1 Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; and 2 Institute of Clinical Biochemistry and Pathobiochemistry, University of Wuerzburg, Wuerzburg 97080, Germany
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ABSTRACT |
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The cGMP-cGMP-dependent
protein kinase (protein kinase G) system plays an important role in the
pathogenesis of mesangial proliferative glomerulonephritis. However,
the molecular mechanisms of the inhibitory effects of the cGMP-protein
kinase G system in the cell cycle progression of mesangial cells are
not well known. To determine the inhibitory pathway of cGMP-protein
kinase G in cultured mesangial cells, we investigated the effects of cGMP- and adenovirus-mediated overexpression of protein kinase G on the
promoter activities of cyclin E, cyclin D1, and cyclin A. 8-Bromo-cGMP
(8-BrcGMP) and overexpression of protein kinase G reduced
[3H]thymidine uptake, reduced the numbers of cells in S
and G2/M phases, and decreased the phosphorylation of
retinoblastoma (Rb) protein. 8-BrcGMP (103 M), protein
kinase G adenovirus (Ad-cGKI
; 1010 plaque-forming
units/ml), atrial natriuretic peptide (ANP), and C-type natriuretic
peptide (CNP) inhibited the promoter activity of cyclin E to 49, 57, 77, and 78%, respectively. On the other hand, the promoter activities
of cyclin D1 and cyclin A were not changed significantly. In Western
blot analysis, 8-BrcGMP, Ad-cGKI
, ANP, and CNP also inhibited cyclin
E protein expression dose and time dependently. The p44/p42
mitogen-activated protein kinase (MAPK) kinase 1-p44/p42 MAPK had no
effect on cyclin E promoter activities, and the cGMP-protein kinase G
pathway did not change MAPK activity. In conclusion, our findings
suggest that the reduction of the cyclin E promoter activity that
downregulates G1/S transition plays a dominant role in the
cGMP- and protein kinase G-induced inhibition of mesangial cell proliferation.
mitogen-activated protein kinase; cyclin A; cyclin D1; atrial natriuretic peptide; C-type natriuretic peptide
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INTRODUCTION |
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MESANGIAL CELL PROLIFERATION is an essential component of glomerulonephritis. In recent studies, many cytokines have been shown to either promote or suppress the cell cycle of mesangial cells. Because the interactions between these factors and regulational mechanisms of the mesangial cell cycle are not well known, learning more about them would be of great help in developing a curative treatment for mesangial proliferative glomerulonephritis.
Atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) play an important role in many physiological functions that change the hemodynamics, such as vasorelaxant activity in vascular smooth muscle cells (VSMC) and promotion of urinary excretion of sodium and water by the kidney (3, 32). These cytokines also exhibit a growth inhibitory response in renal mesangial cells, VSMC, and endothelial cells that is mediated by the intracellular second messenger cGMP via an involvement of the cGMP-dependent protein kinase, protein kinase G (4, 9, 10, 24). Former investigations suggested that these cytokines and the cGMP-protein kinase G pathway have important roles in mesangial cell proliferation (6). For example, in in vitro studies, ANP and CNP inhibited mesangial cell proliferation (1, 2, 18), and in an in vivo study, continuous infusion of CNP prevented the proliferation of mesangial cells in a rat mesangioproliferative anti-Thy1.1 model (5).
However, inhibitory signaling pathways of cGMP-protein kinase G are not
well known. In recent studies, several different pathways have been
proposed, for example, the inhibitory pathways via upregulation of the
Gax-p21cip1 (11, 27) and transforming growth
factor (TGF)- (25, 35), reduction of cyclin A
(14), and suppression of the mitogen-activated protein
kinase (MAPK) cascade (7, 15).
In the present study, to determine the stage of cell cycle progression that is suppressed by protein kinase G in mesangial cells, we investigated the effects of adenovirus-mediated overexpression of protein kinase G on G1 cyclin promoter activities. We found that protein kinase G overexpression decreased cyclin E expression transcriptionally and inhibited the cell cycle at the G1/S transition in mesangial cells. We conclude that the reduction of cyclin E promoter activity plays a dominant role in cGMP- and protein kinase G-induced inhibition.
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METHODS |
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Mesangial cell culture.
Mesangial cell strains from male Sprague-Dawley rats were isolated and
characterized as previously reported (23). Cells were
cultured in RPMI 1640 medium containing 20% FBS, 100 U/ml penicillin,
100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, and
5 ng/ml selenite at 37°C in a 5% CO2 incubator. The
cells were seeded in 10-cm dishes for all experiments except the
experiment on [3H]thymidine incorporation in which
24-well dishes were used. When the cells reached 80% confluence, they
were either treated with an indicated dose of 8-bromo-cGMP (8-BrcGMP),
8-bromo-cAMP (8-BrcAMP) in 20% FCS medium for 48 h, or infected
with either protein kinase G adenovirus (Ad-cGKI) or Ad-LacZ in 20%
FCS medium for 48 h. For the experiment in Fig. 5, when the cells
reached 80% confluence they were incubated in 0.5% FCS starved medium
for 48 h and then changed to the 0.5% FCS medium containing 25 ng/ml platelet-derived growth factor (PDGF)-BB with 8-BrcGMP or
infected with Ad-cGK
I for 48 h. In our preliminary experiments,
we confirmed that the magnitude of PDGF-BB stimulation in the mesangial
cell proliferation at 80% confluency was not different from that at
60% confluency.
Antibodies.
Antibodies against anti-mouse-cyclin D1, anti-rabbit-cyclin A, and
anti-rabbit-cyclin E were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against anti-p44/p42 MAPK,
anti-phospho-p44/p42 MAPK, and human retinoblastoma (Rb) protein were
purchased from New England Biolabs (Beverly, MA). Anti-myc
antibody was purchased from Invitrogen (Carlsbad, CA). Antibody against
human cGKI (protein kinase G type I
) was described previously
(20).
Construction of adenovirus.
Ad-cGKI, a replication-defective, recombinant adenovirus driven by
cytomegalovirus promoter, was described previously (34). The recombinant adenovirus Ad-LacZ was provided by Dr. I. Saito (17). Each adenovirus preparation was titrated by plaque
assay on 293 cells.
Reporter constructs and plasmids.
The cyclin D1 reporter construct used for luciferase assays contained
human cyclin D1 promoter from residues 944 to +139 cloned upstream of
the luciferase gene (generous gift of Dr. M. Eilers; see Ref.
28). The cyclin A reporter construct contained human
cyclin A promoter from residues
924 to +245 (generous gift of Dr. J. Sobczak-Thepot; see Ref. 8), and the cyclin E reporter construct contained human cyclin E promoter from residues
1,195 to
+79 cloned upstream of the luciferase gene (generous gift of Dr. K. Ohtani; see Ref. 22). Cyclin E plasmid construct was provided by Dr. S. Coats (12). Wild-type p44/p42 MAPK
kinase 1 (MKK1), dominant-active MKK1 S222E, and dominant-negative MKK1 S222A were generous gifts of Dr. E. G. Krebs (27).
[3H]thymidine incorporation.
Mesangial cells were plated in 24-well plates and either incubated in a
20% FCS medium containing 8-BrcGMP (103 M
~10
5 M) or infected with Ad-cGKI
or Ad-LacZ in 20%
FCS medium for 48 h. For the last 4 h, 1 µCi
[3H]thymidine (Amersham) was added to the medium. The
cells were washed three times in 4°C PBS, cold 10% TCA was added to
precipitate protein and DNA, and the mixture was redissolved in 0.5 M
NaOH. Aquasol-2 scintillation cocktails (NEN Research Products, Boston, MA) were counted in a scintillation counter. The measurement was made
at 48 h, the point at which we observed maximum inhibition of
cyclin E protein expression, as described later in RESULTS. In a preliminary experiment, we also observed maximum inhibition of
[3H]thymidine at 48 h.
Cell cycle analysis by flow cytometry. Mesangial cells were cultured in 10-cm dishes. The cells were incubated in 20% FCS medium with indicated doses of 8-BrcGMP for 48 h. The samples were washed two times with PBS and then resuspended in 70% ethanol for 1 h at 4°C. Fixed and permeated cells were collected by centrifugation, washed with PBS, treated with RNase, and stained with propidium iodide. The numbers of cells in the G1, S, and G2/M phases were analyzed by flow cytometry using a fluorescence-activated cell sorter (FACS) calibur (Becton-Dickinson, San Jose, CA) as described previously (31, 33).
Cyclin E-dependent kinase assay.
Immune complex kinase assay was performed using essentially the same
methods as previously described by Terada et al. (31). Mesangial cells were treated with 8-BrcGMP or infected with Ad-cGKI for 48 h. The cell lysates were clarified by centrifugation at 10,000 g for 5 min. After incubation of the supernatants for
2 h at 4°C with 10 µl of anti-cyclin E antibody and then for
1 h with 30 µl of protein G-agarose (Boehringer Mannheim,
Mannheim, Germany), immune complexes were recovered by centrifugation.
Immunoprecipitated proteins were suspended in 30 µl of kinase buffer
[50 mM HEPES (pH 7.5), 10 mM MgCl2, and 1 mM
dithiothreitol] containing 0.2 µg of histone H1 (Boehringer
Mannheim) and 2.5 mM EGTA, 10 mM
-glycerophosphate, 1 mM NaF, 20 µM ATP, and 10 µCi of [
-32P]ATP (NEN). After
incubation for 30 min at 30°C with occasional mixing, the samples
were boiled in polyacrylamide gel sample buffer containing SDS and
separated by electrophoresis. Phosphorylated proteins were analyzed by
a BAS station (Fuji, Tokyo, Japan).
Transient transfection and luciferase assay.
Mesangial cells were transfected by the electroporation method with 4 µg of -galactosidase construct and 20 µg of cyclins E, D1, or A
promoter construct. After transfection, the cells were cultured in
medium containing 20% FCS for 12 h and then either changed to a
20% FCS medium containing an indicated dose of 8-BrcGMP or infected
with Ad-cGKI
or Ad-LacZ in a 20% FCS medium for 48 h as a
control. Luciferase and
-galactosidase activities were measured
according to the protocols of the manufacturer (Promega, Madison, WI).
Luciferase enzyme units were normalized to
-galactosidase. Our
previous report demonstrated that the transfection efficiency of our
electroporation method in mesangial cells was around 30% (21).
Generation of cyclin E-expressing clones. Twenty micrograms of cyclin E plasmid containing the neo resistant gene and myc gene were transfected into mesangial cells by electroporation. The cells were cultured in medium containing 20% FCS and G418 (200 µg/ml; GIBCO-BRL, Grand Island, NY) to expand the G418-resistant clones. The exogenous cyclin E expression of each clone was examined by Western blot analysis using anti-myc antibody (data not shown). The selected clones found to express exogenous cyclin E protein expression were maintained in medium containing G418 to preserve their indelibility and used for the experiment (26).
Western blot analysis. Whole cell lysates were extracted from the cultured mesangial cells and lysed in the buffer (50 mM HEPES, (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM sodium orthovanadate) at 4°C. After incubation for 5 min, lysates were centrifuged at 4°C for 15 min at 10,000 g. The soluble lysates were mixed 1:4 with 5× Laemmli buffer and heated for 5 min at 95°C. Forty micrograms of protein extracts were subjected to Western blotting with anti-cyclin E, anti-cyclin D1, anti-cyclin A, anti-Rb, anti-p44/p42 MAPK, anti-phospho-p44/p42 MAPK, or anti-protein kinase G. Proteins were separated in SDS-10% polyacrylamide gels and transferred to an Immobilon-P membrane (Daiichikagaku, Tokyo, Japan). Blots were incubated in 5% nonfat dry milk (in 1× PBS, 0.1% Tween 20) for 1 h at room temperature and then incubated with the indicated antibodies (1:1,000 dilution in 5% nonfat dry milk, 0.1% Tween 20 in 1× PBS) for 2 h. After three washes in 0.1% Tween 20 in 1× PBS (15 min, 5 min, 5 min), blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,500 dilution) for 1 h. After three more washes using the method described before, membranes were visualized by the Amersham enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Statistics. All values were expressed as means ± SE. The differences were tested using ANOVA. P < 0.05 was considered significant.
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RESULTS |
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cGMP and overexpression of protein kinase G decreased
[3H]thymidine incorporation and the cell numbers of S and
G2/M phases in cultured mesangial cells.
We first examined the effects of cGMP and protein kinase G
overexpression on mesangial cell proliferation by measuring
[3H]thymidine uptake. Mesangial cells were treated with
8-BrcGMP (103 M ~10
5 M) or Ad-cGKI
[1010 plaque-forming units (pfu)/ml] for 48 h.
[3H]thymidine was pulsed for the last 4 h. Figure
1A shows a dose-dependent reduction of [3H]thymidine uptake by 8-BrcGMP and
Ad-cGKI
transfection compared with the control. In the Ad-cGKI
experiments, we used Ad-LacZ-infected cells as a control. In a previous
study, we showed that the transfection efficiency of adenovirus
(Ad-LacZ) was nearly 99% in mesangial cells (33).
Overexpression of protein kinase G protein was confirmed by Western
blotting (see Fig. 3A). These data indicate that cGMP and
protein kinase G inhibit mesangial cell proliferation. To verify the
inhibition of cell proliferation by cGMP, we performed cell cycle
analysis by FACS. Mesangial cells were cultured in 10-cm dishes and
incubated in 20% FCS medium with 8-BrcGMP for 48 h (Fig.
1B). 8-BrcGMP reduced the cell numbers of S and
G2/M phases in a dose-dependent manner. These data suggest
that 8-BrcGMP halts the cell cycle at the G1 phase and
prevents its progression to the S phase.
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cGMP and protein kinase G reduced the phosphorylation of Rb
protein.
To examine the inhibitory mechanism of cGMP and protein kinase G in the
G1/S transition, we examined the phosphorylation state of
Rb protein by Western blot analysis using an antibody specific to Rb
proteins. Cells were treated with 8-BrcGMP (103 M) or
infected with Ad-cGKI
(1010 pfu/ml) for 48 h, and
whole cell lysates were used for analysis. In Fig.
2, two parallel bands were observed, and
the top band represented phosphorylated Rb protein. 8-BrcGMP
and Ad-cGKI
decreased the phosphorylation of Rb proteins.
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cGMP and protein kinase G decreased the protein expression of
cyclin E but not of other cyclins.
We next examined the protein expressions of G1/S phase
cyclins (cyclin E, cyclin D1, and cyclin A) by Western blot analysis. 8-BrcGMP and Ad-cGKI both significantly decreased the cyclin E
protein level in a dose-dependent manner (Fig.
3A). We also confirmed that
there were no differences in cyclin E protein expressions between
control cells and Ad-LacZ-infected cells. Figure 3B shows the time course of the inhibitory effect of 8-BrcGMP on cyclin E
protein expression, where the maximum effect was observed at 48 h.
In contrast, neither cGMP nor protein kinase G affected cyclin D1 and A
expressions. Furthermore, we examined protein kinase G protein
expression to confirm the successful gene transfer of protein kinase G
to mesangial cells (Fig. 3A).
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cGMP induced dose-dependent decreases in the promoter activity of
cyclin E but not of other cyclins.
To investigate the role of the cGMP-protein kinase G signaling pathway
in the transcriptional regulation of cyclin E, cyclin D1, and cyclin A,
we measured promoter activity using plasmids containing cyclin E, D1,
and A promoter regions and luciferase reporter genes. Mesangial cells
transfected with cyclin E promoter construct reduced the activity by
8-BrcGMP dose dependently. 8-BrcGMP (104 M) reduced
cyclin E promoter activity to 55% and 10
3 M 8-BrcGMP
reduced the activity to 49% compared with the control (Fig.
4). The cyclin E promoter activity of
Ad-cGKI
-infected cells was reduced to 57% compared with that of the
Ad-LacZ-infected cells (control). In contrast, the promoter activities
of the cells transfected with cyclin D1 and A promoter constructs did
not show any statistically significant changes by 8-BrcGMP and
Ad-cGKI
. These data suggest that the reduction of the cyclin E
promoter activity that downregulates the G1/S transition is
a dominant factor for the cGMP- and protein kinase G-induced inhibition
of mesangial cell proliferation.
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cGMP and protein kinase G inhibited the mesangial cell
proliferation induced by PDGF-BB and decreased cyclin E transcription.
We next examined if the inhibitory effect of the cGMP-protein kinase G
pathway was altered by different proliferative stimuli. Before addition
of PDGF-BB, the cell quiescence was achieved by incubation for 48 h in RPMI 1640 with 0.5% FCS. The cell were then exposed to the 0.5%
FCS medium containing PDGF-BB (25 ng/ml) for 48 h.
[3H]thymidine uptake, cyclin E promoter activity, and
cyclin E protein expression were examined by the same processes as
described previously. cGMP and protein kinase G decreased the
PDGF-BB-induced [3H]thymidine uptake, cyclin E promoter
activities, and cyclin E protein expression of the mesangial cells
(Fig. 5).
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cGMP and protein kinase G decreased cyclin E-dependent kinase
activity.
To further determine the involvement of cyclin E in the inhibitory
effect of cGMP-protein kinase G on mesangial cell proliferation, we
measured cyclin E-dependent kinase activity using histone H1 as
substrate. Figure 6 shows that 8-BrcGMP
(103 M) and the overexpression of protein kinase G
inhibited the cyclin E-dependent kinase activities. These data were
consistent with the reduction of Rb protein phosphorylation by the
cGMP-protein kinase G system.
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ANP and CNP decreased [3H]thymidine incorporation,
cyclin E promoter activity, and the cyclin E protein level.
ANP and CNP were reported to increase cGMP in mesangial cells (1,
2). Thus we next examined whether these peptide hormones also
inhibit the mesangial cell proliferation and change cyclin E
expression. The cells were treated with indicated doses of ANP or CNP
for 48 h. [3H]thymidine was pulsed for the last
4 h, and the [3H]thymidine uptake was measured. ANP
and CNP reduced the uptake in a dose-dependent manner (Fig.
7A). Mesangial cells were
transiently transfected with cyclin E promoter plasmid, treated with
ANP or CNP for 48 h, and assayed for luciferase activities. Both
ANP and CNP reduced cyclin E promoter activities dose dependently (Fig.
7B). Next, the cells were treated with ANP and CNP for
48 h, and then the whole cell lysates from these cells were
analyzed by Western blotting. Cyclin E protein levels were decreased by ANP and CNP in a dose-dependent manner (Fig. 7C). ANP and
CNP inhibited cyclin E at both transcriptional and protein levels, and
these data are compatible with the results from former experiments.
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cAMP did not inhibit mesangial cell proliferation or decrease
cyclin E protein expression.
Mesangial cells were treated with indicated doses of 8-BrcAMP for
48 h. [3H]thymidine was pulsed for the last 4 h, and [3H]thymidine uptake was measured. 8-BrcAMP did
not have an anti-mitogenic effect on the mesangial cells (Fig.
8A). We also examined the effect of 8-BrcAMP on cyclin E protein expression by Western blot analysis. 8-BrcAMP did not decrease the cyclin E protein levels (Fig.
8B).
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Overexpression of cyclin E reversed the anti-mitogenic effect of
cGMP.
To confirm that the suppression of cyclin E transcription is essential
to the anti-mitogenic effect of cGMP, we examined whether the
overexpression of cyclin E overcame the inhibitory effect of cGMP by
analyzing the cell cycle by flow cytometry. Rat mesangial cells were
transfected with 20 µg of cyclin E plasmid and cultured in medium
containing G418. G418-resistant clones that expressed exogenous cyclin
E were selected for the experiment. Original mesangial cells and
mesangial cells expressing exogenous cyclin E were cultured at 80%
confluence with or without 8-BrcGMP for 48 h and examined by flow
cytometry for cell cycle analysis (Fig. 9A). In original mesangial
cells, 8-BrcGMP reduced the percentage of S and G2/M phases
to 10 and 33%, respectively. However, in the mesangial cells that
expressed exogenous cyclin E, the reduction of the percentage of S and
G2/M phases was attenuated and recovered to the control
level (Fig. 9B). The data suggested that cyclin E could
overcome the anti-mitogenic effect of cGMP and that the inhibitory
effect of cGMP worked through cyclin E.
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The MKK1-p44/p42 MAPK pathway did not significantly change cyclin E
promoter activity.
To examine the involvement of the MKK1-p44/p42 MAPK pathway in cyclin E
promoter activity, rat mesangial cells were cotransfected with cyclin E
promoter plasmid construct and either wild-type MKK1, dominant-active
MKK1 S222E, dominant-negative MKK1 S222A, or dominant-active MKK1 S222E
with PD-98059 (106 M). After 48 h of incubation, the
luciferase activities were assayed. Cyclin E promoter activities were
not changed significantly by transfection with either constitutive
active or negative MKK1 constructs (Fig.
10A). These results
suggested that the MKK1-p44/p42 MAPK pathway did not significantly
change cyclin E promoter activity in our experimental condition. To
confirm the effectiveness of the dominant-negative mutants and PD-98059
used in this experiment, we examined the protein expressions of p44/p42
MAPK and phospho-p44/p42 MAPK in the mesangial cells transfected with
either wild-type MKK1, dominant-active MKK1 S222E, dominant-negative
MKK1 S222A, or dominant-active MKK1 S222E with PD-98059
(10
6 M). The transfections of dominant-negative MKK1
S222A and dominant-active MKK1 S222E with PD-98059 (10
6
M) to the mesangial cells reduced the phosphorylation of p44/p42 MAPK
without changing the total amount of p44/p42 MAPK (Fig.
10B). The results confirmed the effectiveness of the
dominant-negative mutants and PD-98059 used in this study.
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DISCUSSION |
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In this study, we show that protein kinase G overexpression by adenovirus decreases cyclin E transcriptionally and inhibits the cell cycle at the G1/S transition in mesangial cells. We observed a reduction of cyclin E promoter activity by protein kinase G and found that the mechanisms of the antiproliferative effect of cGMP-protein kinase G were associated with a decrease in the transcriptional level of cyclin E expression.
These results were also confirmed by other experiments. First, cGMP and protein kinase G overexpression decreased [3H]thymidine incorporation, delayed the progression from the G0/G1 phase to the S and G2/M phases, and reduced the phosphorylation of Rb proteins. This evidence suggested that G1 cyclins are important factors for inhibition (19). Second, in Western blot analysis, cyclin E was the only G1 cyclin to be suppressed by cGMP and protein kinase G. Third, cGMP and overexpression of protein kinase G reduced the cyclin E-dependent kinase activity, thereby inhibiting the progression of the cell cycle at the G1 phase. Fourth, overexpression of cyclin E overcame the anti-mitogenic effect of cGMP, indicating that the inhibitory effect of cGMP worked through cyclin E. Finally, ANP and CNP, the upstream signals of cGMP-protein kinase G, also transcriptionally inhibited cyclin E gene expression. All of these observations were compatible with the inhibition by the cGMP-protein kinase G pathway observed earlier.
As far as we know, this is the first report demonstrating that transcriptional inhibition of the cyclin E gene is involved in cell cycle inhibition by cGMP and protein kinase G. Compared with former investigations, this study took a more direct approach to the mechanisms of inhibition in two ways. First, we directly examined the promoter activities of G1 cyclins and elucidated that the inhibition occurred at a transcriptional level. Second, we directly used the adenovirus-mediated overexpression of protein kinase G. This method skirted the problem of the involvement of other signal transductions via processes not involving protein kinase G, such as cross-activation via cAMP. Using this method, we also sidestepped the potential risk of inhibition by the toxic effect of the cGMP analog (2). We also used 8-BrcAMP instead of 8-BrcGMP in the investigations of thymidine incorporation, cyclin E promoter activity, and cyclin E protein expression to examine the cross-activation between these two messengers; however, in our experiments, 8-BrcAMP did not change either thymidine uptake, the promoter activities, or the protein expression.
According to former investigations, several antiproliferative pathways
were reported downstream of protein kinase G, for example, pathways via
cyclin A (14), Gax-p21cip1 (27),
c-fos (13, 16), and TGF- (25,
35). This diversity may have been due to the difference of cell
type or experimental conditions. Several studies demonstrated that cGMP
suppressed the cell cycle via reduction of MAPK activity in VSMC
(36) and rat mesangial cells (15, 30). To
clarify this, we examined cyclin E promoter activity after transfecting
dominant-active and dominant-negative forms of MKK1 plasmid constructs.
From the results under our experimental condition, cyclin E promoter
activity was not affected by MAPK and its inhibitor PD-98059 (Fig. 10); however, it will be necessary to examine several experimental conditions to confirm the effects of the MAPK pathway on the cGMP signaling system. Further studies may be necessary to clarify the
complex signaling pathways involved in the regulation of the mesangial
cell cycle.
In summary, we demonstrated that the inhibition of cyclin E transcription is the main factor of suppression of the cell cycle by cGMP-protein kinase G in mesangial cells. Determination of the functional roles of the cGMP-protein kinase G pathway is important for understanding the mechanisms of mesangial cell proliferation. Our findings may permit the development of new strategies to understand the mechanism of mesangial proliferative glomerulonephritis and ways to inhibit it.
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ACKNOWLEDGEMENTS |
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We thank Drs. E. G. Krebs, M. Eilers, J. Sobczak-Thepot, K. Ohtani, and S. Coats for providing plasmids.
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FOOTNOTES |
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This research was supported by Deutsche Forschungsgemeinschaft Grant SFB355 (to S. M. Lohmann).
Address for reprint requests and other correspondence: Y. Terada, Second Dept. of Internal Medicine, Tokyo Medical and Dental Univ., 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: yterada.kid{at}tmd.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 February 2000; accepted in final form 8 January 2001.
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