(Received for publication, September 27, 1996, and in revised form, December 17, 1996)
From the Vascular Biology Unit, Departments of Medicine and Vascular Surgery, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215
Activation of protein kinase C (PKC) induces
angiogenesis, migration, and proliferation of endothelial cells (EC),
but can also prevent growth factor-induced EC proliferation. To
determine whether these disparate effects are mediated by substrates of individual PKC isoenzymes, PKC and PKC
were overexpressed in rat
microvascular EC. Basal and stimulated migration were enhanced in
PKC
EC compared with either PKC
or control EC. Serum-induced growth of PKC
EC was decreased, while that of PKC
cells was similar to control EC. Phorbol ester markedly inhibited PKC
EC growth but enhanced growth of PKC
and control EC. To determine possible causes for this altered proliferation, the effect of PKC
on
adhesion, mitogen-activated protein kinase activity, and cell cycle
progression was measured. Adherence of PKC
EC to vitronectin was
significantly enhanced. Serum-induced extracellular signal-regulated kinase-2 activity was increased equally in both PKC
and PKC
EC
above that of control, while extracellular signal-regulated kinase-1
activity was similar in all EC. Cell cycle analysis suggested that
PKC
EC entered S phase inappropriately and were delayed in passage
through S phase. Thus, PKC
may mediate some proangiogenic effects of
PKC activation; conversely, PKC
may direct antiangiogenic aspects of
overall PKC activation, including slowing of the cell cycle
progression.
The formation of new blood vessels and the repair of those damaged by disease or injury depend upon endothelial migration and proliferation (1, 2). Several external agents that promote or inhibit proliferation and migration have been identified (3, 4), but the intracellular messengers that mediate these processes are less clear.
Activation of the serine-threonine kinase protein kinase C (PKC)1 by phorbol esters induces migration, proliferation (5), and tube formation of cultured endothelial cells (6, 7) and causes angiogenesis in vivo (7-9). In addition, chemical inhibitors of PKC or the down-regulation of PKC by prolonged treatment with phorbol esters abrogates the proliferative effects induced by growth factors and mitogens (10, 11) and also enhances endothelial permeability (12, 13) and alters the expression level of several fibrinolytic enzymes and their inhibitors (14). In contrast, treatment of endothelial cells with direct activators of PKC alters some responses that are usually associated with stimulation by physiologic agonists (15) and, under some conditions, can prevent growth factor-induced proliferation (16). This apparent paradox might be explained by the fact that the PKC family is composed of related but structurally distinct isoenzymes, each a product of separate genes and with discrete cofactor requirements, substrate specificity, and tissue distribution (16-18). Since phorbol esters activate multiple isoenzymes of PKC, the possibility is raised that each PKC isoenzyme may selectively mediate separate, and perhaps opposing, effects within stimulated endothelial cells.
Preliminary studies in our laboratory revealed that rat capillary
endothelial cells expressed several isoenzymes of PKC, including PKC, -
, -
, -
, and -
. Of these isoenzymes, previous
investigations have found that overexpression of PKC
or PKC
in
various cultured cells could affect their proliferation (19-21).
Overexpression of PKC
in fibroblasts promoted their proliferation,
while proliferation was inhibited in human breast cancer cells and
other cell lines (20, 22-24); similar alterations of cell growth have
been observed in cells overexpressing PKC
(20, 21, 25). Thus, the
possibility exists that activation of either of these isoenzymes
mediates the inhibitory component of PKC activation on endothelial
growth, while the other promotes one or more processes essential to
endothelial repair and angiogenesis. Such an effect would be presumably
mediated by isoenzyme-specific substrates, of which a few have been
identified (e.g. elongation factor eEF-1
(26)). In the
present study, we began by testing the effect of overexpression of
PKC
and PKC
on endothelial migration and proliferation, both of
which are essential processes for angiogenesis and wound healing. When
initial experiments revealed an inhibitory effect of PKC
on
endothelial proliferation, we then examined potential underlying
causes.
The RFPEC were a
generous gift from R. D. Rosenberg (MIT) (27, 28) and were propagated
in M199 medium (Life Technologies, Inc.) supplemented with 2 mM L-glutamine, penicillin (10 units ml1), streptomycin (10 units ml
1), and
amphotericin B (250 ng ml
1). To obtain the RFPEC that
stably express vector (control), PKC
, or PKC
, pcDNA-Neo
(Invitrogen, Inc.), pcDNA-bPKC
, or pcDNA-hPKC
constructs,
respectively, were transfected into early passage RFPEC cells by the
calcium phosphate precipitation technique. Following selection for
resistance to Geneticin, a number of vector-transfected, PKC
-transfected, and PKC
-transfected clones of endothelial cells (designated as control EC, PKC
EC, and PKC
EC) were isolated and
expanded, and the mRNA was examined by Northern blot analysis for
expression of the respective transcripts.
Cells were
removed from subconfluent cultures of stably transfected RFPEC by
trypsin and washed with PBS. Total cell counts were determined using a
Coulter counter. Equivalent numbers of cells were pelleted, resuspended
in homogenization buffer (50 mM Tris, pH 7.5, 5 mM EDTA, 10 mM EGTA, 50 mg ml1
N
-p-tosyl-L-chloromethyl
ketone, 100 mg ml
1
N-tosyl-L-phenylalanine chloromethyl
ketone, 100 mg ml
1 phenylmethylsulfonyl fluoride, 2 mg
ml
1 leupeptin, 0.3%
-mercaptoethanol), and briefly
sonicated. The cytosolic and cytoskeletal (the latter defined by its
lack of solubility in 1% Triton X-100) fractions were obtained by
differential centrifugation. PKC kinase activity was determined by
measuring phosphorylation of a PKC-specific peptide substrate, based on a conserved region of the epidermal growth factor receptor, using the
protein kinase C enzyme assay system purchased from Amersham Life
Science, Inc.
The EC were removed from the culture dish with
trypsin and washed with PBS. The cell pellet was resuspended in lysis
buffer (140 mM NaCl, 1.5 mM Mg2Cl,
0.5% Triton X-100, 15 mM Tris, pH 8.3), vortexed for
30 s, and incubated on ice for 10 min. The nuclei were pelleted,
and the supernatant was transferred to a fresh tube. An equivalent
volume of buffer containing 25 mM EDTA, 300 mM
NaCl, 2% SDS, 200 mg ml1 proteinase K, and 200 mM Tris, pH 7.5, was added to the supernatant and incubated
at 65 °C for 1 h. Total RNA was extracted with
phenol/chloroform, precipitated with ethanol, and resuspended in
DEPC-treated double distilled H2O. For Northern transfer
analysis, 20 µg of total RNA was loaded per lane, subjected to
electrophoresis on a 2% formaldehyde-agarose gel, and transferred to
GeneScreen Plus membrane according to the manufacturer's
recommendations. The blot was UV-cross-linked (Stratalinker,
Stratagene) and hybridized with random primed cDNA probes at
65 °C for 3 h in Quik-Hyb solution (Stratagene). Blots were
washed under high stringency conditions. Audioradiographs were analyzed
using a scanning densitometer.
Vector control, PKC, and PKC
EC
were removed from the culture dish with trypsin and washed with PBS.
The cell pellet was resuspended in dissociation buffer (20 mM Tris-Cl, pH 6.8, 50 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate, 50 mg ml
1
N
-p-tosyl-L-chloromethyl
ketone, 100 mg ml
1
N-tosyl-L-phenylalanine chloromethyl
ketone), sonicated briefly, and incubated at 37 °C for 30 min. Total
crude cellular protein was suspended in 4 × Laemmli buffer (1 × Laemmli buffer 0.25 M Tris-Cl, pH 6.8, 8% SDS, 40%
glycerol, 10%
-mercaptoethanol, 0.004% bromphenol blue) and
resolved on a 10% SDS-polyacrylamide separating gel with a 4%
polyacrylamide stacking gel. The proteins were subsequently transferred
to Immobilon-P membranes (Millipore, Inc.) according to the
manufacturer's recommendations. Immunoblot analysis of the stably
expressing clones was performed using the polyclonal antibodies for
PKC
and PKC
obtained from Santa Cruz Biotechnology, Inc.
Stably transfected cells were
propagated on coverslips placed in 12-well dishes and grown in the
absence of serum for 24 h prior to immunofluorescence analysis of
the PKC isoenzymes. The cells were washed with PBS and fixed in 2%
paraformaldehyde in PBS for 10 min. The cells were then washed with PBS
supplemented with 1% bovine serum albumin (PBS-BSA) and rendered
permeable in 0.1% Triton X-100, PBS-BSA solution for 10 min. The cells
were then washed and incubated in 10% goat serum, PBS-BSA solution for
30 min at room temperature. The solution was removed, and the cells
were incubated in the presence of an optimal concentration of primary
antibody (a 1:50 dilution of the PKC, PKC
(Santa Cruz
Biotechnology), or histone (Accurate Biochemicals, Inc.) antibodies) in
2% goat serum, PBS-BSA solution for 1 h. The cells were washed
with 0.05% Triton X-100 in PBS-BSA. The biotinylated anti-rabbit or
anti-mouse IgG (Vector Laboratories) was added at a 1:200 final
dilution and incubated at room temperature for 1 h. The cells were
washed, and streptavidin-Texas Red (Amersham Life Sciences) was added
at a 1:200 dilution and incubated for 30 min at room temperature. Cells
stained for the Golgi apparatus were treated with 0.5 µM
BODIPY TR ceramide (Molecular Probes) for 1 h. Cells were washed
once, and the coverslips were placed face-down on a slide with
FluorSave (Calbiochem). The cells were visualized under × 40 magnification by fluorescence microscopy.
Stably transfected cells were grown to
confluency and then incubated in serum-free M199 for 24 h prior to
the start of the assay. The cells were removed from the culture dish
with trypsin, washed once in PBS, and resuspended in serum-free M199 at
a final concentration of 1 × 106 cells/ml of medium.
Chemotactic agents were placed in the lower wells of a 48-well
microchemotaxis chamber (Neuro Probe, Inc.) at indicated
concentrations. An 8-µm porous, polyvinylpyrrolidone free,
polycarbonate membrane (Poretics Corp.), precoated with collagen type
I, was placed between the upper and lower wells of the chemotaxis
chamber. The cell suspension was then added to the upper wells of the
chamber at a density of 5 × 104 cells/well.
Chemotaxis was assayed over 4 h at 37 °C in a CO2 incubator, under both unstimulated (basal) and stimulated (25 ng of
hepatocyte growth factor/scatter factor per ml of medium) conditions.
The membrane was removed from the chamber, fixed in 70% ethanol for 20 min at 20 °C, and stained in hematoxylin overnight. The upper
surface of the stained membrane was scraped using a cotton swab,
leaving only the cells that migrated to the undersurface. Migration was
assessed by counting the number of cells on the lower surface of the
membrane at a × 200 magnification by light microscopy.
Endothelial cells were seeded at equivalent densities in six-well culture dishes and allowed to adhere overnight in complete medium. Following a wash with PBS, the cells were incubated for 24 h in M199 without serum. The cells were subsequently stimulated by the addition of enriched serum concentrations (either 1 or 15%) in the presence or absence of 1 µM phorbol 12-myristate 13-acetate (PMA). At the indicated times following stimulation, the cells were treated with trypsin, and the cells were counted with a Coulter counter.
Cell Adhesion AssayThe determination of endothelial cell
adhesion was performed as described previously (29) with some
modification. Briefly, Corning 96-well enzyme-linked immunosorbent
assay plates were coated with 1 mg of purified human vitronectin
resuspended in Ca2+, Mg2+-free PBS (pH 7.4) for
1 h at 37 °C. The plates were rinsed with the same buffer,
coated with 1% heat-denatured BSA in the same buffer, and incubated at
room temperature for 30-60 min. Actively growing (50-80% confluent)
cultures of RFPEC were removed from the culture plate with trypsin,
washed, and resuspended in M199 supplemented with 0.5% BSA. Cells were
plated in each matrix-coated well at 2.5 × 104
cells/well and incubated at 37 °C for 60-90 min. The unadhered cells were removed, and the wells were gently washed. Adherent cells
were detected by incubating in the presence of 6 mg ml1
p-nitrophenylphosphate (Sigma) in 50 mM sodium acetate (pH 5), 1% Triton X-100 for 1 h at
37 °C, followed by the addition of 1 N NaOH, and the
absorbance was determined at 405 nm using an enzyme-linked
immunosorbent assay plate reader.
The cells were seeded, synchronized by serum deprivation for 72 h, and stimulated as described for the proliferation assay. DNA flow cytometric analysis of stably overexpressing EC cells was performed using a technique described by Tennenbaum et al. (30). The endothelial cells were removed from the culture plate by treating with 0.003% trypsin in sample buffer (3.4 mM sodium citrate, 0.1% Nonidet P-40, 1.5 mM spermine HCl, 0.5 mM Tris-Cl, pH 7.6). The reaction was stopped by the addition of 0.05% trypsin inhibitor, 0.01% ribonuclease A in sample buffer. The cells were subsequently treated with ice-cold 0.042% propidium iodide, 0.116% spermine HCl in sample buffer. The cells were kept on ice and in aluminum foil until analyzed. Flow cytometry was performed on a FACStar plus flow cytometer at an excitation of 488-nm wavelength and 630DF22 emission, and the data were analyzed using the Verity MODFIT software.
Stably transfected control, PKC, and PKC
EC were removed with
trypsin from the culture plate and resuspended in PBS at 5 × 106 cells ml
1 for the flow cytometric
analysis of the surface expression of
3 or
5 integrins. Fifty-microliter aliquots of the cell
suspensions were incubated in the presence of optimal concentrations of
the primary antibody (1 µg of rabbit anti-rat
3 IgG
fluorescein isothiocyanate-conjugated (Pharmingen, Inc.) or 0.5 µl of
rabbit anti-human
5 polyclonal antibody (Chemicon, Inc.)
per 50 µl reaction) at room temperature for 15 min. The cells were
pelleted at 1000 × g for 5 min at 4 °C. When
necessary, the cells were resuspended in 50 µl of PBS and incubated
with appropriate volumes of the secondary antibody (1.2 mg
ml
1 donkey anti-rabbit IgG fluorescein
isothiocyanate-conjugated (Jackson Immunochemicals, Inc.)) for 15 min
at room temperature. The cells were washed twice with PBS and
resuspended in 200 µl of PBS. Fluorescence bound to cells was
detected with a FACStar plus flow cytometer set at a 488-nm
excitation wavelength and 530DF30 emission.
Vector control, PKC, and
PKC
EC were rendered quiescent for 24 h prior to the assay. The
cells were stimulated with M199 supplemented with 15% FBS or 1 µM PMA for the indicated times. The cells were then
harvested by removing the medium, washing once with ice-cold PBS, and
incubating in radioimmune precipitation buffer (50 mM
Hepes, pH 7.4, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 20 mM NaF, 20 mM sodium
pyrophosphate, 1% Triton X-100, 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride) on a rocking incubator
at 4 °C for 30 min. The lysed cells were scraped from the culture
dish and transferred to a microcentrifuge tube. Large cellular debris
was removed from the protein suspension by centrifugation at 15800 × g for 5 min at 4 °C. The cleared protein extracts were transferred to a fresh microcentrifuge tube, and total protein concentration was determined by means of the bicinchoninic acid assay
(Pierce). MAP kinase was immunoprecipitated using extracellular signal-regulated kinase (ERK)-1 or ERK-2 antibody (Santa Cruz Biotechnology) from extract containing equal amounts of protein. After
the sample volumes were adjusted with radioimmune precipitation buffer,
the ERK-1 or ERK-2 antibody (1 µg of antibody/250 µg total protein)
was added. The extracts were incubated on a rocking incubator at
4 °C overnight. The immune complexes were pelleted with protein A-agarose (Life Technologies, Inc.), washed three times in radioimmune precipitation buffer, and suspended in 50 µl of kinase buffer (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 10 mM MgCl2). ERK-2 activity was assayed by
incubating 20 µl of each sample with 20 µl of the reaction mixture
(8 mg of myelin basic protein (Sigma), 0.5 µCi of
[
-32P]ATP (specific activity 3000 mCi/mmol) (DuPont
NEN), and 10 µM ATP) for 30 min at 25 °C. The reaction
was quenched with 15 µl of 4 × Laemmli buffer. The
phosphorylated myelin basic protein was then resolved on a 15%
SDS-polyacrylamide separating gel with a 4% polyacrylamide stacking
gel and visualized by autoradiography.
To investigate the role of PKC and PKC
isoenzymes in
relation to angiogenesis, stably transfected microvascular RFPEC were established that overexpressed the eukaryotic expression vector pcDNA-Neo containing the complete cDNA sequence of PKC
isoenzyme PKC
or PKC
or, as a control, the expression vector
without an inserted gene (PKC
EC, PKC
EC, and control EC,
respectively). The stably expressing RFPEC cell lines were selected by
neomycin resistance and screened by Northern blot analysis for gene
expression (Fig. 1).
The stably transfected RFPEC displayed the cobblestone morphology
typical of endothelial cells and were not visibly altered by
transfection or overexpression. Immunoblot analysis demonstrated increased protein production of the corresponding PKC isoenzymes. The
enzymatic activity of PKC was determined in several cell lines. Following this initial analysis, two clones of each type (PKC EC and
PKC
EC) were chosen for further study on the basis of similar levels
of total kinase activity. Table I summarizes the PKC
activity of the control and the two selected PKC
and PKC
EC
lines, revealing total PKC activity that was increased in both the
cytosolic and cytoskeletal fractions and was comparable between PKC
and PKC
EC.
|
To ensure that overexpression of PKC and PKC
isoenzymes in the
endothelial cells did not cause abnormal subcellular localization, we
assessed the intracellular location of these PKC isoenzymes by
immunofluorescence in both quiescent and PMA-treated EC. Experiments in
PMA-treated EC were performed because activation of some PKC isoenzymes
is associated with their redistribution into distinct subcellular
locations (31). In quiescent control EC, PKC
could be detected
primarily within the cytoplasm and nucleus (Fig.
2A). Staining of these cells with antibodies
directed against histone proteins or with a fluorescently tagged
ceramide confirmed the nuclei and Golgi apparatus structures (Fig. 2,
I and J), respectively. Following a 10-min
incubation with PMA, PKC
could still be seen in the nucleus, but
also at the periphery of the cell along the plasma membrane (Fig.
2B). Interestingly, PKC
translocated primarily to regions
of the plasma membrane in which there was cell-cell contact. A similar
cellular distribution of PKC
was noted in both the PKC
EC (Fig.
2, C and D) and in the PKC
EC (data not shown). Immunofluorescent staining for PKC
demonstrated the nuclear and cytosolic location for this isoenzyme in serum-starved control EC
(Fig. 2E). PKC
redistributed to the plasma membrane and
the nuclear membrane upon activation with PMA (Fig. 2F). A
similar pattern of staining for PKC
was noted in the PKC
(data
not shown) and PKC
(Fig. 2, G and H) EC. Thus,
constitutive overexpression of PKC
or PKC
did not affect normal
cellular localization in either stimulated or quiescent endothelial
cells, although enzymatic activity was increased.
Effect of PKC Isoenzymes on Endothelial Cell Migration
To
determine the role of PKC and
in endothelial cell migration, the
respective stably transfected cell lines were seeded in a
microchemotaxis chamber, and the number of endothelial cells that
migrated through the polycarbonate membrane was determined as described
under "Experimental Procedures." When hepatocyte growth factor (HGF
or scatter factor), a powerful stimulus for migration and angiogenesis
(32, 33), was utilized as the agonist, PKC
EC traversed the membrane
at a significantly greater rate than did PKC
or control EC (Fig.
3), suggesting a migratory response mediated by PKC
to this stimulus. The basal rate of migration (i.e. that
occurring in the absence of any chemotactic agent) of PKC
was also
consistently greater than that of the control EC or PKC
EC (Fig. 3),
further implicating a specific role for PKC
in enhancing endothelial
cell migration. Thus, both basal and agonist-stimulated endothelial
migration differed between PKC
and PKC
EC, and PKC
EC
migration was enhanced from the response seen in control EC.
Effect of PKC
Stimulation of quiescent PKC EC with low (1%)
concentrations of serum induced a growth rate similar to that of the
vector control EC (Fig. 4A). In contrast,
PKC
EC exhibited much less proliferation in response to serum
stimulation than did either the control or PKC
EC. Proliferation of
the control EC and PKC
EC in response to PMA was mildly enhanced
(7.5%) above EC treated with serum alone at 72 h (Fig.
4B), but the growth rate of PKC
EC was significantly
inhibited further by PMA, with an inhibition of 46.2 ± 8.8%
below that of non-PMA-treated PKC
EC at 72 h (p < 0.05) (Fig. 4C). This inhibition of serum-induced growth
to serum stimulation was noted in both clones of PKC
EC tested; neither of the PKC
EC clones exhibited altered growth. Qualitatively similar responses were seen when quiescent cells were stimulated with
higher (15%) serum concentrations in the presence or absence of PMA
(data not shown). Thus, overexpression and stimulation of one isoenzyme
(PKC
) blocked endothelial proliferation, a response not seen when
PKC
was overexpressed to a similar degree.
Effects of PKC
In order to better understand the cause of the decreased
proliferation in PKC EC, we next asked whether PKC would lessen adhesion to extracellular matrices, prevent mitogen-activated protein
kinase (MAP kinase) activation, or alter cell cycle progression in EC.
To determine whether integrin-mediated endothelial adhesion, an event
that is required for endothelial proliferation and angiogenesis (29,
34, 35), was lessened by PKC
, we examined the adhesion of
subconfluent cultures of control, PKC
, and PKC
EC seeded on
vitronectin-coated plates. Rather than being diminished, the ability of
the PKC
EC to adhere to the extracellular matrix was significantly
enhanced above that seen with the control EC (Fig. 5),
with a mean increase in adherence of 32.4% (p
0.005). In contrast, increased PKC
expression did not significantly
alter the ability of the endothelial cells to adhere to vitronectin. Preincubation of these cells with a synthetic peptide, GRGDSP, which
corresponds to the vitronectin protein sequence that directly interacts
with the integrin receptor binding domain, abolished adherence of these
cells, thus demonstrating that the base line adherence of the cells,
plus that enhanced in PKC
EC, was specific for the integrin
receptors. To determine whether the enhanced adhesion resulted from
increased expression of integrin receptors on the cell surface, we
analyzed the cellular surface expression level of
v
3 and
v
5
by immunofluorescence using flow cytometry. Neither the overall
cellular surface expression level of
3 nor that of
5 was significantly enhanced in PKC
EC as compared
with PKC
or control EC (data not shown). Thus, enhanced adhesion in PKC
EC most probably resulted from increased affinity modulation of
the integrin receptors. These results, therefore, demonstrate that the
reduction in cell growth in PKC
did not result from impaired
adhesion to extracellular matrices.
Serum-induced ERK-2 Activation in PKC
We
next tested the possibility that impaired PKC EC growth resulted
from impaired activation of one of the MAP kinases, ERK-1 or ERK-2,
that are known to be activated following overall PKC activation (36,
37) in EC.
Serum stimulation of vector (control) EC that had been rendered
quiescent demonstrated a rapid increase in ERK-2 activity by 10 min,
with a gradual diminution of the kinase activity by 2-4 h after
stimulation (Fig. 6, A and B).
ERK-2 activity also increased within 10 min following serum stimulation
of the PKC and PKC
EC; however, the activity was enhanced above
control and remained elevated above the basal kinase activity even at 4 h following stimulation. Phorbol ester treatment of the stably transfected cells resulted in similar levels of ERK-2 activity in
PKC
or PKC
EC as compared with control, with the overall ERK-2
activity diminishing at a more rapid rate in control EC (i.e. 4 h) (Fig. 6, A and C).
Similar analyses demonstrated very low overall ERK-1 activity in all
cells tested. Serum stimulation resulted in mild enhanced ERK-1
activity; however, there were no noticeable differences between the
control, PKC
, and PKC
EC (data not shown). Thus, PKC
and
PKC
appeared to be equally effective in activating ERK-2
, and
thus the decrease in proliferation in PKC
EC appeared to result from
a mechanism independent of ERK-1 or ERK-2 activity.
Effect of PKC
To
determine if an alteration in cell cycle progression might explain the
decrease in cell growth in PKC EC, cell cycle analysis was performed
on serum-deprived and stimulated PKC
, PKC
, and control EC. After
72 h of serum starvation, 19.8 ± 4.2% of control EC and
17.1 ± 0.4% PKC
EC were in S phase, with 74.2 ± 5.5%
and 77.3 ± 0.9% in G0/G1, respectively
(Fig. 7A). In contrast, 26.3 ± 2.2% of
the PKC
cells were in S phase, with 66.5 ± 1.9% in G0/G1. These data indicate that an abnormally
high percentage of PKC
EC entered S phase inappropriately,
i.e. under conditions of serum deprivation. Stimulation with
serum caused control EC and PKC
EC to reenter the cell cycle
normally (Fig. 7, B and C). PKC
EC, on the
other hand, after an initial increase in the percentage of cells in
G2/M phase at 6 h, followed by an increase in cells in
G0/G1 at 12 h after serum, returned to a
very high percentage of cells in S phase up to 60 h (Fig.
7D). The prolonged time that a high percentage of PKC
EC
could be found in S phase suggested that these cells required an
abnormal amount of time to complete S phase.
The two major findings of this study are that overexpression of
two different PKC isoenzymes normally expressed in microvascular endothelial cells exert distinct effects on endothelial proliferation, migration, and adhesion to extracellular matrix and that
PKC-mediated inhibition of endothelial growth results from a defect
in S phase of the endothelial cell cycle. The observation that
overexpression of PKC
, but not PKC
, prevents proliferation of
microvascular endothelial cells, while PKC
enhances their migration
in response to HGF (scatter factor), suggests that these isoenzymes
phosphorylate different substrates in these cells with different
physiologic effects. The disparity between the effects of
overexpression of the two isoenzymes is heightened by PKC-activating
phorbol esters in that treatment of PKC
EC exerts a mitogenic effect
similar to that in control cells, while PKC
EC were even more
strongly inhibited by similar treatment. Thus, these data suggest that activation of each of these isoenzymes by angiogenic stimuli, such as
HGF or those contained in serum, may mediate distinct aspects of
several processes that are required for vascular repair and
angiogenesis. Stimulation of endothelium with phorbol esters, which
activate both PKC
and PKC
, as well as several other isoenzymes expressed in endothelium (5, 38), has been noted to have both
stimulatory and inhibitory effects on endothelial proliferation and
angiogenesis (5, 10, 39, 40) that can be temporally dispersed within
the same cells (41). The results from this study suggest that PKC
might mediate those aspects of PKC activation that are inhibitory for
endothelial repair and angiogenesis, while an important component of
the proangiogenic effect, endothelial migration, might be mediated by
PKC
.
Repair of small defects in endothelium are accomplished largely by
endothelial migration, rather than proliferation (3, 42). Migration is
an important component of angiogenesis as well (3). The present
study's finding that endothelial migration is enhanced in PKC EC
might result from enhancement of cytoskeletal reorganization in
response to stimuli, which is a necessary component of cell locomotion;
overall PKC stimulation has been associated with promotion of
cytoskeletal reorganization of endothelial cells (3, 43). Our results
suggest that PKC
, but not PKC
, may be at least one mediator of
migration response to HGF (scatter factor), a powerful angiogenic agent
that is present along with its receptor in a substantial amount in the
vasculature (4, 32, 44, 45).
We considered several possible explanations for the PKC-mediated
inhibition of endothelial growth, including a reduction in endothelial
adhesion to matrix, a failure to activate downstream mediators such as
ERK-1 or ERK-2, and a defect in progression of endothelial cells
through the cell cycle. Of these explanations, only the latter appears
to be the case. Regarding adhesion, both endothelial cell growth and
migration are thought to require attachment of the cell to matrix via
its integrin surface receptors (42, 46, 47). Blocking the vitronectin
integrin receptors
v
3 or
v
5 inhibits neovascularization in the
cornea or chick chorioallantoic membrane models (35), suggesting the
importance of these two integrins for endothelial cell proliferation.
In this study, microvascular endothelial cells in which PKC
was
overexpressed demonstrated markedly enhanced adhesion to vitronectin or
fibrinogen matrices, and thus a decrease in integrin mediated adhesion
was not the cause of the decrease in proliferation of PKC
EC. This
enhancement of adhesion could result from either a PKC-mediated
alteration of the number of these receptors or an increase in avidity
by either a direct effect on integrin conformation (so-called
"inside-out" signaling) (48) or as an amplification step following
cell adhesion ("outside-in") that prevents detachment (48). Overall
PKC activation has been linked with promotion of integrin avidity for
soluble fibrinogen and solid matrices in several cell types (49, 50). Neither PKC
EC nor PKC
EC demonstrated increased expression of
these receptors when compared with control; thus, a PKC
-mediated effect on affinity of these integrins for vitronectin is a likely explanation for our results.
A cascade of signaling events merging at the MAP kinase family of
proteins, ERK-1 and ERK-2, is involved in many of the intracellular signaling pathways that lead to endothelial cell growth, migration, and
adhesion (51). Activation of overall PKC leads to activation of MAP
kinase; PKC, at least, has been shown to phosphorylate Raf kinase
(40), an upstream mediator of MAP kinase (36). In our studies, both
PKC
and PKC
enhance ERK-1 and ERK-2 kinase activity with a
resultant prolongation of ERK-2 activity in stably transfected
endothelial cells. The pathway by which PKC
blocks proliferation and
cell cycle progression, however, must be distal or parallel to that
leading to ERK-2 or ERK-1. In addition, our results suggest that
effective activation of ERK-2 activity by PKC
is not sufficient for
endothelial cell proliferation. This finding bolsters those of Hirai
et al. (25), who found that PKC
inhibited growth of
Ras-transformed NIH 3T3 cells despite activating AP-1, a component of
the signaling pathway downstream from MAP kinase.
Inhibition of endothelial cell proliferation by PKC appears to
result from a specific defect in endothelial cell cycle progression, in
which the cells enter S phase inappropriately and require additional time to complete this phase. Because of the distal nature of this defect and the intranuclear location of PKC
, it seems likely that
the isoenzyme interacts directly with one of the
cyclin-cyclin-dependent kinase/inhibitor complexes that regulates entry
and completion of S phase. Inappropriate S phase entry has been
associated with apoptosis of vascular smooth muscle cells treated with
basic fibroblast growth factor antisense oligonucleotides (52), but
such apoptosis was not found in PKC
EC.2
Manipulation of PKC activity has not been previously associated with
this S phase defect and only rarely with any abnormality of cell cycle
function. Overexpression of PKC
, but not PKC
or PKC
, in
Chinese hamster ovary fibroblasts causes an arrest in G2/M
phase of the cell cycle, but only after activation with PMA (21); no
defects were seen in S phase. In that study, inhibition of cell cycle
progression by PKC
was attributed to an isoenzyme-specific effect;
since the PKC enzymatic activity was much higher in the PKC
-transfected cells than in the other transfectants, however, it
is possible that the observed effect merely reflected an increase in
overall enzyme activity. In this study, however, the similarities of
enzymatic activity make it likely that the defect in cell cycle progression, as well as differences in adhesion and migration, resulted
from interaction of PKC
with isoenzyme-specific substrates. Thus, in
addition to its isoenzyme-specific character, this arrest in S phase
appears to be somewhat specific for endothelial cells.
Specific effects of individual PKC isoenzymes on proliferation are
known to vary according to cell types. For example, overexpression of
PKC in murine fibroblast cells inhibits proliferation and does not
lead to cell transformation (53, 54), but in NIH 3T3 and human breast
cancer cell lines, increases in PKC
expression altered the cell
morphology, enhanced proliferation, and increased tumorigenicity (23,
55). Thus, the tissue-specific effects of individual PKC isoenzymes are
likely to be mediated by substrates or effectors with restricted tissue
or subcellular expression. Even within an individual cell type, the
substrates with which each isoenzyme interacts differ. A full
understanding of the mechanism by which these two individual PKC
isoenzymes mediate either enhanced adhesion or migration or decrease
proliferation of endothelial cells will require identification of their
selective downstream targets. Such identification, together with the
assignment of selective endothelial functions to individual PKC
isoenzymes provided by this study, would provide essential details
critical to our understanding of reendothelialization and
angiogenesis.
We thank Malay Raychowdhury, James D. Chang, and Masao Yukawa for technical assistance and advice.