fixed and stained with PROTOCOL HEMA3 solutions
(Fisher Scientific Company L.L.C., Kalamazoo, MI).
A Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan)
equipped with a DP71 digital camera and DP Controller
software (Olympus, Tokyo, Japan) was used to take
photomicrographs.
Cell length measurement
To quantify cell morphologic change, cell length was
measured using ImageJ 1.33u software, NIH. A total of 50
cells for each condition was measured. Data are presented
as percent of control.
Immunoblot
Following treatment, cells were washed with ice-cold PBS
and total protein was extracted with lysis buffer (35 mM
Tris–HCl, pH 7.4, 0.1% Triton X-100, 0.4 mM EGTA,
10 mM MgCl2) containing a protease inhibitor cocktail
(Sigma-Aldrich). Lysates were centrifuged at 13,000g for
10 min and the protein concentration was measured using
the BIO-RAD Protein Assay Kit (Bio-Rad, Hercules, CA).
Lysates were diluted with 29 sample buffer (125 mM
Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromo-
phenol blue, 10% b-mercaptoethanol) and heated at 95�C
for 5 min. Five micrograms of proteins were loaded, sep-
arated by electrophoresis in 7.5% SDS–polyacrylamide
gels, and transferred to a PVDF membrane (Bio-Rad). The
234 N. Kanaji et al.
123
membrane was blocked in 5% skim milk for 1 h at room
temperature and incubated with proper concentrations of
first antibody at 4�C overnight. Targeting proteins were
subsequently detected using horseradish peroxidase con-
jugated IgG with an enhanced chemiluminescence plus
detection system (ECL plus) and Typhoon Scanner
(Amersham Pharmacia Biotech, Little Chalfont, Bucking-
hamshire, UK).
Visualization of F-actin
Cells were plated in a 48-well plate and treated with or
without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) in
1:1 EBM for 2 days. Cells were fixed with 3.7% formal-
dehyde for 15 min at room temperature and permeabilized
with 0.5% Triton X-100 in PBS for 15 min. After washing
with PBS, cells were blocked with 5% BSA, and then
incubated with FITC–phalloidin (1 lg/ml) at room tem-
perature for 1 h.
RNA interference
To selectively silence each isoform of p38 MAPK and
NF-jB p65, RNA interference was performed. Briefly,
cells were seeded at a cell density of 1 9 105 cells/ml. The
next day, cells were transfected with siRNA targeting
individual p38 MAPK isoforms, p65 or with non-targeting
control siRNA (final concentration of siRNA was 50 nM)
in OptiMEM I (Invitrogen, Carlsbad, CA) using Lipofect-
amine2000 (Invitrogen). After 8 h of incubation, media
were changed to complete EBM. Cell lysates were extracted
on day 4 and the efficacy of RNA interference was assessed
by immunoblot.
Collagen gel contraction assay
Native type I collagen gels were prepared by mixing a
solution of rat tail tendon collagen (approximately 3 mg/ml),
complete EBM and cell suspension so that the final
mixture resulted in 0.75 mg/ml collagen and 3 9 105
cells/ml. A 500 ll aliquot of the resulting solution was
then cast into each well of a 24-well culture plate and
allowed to polymerize. After gelation was completed, in
about 20 min at room temperature, the gels were gently
released into 60-mm dishes (three gels in each dish)
containing 5 ml of 1:1 EBM with or without p38 inhibi-
tors (5 lM) and/or IL-1b (0.5 ng/ml) and incubated at
37�C for 4 days. Gel size was measured with the Optomax
V image analyzer (Optomax, Burlington, MA). Data are
expressed as the percentage of gel area compared with the
original gel size.
Wound closure assay
Cells were pretreated with or without p38 inhibitors (5 lM)
and/or IL-1b (0.5 ng/ml) in a 1:1 EBM for 2 days. Fixed
width linear wounds were then created using a cell scraper
(Corning Inc., Corning, NY), and the remaining cells were
incubated in a 1:4 mixture complete EBM to EBM-2 for
16 h in the presence or absence of p38 inhibitors (5 lM)
and/or IL-1b (0.5 ng/ml). Cells were then fixed, stained
and photographed. To quantify cell migration, a grid was
overlaid on each image at 0 and 16 h after wounding using
Photoshop Elements 2.0 software (Adobe Systems Inc.,
San Jose, CA) to make 1,064 tiny squares. The number of
squares that contained migrated cells was counted. Three
fields for each condition were quantified. Data are pre-
sented as percentage of squares occupied by migrated cells
after wounding.
Profiling of DNA content by flow cytometry
Nearly confluent cells were treated with or without p38
inhibitors (5 lM) in EBM-2 only (serum and growth factor
free medium) for 2 days. Both attached and floating cells
were collected and fixed with cold 70% ethanol in PBS
at 4�C for 30 min. Cells were then pelleted by centrifu-
gation and re-suspended in the staining solution (50 lg
propidium iodide, 100 lg RNase A in 1 ml PBS). After 1 h
staining at 4�C, flow cytometric DNA content profiling was
performed.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). An
unpaired two-tailed Student’s t test was used for single
comparisons. For group test, one-way ANOVA followed
by Tukey’s test was used with the PRISM4 program.
p values \0.05 were considered as statistically significant.
Results
Suppression of p38 results in endothelial cell
morphologic change into spindle-shaped fibroblast-like
cells
Under normal culture conditions, HPAECs grow as a
monolayer of polygonal shaped cells. In the presence of the
pro-inflammatory cytokine IL-1b, HPAECs undergo spin-
dle-shaped morphologic alteration (Fig. 1a) along with
activation of p38 MAPK (Fig. 1b, c). We next investigated
if pharmacologic p38 inhibitors could block the cell mor-
phologic change induced by IL-1b. Surprisingly, inhibition
of p38 MAPK by p38 inhibitors (SB203580 or SB220025)
p38 and tissue repair on endothelial cells 235
123
per se induced cell morphologic alteration when added
alone and further augmented the inflammatory cytokine-
induced change (Fig. 2a). This was evidenced by micro-
photography of the cells, measurement of the cell length
(Fig. 2a), and by the polymerization of F-actin (Fig. 2b).
Next, we examined if p38 inhibitors can reduce endothelial
cell markers and/or induce mesenchymal cell markers.
However, the endothelial cell marker, VE–cadherin, was
not significantly altered by p38 inhibitor (data not shown).
Furthermore, none of the tested mesenchymal cell markers
including N-cadherin, vimentin, or fibronectin expression
was affected by p38 inhibitors (data not shown). Similar to
HPAEC, the p38 inhibitor-induced spindle-shaped mor-
phologic alteration was also observed in human dermal
microvascular endothelial cells (data not shown). In addi-
tion, the effect of the p38 inhibitors on endothelial cell
morphologic change was reversible following removal of
the reagents (data not shown).
To further confirm the effect of the pharmacologic
inhibitors of p38 and to identify the p38 isoforms that
modulate HPAEC morphology, siRNAs targeting individ-
ual isoforms of p38a, b, c or d were transfected into the
cells. As shown in Fig. 3a, the siRNA targeting each p38
isoform could significantly and specifically silence their
respective target. Suppression of p38a, c or d resulted in
cell morphologic change (Fig. 3b) and elongation in cell
length (Fig. 3b, p \ 0.05 compared to control siRNA).
Polymerization of F-actin was altered only by suppression
of p38c or d (Fig. 3c). In contrast, p38b-siRNA did not
affect cell morphology and cell length (p [ 0.05), or
F-actin polymerization (Fig. 3b, c). Although the inhibition
of p38a led to a slight decrease in VE–cadherin expression,
suppression of any one of the p38 isoforms did not alter
expression of N-cadherin or vimentin (data not shown).
Inhibition of p38 prevents cell death from deprivation
of serum and growth factors
HPAECs were cultured to sub-confluence in complete
EBM. When the cells were subsequently cultured in basal
medium without serum and growth factors, the majority of
the cells detached and were lost within 4 days (Con-
trol ? DMSO panel, Fig. 4a). An average of 22.5 ± 5.3%
of the cells survived after 4 days under this condition
(Fig. 4a insert). In the presence of either SB203580 or
SB220025, however, a significantly higher number of cells
survived (53.4 ± 14.3% or 43.8 ± 9.3%, respectively,
p \ 0.05, both comparisons) and the majority of these
surviving cells underwent morphologic change (Fig. 4a).
Interestingly, the elongated fibroblast-like appearance was
also observed in the few surviving cells under the control
condition (Fig. 4a). Furthermore, the protective effect of
p38 inhibitors on cell survival was also observed in the
presence of IL-1b (Fig. 4a); that is, nearly all of the cells
survived in the presence of p38 inhibitors plus IL-1b (81.7 ±
8.1% SB203580 plus IL-1b; 75.2 ± 2.5% SB220025 plus
IL-1b vs. 57.1 ± 7.5% of IL-1b alone, p \ 0.01, both
comparisons).
Fig. 1 Effect of IL-1b on cell
morphology and activation of
p38 MAPK in HPAECs. a Cell
morphology. Human pulmonary
artery endothelial cells
(HPAECs) were treated with
2 ng/ml of IL-1b for 2 days.
Cells were then stained and
photographed under microscope.
Scale bar 200 lm. b Time-
dependent p38 activation. Total
cell proteins were extracted after
0, 20, 40, 60 min of cytokine
treatment (2 ng/ml), and then
immunoblotted for
phosphorylated-p38 MAPK and
b-actin. c Semi-quantification of
phosphorylated p-38 versus
b-actin. Vertical axis density
versus control; horizontal axistime of treatment. Data
presented is an average of three
separate experiments. *p \ 0.05
compared to control by one way
ANOVA followed by Tukey’s
correction
236 N. Kanaji et al.
123
Similar to the effect of pharmacologic p38 inhibitors,
RNA interference targeting p38 isoforms also prevented
cells from death induced by serum and growth factor
withdrawal (Fig. 4b). Although all of the four siRNAs
targeting individual isoforms showed a protective effect on
cell survival compared to control siRNA (surviving cells:
15.9 ± 4.1%), the p38a-siRNA had a strongest effect
(95.4 ± 9.4%, p \ 0.01) and others had a more modest but
statistically significant effect (48.2 ± 7.7% b-siRNA, p \0.01; 40.5 ± 6.8% c-siRNA, p \ 0.01; 53.2 ± 4.4%
d-siRNA, p \ 0.01).
To evaluate if the cells were undergoing apoptosis or
necrosis following deprivation of growth factors, DNA
content profiling by FACS analysis was conducted. Con-
sistent with the microscopic observation and cell number
count, DNA content profiling assay demonstrated that an
average of 26.4 ± 3.2% of the cells underwent apoptosis
after 2 days culture in the basal medium only (DMSO
panel, Fig. 4c). This apoptosis was significantly blocked by
either SB203580 or SB220025 (16.6 ± 2.0% or 13.3 ±
3.2%, respectively, p \ 0.05, Fig. 4c).
Inhibition of p38 modulates tissue repair functions
To investigate if inhibition of p38 MAPK alters tissue
repair functions in HPAECs, the gel contraction assay (an
in vitro model of remodeling of extracellular matrix) and
the wound closure assay (an in vitro wound healing model)
were performed. Under control conditions, normal HPAECs
were able to contract native type I collagen gels when the
cells were cast into the gels. Gel size decreased gradually
and reached 75.2 ± 1.5% of initial size on day 4 (Fig. 5a).
SB203580 and SB220025 augmented collagen gel con-
traction by HPAECs (69.6 ± 1.7% and 60.7 ± 2.6% of
initial size on day 4, respectively, p \ 0.05, Fig. 5a).
The inflammatory cytokine IL-1b also augmented gel
contraction mediated by HPAEC (67.1 ± 2.5%, p \ 0.05,
Fig. 5a). The cytokine effect was further augmented in the
Fig. 2 Effect of pharmacologic
p38 inhibitors on cell
morphology and F-actin
cytoskeletal arrangement. a Cell
morphology and cell length.
Cells were treated with or
without p38 inhibitors
(SB203580 or SB220025,
5 lM) in the presence or
absence of IL-1b (0.5 ng/ml) for
2 days. Cells were then fixed,
stained and photographed. The
cell length was measured as
described in the methods
(inserts). Values are
means ± SD of three separate
experiments. *p \ 0.05
compared to DMSO without
IL-1b. **p \ 0.05 compared to
DMSO with IL-1b. b F-actin
polymerization. F-actin was
visualized by FITC–phalloidin
as described in the methods
p38 and tissue repair on endothelial cells 237
123
P38
p38
p38
P38
-actin
NT siRNA targeting p38
Control siRNA p38 -siRNA p38 -siRNA
p38 -siRNA p38 -siRNA
Cell length 100% 129.7±9.6% * 99.1±2.9%
141.7±12.5% * 128.8±9.6% *
Control siRNA p38 -siRNA p38 -siRNA
p38 -siRNA p38 -siRNA
A
C
B
Fig. 3 Effect of siRNAs
targeting individual p38
isoforms on cell morphology
and F-actin polymerization.
a Suppression of p38 isoforms
by siRNA. Sub-confluent
HPAECs were transfected with
siRNAs using Lipofectamine
2000 described in the method.
On day 4, total proteins were
extracted and immunoblotted
for each p38 MAPK isoform.
Five micrograms of total protein
was loaded and b-actin was
blotted as a loading control.
b Cell morphology. On day 0,
cells were transfected with
siRNAs. On day 4, cells were
fixed, stained and photographed.
Cell length was measured as
described in the methods
(inserts). Values are
means ± SD of three separate
experiments. *p \ 0.05
compared to control siRNA.
c F-actin polymerization. After
4 days of transfection of
siRNAs, F-actin was visualized
by FITC–phalloidin
238 N. Kanaji et al.
123
control siRNA p38 -siRNA p38 -siRNA
p38 -siRNA p38 -siRNA
day 0 day 3 (serum and growth factor free)
control siRNA
100% Survived cells 15.9±4.1% 95.4±9.4% * 48.2±7.7% *
40.5±6.8% * 53.2±4.4% *
A
B
C
Fig. 4 Effect of p38
suppression on cell survival
following withdrawal of serum
and growth factors. a Effect of
pharmacologic p38 inhibitors on
cell survival. HPAECs were
cultured until sub-confluent
with complete medium. The
cells were then treated with
either p38 inhibitors or IL-1b(0.5 ng/ml) or both in a basal
medium without serum or any
growth factors. After 4 days,
attached cells were fixed,
stained and photographed. Cell
number was counted (inserts).
Values are means ± SD of
three separate experiments.
*p \ 0.05 compared to DMSO
without cytokine. **p \ 0.05
compared to DMSO with IL-1balone. b Effect of siRNAs
targeting p38 isoforms on cell
survival. After transfection with
siRNAs, cells were fed with a
basal medium containing no
serum or growth factors. After
3 days, attached cells were
fixed, stained and photographed.
Cell number was counted
(inserts). Values are
means ± SD of three separate
experiments. *p \ 0.01
compared to control siRNA.
c Evaluation of apoptosis by
DNA content profiling. Cells
were treated with or without p38
inhibitors for 2 days in a basal
medium. Both floating and
attached cells were harvested
and used for DNA content
profiling. Values are
means ± SD of three separate
experiments. *p \ 0.05
compared to DMSO
p38 and tissue repair on endothelial cells 239
123
presence of either of the p38 inhibitors, SB203580 or
SB220025 (61.0 ± 2.0% or 47.6 ± 3.2%, respectively,
p \ 0.05, Fig. 5a). Augmentation of collagen gel contrac-
tion by p38 inhibitors was further confirmed by siRNAs.
While the cells transfected with control siRNA contracted
the gels to 75.8 ± 3.0% of initial size on day 4, either
p38a- or d-siRNA led to augmentation of gel contrac-
tion (54.8 ± 7.9% or 53.0 ± 5.0%, respectively, p \ 0.05,
Fig. 5b). In contrast, p38b- and c-siRNAs did not affect gel
contraction (p [ 0.05).
Next, we evaluated wound closure in the presence or
absence of p38 inhibitors. Under control conditions, nearly
half (46.0 ± 4.0%) of the wounded area was covered by
the migrated cells (Control ? DMSO, Fig. 5c). Interest-
ingly, the two p38 inhibitors had opposite effects on wound
closure; that is, SB203580 stimulated wound closure
(66.5 ± 2.6%, p \ 0.05), and SB220025 inhibited wound
closure (29.5 ± 4.4%, p \ 0.05, Fig. 5c). Furthermore,
SB203580 augmented the cytokine effect on wound
closure (70.3 ± 4.2% of IL-1b alone vs. 94.1 ± 0.9% of
IL-1b ? SB203580, p \ 0.05), while SB220025 signifi-
cantly blocked the effect of cytokine-augmented wound
closure (49.7 ± 7.9% of IL-1b ? SB220025, p \ 0.05,
Fig. 5c). In addition, we examined the wound closure assay
after RNA interference (Fig. 5d). Interestingly, transfection
with p38a-siRNA resulted in greater wound closure com-
pared to control siRNA (71.9 ± 9.6% vs. 48.4 ± 4.5%,
p \ 0.05), while suppression of other p38 isoforms slightly
but significantly inhibited wound closure (p38b-siRNA;
35.9 ±
3.3%, p38c-siRNA; 32.3 ± 6.5%, and p38d-siRNA; 31.2 ±
6.9%, p \ 0.05). Similar to the disparate effects of the p38
inhibitors on wound closure, SB203580 increased while
SB220025 decreased trans-endothelial electrical resistance
measured with electric cell-substrate impedance sensing
(data not shown).
Interaction between NF-jB and p38 MAPK signaling
pathways
Since NF-jB is a major signal transduction mediator of
the effect of IL-1b in a variety of cells including endo-
thelial cells, the role of NF-jB p65 activation in p38
inhibitor-induced cell morphologic alteration was exam-
ined. To accomplish this, first, effect of p38 inhibitor
(SB203580) on p65 phosphorylaton and p38 phosphory-
lation was evaluated by immunoblot. As shown in Fig. 6a,
SB203580 appeared to block IL-1b-induced p38 phos-
phorylation. Surprisingly, however, SB203580 further
enhanced IL-1b-induced p65 phosphorylation. Second, the
NF-jB signaling pathway was blocked by RNA interfer-
ence targeting NF-jB p65 followed by treatment with p38
inhibitors and/or cytokines. While suppression of p65 by
siRNA (Fig. 6b) prevented cell morphologic change in
response to IL-1b, it did not affect the morphologic
change in response to p38 inhibitor (SB203580) as shown
by microphotography (Fig. 6c) and measurement of cell
length (Fig. 6c insert), indicating p38 inhibitor-induced
HPAEC morphologic change is not mediated by p65
subunit of NF-jB.
Discussion
The present study demonstrates that IL-1b induces p38
phosphorylation and alteration of cell morphology.
Suppression of p38 MAPK, however, does not block the
IL-1b effect, but enhances IL-1b-induced cell morphologic
change. Furthermore, p38 suppression per se induces an
elongated and spindle-shaped cell morphologic change
along with the organization of the F-actin cytoskeleton.
Moreover, inhibition of p38 MAPK per se also prevents
HPAECs from undergoing apoptosis induced by depriva-
tion of serum and growth factors. In addition, suppression
of p38a or d increases contraction of type I collagen gels by
the HPAECs. Suppression of p38a increases wound clo-
sure, while suppression of p38b, c or d decreases it. Finally,
both NF-jB p65 and p38 are activated by IL-1b, but sup-
pression of p65 does not alter either p38 activation or the
Fig. 5 Effect of p38 suppression on gel contraction and cell migration.
a Effect of pharmacologic p38 inhibitors on collagen gel contraction
mediated by HPAECs. Cells were cast into collagen gels and treated
with or without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) for
4 days. Gel size was measured with an image analyzer on day 4.
Vertical axis gel size expressed as percent of initial size (%). Horizontalaxis treatment with or without IL-1b. Open bars DMSO only; hatchedbars SB203580; solid bars SB220025. Data presented are means ± SD
of three separate experiments. *p \ 0.05 compared to DMSO without
cytokines. **p \ 0.05 compared to DMSO with same cytokine
treatment. b Effect of siRNAs targeting p38 isoforms on collagen gel
contraction. After transfection with siRNAs, the cells were cast into
collagen gels (day 0) and allowed to contract for 4 days. Gel size was
measured with an image analyzer. Vertical axis gel size expressed as
percent of initial size (%) on day 4. Horizontal axis siRNA treatment.
Data presented are means ± SD of three separate experiments.
*p \ 0.05 compared to control siRNA. c Effect of pharmacologic
p38 inhibitors on wound closure. HPAECs were pre-treated with or
without p38 inhibitors (5 lM) and/or IL-1b (0.5 ng/ml) for 2 days. The
cell layer was then wounded with a cell scraper followed by incubation
with same p38 inhibitors with or without IL-1b for additional 16 h.
Wound repair was quantified by counting the number of migrated cells
as described in the method (inserts). Data presented are means ± SD of
three separate experiments. *p \ 0.05 compared to DMSO without
cytokine. **p \ 0.05 compared to DMSO with IL-1b treatment.
d Effect of siRNAs targeting p38 isoforms on wound closure. After
3 days of transfection with siRNAs, the cells were wounded and
allowed to repair for 16 h. Wound repair was quantified as described
above (inserts). Data presented are means ± SD of three separate
experiments. *p \ 0.05 compared to control siRNA
c
240 N. Kanaji et al.
123
effect of p38 inhibitors on HPAECs, suggesting the effect
of p38 on HPAECs is NF-jB independent.
Pulmonary vascular endothelial cells have been reported
to be involved in the pathogenesis of inflammatory lung
diseases including chronic obstructive pulmonary disease
and pulmonary hypertension [16]. Inflammatory cytokines
such as IL-1b can alter endothelial cell functions such
as cell migration and susceptibility to apoptosis [17, 18].
p38 and tissue repair on endothelial cells 241
123
The current study provides new mechanistic insight into
IL-1b modulation of endothelial cell function. Specifically,
p38 MAPKs negatively modulate IL-1b effects on endo-
thelial cells. In addition, p38 MAPKs alone can regulate
endothelial cell functions. The four isoforms (a, b, c and d)
of p38 MAPKs share only about 60% amino acid sequence
identity, suggesting they may have highly diverse functions
[4, 19]. In the current study, we not only confirmed the
expression of all four p38 isoforms in HPAECs, but also
determined that the isoforms play different roles in regu-
lating HPAEC morphology, survival, and cell functions
related to tissue repair.
MAPKs are associated with cell differentiation [4]. In
this content, several reports have shown a positive role
of p38 MAPK in epithelial cells undergoing epithelial-
mesenchymal transition (EMT), that is, a p38 inhibitor
(SB203580, SB202190 or 2-(4-chlorophenyl)-4-(4-fluo-
rophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one) or a
dominant negative-p38a blocked transforming growth
factor-b1-induced EMT in several types of epithelial cells
[20–22]. In contrast to these reports, our findings are dif-
ferent in several respects. First, the p38 inhibitors not only
augmented inflammatory cytokine-induced endothelial cell
morphologic change, but also these inhibitors per se
induced endothelial cells to undergo morphologic change
from typical polygonal-shaped endothelial cells into spin-
dle-shaped fibroblast-like cells. Second, none of the tested
mesenchymal markers including vimentin, N-cadherin or
fibronectin was altered by the p38 inhibitors in HPAECs.
These results suggest that the p38 signal pathway plays a
different role in endothelial cells compared to epithelial
cells. We can not conclude that the changes observed by
inhibition of p38 are consistent with endothelial-mesen-
chymal transition (EndMT). However, the current study
demonstrates that HPAECs lacking p38 MAPK undergo
morphologic change to become more contractile and are
A B
C
Fig. 6 Role of p65 in mediating IL-1b- or SB203580-induced cell
morphologic change. a Effect of IL-1b and SB203580 on p65 and p38
phosphorylation. Cells pretreated with SB203580 (5 lM) for 30 min
followed by stimulation with IL-1b (2 ng/ml, 20 min). Whole cell
lysate was immunoblotted for phosphorylated p65 or p38, and b-actin.
C control; S SB203580. b Immunoblot of p65 following siRNA
transfection. Cells were transfected with either a control siRNA or a
siRNA targeting NF-jB-p65 using lipofectamine 2000 as described in
the methods. Whole cell lysates were subjected for immunoblot of
phosphorylated p65 and b-actin. c Effect of p65 suppression on cell
morphologic change in response to p38 inhibitor or IL-1b stimulation.
Following siRNA transfection, the cells were treated with or without
SB203580 (5 lM) and/or IL-1b (2 ng/ml) for 2 days. The cells were
then stained and photographed. Cell length was measured as
described (inserts). Values are means ± SD of three separate
experiments. *p \ 0.05 compared to cells transfected with control
siRNA and treated with DMSO only. **p \ 0.05 compared to the
cells transfected with control siRNA and treated with IL-1b.#p \ 0.05 compared to the cells transfected with siRNA targeting
p65 and treated with DMSO only
242 N. Kanaji et al.
123
resistant to apoptosis. In addition, suppression of p38
MAPK further enhanced the augmentative effect of IL-1bon these functions. These results suggest that endothelial
cells can contribute to tissue repair whether or not the
criteria for EndMT are met.
Besides regulating cell differentiation, p38 MAPK is also
involved in modulating cell survival and death. There is
increasing evidence showing that inhibition of p38 MAPK
protects endothelial cells from apoptosis [23, 24]. Consis-
tent with these reports, the current study also demonstrated
that HPAEC survival is negatively regulated by the p38
signal pathway. Interestingly, it has been also reported that
activation of p38b promoted cell survival, whereas activa-
tion of p38a resulted in cell death in cardiac myocytes,
Jurkat cells and HeLa uterine cervical adenocarcinoma cells
[25–27]. Similarly, the anti-apoptotic effect of heme oxy-
genase-1 was dependent on the degradation of p38a and on
the expression of p38b in human umbilical vein and bovine
aortic endothelial cells [28]. These results suggest that p38aand p38b have opposite effects in controlling cell viability
of certain cell types, i.e., p38a is pro-apoptotic, whereas
p38b is anti-apoptotic. Unlike these reported data, the
current study demonstrated that inhibition of any one of the
p38 isoforms including p38b prevented HPAECs from
apoptosis and inhibition of p38a had the strongest effect.
These findings suggest the role of p38b isoform on cell
survival might vary among cell types.
Both cell morphologic change and cell viability are key
features of tissue repair following injury. Thus, we further
determined the role of p38 MAPK in regulating endothelial
cell tissue repair functions by evaluating collagen gel
contraction and wound closure. We demonstrated that the
pharmacologic p38 inhibitors significantly augmented
collagen gel contraction mediated by HPAECs, which is
opposite to the result Meyer-Ter-Vehn et al. [29] reported
using fibroblasts, that is, suppression of p38 MAPK
inhibited collagen gel contraction mediated by fibroblasts.
In our study, the effect of pharmacologic inhibitors on
collagen gel contraction was confirmed by RNA interference.
Suppression of p38a or d by siRNA also resulted in aug-
mentation of collagen gel contraction mediated by HPAEC.
Inhibition of p38c, however, did not alter gel contraction in
spite of its ability to induce fibroblast-like cell morphology,
suggesting that cell morphologic alteration is not always
parallel to contraction of collagen gels.
Several studies have reported that cell migration was
blocked by inhibition of p38 MAPK in epithelial, mesen-
chymal and endothelial cells [12, 20, 30, 31]. In the current
study, the two pharmacologic p38 inhibitors showed
opposite effect on endothelial wound closure, that is,
SB203580 enhanced endothelial wound closure while
SB220025 inhibited wound closure. SB203580 is a pyridyl
imidazole compound, and it inhibits p38a and b isoforms,
but not p38c or d [4, 19]. In the current study, only p38asuppression by siRNA resulted in enhancement of wound
closure, while suppression of the other three isoforms
slightly but significantly decreased wound closure. This
result indicates that p38a negatively, while other p38 iso-
forms positively modulates endothelial cell-mediated
wound closure. It has not been reported which p38 iso-
forms SB220025, a pyrimidinyl imidazole compound,
inhibits. Our data suggest that the inhibitory effect of
SB220025 may be more potent on p38d than on p38a.
IL-1b is known to activate the p38 signaling pathway,
which is also confirmed in the current study. Interestingly,
however, the current study demonstrated that suppression
of p38 by either pharmacologic inhibitors or specific siR-
NAs could dramatically enhance IL-1b effect on endothelial
cells, that is, alteration of morphology, resistance to
apoptosis and enhancement of collagen gel contraction.
This suggests that p38 activation could serve as an internal
feedback inhibitory role following IL-1b stimulation. To
our knowledge, these are novel findings for the function
of p38 in endothelial cells. In addition, SB203580, an
inhibitor of p38 phosphorylation in response to IL-1b stimu-
lation, augments IL-1b-induced p65 phosphorylation.
Furthermore, the suppression of NF-jB p65 did not affect
SB203580 or SB220025 induced endothelial cell morpho-
logic change while it did block the effect of IL-1b. Thus,
we propose that both p38 MAPK and NF-jB p65 mediate
signal transduction of IL-1b in endothelial cells and that
p38 MAPK negatively while NF-jB p65 positively medi-
ates the responses of endothelial cells evaluated in the
current study.
In the current study, we have demonstrated that p38
suppression by either a pharmacologic inhibitor or a siRNA
could not only lead to HAPEC morphologic change under
normal culture condition, but also result in cell survival in
the absence of growth factors. While p38 seems negatively
regulate cellular functions in HPAECs, the downstream
mechanism of p38 on cell morphologic change and cell
survival, however, remains to be further defined.
In summary, suppression of p38 MAPK induces
HPAECs to undergo a morphologic change from a polyg-
onal-shape into spindle-shaped fibroblast-like cells. The
cells in which p38 MAPK is inhibited are resistant to
apoptosis in response to serum and growth factor depri-
vation, and are more robust in contracting collagen gels.
IL-1b activates p38 MAPK but inhibition of p38 MAPK
further augments IL-1b-induced morphologic change, cell
survival, and gel contraction. Inhibition of p38a enhances
wound closure, while inhibition of other isoforms decrea-
ses it. These findings suggest that p38 MAPK may
modulate tissue remodeling mediated by endothelial cells,
especially in an inflammatory milieu where inflammatory
cytokine IL-1b is present.
p38 and tissue repair on endothelial cells 243
123
Acknowledgments The authors thank Ms. Lillian Richards for the
excellent secretarial support of this manuscript. Funding: Larson
Endowment, University of Nebraska Medical Center and LB506.
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