Molecular Biology of the Cell
Vol. 17, 1995–2008, April 2006
Protein Kinase C? Attenuates Hypoxia-induced
Proliferation of Fibroblasts by Regulating MAP Kinase
Megan D. Short, Stephanie M. Fox, Ching F. Lam, Kurt R. Stenmark,
and Mita Das
Developmental Lung Biology Research Laboratory, Department of Pediatrics, University of Colorado Health
Sciences Center, Denver, CO 80262
Submitted September 20, 2005; Revised January 5, 2006; Accepted January 31, 2006
Monitoring Editor: Richard Assoian
We have previously found that hypoxia stimulates proliferation of vascular fibroblasts through G?i-mediated activation
of ERK1/2. Here, we demonstrate that hypoxia also activates the atypical protein kinase C? (PKC?) isozyme and stimulates
the expression of ERK1/2-specific phosphatase, MAP kinase phosphatase-1 (MKP-1), which attenuates ERK1/2-mediated
proliferative signals. Replication repressor activity is unique to PKC? because the blockade of classical and novel PKC
isozymes does not affect fibroblast proliferation. PKC? is phosphorylated upon prolonged (24 h) exposure to hypoxia,
whereas ERK1/2, the downstream kinases, are maximally activated in fibroblasts exposed to acute (10 min) hypoxia.
However, PKC? blockade results in persistent ERK1/2 phosphorylation and marked increase in hypoxia-induced repli-
cation. Similarly prolonged ERK1/2 phosphorylation and increase in hypoxia-stimulated proliferation are also observed
upon blockade of MKP-1 activation. Because of the parallel suppressive actions of PKC? and MKP-1 on ERK1/2
phosphorylation and proliferation, the role of PKC? in the regulation of MKP-1 expression was evaluated. PKC?
attenuation reduces MKP-1 expression, whereas PKC? overexpression increases MKP-1 levels. In conclusion, our results
indicate for the first time that hypoxia activates PKC?, which acts as a terminator of ERK1/2 activation through the
regulation of downstream target, MKP-1 expression and thus serves to limit hypoxia-induced proliferation of fibroblasts.
Fibroblast proliferation is associated with various forms of
vascular diseases (Sartore et al., 2001), different fibrotic con-
ditions (Atamas, 2002) and cancer (Bhowmick et al., 2004).
Hypoxia is the critical contributor to the pathophysiological
conditions of these diseases. We have found that cultured
vascular adventitial fibroblasts have the distinct capability to
proliferate directly in response to hypoxia in the absence of
any exogenous growth factors (Das et al., 2001). Intracellular
signaling intermediates, e.g., protein kinase C (PKC) and
MAP kinase families are the major mediators of hypoxic
signal stimulating replication of cells (Das et al., 2000, 2001;
Sodhi et al., 2000). However, cellular proliferation is tightly
regulated by proper exit from the cell cycle to maintain
normal physiological conditions. The molecular pathways
that direct attenuation of hypoxia-induced proliferative sig-
nals in fibroblasts remain unknown.
PKC, a family of serine/threonine kinases, have been
divided into three distinct groups: the conventional: calcium-,
phospholipid-, and diacylglycerol-dependent PKC isozymes
(cPKC?, ??, ???, ?); the novel: calcium-independent PKC
isozymes (nPKC?, ?, ?, ?); and the atypical PKC isozymes
(aPKC?, ?, ?), which are calcium-, phospholipid-, and
diacylglycerol-independent (Nishizuka, 1992; Hug and
Sarre, 1993). PKC? can be activated directly or indirectly by
a variety of important signaling molecules including cer-
amide (Powell et al., 2004), phosphatidic acid (Le Good et al.,
1998), phosphoinositide 3-kinase lipid products and activa-
tion of the p21Ras pathway (Pal et al., 2001). PKC? has
emerged as a critical regulator of a number of cellular func-
tions including proliferation, differentiation, and apoptosis
(Hirai and Chida, 2003). This isozyme mediates proliferation
in NIH3T3 cells (Berra et al., 1993; Kim et al., 1997), endothe-
lial cells (Kent et al., 1995), and smooth muscle cells (Yano et
al., 1999). In contrast, cytokine- and ceramide-induced acti-
vation of PKC? leads to inhibition of proliferation and
growth arrest in vascular smooth muscle cells, respectively
(Bourbon et al., 2002; Hussain et al., 2002). Therefore, the
biological functions of PKC? in cellular responses are cell-
type and stimulus specific. The mechanisms responsible for
diverse physiological functions of PKC? at the cellular level
are not known.
A recent report has demonstrated that phosphorylation of
the Na,K-ATPase ?1subunit in lung alveolar epithelial cells
under hypoxic conditions is mediated through PKC? (Dada
et al., 2003). Datta et al. (2004) have found that PKC? partic-
ipates in the activation of hypoxia-inducible factor-1? (HIF-
1?) by inhibiting the expression of asparagine hydroxylase
(enzyme inhibitor of HIF-1), thereby promoting the tran-
scription of hypoxia-inducible genes such as vascular per-
meability factor and vascular endothelial growth factor. De-
spite the importance of PKC? in cellular signaling under
hypoxic conditions, it is unknown whether PKC? is a pro-
liferative stimulator or suppressor in fibroblasts under hy-
This article was published online ahead of print in MBC in Press
on February 8, 2006.
Address correspondence to: Mita Das (Mita.Das@uchsc.edu).
© 2006 by The American Society for Cell Biology1995
Another set of protein kinases that plays an important role
in transducing signal from intracellular PKC isozymes to the
cell nucleus is MAP kinase family (Kim et al., 1997; Corbit et
al., 2000; Mas et al., 2003). Previously, we have demonstrated
that hypoxia induces transient activation of ERK1/2, one
member of the MAP kinase family, and that ERK1/2 activa-
tion mediates replication of hypoxic fibroblasts (Das et al.,
2001). PKC? acts as an upstream regulator of ERK1/2 acti-
vation in response to various stimuli in different cell types
(Hirai and Chida, 2003). However, the functional role of
PKC? in the regulation of hypoxia-induced activation of
ERK1/2 in fibroblasts is not known.
Once activated, ERK1/2 can be rapidly inactivated through
dephosphorylation by phosphatases known as dual specificity
MAP kinase phosphatases (MKPs; Keyse and Emslie, 1992).
The existence of at least eleven MKPs in mammals implies
a considerable complexity in the regulation of MAP ki-
nase signaling by these enzymes. Among these phospha-
tases, MKP-1 is encoded by an immediate early gene
(Noguchi et al., 1993). Though MKP-1 is identified as a
hypoxia-responsive gene (Laderoute et al., 1999; Seta et al.,
2001; Liu et al., 2003), the role of this phosphatase in cellular
responses under hypoxic conditions, is poorly understood. It
is important to understand the mechanisms regulating MKP
expression because the physiological functions of MKPs are
largely determined by their expression patterns. Multiple
pathways, e.g., ERK1/2, c-Jun N-terminal kinase (JNK), p38
MAP kinase and Ca2?-dependent pathways regulate MKP-1
expression (Reffas and Schlegel, 2000; Slack et al., 2001). PKC
is also implicated as an important regulator in the expres-
sion of these phosphatases in various cell types (Stawowy et
al., 2003). However, the signaling mechanisms that control
hypoxia-stimulated MKP-1 expression in fibroblasts remain
The current study was undertaken to determine whether
PKC? activation and MKP-1 expression are required for the
attenuation of ERK1/2 activation and proliferation in re-
sponse to hypoxia in cultured vascular fibroblasts and
whether PKC? plays a role in the expression of MKP-1.
Collectively, we hypothesized that PKC?-induced up-regu-
lation of MKP-1 mediates replication repressor activity of
PKC? to suppress ERK1/2 activation in fibroblasts upon
MATERIALS AND METHODS
Eagle’s MEM, bromodeoxyuridine (BrdU), monoclonal antibody (mAb)
against ?-tubulin and protease inhibitor cocktail were purchased from Sigma-
Aldrich Chemical Company (St. Louis, MO). mAb against BrdU was obtained
from Becton Dickinson Immunocytometry Systems (San Jose, CA). Fetal
bovine serum (FBS) and porcine serum were from Gemini Bio-Products
(Woodland, CA). Other antibodies were purchased from the following com-
panies: polyclonal and monoclonal antibodies against PKC? and PKC? from
Santa Cruz Biotechnology (Santa Cruz, CA), biotinylated anti-mouse IgG,
streptavidin-conjugated Alexa 488– and Alexa 594–conjugated anti-rabbit
IgG from Molecular Probes (Eugene, OR), phosphoERK1/2 and phos-
phoPKC?/? (Thr410) from Cell Signaling (Beverly, MA). Polyclonal antibody
against MKP-1, goat anti-rabbit and goat anti-mouse IgG conjugated with
alkaline phosphatase were obtained from Santa Cruz and Upstate Biotech-
nology (Waltham, MA). Mounting medium containing 4?,6-diamino-2-phe-
nylindole dihydrochloride (DAPI) was obtained from Vector Laboratories
(Burlingame, CA). Myristoylated pseudosubstrate peptide inhibitors of PKC?
and PKC classical and novel isozymes (PKC inhibitor) were purchased from
Biomol (Plymouth, PA). Sodium vanadate was obtained from Fisher Scientific
(Pittsburgh, PA). Additionally, PKC? antisense phosphorothioate oligonucle-
otides were obtained from Integrated DNA Technologies (Skokie, IL) and
Oligos Etc. (Wilsonville, OR). The rabbit PKC? sequences used are as follows:
rabbit PKC? antisense (5?-GCTGCGCCGGCCTCACACG-3?); PKC? scram-
bled antisense (5?-ACCCCGTCGGCCCATGGCG-3?). Plasmids containing
phosphatase inactive mutant (Cys258to Ser, pSG5-MKP-1-CS) of MKP-1
(MKP-1PI) was kindly provided by Dr. N. K. Tonks (Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY). Vector (pCMV6) containing constitu-
tively active PKC? (MyrPKC?) was a gift of Dr. A. Toker (Harvard Medical
School, Boston, MA).
Main pulmonary artery adventitia was harvested from the 15-d-old neonatal
control calves, carefully dissected free of blood vessels and fat under a
dissecting microscope and then cut into small pieces. Fibroblasts from the
tissue were isolated, characterized, and maintained according to previously
described methods (Das et al., 2002). Cells were maintained in MEM, pH 7.4,
supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin,
l-glutamine and incubated in a humidified atmosphere with 5% CO2at 37°C.
Medium was changed twice per week and cells were harvested with trypsin
(0.2 g/L) containing EDTA (0.5 g/L). Passages ranging from 3 to 12 were used
for all experiments. The growth characteristics and the appearance of the cells,
examined by light microscopy, did not change for the passages studied.
Cell Proliferation Assay
Cells were plated at a density of 20,000/well in a 24-well plate in 10%
FBS-containing medium and growth arrested for 72 h according to the pre-
viously described method (Das et al., 2001). Quiescent cells were transiently
transfected with Optimem medium (Invitrogen, Carlsbad, CA) by combining
2 ?g/ml of lipofectin (Invitrogen) with DNA for 5–6 h at 37°C. We used
MyrPKC?, MKP-1PI, PKC? antisense and scrambled oligonucleotides for
different transfection experiments. The cells were allowed to recover from
transfection either overnight (PKC? antisense experiments) or 48 h (myrPKC?
and MKP-1PI experiments) and then exposed to either normoxia (21% O2) or
hypoxia (1% O2) in the presence of BrdU (10 ?M) for 24 h. For the hypoxic
experiments, cells were placed either in sealed humidified gas chambers as
previously described (Das et al., 2002) or Bactron X (Sheldon Manufacturing,
Cornelius, OR) environmental hood. At the end of the treatment, cells were
fixed with cold 0.3% H2O2in methanol for 15 min, treated with 2 N HCl for
20 min, and then incubated with anti-BrdU antibody. VectaStain DAB kit
(Vector Laboratories, Burlingame, CA) was used to visualize nuclei. The
BrdU-positive and -negative nuclei were counted in five randomly chosen
areas in each well and reported as a percent of control.
For the studies with PKC? inhibitor and the inhibitor of PKC classical and
novel isozymes, quiescent cells were preincubated with the pseudosubstrate
peptide inhibitors for 1 h at 37°C and then exposed to either normoxia or
hypoxia for 24 h in the presence of BrdU. Cells were processed for BrdU
immunostaining as described above.
PKC? Activation Assay
Cells were plated at a density of ?0.5–1 ? 106cells/100-mm Petri dish in
medium containing 10% FBS, and growth was arrested for 72 h. Quiescent
cells were exposed to hypoxia (1% O2) for either 10 min or 24 h. Calyculin A
(100 nM) was added to the cells to block the serine/threonine phosphatases.
Fibroblasts were exposed to calyculin A for 10 min. For 24-h hypoxic expo-
sure, cells were treated with calyculin A for last 10 min of the experimental
period. At the end of the exposure, cells were harvested with lysis buffer (Cell
Signaling) containing protease inhibitor cocktail (1:100), 0.1% SDS, and 1%
sodium deoxycholate, freeze-thawed to disrupt cell membranes, and centri-
fuged at 10,000 ? g. Lysates were collected and protein concentrations were
estimated using the Bradford method (BIORAD, Hercules, CA). To evaluate
PKC? activation, 500–700 ?g protein was incubated with anti-PKC? antibody
(1:200, monoclonal) overnight at 4°C and then with AG agarose beads (Santa
Cruz Biotechnology and Upstate Biotechnology) for 2 h at 4°C with agitation.
The antigen–antibody complex was isolated by centrifugation at 10,000 ? g
for 1 min. The supernatant was discarded and the beads were washed
multiple times with lysis buffer. The recovered protein was separated by gel
electrophoresis, transferred to polyvinylidene difluoride membranes (PVDF;
Amersham Pharmacia Biotech, Piscataway, NJ), and probed with an antibody
against phosphoThr-PKC? (1:1000). Immunoreactivity was detected on x-ray
film using chemiluminescent reagents (Amersham Pharmacia Biotech). Im-
munoprecipitation followed by immunoblotting using the anti-PKC? anti-
body (1:1000) served as a control to monitor any changes in total PKC? protein
levels. Because the mAb against PKC? may cross-react with PKC?/? isozyme,
immunoprecipitates were also immunoblotted against PKC? using PKC?-
specific antibody (1:1000). Images of Western blots were scanned using EPSON
TWAIN software with EPSON PERFECTION 2450 PHOTO scanner. Densito-
metric quantitation of the scanned bands was performed using the National
Institutes of Health (NIH) Image 1.58 program. The area under the curve (AUC)
for each band was determined and represents the band intensity in arbitrary
units. The AUC at 0-h time was considered as 100% and percent increases in
hypoxic fibroblasts were calculated with respect to 0 h.
For the examination of PKC? and PKC? expression in the presence of either
MyrPKC? or PKC?-specific antisense oligonucleotides, vascular fibroblasts
were plated at the density of 50,000–100,000 cells/well in a six-well plate in
M. D. Short et al.
Molecular Biology of the Cell1996
10% FBS-containing medium and growth was arrested for 72 h. Quiescent
cells were transiently transfected with the corresponding DNA according to
the method described above for BrdU incorporation. Transfected cells were
exposed to either normoxia or hypoxia for 24 h. Cells were harvested and
processed for the evaluation of PKC? and PKC? expression at the end of the
For the evaluation of ERK1/2 activation, cells were plated and growth-
arrested according to the abovementioned method. In one set of experiments,
the cells were transiently transfected with PKC? scrambled and antisense
oligonucleotides and exposed to either normoxia or hypoxia for 10 min and
24 h. In the other set of experiments, the cells were pretreated with PKC?
pseudosubstrate peptide inhibitor for 1 h and then exposed to either nor-
moxia or hypoxia for 24 h. In both instances, the cells were harvested with
lysis buffer containing protease inhibitor/deoxycholate/SDS and freeze-
thawed, and lysates were recovered by centrifugation at 10,000 ? g. Proteins
were separated by gel electrophoresis, transferred to a PVDF membrane, and
probed with anti-phosphoERK1/2 antibody (1:1000).
MKP-1 expression was also evaluated by immunoblotting techniques. An-
tibody against MKP-1 was used at a concentration of 1:200–1:1000. The time
course of MKP-1 expression was determined at 0, 24, 48, and 72 h of hypoxic
exposure. ?-Tubulin expression, used as an internal control, was detected by
incubating the PVDF membrane with a mAb against ?-tubulin (1:1000).
Fibroblasts were plated in medium containing 10% FBS at a density of
?20,000/well in eight-well glass chamber slides (Life Sciences, Denver, CO).
After allowing to attach overnight, cells were growth-arrested with medium
containing 0.1% FBS for 72 h. Quiescent cells were then exposed to either
normoxia or hypoxia for 24 h. After treatment, cells were fixed with 4%
paraformaldehyde at 4°C, permeabilized with 0.5% Triton X-100, blocked
with porcine serum, and incubated with either an anti-PKC? antibody (1:25
dilution) or anti-MKP-1 antibody (1:50 dilution). Immunofluorescent staining
of PKC? was completed by incubating cells with biotin-conjugated anti-
mouse IgG (1:300) and streptavidin-conjugated Alexa 488 (1:2000). For
MKP-1, antigen–antibody complexes were visualized by incubating the cells
with Alexa 594–conjugated anti-rabbit secondary antibody (1:500). Nuclei
were stained with Hoechst dye (1:1000). Slides were mounted with H1000
mounting medium. All steps were done at room temperature unless other-
wise stated. Images were captured using an Olympus Infinity microscope
(Melville, NY) coupled to a Photometrics Quantix cooled CCD camera
(Tucson, AZ) and Deltavision digital deconvolution software.
All data are expressed as arithmetic means ? SE; n equals the number of
replicate wells per test condition in representative experiments. One- and
two-way analyses of variance, followed by the Student-Newman-Keuls mul-
tiple-comparisons tests, were conducted within and between groups of data
points. Data are considered significantly different if p ? 0.05.
PKC? Attenuation Greatly Up-regulates Hypoxia-induced
Proliferation of Fibroblasts
We previously demonstrated that hypoxia stimulates an
increase in DNA synthesis, which results in cell division in
vascular fibroblasts (Das et al., 2001). PKC is an important
intracellular regulator of hypoxia-induced proliferation of
fibroblasts (Das et al., 2000). To evaluate the role of atypical
PKC? isozyme in these proliferative responses, we first used
the isozyme-specific myristoylated peptide inhibitor derived
from the pseudosubstrate region. The peptide inhibitor
mimics the substrate and maintains PKC? in its inactive
form (Hirai and Chida, 2003). Figure 1 depicts concentra-
tion-dependent effects of peptide inhibitor on the prolifera-
tive responses of fibroblasts. The inhibitor at low dose (1
?M) did not affect the DNA synthesis (Figure 1). However,
BrdU incorporation in the quiescent cells was significantly
increased in the presence of 10 ?M inhibitor (Figure 1A).
Hypoxia alone stimulated a twofold increase in DNA syn-
thesis, whereas BrdU incorporation in hypoxia-exposed fi-
broblasts which were treated with 10 ?M inhibitor was
up-regulated by sevenfold (Figure 1A).
The role of PKC? in hypoxia-induced proliferation was
compared with that of basic FGF (bFGF) stimulation, a well-
known fibroblast mitogen (Figure 1B). bFGF-induced DNA
synthesis in the presence of the PKC? inhibitor (10 ?M) was
again significantly greater than that induced by bFGF or
inhibitor alone (Figure 1B).
To confirm the selectivity of PKC? inhibitor-mediated
stimulatory effects on BrdU incorporation, we also used a
myristoylated pseudosubstrate peptide inhibitor, which was
designed against classical and novel isozymes of PKC (PKC
inhibitor). Neither hypoxia-induced nor bFGF-stimulated
DNA synthesis was affected by PKC inhibitor (Figure 2, A
and B). In contrast, and as shown independently above
(Figure 1, A and B), PKC? inhibitor selectively augmented
hypoxia-stimulated and bFGF-induced BrdU incorporation
(Figure 2, A and B). However, one can interpret, by contrast-
ing hypoxia-induced and bFGF-stimulated BrdU incorpora-
tion in the presence of PKC? inhibitor, that PKC? attenuation
stimulates DNA synthesis in vascular fibroblasts. (A) BrdU incor-
poration in hypoxic fibroblasts is greatly augmented by PKC? in-
hibitor. For all the experiments where BrdU incorporation was
evaluated, cells were plated at the density of 20 ? 103/well in
24-well plates and growth-arrested with 0.1% FBS/MEM for 72 h.
Quiescent fibroblasts were preincubated with different concentra-
tions of PKC? inhibitor at 37°C for 1 h and then exposed to either
normoxia (21% O2) or hypoxia (1% O2) in the presence of BrdU (10
?M) for 24 h. At the end of the treatment, cells were fixed with cold
0.3% H2O2/methanol and processed for immunoperoxidase stain-
ing with anti-BrdU antibody. BrdU-positive and total nuclei were
counted at five different randomly selected areas in individual well.
n ? 4 replicate wells. * p ? 0.05 compared with normoxic control
data. ** p ? 0.05 compared with the hypoxic data. Similar results
were obtained in three independent experiments with cell popula-
tions isolated from three independent animals. (B) PKC? peptide
inhibitor also up-regulates bFGF-stimulated DNA synthesis. Quies-
cent fibroblasts were treated with PKC? inhibitor as mentioned
above and then stimulated with bFGF (30 ng/ml) for 24 h in the
presence of BrdU. n ? 4 replicate wells. * p ? 0.05 compared with
the unstimulated control value. ** p ? 0.05 compared with bFGF-
stimulated data. Similar results were reproduced with at least two
other cell populations.
Myristoylated PKC? pseudosubstrate peptide inhibitor
Hypoxia-induced Dephosphorylation of ERK1/2
Vol. 17, April 20061997
unmasked proliferative responses in greater extent in hy-
poxic cells (Figure 1, 3-fold, and Figure 2, 7.6-fold, over
hypoxia-induced DNA synthesis) than bFGF-stimulated fi-
broblasts (Figure 1, 1.3-fold, and Figure 2, 4-fold, compared
with bFGF-stimulated proliferation). Therefore, functional
role of PKC? might be more critical during hypoxic exposure
than bFGF-stimulated fibroblasts. Taken together, our data
suggest that replicative up-regulation by myristoylated
PKC? pseudosubstrate peptide inhibitor is specific to this
peptide and that PKC? might act as a proliferation suppress-
ing kinase in fibroblasts.
To confirm the role of PKC? as a replication repressor, we
then used antisense oligonucleotides specific for the mes-
sage encoding PKC?. Transient transfection of the cells with
PKC? antisense oligonucleotides selectively reduced the
PKC? expression, but not the PKC? levels (Figure 3, A and
B). For detection of the PKC? level in our cells by immuno-
blotting techniques, we used 4–5 times higher protein con-
centration than that used for the evaluation of the PKC?
levels. In addition, x-ray film was exposed up to 30 min for
the detection of the PKC? signal. Collectively, these obser-
vations suggest that a very low level of PKC? is present in
vascular fibroblasts. A recent report (Cogolludo et al., 2005)
also supports the notion that PKC?, but not PKC?, is the
major atypical isozyme of pulmonary vasculature.
Proliferative responses of fibroblasts in the presence of
PKC? antisense oligonucleotides were then evaluated by
measuring the BrdU incorporation. Marked increase in DNA
synthesis was observed upon attenuation of PKC? expres-
sion with antisense oligonucleotides in normoxic fibroblasts
(Figure 3C). Hypoxia stimulated BrdU incorporation in fi-
broblasts both in the presence of scrambled and PKC?-spe-
cific antisense oligonucleotides (Figure 3C). Equal magni-
tude in the up-regulation of DNA synthesis upon blockade
of PKC? expression with antisense oligonucleotides under
both normoxic and hypoxic conditions suggests that prolif-
erative responses are primarily regulated by PKC?-mediated
signaling pathways in fibroblasts in response to hypoxic
exposure. Collectively, these results further support the role
of PKC? as a proliferative repressor for vascular fibroblasts.
PKC? Overexpression Attenuates Proliferation
of Hypoxic Fibroblasts
The myristoylated pseudosubstrate peptide inhibitor can
block the activation of both PKC? and PKC? because se-
novel isozymes, stimulates proliferation of vascular fibroblasts. (A)
Hypoxia-induced DNA synthesis is selectively up-regulated by
PKC? inhibitor. Quiescent fibroblasts were preincubated with pep-
tide inhibitors of PKC classical and novel isozymes (10 ?M) and
PKC? isozyme (10 ?M) at 37°C for 1 h. BrdU incorporation in both
normoxic and hypoxic fibroblasts was evaluated. n ? 4 replicate
wells. * p ? 0.05 compared with normoxic control results. ** p ? 0.05
compared with hypoxic data. (B) PKC inhibitor of classical and
novel isozymes does not affect bFGF-stimulated DNA synthesis.
Cells were preincubated with inhibitors as mentioned above and
then stimulated with bFGF (30 ng/ml) in the presence of BrdU (10
?M) for 24 h. n ? 4 replicate wells. * p ? 0.05 compared with
nonstimulated control results. ** p ? 0.05 compared with bFGF-
induced data. Similar results were obtained from three independent
experiments using fibroblasts isolated from three different animals.
PKC? inhibitor, but not PKC inhibitor of classical and
ulates BrdU incorporation in vascular fibroblasts. (A) PKC? expres-
sion is blocked by antisense oligonucleotides in fibroblasts. For all
the transfection experiments, cells were plated at a density of 50–
100 ? 103/well in six-well plates in 10% FBS/MEM and growth-
arrested with 0.1% FBS/MEM for 72 h. Quiescent cells were tran-
siently transfected with PKC? scramble and antisense oligonucleotides.
After overnight recovery from transfection, cells were harvested with
the lysis buffer. Whole cell lysates were processed for immunoblot
analysis with anti-PKC? mAb. (B) PKC? antisense oligonucleotides
have no effects on PKC? expression. The abovementioned cell lysates
were processed for Western blot analysis for PKC? detection using
mAb against PKC?. (C) BrdU incorporation in fibroblasts is greatly
up-regulated by PKC? antisense oligonucleotides. Quiescent cells were
transiently transfected with PKC? scramble and antisense oligonucle-
otides. Transfected cells were incubated at 37°C for overnight. Then
BrdU (10 ?M) was added to the cells and exposed to either normoxia
or hypoxia for 24 h at 37°C. n ? 4 replicate wells. * p ? 0.05 compared
with the data in the presence of scramble oligonucleotides under
normoxic conditions. Similar results were reproduced in at least two
different cell populations isolated from two different animals.
PKC? attenuation with antisense oligonucleotides stim-
M. D. Short et al.
Molecular Biology of the Cell1998
quence homology exists in the pseudosubstrate region be-
tween these two atypical isozymes (Toker, 1998). Therefore,
to confirm that the proliferation stimulatory effects of the
inhibitor (Figures 1 and 2) are due to PKC? inhibition and
not attenuation of PKC? activity, PKC? was overexpressed in
fibroblasts by transient transfection with constitutively ac-
tive PKC? (MyrPKC?). Increased expression of PKC?, but
not PKC?, in the presence of MyrPKC? was confirmed by
Western blot analysis (Figure 4, A and B).
We then evaluated BrdU incorporation in the presence of
MyrPKC?. Hypoxia induced a 2.5-fold up-regulation of
BrdU incorporation in vector-transfected cells (Figure 4C).
However, marked down-regulation (6-fold) in DNA synthe-
sis was observed in PKC?-overexpressing cells compared
with the vector-transfected fibroblasts under hypoxic condi-
tions (Figure 4C). These data strongly suggest that PKC? acts
as a proliferative repressor in fibroblasts under hypoxic
Hypoxia Up-regulates PKC? Phosphorylation
PKC? phosphorylation at the Thr410 residue in the activa-
tion loop is required for its activity in response to a stimulus
(Hirai and Chida, 2003). To evaluate hypoxia-induced PKC?
phosphorylation at the Thr410 site, PKC? was immunopre-
cipitated from the lysates of control and hypoxia-exposed
fibroblasts and immunoblotted with an anti-phosphoThr410
antibody. PKC? phosphorylation levels were significantly
higher in prolonged (24 h) hypoxia-exposed cells compared
with that of the control fibroblasts (Figure 5A). Equal
amounts of PKC? precipitation from all the lysates were
confirmed by immunoblotting the immunoprecipitates
against PKC? (Figure 5B). Quantitative data of the hypoxia-
induced increase in PKC? phosphorylation are presented in
We used an mAb directed toward the C-terminus of PKC?
to immunoprecipitate the isozyme. This antibody may cross-
react with the other atypical isozyme, i.e., PKC?. To rule out
that possibility, PKC? immunoprecipitates were immuno-
blotted for PKC?. PKC? signal was not detected in the PKC?
precipitate (our unpublished observation), suggesting the
antibody against PKC? does not cross-react with PKC? in
vascular fibroblasts. Therefore, our data suggest that hyp-
oxia stimulates phosphorylation of PKC? (Thr410) in vascu-
lar fibroblasts and is consistent with our previous observa-
tion of hypoxia-induced increase in PKC?-specific activity in
fibroblasts (Das et al., 2000).
cular fibroblasts. (A) PKC? expression is augmented in fibroblasts
transfected with constitutively active PKC? (MyrPKC?). Quiescent
cells were transiently transfected with vector containing MyrPKC?
and empty vector (PCMV5). After 48 h of transfection, cells were
exposed to either normoxia or hypoxia for 24 h and harvested with
lysis buffer. Cell lysates were processed for PKC? detection by
immunoblot analysis. (B) MyrPKC? does not affect PKC? expression
in fibroblasts. In the abovementioned cell lysates, PKC? expression
was evaluated by Western blot analysis using PKC?-specific mAb.
(C) BrdU incorporation in hypoxic fibroblasts is attenuated by Myr-
PKC?. Transfected fibroblasts were exposed to either normoxia or
hypoxia in the presence of BrdU for 24 h. n ? 4 replicate wells. * p ?
0.05 compared with PCMV5-transfected normoxic results. ** p ?
0.05 compared with the results of PCMV5-transfected hypoxic cells.
Similar results were obtained from three independent experiments
using fibroblasts isolated from three different animals.
PKC? overexpression attenuates DNA synthesis in vas-
broblasts. (A and B) Phosphorylation of the Thr410 residue of PKC?
is increased by hypoxic exposure. Growth-arrested cells were ex-
posed to hypoxia for 10 min and 24 h. Whole cell lysates (0.5–0.7 mg
total protein) were immunoprecipitated with monoclonal anti-PKC?
antibody and the immunocomplexes were analyzed by immunoblot
analysis with antibodies specific for either phosphoPKC? (A) or
total PKC? (polyclonal; B). Representative immunoblot of three
independent experiments is shown. (C) Quantitative data of hypoxia-
induced PKC? phosphorylation in vascular fibroblasts. * p ? 0.05 com-
pared with the data of normoxic condition.
Hypoxia induces PKC? phosphorylation in vascular fi-
Hypoxia-induced Dephosphorylation of ERK1/2
Vol. 17, April 20061999
Hypoxia Stimulates ERK1/2 Activation
PKC? has been demonstrated to regulate the downstream
ERK1/2 activation in response to various stimuli in different
cell types (Berra et al., 1993). We, therefore, evaluated the
activation of ERK1/2 as a possible downstream target of
PKC? in hypoxic fibroblasts. In contrast to the abovemen-
tioned up-regulation of PKC? phosphorylation in fibroblasts
exposed to prolonged hypoxia (Figure 5A), increase in
ERK1/2 phosphorylation was evident in both acute (10 min)
and prolonged (24 h) hypoxia-exposed fibroblasts. Greatest
magnitude of hypoxia-stimulated ERK1/2 phosphorylation
was observed in acute (10 min) hypoxia-exposed fibroblasts
(Figure 6A), which is consistent with our previous observa-
tions (Das et al., 2001). In that report, we have also demon-
strated that the activation of ERK1/2 mediates hypoxia-
induced proliferation of fibroblasts. With prolonged (24 h)
hypoxic exposure, the increase in phosphorylated ERK1/2
was significantly reduced compared with that of acute (10
min) hypoxic exposure (Figure 6C). Western blot analysis of
these lysates for total ERK1/2 confirmed equal protein load-
ing among all samples (Figure 6B). Quantitative data of
hypoxia-induced increase in phosphoERK1/2 levels are pre-
sented in Figure 6C. These results suggest that hypoxia
stimulates ERK1/2 phosphorylation in vascular fibroblasts.
PKC? Inhibition Up-regulates ERK1/2 Phosphorylation
To evaluate a possible role of PKC? in hypoxia-induced
ERK1/2 activation, ERK1/2 phosphorylation was examined
upon blockade of PKC? expression. First, attenuation of
PKC? level with antisense oligonucleotides was confirmed
by Western blot analysis (Figure 7A). Acute (10 min) hy-
poxia-induced ERK1/2 phosphorylation was observed in
the presence of scrambled oligonucleotides (Figure 7B). To
our surprise, ERK1/2 were also significantly phosphory-
lated in both normoxic and hypoxic fibroblasts upon down-
regulation of PKC? expression with antisense oligonucleo-
tides, implicating that PKC? might be a suppressor of
hypoxia-induced ERK1/2 phosphorylation (Figure 7B). The
quantitative data of acute hypoxia-induced ERK1/2 activa-
tion in the presence of PKC? antisense and scrambled oligo-
nucleotides are presented in Figure 7D.
With prolonged (24 h) hypoxic exposure, phosphoERK1/2
levels were significantly decreased compared with the
acute (10 min) hypoxia-exposed fibroblasts in the pres-
ence of scrambled oligonucleotides (Figure 7C), which is
consistent with the time course of hypoxia-induced ERK1/2
activation data (Figure 6). However, ERK1/2 phosphoryla-
tion was markedly up-regulated upon PKC? blockade with
antisense oligonucleotides in both normoxic and hypoxic
fibroblasts (Figure 7, C and E). Persistent ERK1/2 phosphor-
ylation upon PKC? attenuation suggests that PKC? is the
terminator of hypoxia-induced ERK1/2 activation in fibro-
The role of PKC? on ERK1/2 dephosphorylation was fur-
ther investigated using myristoylated PKC? pseudosub-
strate peptide inhibitor. Significant up-regulation of ERK1/2
phosphorylation has occurred in the presence of PKC? in-
hibitor (Figure 8A). We then used PKC? inhibitor to evaluate
phopshoERK1/2 localization in response to hypoxic stimu-
lation. We were not able to detect any signal for activated
ERK1/2 in quiescent fibroblasts by immunofluorescent
staining (Figure 8C). In cells exposed to prolonged hypoxia
(24 h), phosphoERK1/2 was expressed as distinct spots out-
side the nucleus (Figure 8C). However, in the presence of the
PKC? inhibitor, the intensity of phosphoERK1/2 immuno-
fluorescent staining was strikingly enhanced (Figure 8C).
Also, phosphoERK1/2 was compartmentalized in the nu-
cleus as well as in the cytoplasm of hypoxic fibroblasts upon
PKC? inhibition (Figure 8C). PhosphoERK1/2 must be in-
side the nuclear compartment to initiate cellular prolifera-
tive responses (Pouyssegur et al., 2002). Therefore, our data
suggest that PKC? inhibition initiates exuberant replication
of hypoxic fibroblasts (Figures 1 and 2) by permitting per-
sistent nuclear localization of phosphoERK1/2 in hypoxic
cells. Taken together the data, we conclude that PKC? is the
regulatory switch of ERK1/2 dephosphorylation status in
fibroblasts in response to hypoxic exposure.
MKP-1 Regulates ERK1/2 Dephosphorylation
A reduction in ERK1/2 phosphorylation can be achieved by
up-regulation of protein phosphatases, which dephosphor-
ylate activated ERK1/2 (Keyse and Emslie, 1992). To explore
the role of phosphatases in ERK1/2 dephosphorylation in
these primary fibroblasts, phosphoERK1/2 levels were eval-
uated in the presence of sodium vanadate, an antagonist of
phosphotyrosine phosphatases. There was marked increase
in phosophorylated ERK1/2 levels (Figure 9, A and B) in
fibroblasts, implicating the role of tyrosine phosphatases in
ERK1/2 dephosphorylation events.
Among potential candidates for such phosphatases, MKP-1
was chosen for investigation because this hypoxia-respon-
sive phosphatase dephosphorylates ERK1/2 in other cell
types (Keyse and Emslie, 1992). We utilized phosphatase
inactive MKP-1 (MKP-1PI), which acts as a dominant nega-
tive MKP-1, and examined the effects of MKP-1 blockade on
ERK1/2 phosphorylation. Overexpression of MKP-1 in cells
fibroblasts. (A and B) ERK1/2 phosphorylation is greatest in acute
(10 min) hypoxia-exposed fibroblasts. Quiescent cells were exposed
to hypoxia for 10 min and 24 h and harvested with lysis buffer.
Whole cell lysates were processed for immunoblot analysis. Phos-
phoERK1/2 (A) and total ERK1/2 (B) were evaluated using phos-
phoERK1/2- and total ERK1/2-specific antibodies. Representative
blots of four independent experiments are shown here. (C) Quantita-
tive data of hypoxia-stimulated ERK1/2 phosphorylation in vascular
fibroblasts. * p ? 0.001 compared with normoxic results. ** p ? 0.001
compared with data of normoxic and acute (10 min) hypoxia-exposed
Hypoxia stimulates ERK1/2 phosphorylation in vascular
M. D. Short et al.
Molecular Biology of the Cell2000
transfected with MKP-1PI was confirmed by Western blot
analysis (Figure 9C). Persistent phosphorylation of ERK1/2
was observed in both normoxic and hypoxic fibroblasts
upon expression of MKP-1PI (Figure 9E). However, ERK1/2
phosphorylation in the vector-transfected cells was up-reg-
ulated only upon acute (10 min) hypoxic exposure (Figure
9E), which is consistent with the time course of ERK activa-
tion in hypoxic cells as demonstrated in Figure 6. Total
ERK1/2 levels were not affected by MKP-1PI in fibroblasts
(Figure 9F). These results suggest that MKP-1 regulates
ERK1/2 dephosphorylation in vascular fibroblasts.
Hypoxia-induced Increase in MKP-1 Levels Represses
MKP-1 is a hypoxia-responsive gene (Laderoute et al., 1999;
Seta et al., 2001; Liu et al., 2003). To evaluate the effects of
hypoxia on MKP-1 expression in vascular fibroblasts, quies-
cent cells were exposed to hypoxia (1% O2) for different
lengths of time and MKP-1 expression was examined by
immunoblot analysis. Growth-arrested cells expressed a sig-
nificant amount of constitutive MKP-1 protein (Figure 10A).
After 24 h of hypoxic exposure, MKP-1 levels were greatly
increased in the cells (Figure 10A). MKP-1 is an early re-
sponse tumor suppressor gene and has a rapid turnover
(Noguchi et al., 1993). MKP-1 might accumulate over time in
both normoxic as well as hypoxic fibroblasts (Figure 10A). In
spite of the high levels of MKP-1 under normoxic conditions,
hypoxic cells always had greater MKP-1 levels (Figure 10A).
Equal protein loading among the samples was verified by
immunoblotting with an antibody against tubulin (Figure
10). Therefore, these data suggest that hypoxia up-regulates
MKP-1 expression in fibroblasts.
We then explored the role of MKP-1 in hypoxia-induced
proliferative responses using MKP-1PI. Marked up-regula-
tion (5-fold) in BrdU incorporation was observed in the
presence of MKP-1PI under normal conditions (Figure 10B).
Hypoxia induced DNA synthesis of the vector-transfected
cells by twofold (Figure 10B). However, MKP-1PI induced
an up-regulation of hypoxia-induced BrdU incorporation in
fibroblasts by 5.5-fold (Figure 10B). Similar magnitude in the
up-regulation of DNA synthesis between the normoxic and
hypoxic fibroblasts by MKP-1PI, suggests that hypoxia-in-
duced proliferation of fibroblasts is primarily regulated
through the MKP-1 pathway (Figure 10B). Collectively,
these data imply that hypoxia-induced increase in MKP-1
expression represses hypoxia-induced proliferation of fibro-
PKC? Regulates MKP-1 Expression
The strong parallel between proliferative up-regulation and
persistent ERK1/2 phosphorylation with PKC? attenuation
(Figures 1, 2, 7, and 8) and MKP-1 blockade (Figures 9 and
10) suggest that PKC? might direct ERK1/2 dephosphory-
lation events by regulating MKP-1. Therefore, the role of
PKC? in MKP-1 expression was first evaluated using PKC?
antisense oligonucleotides. In the presence of scrambled
oligonucleotides, MKP-1 levels were up-regulated in hy-
poxic fibroblasts (Figure 11A). PKC? blockade with anti-
sense oligonucleotides induced marked reduction in MKP-1
expression (Figure 11A). Preincubation of fibroblasts with
ERK1/2 activation in vascular fibroblasts. (A) Tran-
sient transfection of quiescent fibroblasts with PKC?
scramble and antisense oligonucleotides induced se-
lective blockade of PKC? expression, but did not alter
PKC? levels. (B) ERK1/2 phosphorylation is increased
by PKC? antisense oligonucleotides in acute (10 min)
hypoxia-exposed cells. Transfected cells were exposed
to either normoxia or hypoxia for 10 min. Total protein
was extracted with lysis buffer. Western blots were
performed on the extracts using an anti-phos-
phoERK1/2 antibody and anti-ERK1/2 antibody to
examine the levels of phosphorylated and total
ERK1/2. Similar results were obtained from three inde-
pendent experiments. Representative immunoblots of
hypoxia-exposed fibroblasts, PKC? blockade results in per-
sistent ERK1/2 phosphorylation. Transfected fibroblasts
end of the experimental period, whole cell lysates were
processed for immunoblot analysis with anti-phos-
phoERK1/2 and total ERK1/2 antibodies. (D) Quantita-
tive measurement of acute (10 min) hypoxia-induced
ERK1/2 phosphorylation in the presence of PKC?
scramble and antisense oligonucleotides. * p ? 0.05 com-
pared with the results of normoxic cells transfected with
scramble oligonucleotides. (E) Quantitative measurement
of ERK1/2 phosphorylation upon PKC? attenuation with
antisense oligonucleotides in prolonged (24 h) hypoxia-
normoxic cells that were transfected with scramble oligo-
nucleotides. **p ? 0.05 compared with the results of hy-
poxic fibroblasts transfected with scramble oligonucleo-
Hypoxia-induced Dephosphorylation of ERK1/2
Vol. 17, April 20062001
myristoylated PKC? pseudosubstrate peptide inhibitor also
resulted in complete blockade of MKP-1 expression under
both normoxic and hypoxic conditions (our unpublished
MKP-1 regulation by PKC? was further confirmed by
using MyrPKC?. Growth-arrested fibroblasts were tran-
siently transfected with empty vector (PCMV5) and Myr-
PKC?. PKC? overexpression stimulated an increase in
MKP-1 levels in fibroblasts (Figure 11B). Collectively, these
data suggest that PKC? regulates MKP-1 expression in vas-
PKC? and MKP-1 Colocalize in the Nucleus
of Vascular Fibroblasts
To evaluate the localization of PKC? and MKP-1, both nor-
moxic and hypoxia-exposed (24 h) cells were subjected to
double immunofluorescent staining for PKC? and MKP-1.
Under normoxic conditions, punctate staining pattern of
both MKP-1 and PKC? was detected in the nuclear compart-
ment of fibroblasts (Figure 12A). Intensity of the immuno-
reactivity of both PKC? and MKP-1 was enhanced in the
nuclear compartment of hypoxic fibroblasts (Figure 12B).
Nuclear localization of MKP-1 in vascular fibroblasts is
consistent with other reports (Reffas and Schlegel, 2000;
Plows et al., 2002; Pouyssegur et al., 2002). However, subcel-
lular localization of PKC? is cell-type dependent (Fields et
al., 1989; Cho and Ziboh, 1995). Specificity of nuclear PKC?
immunofluorescent staining in fibroblasts was therefore
confirmed with the peptide against which PKC?-specific
antibody was raised. Anti-PKC? antibody was preincubated
with the peptide and the mixture then was used for PKC?
detection. Punctate staining in the nuclear compartment was
completely abolished in the presence of this antigen–anti-
body complex, (Figure 12C) confirming that PKC? resides in
well-defined foci and colocalizes with MKP-1 in the nucleus
of vascular fibroblasts.
Fibroblasts were then preincubated with PKC?-specific
peptide inhibitor and exposed to either normoxia or hypoxia
for 24 h. MKP-1 immunofluorescent staining in the nucleus
was disrupted upon PKC? inhibition (Figure 12D). Nuclear
effects of myristoylated PKC? pseudosubstrate peptide in-
hibitor are neither surprising nor unique for this cell type.
Theodore et al. (1995) have reported that myristoylated PKC
pseudosubstrate peptide inhibitor is capable of translocating
stimulates prolonged ERK1/2 phosphorylation in the
nuclear compartment of vascular fibroblasts. (A)
Western blot analysis of phosphoERK1/2 in the pres-
ence of PKC? inhibitor. Quiescent fibroblasts were
preincubated with PKC? inhibitor (10 ?M) for 1 h at
37°C, exposed to either normoxia or hypoxia for 24 h,
and then harvested with lysis buffer. Whole cell ly-
sates were separated by Western blot analysis and
probed with anti-phosphoERK1/2 and total ERK1/2
antibodies. Similar results were obtained in three dif-
ent animals.A representative
phoERK1/2 is shown. (B) Representative blot of total
ERK1/2. (C) PKC? inhibition induces nuclear localiza-
tion of activated ERK1/2 in fibroblasts. Representative
photographs of phosphoERK1/2 compartmentaliza-
tion in hypoxic fibroblasts. Cells were plated at the
density of 20 ? 103/well/0.5 ml of 10% FBS/MEM in
eight-well glass chamber slides, allowed to attach
overnight, and then growth-arrested with 0.1% FBS/
MEM for 72 h. Fibroblasts were incubated with PKC?
inhibitor and then exposed to either normoxia or hyp-
oxia according to the abovementioned method. At the
end of the treatment, cells were fixed with cold 4%
paraformaldehyde and indirect immunofluorescent
staining of phosphoERK1/2 was performed. Nuclei
were stained with Hoechst dye. Magnification, ?100.
Similar results were obtained in two other experi-
ments using different cell populations.
PKC? pseudosubstrate peptide inhibitor
for the three
M. D. Short et al.
Molecular Biology of the Cell 2002
across biological membranes to accumulate in all compart-
ments of neuronal cells. Therefore, our results show that
MKP-1 expression is tightly regulated by PKC? in the nu-
cleus of vascular fibroblasts.
Despite our previous studies on signaling pathways that
mediate hypoxia-induced proliferation of fibroblasts (Das et
al., 2000 and 2001), it remains unclear how replication sup-
pressors, which simultaneously coexist with proliferative
stimulators, work collaboratively to attenuate normal pro-
liferation of hypoxic fibroblasts. In the present study, we
report for the first time that PKC? is the master regulator
of hypoxia-induced ERK1/2 dephosphorylation events
through the regulation of MKP-1 expression and thereby
limits proliferation of hypoxic fibroblasts (Figure 13). PKC?
attenuation leads to striking up-regulation in proliferation as
well as ERK1/2 phosphorylation in hypoxic fibroblasts. In
contrast, PKC? overexpression induces a significant down-
regulation of replication in hypoxic cells. PKC? regulates
hypoxia-induced MKP-1 expression in fibroblasts. MKP-1
blockade mimics the results of PKC? attenuation on hy-
poxia-stimulated ERK1/2 phosphorylation and prolifera-
tion. These results strongly support the idea that PKC? acts
as a replication repressor through its regulation of MKP-1
expression in hypoxic fibroblasts.
fibroblasts. (A and B) Significant up-regulation in phosphoERK1/2
levels is observed in the presence of sodium vanadate, an antagonist
of tyrosine phosphatases. Growth-arrested cells were preincubated
with sodium vanadate (200 ?M) for 24 h. Whole cell lysates were
separated by Western blot analysis and probed with anti-phos-
phoERK1/2 and total ERK1/2 antibodies. Similar results were ob-
tained in another independent experiment using different cell pop-
ulations. (C and D) MKP-1 is overexpressed in fibroblasts
transfected with phosphatase inactive MKP-1 (MKP-1PI). Quiescent
fibroblasts were transiently transfected with MKP-1PI or empty
vector. Cells were harvested with lysis buffer after 48 h of transfec-
tion. Whole cell lysates were separated by immunoblot analysis for
the evaluation of MKP-1 and tubulin expression. (E and F) Phos-
phoERK1/2 levels are greatly higher in the cells transfected with
MKP-1PI than in those transfected with empty vector. MKP-1PI-
and empty vector-transfected cells were exposed to either normoxia
or hypoxia (10 and 60 min). Proteins from the cell lysates were
separated by Western blot analysis and probed with anti-phos-
phoERK1/2 and total ERK1/2 antibodies. Similar results were ob-
tained in three different experiments using three different cell pop-
MKP-1 regulates ERK1/2 dephosphorylation in vascular
liferation of hypoxic fibroblasts. (A) Hypoxia up-regulates MKP-1 ex-
pression. Quiescent fibroblasts were exposed to either normoxia or
hypoxia for 24–72 h and harvested with lysis buffer. Western blot
analysis of the cell lysates was performed using anti-MKP-1 and tubu-
lin antibodies. Representative blot of the three independent experi-
ments is shown. (B) Transfection of fibroblasts with MKP-1PI stim-
ulates BrdU incorporation in the cells. Growth-arrested cells were
transiently transfected with empty vector and MKP-1PI. After 48 h of
transfection, cells were exposed to either normoxia or hypoxia in the
presence of BrdU for 24 h. n ? 4 replicate wells. * p ? 0.05 compared
with vector transfected normoxic results. ** p ? 0.05 compared with
the vector-transfected hypoxic data. Similar results were obtained from
three independent experiments using fibroblasts from three different
Hypoxia-induced increase in MKP-1 levels terminates pro-
Hypoxia-induced Dephosphorylation of ERK1/2
Vol. 17, April 20062003
Our data in vascular fibroblasts contrast with the majority of
published reports where PKC? is described as a proliferative
mediator in a variety of cells (Hirai and Chida, 2003). Recently,
Braun and Mochly-Rosen (2003) have demonstrated that PKC?
is required for TGF?1-induced proliferation of neonatal pri-
mary cardiac fibroblasts. PKC? attenuation also inhibits plate-
let-derived growth factor (PDGF)-induced proliferation of hu-
man airway smooth muscle cells (Carlin et al., 2000). However,
a recent report also demonstrates that PKC? blockade
does not inhibit rabbit vascular smooth muscle cell prolif-
eration (Hussain et al., 2002). In fact, PKC? inhibition in-
creases growth factor– and cytokine-induced proliferation,
which supports the notion that PKC? is an antiproliferative
kinase under specific circumstances and is in agreement
with our results describing the role of PKC? as proliferative
repressor of hypoxic fibroblasts. Therefore, the role of PKC? in
proliferation is not only cell type specific, but also species
specific and hence, the function of this particular atypical
isozyme in cellular proliferative responses requires more rig-
PKC? plays an important functional role in mitogenic
signaling by initiating the activation of the downstream
MAP kinases such as MEK and ERK family proteins (Berra
et al., 1993). Activation of the ERK cascade is known to be
associated with cellular proliferation (Chang et al., 2003).
Previously, we have also reported that proliferation of hy-
poxic fibroblasts is regulated by hypoxia-induced ERK1/2
activation (Short et al., 2004). However, in the present study,
PKC? attenuation results in persistent ERK1/2 phosphory-
lation, which contributes to the exuberant proliferation of
hypoxic fibroblasts. Another important point is that PKC?
blockade induces accumulation of activated ERK1/2 in the
nucleus, which is consistent with the fact that presence of
activated ERK1/2 in the nucleus is necessary for the induc-
tion of cell proliferation (Pouyssegur et al., 2002). PKC?-
induced termination of ERK phosphorylation observed in
the present studies is in contrast to the previously published
reports demonstrating that PKC? is the upstream kinase of
MEK and ERK in other cell types (Berra et al., 1993). Also, in
spite of the similar increases in proliferation of our cells and
rabbit vascular smooth muscle cells (Hussain et al., 2002)
upon PKC? inhibition, the role of PKC? in ERK1/2 activa-
tion is different between the two cell types. In the vascular
fibroblasts, PKC? is the master regulatory switch of ERK1/2
dephosphorylation events, whereas in rabbit smooth muscle
cells ERK1/2 activation is independent of PKC? activation
(Hussain et al., 2002). Therefore, the functional role of PKC?
in the regulation of ERK1/2 activation upon a stimulus is a
highly cell- and condition-specific event.
Because of the critical importance of ERK1/2 in cellular
signaling, the activities of ERKs must be tightly regulated
and this can be achieved by ERK-specific phosphatases, e.g.,
MKP family members. There are at least 11 MKPs in mam-
mals, which imply the existence of a complex regulatory
network for MAP kinase signaling. MKPs differ by proper-
ties such as tissue-specific expression, differential regulation
in response to various stimuli, distinct subcellular localiza-
tion and substrate specificity. MKP-1 has also been identi-
fied as a hypoxia-responsive gene in a variety of cell types
(Keyse and Emslie, 1992; Noguchi et al., 1993; Laderoute et
al., 1999) and is an immediate-early gene product, chara-
cterized by rapid transcriptional induction after MAP kinase
family activation, short half-life, and rapid destruction by
the 26S proteasome (Keyse and Emslie, 1992; Keyse, 1995).
MKP-1 participates in the determination of the kinetics and
thus the cellular outcome of MAP kinase signaling (e.g.,
proliferation vs. differentiation; Charles et al., 1993; Nishida
and Gotoh, 1993; and survival vs. apoptosis; Xaus et al.,
2001). The interaction between MKP-1/MAP kinase signal-
ing also exhibits cell type specificity (Zhang et al., 2003).
In the present study, hypoxia-induced up-regulation of
MKP-1 provides a novel mechanism, to account for the
inhibitory effects of PKC? on ERK1/2 phosphorylation and
proliferation in hypoxic fibroblasts. Marked prolongation of
ERK1/2 phosphorylation in cells treated with either PKC?
antisense oligonucleotides or myristoylated PKC? peptide
inhibitor correlates with the inhibition of MKP-1 expression
upon PKC? attenuation. Consequently, our results allow us
to conclude that PKC? is the suppressor of ERK1/2 phos-
phorylation by regulating MKP-1 expression. Prolonged ac-
tivation of PKC? in hypoxic fibroblasts (Figure 5) might be
required by the system to maintain the level of MKP-1 upon
hypoxic exposure. MKP-1 overexpression inhibits ERK-reg-
ulated reporter gene expression, Ras-induced DNA synthe-
sis, and growth-factor–stimulated entry into the S phase in
fibroblasts (Brunet et al., 1995). As a result, MKP-1 expres-
sion constitutes a control mechanism for attenuation of mi-
togenic signaling pathways. Our data further suggest that
blasts. (A) PKC? blockade attenuates MKP-1 expression. Quiescent
fibroblasts were transiently transfected with PKC? scramble and
antisense oligonucleotides. After allowing overnight recovery from
transfection, cells were exposed to either normoxia or hypoxia for
24 h. Whole cell lysates were separated by gel electrophoresis and
probed with anti-MKP-1, PKC?, and tubulin antibodies. (B) Myr-
PKC? stimulates MKP-1 expression in fibroblasts. Growth-arrested
fibroblasts were transiently transfected with PCMV5 and MyrPKC?.
Cells were harvested with lysis buffer after 48 h of transfection.
MKP-1, PKC? and tubulin were detected in the cell lysates by
Western blot analysis. Similar results were obtained in three inde-
pendent experiments using three different fibroblast populations
isolated from three different animals.
PKC? regulates MKP-1 expression in vascular fibro-
M. D. Short et al.
Molecular Biology of the Cell 2004
PKC?-mediated MKP-1 expression plays a crucial role in the
proper termination of ERK1/2 activation during the hypoxic
exposure and contributes to “switching off” the signals di-
recting the proliferation of hypoxic fibroblasts.
Evidence is accumulating to indicate that PKC is associ-
ated with nuclear events both in resting cells as well as in
actively dividing cells (Capitani et al., 1987; Chiarugi et al.,
1990; Buchner et al., 1992; Neri et al., 1994). In the present
study, PKC? localization in the nuclear compartment is con-
sistent with the report demonstrating its expression in the
nuclei of unstimulated adipocytes (Lacasa et al., 1995). Stim-
ulation of the adipocytes with insulin or serum caused a
rapid increase in nuclear PKC? activity, suggesting that
PKC? could directly phosphorylate structural and/or regu-
latory nuclear proteins. Nucleolin and heterogeneous nu-
clear ribonucleoprotein are the substrates of PKC?, suggest-
ing that PKC? may play an important role in nuclear signal
transduction (Tuteja and Tuteja, 1998). Nuclear PKC? might
compartment of vascular fibroblasts. (A) Representa-
tive photographs of MKP-1 and PKC? in normoxic
fibroblasts. Quiescent cells were fixed with cold 4%
paraformaldehyde and permeabilized with 0.5% Tri-
ton X-100. Double immunofluorescent staining was
performed with anti-MKP-1 and PKC? antibodies.
Magnification, ?100. (B) Representative photographs
of nuclear localization of MKP-1 and PKC? in hypoxic
fibroblasts. Growth-arrested cells were exposed to
hypoxia for 24 h. At the end of hypoxic exposure,
MKP-1 and PKC? were visualized by double immu-
nofluorescent staining. (C) Nuclear staining of PKC? is
abolished in the presence of PKC? peptide and anti-
PKC? antibody. To confirm the specificity of nuclear
PKC? staining, the PKC? peptide was preincubated
with the anti-PKC? antibody overnight at 4°C. Immu-
nofluorescent staining of PKC? was performed with
this antigen–antibody complex. (D) Myristoylated
PKC? pseudosubstrate peptide inhibitor induces dis-
organization of defined nuclear localization of MKP-1
in fibroblasts. Growth-arrested fibroblasts were prein-
cubated with PKC?-specific peptide inhibitor (10 ?M)
for 1 h at 37°C and then exposed to either normoxia or
hypoxia for 24 h. At the end of the treatment, cells were
fixed and processed for indirect immunofluorescent
staining of MKP-1. Similar results were reproduced in
two other fibroblast populations. Magnification, ?100.
n ? 4 replicate wells. A representative micrograph of the
three independent experiments is shown in each case.
MKP-1 and PKC? colocalize in the nuclear
Hypoxia-induced Dephosphorylation of ERK1/2
Vol. 17, April 20062005
be the critical mediator of the hypoxic responses in fibro-
blasts because PKC? transactivates HIF-1? by blocking the
expression of a factor inhibiting HIF-1 in renal cancer cells
(Datta et al., 2004). Interestingly, MKP-1 colocalizes with
PKC? in the nuclear compartment of fibroblasts. PKC? asso-
ciation with MKP-1 in the nucleus may constitute the critical
replication repressor system in fibroblasts.
Manipulation of PKC? levels, either by inhibition or over-
expression, leads to subsequent alteration in MKP-1 levels,
suggesting that PKC? tightly regulates MKP-1 expression in
fibroblasts. MKP-1 expression is also regulated by PKC,
albeit by a different isozyme, PKC?, in bone marrow mac-
rophages (Valledor et al., 2000). In H41E rat hepatoma cells,
insulin-induced MKP-1 expression is blocked by the myris-
toylated PKC? pseudosubstrate peptide inhibitor (Lornejad-
Schafer et al., 2003). However, in that report PKC?-mediated
MKP-1 expression is not correlated with any cellular re-
sponse. We believe that our data are the first demonstration
of nuclear colocalization of MKP-1 and PKC?, which to-
gether function as terminators of proliferative signals in
hypoxic fibroblasts. Our future studies will focus on evalu-
ating the mechanism of PKC?-mediated regulation of
MKP-1 expression in vascular fibroblasts.
Excessive proliferation of fibroblasts is associated with a
number of vascular diseases (Stenmark et al., 1987; Stenmark
and Mecham, 1997; Stenmark et al., 2000; Rey and Pagano,
2002), asthma, chronic obstructive pulmonary diseases, pulmo-
nary fibrosis (Zhong et al., 2005), and also cancer (Bhowmick et
al., 2004). Multiple factors including growth factors, cytokines,
mechanical stress, hypoxia, neurotransmitters, and hormones
are believed to contribute to the processes leading to fibroblast
proliferation (Sartore et al., 2001), wherein PKC? might serve as
a common second messenger mediating the termination sig-
nals for proliferative responses. One of the primary steps in the
orchestrated “emergency stop” cascade may be the up-regula-
tion of MKP-1 expression that dephosphorylates ERK1/2 and
consequently attenuates proliferation. PKC? exerts its sup-
pressing effect on ERK activation and proliferation in vascular
fibroblasts primarily through interactions that involve MKP-1.
Thus, we postulate that the balance between PKC?-mediated
MKP-1 expression and ERK1/2 activities stimulated by hyp-
oxia is critical for maintaining cellular homeostasis. Further
understanding of the mechanisms by which hypoxia stimu-
lates the PKC?-mediated induction of MAP kinase phospha-
tase might lead to strategies for the prevention and treatment
of fibroproliferative diseases resulting from chronic hypoxic
We thank Steve Hofmeister and Sandi Walchak for harvesting bovine pul-
monary artery tissue and Dr. Michael Zawada, Dr. Mary Reyland, and Dr.
Raphael Nemenoff for critical review of the manuscript. This study was
supported by National Heart, Lung, and Blood Institute Grant HL 64917 (M.
Das), HL57144-09, and HL14985-33 (K.R. Stenmark).
Atamas, S. P. (2002). Complex cytokine regulation of tissue fibrosis. Life Sci.
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J., Chapkin, R. S., and Moscat, J. (1993). Protein kinase C zeta isoform is critical
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