Selected isozymes of PKC contribute to augmented growth of
fetal and neonatal bovine PA adventitial fibroblasts
Mita
Das1,
Kurt R.
Stenmark1,
Laura J.
Ruff1, and
Edward C.
Dempsey1,2
1 Cardiovascular Pulmonary and
Developmental Biology Research Laboratories, University of Colorado
Health Sciences Center, Denver 80262; and
2 Denver Veterans Administration
Medical Center, Denver, Colorado 80220
 |
ABSTRACT |
We sought to determine which isozymes of
protein kinase C (PKC) contribute to the increased proliferation of
immature bovine pulmonary artery (PA) adventitial fibroblasts. Seven
were identified in lysates of neonatal PA fibroblasts by Western blot:
three Ca2+ dependent (
,
I, and
II) and four
Ca2+ independent (
,
,
, and µ). Four isozymes
(
,
,
, and
) were not detected in fibroblasts isolated at
any developmental stage. Of the seven detected isozymes, only PKC-
and -
II protein levels were higher in fetal and neonatal cells
compared with adult fibroblasts. Their role in the enhanced growth of
immature fibroblasts was then evaluated. The isozyme nonselective PKC
inhibitor Ro-31-8220 was first compared with GF-109203X, a structural
analog of Ro-31-8220 with relative specificity for the
Ca2+-dependent isozymes of PKC. GF-109203X selectively
inhibited the growth of immature cells and was nearly as potent as
Ro-31-8220. Go-6976, a more specific inhibitor of the
Ca2+-dependent isozymes, mimicked the antiproliferative
effect of GF-109203X. PKC downregulation with 1 µM phorbol
12-myristate 13-acetate had the same selective antiproliferative effect
on immature fibroblasts as GF-109203X and Go-6976. The protein levels of PKC-
and -
II, but not of PKC-
I, were completely degraded in
response to phorbol 12-myristate 13-acetate pretreatment. These results
suggest that PKC-
and -
II are important in the augmented growth
of immature bovine PA adventitial fibroblasts.
protein kinase C-
; protein kinase C-
II; protein kinase C-
; protein kinase C-µ; Go-6976; GF-109203X; Ro-31-8220; phorbol
12-myristate 13-acetate-induced downregulation; pulmonary artery
 |
INTRODUCTION |
ADVENTITIAL THICKENING of the pulmonary arteries is an
important component of the structural changes observed in various forms of chronic pulmonary hypertension (17, 24, 26, 30). This thickening is
due, at least in part, to proliferation of resident fibroblasts. The
changes in proliferation of adventitial fibroblasts in response to
injury appear more impressive in the neonatal than in the adult
pulmonary circulation (9, 24, 26, 27, 30). In addition, recent
experiments have demonstrated that the developmental differences in
growth potential of bovine pulmonary artery (PA) adventitial
fibroblasts (fetal > neonatal > adult) are also retained by
isolated cells in vitro (2). However, the mechanisms contributing to
the increased growth potential of immature adventitial fibroblasts remain poorly understood.
Protein kinase C (PKC) plays a key role in the regulation of cell
proliferation, differentiation, and maturation (3, 6, 20). This pathway
has been shown to be developmentally regulated (3, 23) and to
contribute to the enhanced growth of neonatal bovine PA smooth muscle
cells (3, 6). Activation of PKC increases responsiveness to
developmentally regulated mitogens (3, 7) and is a requisite step for
vascular cells to respond directly to hypoxia (4). In addition, this
pathway has recently been implicated in the enhanced growth of fetal
and neonatal PA adventitial fibroblasts by experiments demonstrating
that immature fibroblasts have increased PKC catalytic activity and
higher susceptibility to the growth-inhibiting effects of PKC
antagonists compared with adult fibroblasts (2). The PKC signalling
pathway, however, is a complex one, with four
Ca2+-dependent and seven
Ca2+-independent isozymes having
been identified (12, 20). Developmental changes in the expression of
individual isozymes have been observed, and increased expression of
selected PKC isozymes has been linked to augmented growth capacity (23,
28, 32). However, the isozymes of PKC expressed by PA adventitial
fibroblasts and their respective importance in the regulation of
developmental differences in growth are not known.
The goals of this study were therefore to determine first the pattern
of PKC isozyme expression that exists in PA adventitial fibroblasts and
then to determine which specific isozymes contribute selectively to the
increased growth potential of immature bovine PA adventitial
fibroblasts (2). Our approach was to identify the PKC isozymes
expressed by neonatal PA adventitial fibroblasts using antibody
screening and then to quantitatively evaluate specific isozyme
expression at different developmental stages. Finally, we used
complementary antagonist strategies to determine whether two
Ca2+-dependent isozymes, which
were found to have increased expression during the fetal and neonatal
period, contributed to the augmented growth of PA adventitial
fibroblasts isolated at the same developmental stages.
Our data demonstrate that neonatal bovine PA adventitial fibroblasts
express 7 of the 11 described isozymes of PKC as follows: three
Ca2+ dependent (
,
I, and
II) and four Ca2+ independent
(
,
,
, and µ). The expression pattern of two isozymes, the
Ca2+-dependent PKC-
and -
II,
paralleled the developmental differences in growth, susceptibility to
PKC inhibitors, and PKC catalytic activity previously observed for PA
adventitial fibroblasts (2). Antagonist strategies implicated these
same Ca2+-dependent isozymes in
the enhanced growth of fetal and neonatal PA fibroblasts. These
observations suggest that PKC-
and -
II contribute to the
augmented proliferative response of immature bovine PA adventitial
fibroblasts.
 |
MATERIALS AND METHODS |
Materials. Minimal essential medium
(MEM), trypsin-EDTA 10× suspension, penicillin, streptomycin, and
amphotericin B were from Sigma Chemical (St. Louis, MO). Fetal bovine
serum was purchased from Hyclone Laboratories (Logan, UT). Leupeptin,
aprotinin, phenylmethylsulfonyl fluoride (PMSF), and mercaptoethanol
were also from Sigma. Anti-PKC-
, -
, -
, and -
antibodies and
blocking peptides were purchased from GIBCO BRL (Gaithersburg, MD).
Anti-PKC-
I, -
II, -
, -
, -
, -
, and -µ antibodies,
blocking peptides, and horseradish peroxidase (HRP)-conjugated goat
anti-rabbit immunoglobulin G (IgG) were from Santa Cruz Biotechnology
(Santa Cruz, CA). Molecular mass markers and nitrocellulose membranes
were obtained from GIBCO BRL and Bio-Rad Laboratories (Richmond, CA),
respectively. Enhanced chemiluminescence detection kits were from
Amersham (Arlington Heights, IL). Reagents for protein determination
were purchased from Bio-Rad Laboratories. Phorbol 12-myristate
13-acetate (PMA), Ro-31-8220, and GF-109203X were obtained from LC
Services (Waltham, MA). Go-6976 was kindly provided by Parke-Davis
Pharmaceutical Research (Ann Arbor, MI). All were dissolved in dimethyl
sulfoxide (DMSO) and diluted to working concentrations in
phosphate-buffered saline (PBS).
Isolation and growth of fetal, neonatal, and adult
bovine PA adventitial fibroblasts. PA adventitial
fibroblasts were isolated from 120- to 180-day-old bovine fetuses, 8- to 14-day-old neonatal calves, and adult cows, grown, and characterized
as previously described (2). All cells were maintained in MEM, pH 7.4, supplemented with 10% serum, 100 U/ml penicillin, and 0.1 mg/ml
streptomycin and incubated in a humidified atmosphere with 5%
CO2 at 37°C. Medium was
changed biweekly. Cells were harvested weekly (i.e., passed) with
trypsin (0.2 g/l)-EDTA (0.5 g/l). Early-passage (passages 1-6) cells were used. During the period of study, the cells
retained stable growth properties and light-microscopic appearance.
Preparation of whole cellular lysates.
Fetal, neonatal, and adult PA adventitial fibroblasts (0.5-2.0 × 106/flask) were plated in
15 ml of 10% serum-containing medium in T75 flasks. Cells
were grown for 4-5 days under serum-stimulated conditions.
Fibroblasts were washed with ice-cold PBS three times and were lysed in
1 ml of homogenization buffer [20 mM
tris(hydroxymethyl)aminomethane, pH 7.5, containing 0.25 M sucrose, 3 mM EDTA, 3 mM ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid, 50 mM mercaptoethanol, 50 µg/ml leupeptin, 50 µg/ml
aprotinin, 1 mM PMSF, and 0.1% Triton X-100] by freezing and
thawing one time. On ice, each monolayer of PA adventitial fibroblasts
was scraped into the homogenization buffer and triturated 10 times.
Homogenates were centrifuged at 2,200 revolutions/min for 10 min at
4°C (Beckman centrifuge GS-6R; Fullerton, CA), and supernatant
aliquots were immediately frozen in liquid nitrogen and stored at
80°C. Protein concentrations for each lysate were determined
by micro-Bradford assay as previously described (3).
Western blot analysis of PKC isozymes.
Twenty micrograms of each lysate were applied per slot for
electrophoresis in each 10% reduced sodium dodecyl
sulfate-polyacrylamide gel and then transferred to a nitrocellulose
membrane. Prestained molecular mass protein markers were also loaded
onto each gel. After a 2-h incubation at room temperature in 5% dry
milk-PBS-0.05% Tween 20 to block nonspecific binding, the
nitrocellulose was probed with a 1:300-500 dilution of primary PKC
isozyme-specific antisera in 5% milk-PBS-0.05% Tween 20 overnight at
4°C. Eleven different PKC isozyme-specific antisera were used. The
nitrocellulose was then washed for 7 min three times with PBS-0.05%
Tween 20. To detect bound primary antibody, blots were incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG at a dilution of 1:5,000 in 5% milk-PBS-0.05% Tween 20. The nitrocellulose was washed again with PBS-0.05% Tween 20 for 5 min three times and
then with PBS for 5 min one time. Blots were developed onto Dupont
reflection autoradiography film (Dupont, Wilmington, DE) using an
enhanced chemiluminescence detection kit. Determination of molecular
mass for each isozyme detected was made by comparison with known
molecular mass markers. Developmental and downregulation-induced changes in isozyme expression were determined by comparing matched cell
lysates run side by side. Band intensity was quantified by scanning
with a Lacie-limited Silverscanner II and by analysis with National
Institutes of Health (NIH) Image Software (NIH, Bethesda, MD). Data
were expressed as a percent of the fetal value.
To confirm the specificity of binding between primary antibody and
immunoreactive protein, Western blots were performed in the presence
and absence of isozyme-specific immunizing peptide. Isozyme-specific
antisera were preincubated with the respective peptide antigen (1:10)
used for immunization for 2 h at room temperature or overnight at
4°C and were then diluted to working concentration as described
above. When an isozyme could not be detected in PA adventitial
fibroblasts isolated at any developmental stage, positive control
lysates were used to establish reactivity of the antibody [e.g.,
rabbit brain (
), rat lung (
), skeletal muscle (
), and bovine
PA smooth muscle cells (
)]. Specificity of the antibody was
then confirmed as described above.
Differences in susceptibility to the antiproliferative effects of a
nonselective PKC antagonist vs. PKC antagonists selective for the
Ca2+-dependent
isozymes.
Cells were sparsely seeded (10 × 103/well of a 24-well plate) in
MEM-10% serum and allowed to attach overnight. On day
1, cell numbers were measured by hemocytometer to
ensure that equal numbers were being compared. Then Ro-31-8220 (3 µM), an isozyme nonselective PKC inhibitor (13), GF-109203X (3 µM),
an inhibitor with relative specificity for the
Ca2+-dependent isozymes (31), or
the appropriate vehicle control was added. In preliminary studies, the
dose-dependent inhibitory effects of the two related
bisindolylmaleimide compounds Ro-31-8220 and GF-109203X were first
compared on the fastest growing cell population (fetal PA adventitial
fibroblasts). Threshold and maximal antiproliferative effects for both
compounds were found with 1 and 5 µM concentrations, respectively.
Therefore, an intermediate (3 µM) concentration was used for the
comparison between different cell populations in the current studies.
This is the same concentration range for these compounds that others
have applied to nonvascular cells (10) and that we have recently used
for studies on PA smooth muscle cells (33). A close correlation between
the extent to which PKC-mediated phosphorylation events are inhibited
and the extent to which the cell response of interest is blocked in the
presence of inhibitor has previously been observed (10). To confirm
that the selective antiproliferative effects of GF-109203X were due to
inhibition of the Ca2+-dependent
isozymes of PKC, 3 µM Go-6976 [a structural analog with greater
specificity for these isozymes (15)] was also tested on the
different cell populations. Mukherjee et al. (18) have recently used
Go-6976 at the same concentration to implicate one of the
Ca2+-dependent isozymes of PKC in
the regulation of phosphatidylethanolamine hydrolysis in MCF7 breast
carcinoma cells. Inhibitors were readded on day
3. Final cell numbers were counted on
day 5. Results are expressed as cell
number times 103 per well.
Differences in susceptibility to the antiproliferative
effects of phorbol ester-induced PKC downregulation and detection of isozyme-specific differences in susceptibility to PKC
downregulation. To detect developmental differences in
susceptibility to the antiproliferative effects of phorbol
ester-induced downregulation of PKC, cells were sparsely seeded (10 × 103/well of a 24-well
plate) in MEM-10% serum and allowed to attach overnight.
Downregulation of PKC was achieved as previously described (2). On
day 1, PA fibroblasts were pretreated
with either 1 µM PMA or vehicle (DMSO) alone for 24 h. Change in cell
number was then measured between days
2 and 5. Because the
interval between PMA pretreatment and final growth measurement was long
(72 h) and normal levels of PKC are gradually restored several hours after removal of PMA, the phorbol ester was routinely left in for the
duration of the study (2). A repeat dose of PMA was applied on
day 3 to be sure adequate levels were
maintained. The results are expressed as cell number times
103 per well.
To determine the effect of PKC downregulation on isozymes of PKC,
1-2 × 106 neonatal
fibroblasts were seeded in 10% serum-containing medium per
T75 flask and were grown for 4-5 days. Cells were then
treated with either 1 µM PMA or vehicle (DMSO) alone for 24 h, and
whole cell lysates were prepared as described above. PKC catalytic
activity was measured in lysates of control and downregulated cells as previously described using myelin basic peptide-(4
14) as substrate, with minor modifications (2). Change in isozyme expression was
determined by Western blotting.
Data analysis. All data are presented
as arithmetic means ± SE. For detection of PKC isozymes
in neonatal PA fibroblasts, representative blots are shown. Results
were reproduced at least three times in three different cell
populations for molecular mass determinations. For studies of
developmental change in expression of PKC isozymes with age,
n equals the number of experiments
done on at least three different populations of cells, each isolated from a different animal. For growth studies,
n equals the number of replicate wells
per test condition in representative experiments. Each observation was
reproduced in cells isolated from at least three different animals.
One-way analysis of variance followed by Student-Newman-Keuls multiple
comparison test was used for individual comparisons within and between
groups of data points. Data were considered significantly different at
P < 0.05.
 |
RESULTS |
Neonatal bovine PA adventitial fibroblasts express
Ca2+-dependent
and -independent isozymes of PKC.
To determine which isozymes of PKC are expressed in neonatal bovine PA
adventitial fibroblasts, cell lysates were analyzed for
immunodetectable proteins using Western blotting techniques and
polyclonal antibodies against 11 isozymes of PKC. Specificity of the
antibodies for each isozyme was demonstrated using corresponding blocking peptides. Neonatal cells were found to express seven isozymes
of PKC as follows: three Ca2+
dependent (
,
I, and
II) and four
Ca2+ independent (
,
,
,
and µ; Fig. 1). PKC-
I, -
, and -µ
were resolved as protein doublets. Their apparent molecular masses were:
, 82 ± 3;
I, 89 ± 4 and 80 ± 3;
II, 89 ± 4;
, 78 ± 1;
, 103 ± 7;
, 85 ± 3 and 69 ± 2; and µ, 113 ± 8 and 106 ± 7 kDa (n = 3). The apparent molecular mass
for each isozyme is in agreement with previously reported values in
cells selectively overexpressing each isozyme (7, 12, 20). The antibody
for PKC-
also detected a specific band of higher than expected
molecular mass (105 kDa) that was extinguished by pretreatment with the
appropriate blocking peptide. Because of the magnitude of the molecular
mass difference (105 vs. 78 kDa), this signal is unlikely to be a
phosphorylated derivative of PKC-
. It is more likely to be an
unrelated peptide sharing the same antigenic determinant.

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Fig. 1.
Neonatal bovine pulmonary artery (PA) adventitial fibroblasts express
Ca2+-dependent ( , I, and
II) and -independent ( , , , and µ) isozymes of protein
kinase C (PKC). Whole cell lysates (20 µg protein) were subjected to
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)
and Western blotting as described in MATERIALS AND METHODS.
Blots were probed with antibodies specific for PKC- , - I, - II,
- , - , - , and -µ isozymes in the absence ( ) or
presence (+) of a corresponding blocking peptide (unique PKC peptide
sequence against which each antibody was raised). Arrows identify major
immunoreactive bands. Positions of the molecular mass (MW) standards
(kDa) are indicated on left. Data are
representative of results from 3 separate experiments, each performed
on neonatal fibroblasts from different calves.
|
|
The following four isozymes of PKC were not detected in neonatal PA
fibroblasts: one Ca2+ dependent
(
) and three Ca2+ independent
(
,
, and
; Fig. 2). In each
instance, activity and specificity of the anti-PKC antibody was
confirmed with an appropriate positive control lysate, application of
blocking peptide, and apparent molecular mass determination. Lysates
prepared from fetal and adult cells were also tested for these four
isozymes. No immunodetectable
,
,
, or
isozyme was found
at any developmental stage.

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Fig. 2.
Neonatal bovine PA adventitial fibroblasts do not express the
Ca2+-dependent or
Ca2+-independent , , and isozymes of PKC. Lysates from neonatal fibroblasts (FIB) and control
tissues [rabbit brain, rat lung, rat skeletal muscle, and PA
smooth muscle cells (SMC)] were subjected to SDS-PAGE and
immunoblot analysis with PKC isozyme-specific ( , , , and )
antibodies. Two bands are detected in the rabbit brain control with
anti-PKC- antibody, but only one band at the appropriate MW was
extinguished by the blocking peptide. Antibodies for PKC- , - , and
- detected single bands in control lysates that were at the
appropriate MW and were extinguished by blocking peptide. Nonspecific
binding to high MW proteins in fibroblast lysates was noted with
anti-PKC- and - antibodies. Positions of the molecular mass
standards (kDa) are indicated on left.
Data are representative of results from 3 separate experiments, each
performed on neonatal fibroblasts from different calves. These isozymes
were also not detected in lysates of fetal and adult cells.
|
|
Expression of the
Ca2+-dependent
and
II isozymes of PKC is higher in
immature PA adventitial fibroblasts than in adult cells.
To determine if there were isozyme-specific changes in expression with
advancing developmental stage that paralleled the differences in growth
and catalytic activity previously observed (2), three sets of matched
fetal, neonatal, and adult cell lysates were directly compared. Each
lysate was from a different animal. There was higher expression of the
Ca2+-dependent
and
II, but
not
I, isozymes of PKC in immature PA fibroblasts compared with
adult cells (Fig. 3,
A and
B). PKC-
II peptide was not
detectable in adult fibroblasts. There was no effect of age on
the expression of Ca2+-independent
PKC-
, -
, and -
in bovine PA adventitial fibroblasts (Fig.
4, A and
B). PKC-µ had increased expression
in adult cells (Fig. 4, A and
B).

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Fig. 3.
Expression of the Ca2+-dependent
and II isozymes of PKC is higher in immature PA adventitial
fibroblasts than in adult cells. A:
representative immunoblots for each
Ca2+-dependent isozyme. Whole cell
lysates (20 µg) of fetal (F), neonatal (N), and adult (A) PA
adventitial fibroblasts were resolved by SDS-PAGE, transferred to
nitrocellulose, and probed with anti-PKC- , - I, and - II
antibodies. B: quantitative analysis
of expression pattern for each
Ca2+-dependent isozyme. Results
are pooled from 3 separate experiments
(n = 3). For each experiment,
cells from different animals at each developmental stage were used.
Values are expressed as percent of the fetal value.
* P < 0.05 compared with fetal
cells and ** P < 0.05 compared
with fetal and neonatal cells.
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Fig. 4.
Expression of Ca2+-independent
µ, but not , , or , isozymes of PKC is regulated by
development in PA adventitial fibroblasts.
A: representative immunoblots for each
Ca2+-independent isozyme.
Immunoblots from fetal (F), neonatal (N), and adult (A) PA adventitial
fibroblasts for PKC- , - , - , and -µ are shown.
B: quantitative analysis of expression
pattern for each Ca2+-independent
isozyme. Results are pooled from 3 separate experiments
(n = 3). For each experiment, cells
from different animals at each developmental stage were used. Values
are expressed as percent of the fetal value.
* P < 0.05 compared with fetal
and neonatal cells.
|
|
The
Ca2+-dependent
isozymes of PKC contribute to the augmented growth of immature bovine
PA adventitial fibroblasts.
To test whether the Ca2+-dependent
isozymes of PKC contribute to the augmented growth of immature PA
adventitial fibroblasts, the antagonistic effects of the specific, but
isozyme-nonselective, PKC inhibitor Ro-31-8220 (3 µM) were compared
with GF-109203X (3 µM), a structural analog with relative specificity
for the Ca2+-dependent isozymes of
PKC (13, 31; Fig. 5,
A and
B). GF-109203X selectively inhibited
serum-stimulated growth of fetal and neonatal, but not adult, cells and
was nearly as potent as Ro-31-8220. To be certain that the selective
antiproliferative effects of GF-109203X were due to inhibition of the
Ca2+-dependent isozymes of PKC,
Go-6976, a more specific inhibitor of these isozymes, was also used
(15, 18). Go-6976 (3 µM) had the same selective inhibitory
effect on growth of the immature cells as GF-109203X (Fig.
6A).

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Fig. 5.
Ca2+-dependent isozymes of PKC
contribute to the augmented growth of immature PA adventitial
fibroblasts; n = 4 replicate wells.
A: antiproliferative effects of
Ro-31-8220, a specific but isozyme nonselective inhibitor of PKC.
B: antiproliferative effects of
GF-109203X, a structural analog with relative specificity for
Ca2+-dependent isozymes of PKC.
GF-109203X (3 µM) selectively inhibited the enhanced growth of
immature fibroblasts and was nearly as potent as Ro-31-8220 (3 µM).
Cell counts were performed on day 5 after the initial application (and 2 days after reapplication) of
either Ro-31-8220 or GF-109203X. Same vehicle [dimethyl sulfoxide
(DMSO)] was used for both inhibitors and all test conditions.
* P < 0.05 compared with
control cells. Similar results were reproduced in 2 other matched sets
of cell populations.
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Fig. 6.
Ca2+-dependent and II
isozymes of PKC contribute to the augmented growth of immature PA
adventitial fibroblasts. A: Go-6976, a
more specific inhibitor of the
Ca2+-dependent isozymes of PKC,
also inhibits growth of immature PA adventitial fibroblasts;
n = 4 replicate wells. Like
GF-109203X, Go-6976 (3 µM) inhibited serum-stimulated growth of fetal
and neonatal, but not of adult, fibroblasts. Cell counts were performed
on day 5 after the initial application
(and 2 days after reapplication ) of the inhibitor. DMSO was used as
the vehicle. * P < 0.05 compared with control cells. Similar results were reproduced with 2 other matched sets of cell populations.
B: pretreatment with 1 µM phorbol
12-myristate 13-acetate (PMA) for 24 h (downregulates PKC- and
- II but not PKC- I) has the same selective antiproliferative
effect as GF-109203X and Go-6976; n = 4 replicate wells. PMA (1 µM) was added to the cells on
day 1, and then growth was assessed
between days 2 and
5. PMA was readded on
day 3 and was left in between
days 2 and
5 to maintain the downregulated state.
* P < 0.05 compared with
control cells. Similar results were reproduced with 2 other matched
sets of cell populations.
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|
The
Ca2+-dependent
and
II isozymes of PKC contribute to
the augmented growth of immature bovine PA adventitial fibroblasts.
Phorbol-induced PKC downregulation was found to have the same selective
inhibitory effect as GF-109203X and Go-6976 on the augmented growth of
immature PA adventitial fibroblasts (Fig. 6B). Isozymes of PKC may differ in
their susceptibility to downregulation (11, 22, 23). Those that are
susceptible can be implicated in the cellular response (i.e., enhanced
growth) blocked by this antagonist strategy. Therefore, we identified
which Ca2+-dependent isozymes in
neonatal PA adventitial fibroblasts were susceptible to degradation
with prolonged exposure to PMA (Fig. 7A).
Phorbol pretreatment resulted in >95% degradation of PKC-
and
-
II, the same Ca2+-dependent
isozymes that had increased expression in the immature PA fibroblasts.
We also confirmed that the prolonged exposure to PMA caused a reduction
in whole cellular PKC catalytic activity by 67 ± 3%. The same
pattern of susceptibility to degradation was also observed for fetal
and adult cells. In contrast, the principal band for PKC-
I was not
susceptible to downregulation. Again, the same resistance to
degradation was observed with fetal and adult cells.

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Fig. 7.
Differential susceptibility of PKC isozymes in PA adventitial
fibroblasts to PMA-induced downregulation.
A: differential effect of pretreatment
with 1 µM PMA for 24 h on expression of
Ca2+-dependent isozymes in
neonatal PA adventitial fibroblasts. There is selective degradation of
the Ca2+-dependent and II
isozymes of PKC in response to 1 µM PMA. The same results were
obtained with a second population of neonatal fibroblasts and also with
fetal and adult fibroblasts. B:
differential effect of pretreatment with 1 µM PMA for 24 h on
expression of Ca2+-independent
isozymes in neonatal PA adventitial fibroblasts. PKC- and - are
susceptible and PKC- and -µ are resistant to PMA-induced
degradation. A shift in phosphorylation state of PKC-µ in response to
the high dose of PMA was also observed. Similar results were reproduced
with a second population of neonatal fibroblasts and also with fetal
and adult fibroblasts. Lanes marked with and + represent
lysates of cells pretreated with either vehicle ( ) or 1 µM PMA
(+).
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Susceptibility of the
Ca2+-independent isozyme PKC-µ
to PMA-induced degradation was investigated next because of changes
observed in expression of this isozyme with advancing developmental
stage (Fig. 7B). However, because
this atypical isozyme is not inhibited by the concentrations of
bisindolmaleimide inhibitors used in this study (12), it seemed
unlikely to be involved in the regulation of PA fibroblast growth. A
shift in the phosphorylation state of immunodetectable PKC-µ but not
degradation was observed in PA adventitial fibroblasts.
Part of the growth response of each fetal, neonatal, and adult
population was not dependent on developmental stage and was inhibited
by Ro-31-8220 but not by GF-109203X-, Go-6976-, or PMA-induced downregulation. Therefore, we also determined which of the
Ro-31-8220-sensitive, Ca2+-independent isozymes (
,
, and
) were resistant to degradation induced by prolonged
pretreatment with PMA (Fig. 7B).
Only PKC-
was not degraded after pretreatment with PMA, suggesting
that this isozyme might contribute to an equal extent to the
serum-stimulated growth of both immature and mature cells (but not to
the enhanced growth of the immature fibroblasts).
 |
DISCUSSION |
In this study, we have demonstrated that neonatal bovine PA adventitial
fibroblasts express the following seven isozymes of PKC: three
Ca2+ dependent (
,
I, and
II) and four Ca2+ independent
(
,
,
, and µ). Four isozymes were not detected at any of the
three developmental stages investigated. Of the isozymes detected, only
two (
and
II: both Ca2+
dependent) had higher levels in immature fibroblasts than in adult
cells. This pattern of expression paralleled the developmental differences in growth and PKC catalytic activity previously observed for adventitial fibroblasts (2). These same isozymes of PKC were
further implicated in the enhanced growth of immature PA fibroblasts by
studies showing that GF-109203X, a PKC inhibitor with relative
specificity for the Ca2+-dependent
isozymes of PKC (31), inhibited the serum-stimulated growth of fetal
and neonatal, but not adult, cells. It was nearly as potent as
Ro-31-8220, a structural analog lacking isozyme specificity (13) that
inhibited growth of all three cell types. The discrete antiproliferative effects of GF-109203X on immature PA adventitial fibroblasts were then reproduced with the same concentration of Go-6976, a related analog and more specific inhibitor of the
Ca2+-dependent isozymes of PKC
(15, 18). Phorbol ester-induced PKC downregulation was found to have
the same selective antiproliferative effect on immature PA adventitial
fibroblasts as GF-109203X and Go-6976. The prolonged exposure to PMA
induced near-complete degradation of immunodetectable PKC-
and
-
II, but not of PKC-
I, in PA adventitial fibroblasts. Thus, based
on initial isozyme detection, selective changes in expression with
advancing developmental stage, and antagonist strategies that target
catalytic activity and expression level, we have implicated both
PKC-
and -
II in the augmented growth of immature bovine PA
adventitial fibroblasts.
Using 11 different isozyme-specific antibodies, we have found that
bovine PA adventitial fibroblasts express more isozymes of PKC than
previously described in other fibroblast cell lines. With a smaller
number of antibodies, rodent fibroblasts were found to express only two
(
and
, but not
I,
II, or
) or at most four (
,
,
, and
, but not
and
) isozymes of PKC (1, 16). Human skin
fibroblasts were also shown to express the same four PKC isozymes (
,
,
, and
, but not
I,
II, or
; see Ref. 25). PKC-
mRNA was not detected in R6 rat embryonic fibroblasts (1). In contrast,
in bovine PA fibroblasts, we have detected both PKC-
I and -
II,
alternative splicing products of the same transcript. Two
Ca2+-independent isozymes, PKC-
and -
, that have been detected in adjacent PA smooth muscle cells
(5) were not detected in adventitial fibroblasts at any developmental
stage. Finally, we report the presence of PKC-µ for the first time in
vascular cells.
We have demonstrated that selective changes in expression of the
Ca2+-dependent isozymes PKC-
and -
II occur in PA adventitial fibroblasts during development. The
pattern parallels previously observed growth properties, susceptibility
to PKC inhibitors, and catalytic activity of PKC for fetal, neonatal,
and adult PA adventitial fibroblasts (2). This observation suggests
that specific isozymes may have unique roles in the control of cell
growth in developing vessels. These same isozymes have been implicated
in specific responses in other cell systems (5, 19). Our work
complements recent studies in the developing rat glomerulus and heart
where the expression of PKC isozymes has also been shown to be
regulated in an age-dependent fashion. Saxena et al. (29) studied the role of PKC-
in the developing rat glomerulus. Differential
expression of PKC-
II was found to parallel the proliferative
behavior of maturing mesangial cells. Like our studies, their work
suggests that PKC-
II expression and activation may play a critical
role in development. Puceat et al. (23) found a selective decrease in
expression of PKC-
in adult rat cardiomyocytes compared with neonatal cells. Rybin and Steinberg (28) reported an age-dependent decline in immunodetectable PKC-
and -
as well as in PKC-
in developing rat heart. Expression of PKC-
and -
did not change in
our bovine PA cells. Thus developmental regulation of PKC isozymes appears to be cell, organ, and perhaps species specific, and different isozymes are probably serving unique roles in different cell
populations.
We have implicated the two
Ca2+-dependent isozymes
and
II in the enhanced growth of PA fibroblasts with complementary
antagonist strategies. First, we compared the antagonistic effects of
Ro-31-8220 and GF-109203X. Ro-31-8220 is a specific but
isozyme-nonselective inhibitor (13), whereas GF-109203X, a related
analog of Ro-31-8220, has relative specificity for the
Ca2+-dependent isozymes of PKC
(31). By comparing the antiproliferative effects of Ro-31-8220 and
GF-109203X on the growth of PA smooth muscle cells, the
Ca2+-dependent isozymes of PKC
have recently been implicated in the enhanced growth of immature smooth
muscle cells (33). In the current study, serum-stimulated growth of
fetal and neonatal PA fibroblasts was selectively inhibited by
GF-109203X. The antagonistic effects of Ro-31-8220 on the immature
cells were only slightly greater than those observed with GF-109203X.
In adult cells, Ro-31-8220, but not GF-109203X, inhibited
serum-stimulated growth. In contrast, adult PA smooth muscle cells were
resistant to both inhibitors (33). Therefore, the effect of Ro-31-8220
on the adult fibroblast is cell-type specific. The differential
antiproliferative effect of GF-109203X on immature PA fibroblasts was
also reproduced with a specific inhibitor of the
Ca2+-dependent isozymes of PKC,
Go-6976 (15, 18). These results suggest that the
Ca2+-dependent isozymes of PKC
play an important role in the enhanced growth of immature PA
fibroblasts. The selective pattern of the growth inhibition also shows
that the results are not due to nonspecific effects of the
bisindolmaleimide derivatives. The fact that one of the compounds,
Ro-31-8220, is a potent inhibitor of all three cell types suggests that
permeability to these lipophilic compounds is similar in all three cell
populations. This is also supported by the observation that the extent
of isozyme downregulation after pretreatment with PMA, another
lipophilic cell-permeable compound, is the same in immature and mature
cells.
PKC downregulation induced by prolonged incubation with PMA also
selectively inhibited the growth of immature fibroblasts. This
treatment is known to inhibit PKC catalytic activity and greatly
reduces immunoreactive protein by proteolytic degradation (11). PKC
isozymes have different susceptibility to this form of proteolytic
degradation (5, 11, 22, 23). We took advantage of this phenomenon to
determine which of the
Ca2+-dependent isozymes of PKC in
PA fibroblasts was depleted by this inhibitory strategy. In PA
fibroblasts, there was selective reduction in the immunoreactive
peptide levels of PKC-
and -
II, but not of PKC-
I, in response
to the high concentration of PMA. Interestingly, these same two
isozymes of PKC are the ones that are increased in immature cells. In
other cell systems, PKC-
has been consistently found to be
susceptible to phorbol ester-induced downregulation, although the
results with PKC-
have been more variable (14, 23).
We have found that expression of the
Ca2+-independent isozyme PKC-µ
is lower in immature cells compared with adult fibroblasts. Because
isozymes of PKC could also exert a negative influence on proliferation,
we considered the possibility that PKC-µ could be important in growth
regulation. Johannes et al. (12) demonstrated that PKC-µ activity was
inhibited by sphingosine but not by staurosporine. In our previous
study, we observed that dihydrosphingosine had the same differential
effect on the growth of fetal, neonatal, and adult fibroblasts (2) as
GF-109203X and Go-6976, which are staurosporine derivatives. There has
been a recent report that very high concentrations of Go-6976 can
partially inhibit the kinase activity of PKC-µ in intact cells
(half-maximal inhibitory concentration = 20 µM; see Ref. 8). However,
no inhibition was observed with 3 µM, the concentration used in our
studies. Therefore, the selective antiproliferative effect of Go-6976
on immature cells cannot be attributed to inhibition of PKC-µ. We also demonstrated that PKC-µ is resistant to PMA-induced
downregulation in vascular fibroblasts. A shift in phosphorylation
state was observed but not degradation. This finding is consistent with a recent report on HeLa and COS cells stably transfected with PKC-µ
as well as a carcinoma cell line expressing endogeneous PKC-µ (12).
Collectively, these results suggest that PKC-µ is not responsible for
the enhanced growth of immature PA fibroblasts.
Although it was not the focus of this paper, we were also able to
compare results with the different antagonist strategies to establish a
role for PKC-
in an additional component of growth of all three cell
types that was not developmentally regulated and was Ro-31-8220
sensitive but GF-109203X, Go-6976, and PMA downregulation resistant.
These results are consistent with the fact that PKC-
lacks a phorbol
ester binding domain and has been implicated in the mitogenic response
of some nonvascular cells (34). Interestingly, this isozyme has been
localized to the nucleus in other cell systems (34), and by
immunostaining we have found the same for PA adventitial fibroblasts
(personal communication).
In summary, using antibodies against the 11 described isozymes of PKC,
multiple Ca2+-dependent and
-independent isoforms have been detected in neonatal bovine PA
adventitial fibroblasts. The levels of two
Ca2+-dependent isozymes, PKC-
and -
II, have been found to be selectively higher in fetal and
neonatal than in adult PA adventitial fibroblasts. The change in
expression pattern of these two isozymes paralleled the differences in
growth, PKC catalytic activity, and susceptibility to PKC inhibitors
previously observed in these vascular cells with advancing
developmental stage. Using complementary antagonist strategies, these
same Ca2+-dependent (
and
II) isozymes of PKC have been implicated in the enhanced growth
capacity of immature PA fibroblasts in vitro. Therefore, PKC-
and
-
II may also be important in the augmented proliferative response of
neonatal bovine PA adventitial fibroblasts observed in vivo in settings
of vascular injury.
 |
ACKNOWLEDGEMENTS |
We thank Steve Hofmeister and Sandi Walchak for harvesting bovine
pulmonary artery tissue; Drs. Anirban Banerjee and Fabia Gamboni-Robertson for helpful advice about antibodies for protein kinase C isozymes; and Drs. John V. Weil, Ivan F. McMurtry, and Joan
Keiser for critical review of this manuscript.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
(NHLBI) Grant PPG HL-14985 and Specialized Center of Research Grant
HL-56481. M. Das was supported by NHLBI Training Grant HL-07171 and by
a Postdoctoral Fellowship from the American Heart Association: Arizona,
Colorado, and Wyoming Affiliates. K. R. Stenmark was supported by an
American Lung Association Career Investigator Award. E. C. Dempsey was
supported by a Roerig/American College of Chest Physicians Research
Award, Veterans Administration Grant, and Giles Filley Research Award
from the American Physiological Society.
Preliminary results of this work were presented in part on April 17, 1996 at a minisymposium on "Injury and Repair of the Developing
Pulmonary Circulation" at the Annual Experimental Biology Meeting in
Washington, DC (FASEB J. 10: A810,
1996), on May 20, 1997 at the American Thoracic Society Annual Meeting
in San Francisco, CA (Am. J. Respir. Crit. Care
Med. 155: A637, 1997), and on June 6, 1997 at the
Thomas Petty Aspen Lung Conference on the Pulmonary Circulation.
Address for reprint requests: M. Das, Cardiovascular Pulmonary and
Developmental Biology Research Laboratories, B-133, Univ. of Colorado
Health Sciences Center, 4200 E. 9th Ave. Denver, CO 80262.
Received 10 July 1997; accepted in final form 5 September 1997.
 |
REFERENCES |
1.
Borner, C.,
S. N. Guadagno,
D. Fabbro,
and
I. B. Weinstein.
Expression of four protein kinase C isoforms in rat fibroblasts.
J. Biol. Chem.
267:
12892-12899,
1992[Abstract/Free Full Text].
2.
Das, M.,
K. R. Stenmark,
and
E. C. Dempsey.
Enhanced growth of fetal and neonatal pulmonary artery adventitial fibroblasts is dependent on protein kinase C.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L660-L667,
1995[Abstract/Free Full Text].
3.
Dempsey, E. C.,
D. B. Badesch,
E. L. Dobyns,
and
K. R. Stenmark.
Enhanced growth capacity of neonatal pulmonary artery smooth muscle cells in vitro: dependence on cell size, time from birth, insulin like growth factor I, and auto-activation of protein kinase C.
J. Cell. Physiol.
160:
469-481,
1994[Medline].
4.
Dempsey, E. C.,
I. F. McMurtry,
and
R. F. O'Brien.
Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L136-L145,
1991[Abstract/Free Full Text].
5.
Dempsey, E. C.,
L. J. Ruff,
K. R. Stenmark,
and
M. Das.
Protein kinase C-
is an important determinant of bovine pulmonary artery smooth muscle cell proliferative response to hypoxia in vitro (Abstract).
Am. J. Respir. Crit. Care Med.
153:
193A,
1996.
6.
Dempsey, E. C.,
K. R. Stenmark,
I. F. McMurtry,
R. F. O'Brien,
N. F. Voelkel,
and
D. B. Badesch.
Insulin-like growth factor I and protein kinase C activation stimulate pulmonary artery smooth muscle cell proliferation through separate but synergistic pathways.
J. Cell. Physiol.
144:
159-165,
1990[Medline].
7.
Dempsey, E. C.,
T. Stevens,
A. G. Durmowicz,
and
K. R. Stenmark.
Hypoxia-induced changes in the contraction, growth, and matrix synthetic properties of vascular cells.
In: Tissue Oxygen Deprivation: Developmental, Molecular, and Integrated Function, edited by G. Haddad,
and G. Lister. New York: Dekker, 1996, chapt. 9, p. 225-274. (Lung Biol. Health Dis. Ser.)
8.
Gschwendt, M.,
S. Dieterich,
J. Rennecke,
W. Kittstein,
H.-J. Mueller,
and
F.-J. Johannes.
Inhibition of protein kinase C µ by various inhibitors. Differentiation from protein kinase C isozymes.
FEBS Lett.
392:
77-80,
1996[Medline].
9.
Haworth, S. G.
Pulmonary hypertension in childhood.
Eur. Respir. J.
6:
1037-1043,
1993[Abstract].
10.
Heikkila, J.,
A. Jalava,
and
K. Eriksson.
The selective protein kinase C inhibitor GF109203X inhibits phorbol ester-induced morphological and functional differentiation of SH-SY5Y human neuroblastoma cells.
Biochem. Biophys. Res. Commun.
197:
1185-1193,
1993[Medline].
11.
Isakov, N.,
P. McMahon,
and
A. Altman.
Selective post-transcriptional down-regulation of protein kinase C isozymes in leukemic T cells chronically treated with phorbol ester.
J. Biol. Chem.
265:
2091-2097,
1990[Abstract/Free Full Text].
12.
Johannes, F.-J.,
J. Prestle,
S. Dieterich,
P. Oberhagemann,
G. Link,
and
K. Pfizenmaier.
Characterization of activators and inhibitors of protein kinase C-µ.
Eur. J. Biochem.
227:
303-307,
1995[Abstract].
13.
Jones, K. T.,
and
G. R. Sharpe.
Staurosporine, a non-specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31-8220, a specific inhibitor, does not.
J. Cell. Physiol.
159:
324-330,
1995.
14.
Lake, F. R.,
E. C. Dempsey,
J. D. Spahn,
and
D. W. H. Riches.
Involvement of protein kinase C in macrophage activation by poly(I·C).
Am. J. Physiol.
266 (Cell Physiol. 35):
C134-C142,
1994[Abstract/Free Full Text].
15.
Martiny-Baron, G.,
M. G. Kazanietz,
H. Mischak,
P. M. Blumberg,
G. Kochs,
H. Hug,
D. Marme,
and
C. Schachtele.
Selective inhibition of protein kinase C isozymes by the indolcarbazole Go6976.
J. Biol. Chem.
268:
9194-9197,
1993[Abstract/Free Full Text].
16.
McCaffrey, P. G.,
and
M. R. Rosner.
Characterization of protein kinase C from normal and transformed cultured murine fibroblasts.
Biochem. Biophys. Res. Commun.
146:
140-146,
1987[Medline].
17.
Meyrick, B.,
and
L. Reid.
Hypoxia and incorporation of 3H-thymidine by cells of the rat pulmonary arteries and alveolar wall.
Am. J. Pathol.
96:
51-70,
1979[Abstract].
18.
Mukherjee, J. J.,
T. Chung,
D. Kirk Ways,
and
Z. Kiss.
Protein kinase C-
is a major mediator of the stimulatory effect of phorbol ester on phospholipase D-mediated hydrolysis of phosphatidylethanolamine.
J. Biol. Chem.
271:
28912-28917,
1996[Abstract/Free Full Text].
19.
Murray, N. R.,
G. P. Baumgardner,
D. J. Burns,
and
A. P. Fields.
Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation.
J. Biol. Chem.
268:
15847-15853,
1993[Abstract/Free Full Text].
20.
Nishizuka, Y.
Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
Science
258:
607-614,
1992[Medline].
21.
Okada, K.,
P. Tsai,
V. A. Briner,
C. Caramelo,
and
R. W. Schrier.
Effects of extra- and intracellular pH on vascular action of arginine vasopressin.
Am. J. Physiol.
260 (Renal Fluid Electrolyte Physiol. 29):
F39-F45,
1991[Abstract/Free Full Text].
22.
Olivier, A. R.,
and
P. J. Parker.
Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.
J. Cell. Physiol.
152:
240-244,
1992[Medline].
23.
Puceat, M.,
R. Hilal-Dandan,
B. Strulovici,
L. L. Brunton,
and
J. H. Brown.
Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes.
J. Biol. Chem.
296:
16938-16944,
1994.
24.
Rabinovitch, M.,
W. J. Gamble,
O. S. Miettinen,
and
L. Reid.
Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery.
Am. J. Physiol.
240 (Heart Circ. Physiol. 9):
H62-H72,
1981[Abstract/Free Full Text].
25.
Racchi, M.,
S. Bergamaschi,
S. Govoni,
W. C. Wetsel,
A. Bianchetti,
G. Binetti,
F. Battaini,
and
M. Trabucchi.
Characterization and distribution of protein kinase C isoforms in human skin fibroblasts.
Arch. Biochem. Biophys.
314:
107-111,
1994[Medline].
26.
Reeves, J. T.,
and
J. Herget.
Experimental models of pulmonary hypertension.
In: Pulmonary Hypertension, edited by E. K. Weir,
and J. T. Reeves. Mt. Kisco, NY: Futura, 1984, p. 361-391.
27.
Rudolph, A. M.
Fetal and neonatal pulmonary circulation.
Annu. Rev. Physiol.
41:
383-395,
1979[Medline].
28.
Rybin, V. O.,
and
S. F. Steinberg.
Protein kinase C isoform expression and regulation in the developing rat heart.
Circ. Res.
74:
299-309,
1994[Abstract].
29.
Saxena, R.,
B. A. Saksa,
K. S. Hawkins,
and
M. B. Ganz.
Protein kinase C-
I and
II are differentially expressed in the developing glomerulus.
FASEB J.
8:
646-653,
1994[Abstract/Free Full Text].
30.
Stenmark, K. R.,
J. Fasules,
N. F. Voelkel,
J. Henson,
A. Tucker,
H. Wilson,
and
J. T. Reeves.
Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4,300 m.
J. Appl. Physiol.
62:
821-830,
1987[Abstract/Free Full Text].
31.
Toullec, D.,
P. Pianetti,
H. Coste,
P. Bellevergue,
T. Grand-Perret,
M. Ajakane,
V. Baudet,
P. Boissin,
E. Boursier,
F. Loriolle,
L. Duhamel,
D. Charon,
and
J. Kirilovsky.
The bisindolylmalemide GF109203X is a potent and selective inhibitor of protein kinase C.
J. Biol. Chem.
266:
15771-15781,
1991[Abstract/Free Full Text].
32.
Ways, D. K.,
C. A. Kukoly,
J. deVente,
J. L. Hooker,
W. O. Bryant,
K. J. Posekany,
D. J. Fletcher,
P. P. Cook,
and
P. J. Parker.
MCF-7 breast cancer cells transfected with protein kinase C-
exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype.
J. Clin. Invest.
95:
1906-1915,
1995[Medline].
33.
Xu, Y.,
K. R. Stenmark,
M. Das,
S. J. Walchak,
L. J. Ruff,
and
E. C. Dempsey.
Pulmonary artery smooth muscle cells from chronically hypoxic neonatal calves retain fetal-like and acquire new growth properties.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L234-L245,
1997[Abstract/Free Full Text].
34.
Zhou, G.,
M. W. Wooten,
and
E. S. Coleman.
Regulation of atypical
-protein kinase C in cellular signaling.
Exp. Cell Res.
214:
1-11,
1994[Medline].
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