Department of Pharmacology, The University of Sydney, New South Wales 2006, Australia
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ABSTRACT |
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Protein kinase C (PKC) is implicated in the
regulation of smooth muscle contractility and growth. We have
previously described the pattern of isoform expression of PKC in canine
airway smooth muscle. This study identified the isoforms present in
human cultured airway smooth muscle cells and also addressed the
question of whether mitogenesis in these cells is associated with
changes in a specific isoform, PKC-. Western blot analysis revealed
the presence of PKC-
, -
I, and -
II of the conventional group;
PKC-
, -
, -
, and -
of the novel group; and PKC-
, -µ,
and -
of the atypical group. There was a significant increase in
density of the Western blot for PKC-
in cells proliferating in
response to 10% fetal bovine serum (FBS) to 372 ± 115% of control
values (P < 0.05;
n = 3 patients) in the cytosolic
fraction. Platelet-derived growth factor (PDGF) produced increases in
PKC-
in both the cytosolic and membrane fractions to 210 ± 49 and 443 ± 227%, respectively, of control values
(P < 0.05;
n = 4 patients). There was no change in expression of PKC-
, -
I, -
II, -
, -
, -
, -
, or
-
in response to the same stimuli.
PGE2 (1 µM) added to the cells
30 min before PDGF reduced incorporation of
[3H]thymidine from
5,580 ± 633 (SE) to 3,980 ± 126 dpm
(P < 0.05; n = 3 patients) and, in addition,
reduced expression of PKC-
in the membrane fraction as determined by
Western blotting from 266 ± 66 to 110 ± 4% of control values
(P < 0.05;
n = 3 patients). PKC-
activity in
stimulated cells (10% FBS), as assessed by immunoprecipitation and
phosphorylation of glycogen synthase peptide, was ~3-fold greater
than that in unstimulated cells, and the amount of PKC-
protein
correlated with isoenzyme activity
(r2 = 0.91; P < 0.02;
n = 4 patients). In conclusion, this
study 1) provides the first
description of which isoforms of PKC are present in human cultured
airway smooth muscle cells and 2)
shows that proliferation of these cells is associated with upregulation of PKC-
. Whether activation of PKC-
is a primary or secondary event in airway smooth muscle cell proliferation remains to be determined.
protein kinase C isoenzymes; atypical protein kinase C; bronchial muscle cells; hyperplasia
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INTRODUCTION |
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PROTEIN KINASE C (PKC) is a key regulatory enzyme involved in the regulation and cross talk between signal transduction pathways associated with various cellular functions. Specifically, in the airways, PKC is important in regulating smooth muscle contractility and growth. It has been shown that PKC phosphorylates a number of key contractile proteins in airway smooth muscle (ASM) (23), and PKC activation appears to be more involved in the sustained rather than in the initial phase of ASM contraction. In human isolated airways, stimulation of PKC produces both contraction (25, 29) and relaxation, and Yang and Black (30) previously reported that contractile responses are dependent on extracellular Ca2+ influx, whereas the relaxation phase involves stimulation of Na+-K+-ATPase.
PKC has also been implicated in the mitogenic response of a number of different cell types including vascular smooth muscle cells (14), fibroblasts (11), and, more recently, ASM cells (21). For example, activation of PKC by phorbol esters stimulates proliferation of porcine and human ASM (20, 22). Moreover, inhibitors of PKC such as Ro-31-8220 and Ro-31-7549 reduce the proliferative response of rabbit ASM to fetal calf serum (9) and the proliferative response of bovine pulmonary arterial adventitial fibroblasts (5).
PKC is not a single protein kinase but rather a family of multiple
isoenzymes with different biochemical characteristics, substrate
specificities, and cofactor requirements (10). The various isoforms of
PKC, which vary in their tissue distribution and function, have been
classified into three main groups (19): group
A, the conventional calcium-dependent isoenzymes
(PKC-, -
I, -
II, and -
); group
B, the calcium-independent (novel) isoenzymes (PKC-
,
-
, -
, and -
); and group C,
the atypical isoenzymes (PKC-
, -µ, -
, and -
).
Donnelly et al. (5) first described the PKC isoforms present in ASM
using canine tissue. They found that PKC-I and -
II from the
conventional group, PKC-
, -
and -
from the novel group, and
PKC-
from the atypical group were all present, whereas PKC-
, -
, and -
were absent.
Togashi et al. (27) followed this with experiments
performed in porcine ASM and reported some differences and some
similarities in isoform expression in this species. Webb et al. (28)
examined homogenates of human trachealis and peripheral lung and found some differences in the expression of isoforms both between these regions of the airways and in the results obtained by Northern and
Western blotting. Thus it has become apparent that significant species
and tissue differences exist in the expression of PKC isoforms in ASM,
and so one of the aims of the present study was to identify the
isoforms present in human cultured ASM. In addition, because evidence
is emerging that different isoforms of PKC have specific functions and,
in particular, that PKC- might be important in mitogenic signal
transduction in a number of different cell types (2), a second aim of
this study was to address the question of whether mitogenesis in human
cultured ASM cells is associated with changes in PKC-
.
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METHODS |
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Human ASM culture. Primary cultures of human ASM were established as reported previously by our group (13).
The protocol was approved by the Human Ethical Review Committee of The
University of Sydney (Australia). Briefly, macroscopically normal human
lung was obtained from patients undergoing lung transplantation or
partial resection (n = 4 with emphysema, n = 1 with
bronchiectasis, n = 1 with cystic
fibrosis, n = 1 with bronchopulmonary
dysplasia, n = 1 with pulmonary
hypertension, and n = 5 with primary
carcinoma). Large bronchi (5- to 15-mm internal diameter) were
dissected from the surrounding parenchyma and dipped in 70% (vol/vol)
ethanol in water to kill any surface organisms. ASM bundles, viewed
with a dissecting microscope, were dissected from the bronchi and
placed in tissue flasks containing 1% Fungizone, 1%
penicillin-streptomycin, and 10% fetal bovine serum (FBS). The smooth
muscle cells grew to confluence in a humidified
CO2 incubator in 16-24 days
and were passaged in 175-cm2
flasks at 7- to 10-day intervals. Pure populations of smooth muscle
cells were confirmed by the presence of positive staining for
-smooth muscle actin.
Cell preparation. Cells from passages 4-7 were seeded at a density of 104 cells/cm2, and the medium was changed from DMEM with 10% FBS to DMEM with 1% FBS for 24 h. It has previously been shown with flow cytometry that >80% of cells are in the G0/G1 phase of the cell cycle (K. Hawker, unpublished observations) after this treatment. Cells were then incubated in DMEM with 10% FBS or DMEM with 1% FBS plus platelet-derived growth factor (PDGF) at 40 ng/ml (13) for 24 h. The cells were then washed in PBS and homogenization buffer containing 30 mM Tris · HCl, 1 mM EDTA, 1 mM EGTA, 1 mM benzamidine, 5 mg/ml of leupeptin, 0.1 mM soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4. The cells were harvested, homogenized, and centrifuged, and the supernatants were taken as the cytosolic fractions. The pellets were washed, sonicated in 0.5% Triton X-100 in 150 µl of homogenization buffer, and centrifuged at 43,000 g for 30 min at 4°C, and the supernatants were retained as the membrane fractions. Protein concentrations in each sample were determined by the method of Bradford (3) with bovine serum albumin as the standard.
Western blot analysis. Equal amounts
of protein from both the membrane and cytosolic fractions were mixed
with the same volume of SDS sample buffer containing 500 mM
Tris · HCl (pH 6.8), 28% glycerol, 6% SDS, 6 mM
EGTA, 24% mercaptoethanol, and 0.0003% bromphenol blue, heated at
100°C for 2 min, and cooled to room temperature. The samples were
subjected to SDS-PAGE (10% wt/vol), as described by Laemmli
(16), with a Mini-PROTEAN II electrophoresis system (Bio-Rad, Sydney,
Australia). Crude extracts of rat brain were loaded onto adjacent lanes
as positive controls. The following high-molecular-mass
protein markers were also loaded onto each gel: 20-kDa myosin, 116-kDa
-galactosidase, 97-kDa phosphorylase b, 66-kDa albumin, and
45-kDa ovalbumin. Electrophoretic separation was carried out in 1%
SDS, 25 mM Tris, and 200 mM glycine (pH 8.4) at 200 V for 45-60
min at room temperature with a Bio-Rad model 1000/500 power supply. A
polyvinylidene difluoride membrane (Immobilon-P,
Millipore, Bedford, MA) was activated in 20% methanol for 1 min. The
proteins were then transferred to the membrane in 25 mM Tris, 192 mM
glycine, and 20% methanol (vol/vol; pH 8.3) at 100 V and 250 mA for 1 h at 4°C. At the conclusion of the transfer process, the
molecular-mass standards were cut off from the membrane, stained with
0.1% amino black in 2% acetic acid for 5 min, and then destained in
40% methanol and 10% acetic acid for 10 min. The membrane was
incubated for 12 h at 4°C in a Tris-buffered saline-Tween 20 solution (TBS-T) containing 10 mM Tris, 0.5 M NaCl, and 0.5% Tween 20, pH 7.4, with 5% (wt/vol) nonfat dried milk. After the blocking
step, the membrane was rinsed in TBS-T with 1% nonfat dried milk, pH
7.4, and divided into strips. Isoform-specific primary antibodies for
all PKC isoforms were diluted 1:2,000 in rinsing solution. Each strip
was incubated with one primary antibody for 2 h on a rocking platform
and washed four times in rinsing solution. All strips were then
incubated with secondary antibody (horseradish peroxidase-conjugated
IgG fraction of goat anti-rabbit IgG) diluted 1:20,000 in TBS-T. The
membrane strips were then washed in TBS-T and developed with an
enhanced chemiluminescence detection kit before exposure onto
Kodak-X-Omat film. The fluorescence ratio values were
calculated for the molecular-mass standards and the bands of interest
on each gel, and the apparent molecular masses were obtained from a
linear plot of the fluorescence ratio versus molecular mass for the
standard markers. Blots were quantified by computerized densitometry
(Molecular Dynamics). The identity of each band detected on the gel was
confirmed by experiments in which the primary antibody was blocked with
the appropriate peptide.
Effect of PGE2 on PKC-
expression: [3H]thymidine
incorporation.
The effect of PGE2 on cell
proliferation was estimated by measuring the incorporation of
[methyl-3H]thymidine
([3H]TdR) as
previously described (13). Briefly, cells were grown in 96-well plates,
and 5 h before the end of a growth period, 1 µCi of
[3H]TdR (specific
activity 20 Ci/mmol) was added to each well containing 100 µl of the
test solution before the plates were returned to the incubator. Stock
solutions of [3H]TdR
were diluted in Hanks' balanced salt solution to obtain 1 µCi/10
µl. Ten microliters of this solution were added to each well
containing 100 µl of the test solution. Thus the final concentration of [3H]TdR was 1 µCi/110 µl. After the 5-h incubation period, the cells were
harvested with distilled water onto Whatman GF-C glass fiber filters
with a Harvard cell harvester. The filters were washed five times with
distilled water to remove unbound
[3H]TdR. The glass
fibers were allowed to dry and were then transferred to 5-ml plastic
scintillation vials that contained 4 ml of scintillant. The vials were
mixed on a vortex and, after a 24-h equilibration period, were counted
on a beta emission counter for 5 min/sample.
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RESULTS |
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Expression of PKC isoforms in human ASM
cells. Western blot analysis of cells from three
patients revealed the consistent presence of PKC- (81 kDa), -
I
(79 kDa), and -
II (80 kDa) of the conventional isoforms; PKC-
(76 kDa), -
(79 kDa), -
(80 kDa), and -
(92 kDa) of the novel
group; and PKC-
(74 kDa), -µ (100 kDa), and -
(75 kDa) of the
atypical group. Detection of the isoforms was not influenced by patient
diagnosis because their presence was consistent across all patient
groups. The results of the immunoblots are shown in Fig.
1.
|
Changes in PKC isoforms in proliferating
cells. In the presence of 10% FBS, which has
previously been shown to cause a 17-fold increase in cell number (13),
there was an approximately threefold increase (372 ± 115%) in the
expression of PKC- as observed by the density compared with that in
control cells (100%) incubated in 1% FBS
(P < 0.05;
n = 3; Fig.
2). PDGF (40 ng/ml) also produced significant increases in PKC-
expression in both the cytosolic and
membrane fractions (210 ± 49 and 443 ± 227%, respectively; P < 0.05, n = 4; Fig. 2). In contrast, there was
no significant change in PKC-
expression in either the membrane or
cytosolic fraction in the presence of 10% FBS or PDGF (Fig.
3) or in the isoforms
1,
II,
,
, µ, or
. The signal for PKC-
was very weak in the cytosol
despite a strong signal in the rat brain standard and in the membrane
was 66 ± 8% of the control value in the presence of PDGF
(P > 0.05;
n = 3).
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PKC- enzyme activity.
PKC-
activity in the cytosolic and membrane fractions of cells
stimulated with 10% FBS was 2.6- and 3.8-fold
(patient 1) and 3.4- and
2.3-fold (patient 2),
respectively, greater than that in unstimulated control cells (1%
FBS). Corresponding values for the amount of PKC-
protein as
measured by Western blotting were 4.8- and 5.6-fold
(patient 1) and 7.5- and
3.9-fold (patient 2)
greater. There was a significant correlation for the relationship between the increase in kinase activity and the increase in expression of PKC-
protein for the two membrane and two cytosolic fractions (r2 = 0.903; P < 0.01;
n = 4). The values obtained in the two
sets of assay controls, i.e., those that omitted GS substrate and those that omitted PS, did not differ significantly from each other.
Effect of PGE2 on cell proliferation and
PKC-.
PGE2 decreased both the
proliferative response of the cells to PDGF and the amount of PKC-
as measured in the membrane fraction by immunoblotting (Fig.
4, left
and right, respectively).
[3H]TdR incorporation
in response to PDGF was 5,580 ± 633 (SE) dpm compared with that in
the presence of 1% FBS (186 ± 5 dpm;
P < 0.05;
n = 3).
PGE2 significantly reduced the
count to 3,980 ± 126 dpm (P < 0.05; n = 3).
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DISCUSSION |
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This study represents, to the best of our knowledge, the first
description of the pattern of PKC isoforms expressed in human cultured
ASM. Isoenzymes from all three groups (conventional, novel, and
atypical) were present, and the pattern of expression was similar but
not identical to that which Donnelly et al. (5) first described in
homogenates of canine ASM. The main difference between the two species
was the presence of PKC- in human but not in canine tissue. In other
respects, the isoforms detected in the two species were qualitatively
identical, but this study did not permit quantitative comparisons of
the relative amounts of each isoform expressed. PKC-
I and -
II of
the conventional isoforms; PKC-
, -
, -
, and -
of the novel
isoforms; and PKC-
, -µ, and -
from the atypical group were
present. There was no evidence of the neuronal isoform PKC-
. Our
results show some similarities to those of Webb et al.
(28), who studied homogenates of both human trachealis and
peripheral lung. These investigators used Western and Northern blotting
to detect PKC isoforms, and the major difference between their findings
and those of the present study lies in the detection of the
muscle-specific isoform PKC-
. The reason that we detected this
isoform and Webb et al. did not may reflect the fact that we studied
cells in culture as opposed to those in tissue homogenates. This is
unlikely, however, because Donnelly et al. (5) also found the PKC-
isoform in homogenates of canine trachealis. Togashi et al. (27),
using porcine trachealis, also found expression of PKC-
and, in
addition to those reported in canine tissue, two other members of the
atypical group,
/
and µ. They did not report the
presence of PKC-
.
The presence of different isoforms of PKC strongly suggests a complex
multifunctional role for PKC in ASM, and it seems possible that
different isoforms may be involved in the pathways regulating ASM
contraction and proliferation. Indeed, previous work by Rossetti et al.
(25) and Standaert et al. (26) has demonstrated a role for PKC in the
contraction of human isolated airways, and it is likely that the
contractile responses are associated with the calcium-dependent group
of PKC isozymes. It was of interest, therefore, to find that the
expression and isoenzyme activity of a calcium-independent isoform,
PKC-, were upregulated in cells that were stimulated to proliferate
with either FBS or PDGF. Evidence that this effect was specific for
PKC-
was provided by the fact that, under similar conditions, no
other PKC isoforms were upregulated during mitogenic stimulation.
An association between PKC activation and ASM cell proliferation has
been established in previous experiments in which PKC inhibitors have
been shown to reduce the proliferative response (5, 9).
The present data extend the observations obtained with nonselective PKC
inhibitors by associating the proliferative response in ASM with a
selective increase in the expression and isoenzyme activity of an
individual PKC isoform, PKC-, but these data do not provide direct
information on whether PKC-
activation is a primary initiating event
or a secondary consequence of ASM growth. It would clearly be desirable
to evaluate the effects of a selective inhibitor of PKC-
on cell
proliferation, but although specific antagonists for other PKC isoforms
are becoming available (12), a selective pharmacological blocker of
PKC-
has not been developed, and there are still concerns about the
specificity of alternative techniques, such as the use of antisense
oligonucleotides, for blocking PKC signaling. Nevertheless, the
possibility that PKC-
may be involved in the initiation of cell
division is supported by previous work (2).
The results obtained in the kinase assay after immunoprecipitation with
anti-PKC- antibody demonstrated that mitogenic stimulation of the
ASM cells is associated with not only an increase in the amount of
PKC-
protein but also an increase in the kinase activity of the
isoform. Moreover, there was a positive correlation between the
increase in enzyme activity and the increased amount of protein expressed in the cytosolic and membrane fractions. We chose GS peptide
as our substrate in the kinase activity assay because, although
alternative substrates are sometimes preferred (15), PKC-
has a good
affinity for GS peptide (15), and this was certainly confirmed by our
findings in the present experiments. It is unlikely that the activity
of any other PKC isoform confounded our immunoprecipitation results
because the addition of EGTA would have excluded activity of any
calcium-dependent isoforms, and in the absence of any PKC activators
such as phorbol esters, there would be minimal activation of the novel
group of isoforms. Moreover, in the kinase assay, we used the same
concentration of PKC-
antibody as that shown to be extremely
specific for PKC-
in the Western blot experiments.
Others have attributed specific cellular functions to specific PKC
isoforms (4, 18), especially PKC-. For example, activation of
PKC-
is not only necessary but is sufficient by itself to activate
maturation in oocytes and stimulate growth in fibroblasts (2). In
addition, stimulation of vascular smooth muscle by angiotensin results
in activation of PKC-
(17), whereas in 3T3/L1 cells, insulin
activates PKC-
, -
, and -
, but only PKC-
is required for
glucose transport (1). Furthermore, PKC-
is particularly abundant in
fetal tissues and adult rat liver, an organ that retains regenerative
capacity, and this would be consistent with the notion that PKC-
plays a contributory role in cell proliferation (7). Conversely,
PKC-
inhibits proliferation in both vascular smooth muscle cells (6)
and fibroblasts (8, 17).
The association between increased activity of PKC- and cell
proliferation in human ASM is consistent with other evidence suggesting
that an increase in intracellular calcium is not an essential
requirement for mitogenesis (19). The fact that PKC-
is an isoform
that is activated independently of calcium and that conventional
calcium-dependent PKC isoforms were not increased in this system would
support the hypothesis that there can be some dissociation between
increased calcium levels and proliferation as previously reported in
other experiments (20).
The association of individual PKC isoforms with specific biological
functions raises the intriguing possibility of the use of selective
inhibition of these isoforms to achieve targeted abolition of unwanted
pathological effects. Indeed, selective inhibition of PKC- with a
novel, orally active inhibitor ameliorates vascular dysfunction in a
rat model of diabetes (12). It is therefore conceivable that, if
further evidence were available linking human ASM proliferation with
specific PKC isoforms, new pharmacological or antisense approaches
could be used to inhibit the ASM hyperplasia that is an unwanted
feature of asthmatic airways.
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ACKNOWLEDGEMENTS |
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We thank the surgical and pathology staffs of the following hospitals for the supply of human lung tissue: Royal Prince Alfred, St. Vincents, Concord, Royal North Shore, and Strathfield Private (Sydney, Australia) and Dr. J. Burn (Strathfield Private Hospital). We acknowledge the collaborative effort of the cardiopulmonary transplant team at St. Vincent's Hospital.
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FOOTNOTES |
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This study was supported by the National Health and Medical Research Council of Australia and the Community Health and Antituberculosis Association.
Present addresses: K. X. F. Yang, Clinical Sciences Bldg., Prince Henry Hospital, Little Bay, New South Wales 2036, Australia; R. Donnelly, School of Medical and Surgical Sciences, Univ. of Nottingham, Derby DE1 2QY, UK.
Address for reprint requests: J. L. Black, Dept. of Pharmacology, Univ. of Sydney, New South Wales 2006, Australia (E-mail: judblack{at}pharmacol.usyd.edu.au).
Received 4 December 1997; accepted in final form 7 December 1998.
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