© 1999 American Heart Association, Inc.
Original Contribution |
Molecular Mechanisms of Endothelin-1–Induced Cell-Cycle Progression
Involvement of Extracellular Signal-Regulated Kinase, Protein Kinase C, and Phosphatidylinositol 3-Kinase at Distinct Points
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan.
Correspondence to Etsu Suzuki, MD, PhD, The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail suzuki-2im{at}h.u-tokyo.ac.jp
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Abstract |
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Abstract—Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1±1.9-fold increase, 8 hours after stimulation, P<0.05), cdk4 kinase activity (2.8±0. 5-fold increase, 12 hours after stimulation, P<0.01), and cdk2 kinase activity (2.1±0.4-fold increase, 16 hours after stimulation, P<0.05) in a time- and dose-dependent manner. ET-1–induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1–induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1–induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased 3H-thymidine uptake in a time-dependent fashion (0 hours, 4216±264 cpm per well; 8 hours, 5025±197 cpm per well; 16 hours, 9239±79 cpm per well, P<0.001 versus 0 hours). ET-1–induced increase in 3H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1–induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.
Key Words: endothelin • kinase, extracellular signal-related • protein kinase C • phosphatidylinositol 3-kinase • cell cycle
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been established that endothelin-1 (ET-1) not only possesses a potent vasoconstrictive activity1 but also has a mitogenic activity2 that appears to be involved in vascular remodeling by stimulating proliferation of vascular smooth muscle cells and endothelial cells. In several reports by Schiffrin et al,3 4 vascular "hypertrophy," in which vessel wall thickening occurs, was attenuated by administration of an ET-receptor antagonist in DOCA-salt hypertensive rats, which suggested the possibility that the mitogenic activity of ET-1 might be involved in the process. ET receptor antagonists have also been shown to decrease the neointimal hyperplasia caused by balloon injury.5 6 These results suggest that ET-1 would be implicated in vascular remodeling partly through its mitogenic activity. However, although ET-1–induced changes in intracellular signaling pathways have been well described, the molecular mechanisms of ET-1–induced cell-cycle progression remain poorly understood.
ET-1 binds to its specific heterotrimeric G-protein–coupled receptors and exerts its biological effects by modulating intracellular signaling pathways, including Ca2+ mobilization, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI-3-K), and extracellular signal-regulated kinase (ERK).7 8 9 10 The molecular mechanisms of ET-1–induced activation of the ERK pathway have been well characterized. ET-1 induces Tyr phosphorylation of Shc, which in turn associates with Sos via the adaptor protein, Grb2. The activated complex then activates p21ras (Ras), and Ras activates ERK through Raf and the mitogen-activated protein kinase kinase (MEK).7 8 However, little is known as to whether ET-1–induced activation of the ERK-, PKC- and PI-3-K–mediated pathways really plays any role in the progression of the cell cycle and as to which parts of the cell-cycle regulatory machinery these pathways modulate.
Cell-cycle progression is regulated by timely upregulation and downregulation of Ser/Thr kinases called cyclin-dependent kinases (cdk), which are positively regulated by association with cyclins and are negatively regulated by cdk inhibitors.11 12 In mammalian cells, cyclin D-cdk4/cdk6, cyclin E-cdk2, cyclin A-cdk2, and cyclin B1-cdc2 are the main cyclin-cdk complexes that regulate the progression of G1, G1/S, S, and G2/M phases, respectively. cdk inhibitors comprise 2 families, the ink4 and cip/kip families. The cip/kip family is composed of p21waf1/cip1, p27kip1, and p57kip213 14 15 16 and inhibits a broad spectrum of cdks, including cdk2, cdk4, and cdk6. Although the molecular mechanisms of cell-cycle regulation have been intensively studied, it is not fully understood how the activation of Ras, ERK, PKC, and PI-3-K is linked to the cell-cycle regulatory machinery.
Several studies, in which a dominant-negative Ras mutant was used, have indicated that the Ras-signaling pathway is involved in the induction of cyclin D1 protein, cdk4 kinase activity, and cdk2 kinase activity and in the downregulation of p27kip1.17 18 19 Recently obtained evidence suggests that constitutively active MEK is sufficient to transform cells or induce differentiation, which is dependent on the cell type.20 21 The results indicate that the activation of ERK is linked to the cell-cycle regulatory machinery. The involvement of PKC in cell-cycle regulation is complicated. PKC stimulates cell proliferation or induces cell-cycle arrest, which depends on the timing of PKC activation during the cell cycle, PKC isozymes expressed in the cells, and cell types.22 23 24 25 It is also suggested that PKC activates the ERK pathway in a Ras-independent or Ras-dependent fashion.26 27 The PI-3-K-mediated pathways also appear to be implicated in cell-cycle progression, because a retrovirus-encoded PI-3-K could transform fibroblasts.28 However, it remains unclear how these signaling pathways are linked to the cell-cycle regulatory machinery.
In this study, we examined whether ET-1 activated the cell-cycle regulatory machinery by measuring protein expression levels of cyclins, cdks, and cdk inhibitors, cdk4 and cdk2 kinase activities, and 3H-thymidine uptake. To examine the effects of a dominant-negative Ras mutant, we used the fibroblast cell line NIH3T3 cells to establish stable cell lines that expressed the dominant-negative Ras. We also studied the effects of the MEK, PKC, and PI-3-K inhibition on ET-1–induced changes in the cell-cycle regulatory machinery.
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Materials and Methods |
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Reagents
NIH3T3 cells were purchased from American Type Culture Collection. Anti-cyclin D1, -cyclin E, -cyclin A, -cdk4, -cdk2, -p21waf1/cip1, -p27kip1, and -ERK1 antibodies were obtained from Santa Cruz Biotechnology. Phosphospecific anti-ERK1/2 antibody that recognizes catalytically active ERK1/2 was obtained from New England BioLabs. PRb (769), which is a glutathione S-transferase fusion protein that encodes amino acids 769 to 921 of the mouse pRb, was obtained from Santa Cruz Biotechnology. Histone H1 and myelin basic protein (MBP) were obtained from Boehringer-Mannheim and Upstate Biotechnology , respectively. Calphostin C and LY294002 were purchased from Sigma Chemical Co, and PD98059 was obtained from New England Biolabs. Human ET-1 was obtained from Peptide Institutes.
Cell Culture
NIH3T3 cells were maintained in DMEM that contained 10% FBS. To
induce quiescence, subconfluent cells were incubated in DMEM that
contained 0.2% FBS for 36 hours unless otherwise specified.
Preparation of Protein Extracts
For Western blot analyses and the cdk2 kinase assay, we
used NP-40 cell lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150
mmol/L NaCl, 1% NP-40) that contained 1 mmol/L
phenylmethylsulfonyl fluoride (PMSF), 2 µg/mL leupeptin, and
2 µg/mL aprotinin. Cells were lysed in NP-40 cell lysis buffer for 30
minutes on ice. After centrifugation, the supernatant
was kept at -80°C. For the cdk4 kinase assay, we prepared protein
extracts according to methods previously reported with slight
modifications.29 In brief, we used Tween-20 cell lysis
buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1
mmol/L dithiothreitol, 0.1% Tween-20, 1 mmol/L EDTA, and 10%
glycerol) that contained 1 mmol/L PMSF, 2 µg/mL leupeptin, and 2
µg/mL aprotinin. Cells were resuspended in Tween-20 cell lysis buffer
and subjected once to freezing in liquid nitrogen and thawing at
37°C. The lysates were kept on ice for 30 minutes and
centrifuged for 10 minutes at 4°C. The cleared supernatant
was used for the cdk4 kinase assay. For the ERK1 kinase assay, we used
Triton X cell lysis buffer (50 mmol/L HEPES, pH 7.5, 150
mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1%
Triton X-100, 10% glycerol) that contained 1 mmol/L PMSF, 2
µg/mL leupeptin, and 2 µg/mL aprotinin. Cells were lysed in the
buffer for 30 minutes on ice and centrifuged for 10 minutes at
4°C. The cleared supernatant was used for the ERK1 kinase assay. An
aliquot of each extract was used to measure the protein concentration
according to the method of Bradford (Bio-Rad).
Western Blot Analysis
Forty micrograms of each protein extract was separated on 10%
SDS-polyacrylamide gels and transferred onto nylon membranes
(Millipore) with a semidry blotting system (Pharmacia Biotech). After
blocking in 1xPBS/5% nonfat dry milk/0.2% Tween-20 at 4°C
overnight, the membranes were incubated with the primary antibodies in
a blocking buffer (1xPBS/2% nonfat dry milk/0.2% Tween-20) for 1
hour at room temperature. Antibodies were used at a dilution of 1:100,
except for phosphospecific anti-ERK1/2 antibody, which was diluted at
1:1000. The membranes were washed 3 times with the blocking buffer and
incubated with secondary antibodies, which were conjugated with
horseradish peroxidase (Amersham) at a final dilution of 1:7000. After
final washes with 1xPBS/0.2% Tween-20, the signals were detected with
ECL chemiluminescence reagents (Amersham).
Immune Complex Kinase Assays
For the cdk2 kinase assay, 50 µg of each protein extract was
precleaned with protein A agarose beads (Boehringer Mannheim)
for 1 hour at 4°C in the NP-40 cell lysis buffer. The extracts were
then incubated with 1 µg of anti-cdk2 antibody for 1 hour at 4°C
and with protein A agarose beads for another hour at 4°C with
constant rocking. After centrifugation, the pellets
were washed twice with the NP-40 cell lysis buffer and then 3 times
with a kinase buffer (50 mmol/L Tris-HCl, pH 8.0, 10 mmol/L
MgCl2, 1 mmol/L dithiothreitol, 1
mmol/L PMSF, 2 µg/mL leupeptin, and 2 µg/mL aprotinin). The pellets
were then incubated in 30 µL of the kinase buffer, which contained 3
µg of histone H1, 10 µmol/L ATP, and 10 µCi of
-32P ATP, for 30 minutes at room temperature.
The reactions were terminated by the addition of 30 µL of 2xSDS
loading buffer. After boiling the samples for 5 minutes, one-half of
them were separated on a 12% SDS-polyacrylamide gel and
exposed to x-ray film with an intensifying screen. The cdk4 kinase
assay was performed basically in the same way: 50 µg of each protein
extract was precleaned in the Tween-20 cell lysis buffer and the
extract was incubated with 1 µg of anti-cdk4 antibody. After
incubation with protein A agarose beads, the beads were washed twice
with Tween-20 cell lysis buffer and 3 times with the kinase buffer. The
pellets were then incubated in 20 µL of the kinase buffer, which
contained 1 µg of pRb (769), 10 µmol/L ATP, and 10 µCi of
-32P ATP, for 30 minutes at room
temperature. Half of the samples were loaded on a 12%
SDS-polyacrylamide gel. The ERK1 kinase assay was also
performed in the same way. Fifty micrograms of each protein extract was
precleaned in the Triton X-100 cell lysis buffer in the presence of
protein A agarose beads, and the extract was incubated with 1 µg of
anti-ERK1 antibody. After incubation with protein A agarose beads, the
beads were washed twice with Triton X-100 cell lysis buffer and 3 times
with the kinase buffer. The pellets were incubated in 20 µL of the
kinase buffer, which contained 1 µg of MBP, 10 µmol/L ATP, and
10 µCi of -32P ATP, for 30 minutes at room
temperature. Half of the samples were separated by electrophoresis with
a 12% SDS-polyacrylamide gel. To confirm that almost equal
amounts of cdk4, cdk2, or ERK1 protein were used for the kinase assays,
the same amounts of the protein extracts were immunoprecipitated with
anti-cdk4, -cdk2, or -ERK1 antibody and immunoblotted with
the same antibody as the internal controls. The gels were exposed to
x-ray films, and the signals were analyzed by densitometry.
Measurement of 3H-Thymidine Incorporation
NIH3T3 cells were serum-starved in DMEM/0.5% FBS for 48 hours
and restimulated with 10-7 mol/L ET-1 or 10%
FBS for 8, 16, and 24 hours. 3H-thymidine (2
µCi/mL, Amersham) was added to each well 2 hours before the end of
the incubation period. Cells were washed twice with ice-cold 1xPBS and
incubated with ice-cold 10% trichloroacetic acid for 30 minutes. After
being washed twice with distilled water, the cells were lysed with 0.2N
NaOH, neutralized with 0.2N HCl, and subjected to liquid scintillation
counting.
Cloning of the Dominant Negative Ras Mutant
Mouse Ras cDNA was isolated by reverse transcription-polymerase
chain reaction. Total RNA was extracted from NIH3T3 cells with TRIzol
LS reagent (Life Technologies) according to the instructions provided
by the manufacturer. One microgram of total RNA was then subjected to
reverse transcription and polymerase chain reaction (PCR) with Ready To
Go/You-Prime First-Strand Beads (Pharmacia Biotech) as instructed by
the manufacturer. The primers used for the RT-PCR are listed below:
Primer for RT: 5'-TCAGGACAGCACACATTTGCA-3' Sense
primer: 5'-GAATTCACAGAATACAAGCTTGTGGTGGT-3' Antisense primer (Ras
reverse): 5'-CTCGAGTCAGGACAGC- ACACATTTGCA-3'
These primers were designed to amplify a segment of the mouse Ras, which corresponded to the second codon up to the stop codon. The PCR conditions were 1 minute at 95°C, 1 minute at 55°C, and 1 minute at 72°C for 35 cycles, with final extension for 10 minutes at 72°C. The PCR-amplified product was digested with EcoRI and XhoI and subcloned in pcDNA3 vector (Invitrogen) in which we designed a hemagglutinin (HA)-epitope tag. The sequence that corresponded to the HA tag was ATGGCTTCTAGCTATCCTTATGACGTGCCTGACTATGCCAGCCTGGGA. To make the dominant negative Ras mutant RasS17N, we used the following primer for the first round PCR. Sense primer: 5'-TGGGAAAGAATGCCCTGACCA-3' Antisense primer: Ras reverse
The PCR-amplified product was subjected to the second round of PCR to make the full-length of the RasS17N mutant with the following primers. Sense primer: 5'-GAATTCACAGAATACAAGCTTGTGGTGGT GGGCGCTGGAGGCGTGGGAAAGAATGCCCTGACCA-3' Antisense primer: Ras reverse
The letters in bold type indicate the nucleotide substitution to make the point mutant. The PCR-amplified product was digested with EcoRI and XhoI and subcloned in the HA-tagged pcDNA3 vector. The nucleotide sequences of the constructs were confirmed by cycle sequencing with an Abi Prism 310 Genetic Analyzer (Perkin-Elmer).
Preparation of Stable Cell Lines Expressing RasS17N in an
Inducible Fashion
We used the Lac repressor system to induce the expression of
RasS17N. The HA-tagged RasS17N was subcloned in the pOPRSVI/MCS vector
(Stratagene) at KpnI and XbaI sites
(pOPRSVI-HA-RasS17N). Two micrograms of pCMVLacI and 10 µg
of pOPRSVI-HA-RasS17N were cotransfected in NIH3T3 cells with
Lipofectamin (Life Technologies) according to the instructions provided
by the manufacturer. The pOPRSVI/MCS vector and pCMVLacI
were also cotransfected in NIH3T3 cells as the negative control.
Positive clones were selected by culture in DMEM/10% FBS that
contained 100 µg/mL hygromycin (Life Technologies) and 200 µg/mL
geneticin (Life Technologies). The expression of RasS17N was induced by
2 mmol/L isopropyl ß-D-thiogalactopyranoside (IPTG) and
confirmed by Western blot analysis with anti-HA antibody
(Boehringer Mannheim).
Statistical Analyses
The values were expressed as the mean±SEM. The effects of ET-1
on cyclins, cdks, cdk inhibitors, kinase activities, and
3H-thymidine uptake were assessed with ANOVA
followed by Student-Neumann-Keul's test. Differences with
P<0.05 were considered statistically significant.
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Results |
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Endothelin-Induced Changes in the Cell-Cycle Regulatory Machinery
We first examined changes in the expression levels of cyclins, cdks, and cdk inhibitors on ET-1 stimulation with Western blot analysis. Cyclin D1 was significantly increased 8 hours after stimulation and remained at high levels until 24 hours after stimulation (5.1±1.9-fold increase, 8 hours after stimulation, P<0.05, Figure 1A
and 1C).
ET-1 increased the level of cyclin D1 in a dose-dependent manner
(Figure 1B). ET-1, at concentrations of
10-8 mol/L and 10-7
mol/L, significantly induced the expression of cyclin D1 compared with
the control. However, the effect of ET-1 was weaker than that of serum
mitogen (Figure 1B). In contrast with cyclin D1, the expression
levels of other cell-cycle regulatory factors, including cyclin E,
cyclin A, cdk2, cdk4, p21waf1/cip1, and
p27kip1 did not change significantly on ET-1
stimulation (Figure 1A), whereas 10% FBS significantly
increased the levels of cyclin A (data not shown). The
immunoblot analysis of cdk2 always showed 2 closely
located bands when NIH3T3 cells were used. We preabsorbed the antibody
with 10 µmol/L of the epitope-encoding peptide used to raise the
antibody and found that both bands disappeared after the preabsorption
test (data not shown). Thus, we concluded that both bands
represented cdk2. It is possible that one of the bands
corresponds to cdk2 undergoing phosphorylation or
glycosylation after translation. The above results suggested that ET-1
might be able to progress early G1 phase. We therefore examined
ET-1–induced changes in cdk4 kinase activity, which we measured with
pRb as the substrate. ET-1 induced cdk4 kinase activity in a
time-dependent manner (Figure 2A). Eight
hours after stimulation, cdk4 kinase activity started to increase,
peaked around 12 hours, and remained increased until 24 hours after the
stimulation (2.8±0.5-fold increase, 12 hours after stimulation,
P<0.01, Figure 2A and 2C). ET-1 also increased cdk4
kinase activity in a dose-dependent fashion (Figure 2B).
However, the effect of ET-1 on cdk4 kinase activity was weaker than
that of serum mitogen (Figure 2A).
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We next examined whether ET-1 could induce cdk2 kinase activity. We
used histone H1 as the substrate to measure cdk2 activity. ET-1 also
induced cdk2 kinase activity in a time-dependent manner. Twelve hours
after stimulation, cdk2 kinase activity started to increase, reached
maximal level around 16 hours, and returned to the basal level 24 hours
after stimulation (2.1±0.4-fold increase, 16 hours after stimulation,
P<0.05, Figure 3A and 3C).
The peak appeared later than that of cdk4 kinase activity, which was
compatible with previous findings that cyclin D–associated cdk4 or
cdk6 kinase activity is required earlier than cdk2 kinase activity for
normal cell-cycle progression.30 ET-1 induced cdk2
kinase activity in a dose-dependent manner (Figure 3B). However,
the effect of ET-1 on cdk2 kinase activity was weaker than that of
serum mitogen (Figure 3A).
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Previous reports have suggested that ET-1 induced ERK1
activity.7 8 10 We therefore examined whether ET-1 induced
ERK1 kinase activity in NIH3T3 cells and compared its effect with that
of angiotensin II (ATII) and serum mitogen. We used MBP as
the substrate to measure ERK1 activity. ET-1 induced ERK1 kinase
activity in a time-dependent manner. ET-1–induced ERK1 kinase activity
peaked around 15 minutes after stimulation (4.7±0.7-fold,
P<0.01, Figure 4A and 4C) and
remained at a high level 30 minutes after the stimulation. However,
ERK1 kinase activity returned to the basal level within 1 hour. ET-1
induced ERK1 kinase activity almost to the same extent observed with
ATII and serum mitogen when their effects at 15 minutes after
stimulation were compared (Figure 4B), although serum mitogen
tended to have stronger effect on ERK1 kinase activity. Thus, ET-1
seemed to possess almost the same capacity as serum mitogen to
activate ERK pathway, although ET-1–induced ERK1 activation
did not last for >1 hour.
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Effects of MEK1, PKC, and PI-3-K Inhibitors on
ET-1–Induced Changes in the Cell-Cycle Regulatory Machinery
Previous reports suggested that serum mitogen–induced cell-cycle
progression is, at least in part, mediated by the Ras signaling
pathway.17 18 To study the effect of this pathway on
ET-1–induced cell-cycle progression, we established stable cell lines
that expressed a dominant negative Ras mutant (RasS17N) in an
IPTG-inducible manner. We examined RasS17N expression in 4 independent
clones with anti-HA antibodies. Three of the clones expressed a
small amount of RasS17N in the basal stage, which was induced 3- to
4-fold by 2 mmol/L IPTG in 8 hours. When IPTG was added
simultaneously, ET-1–induced cdk4 and cdk2 kinase
activities were inhibited to the basal level in the clones 12 hours and
16 hours after stimulations, respectively, whereas ET-1–induced cdk4
and cdk2 kinase activities were not inhibited by IPTG treatment in
the control clones transformed with the vector alone (data not
shown), which was compatible with the results of previous reports.
However, we observed that the inhibition by RasS17N of ET-1–induced
activation of cdk4 and cdk2 kinases occurred even in the absence of
IPTG (noninduced stage), probably because of leakage of the promoter
activity in the basal condition, which resulted in RasS17N expression
in the noninduced stage. Therefore, to confirm that the Ras-dependent
pathway was involved in ET-1–induced cell-cycle progression and to
study more specifically the roles of ERK-, PKC- and PI-3-K-dependent
pathways on ET-1–induced cell-cycle progression, we used the MEK1/2
inhibitor PD98059, the PKC inhibitor calphostin
C, and the PI-3-K inhibitor LY294002. Neither PD98059 nor
calphostin C, alone or in combination, had significant effects on
ET-1–induced upregulation of cyclin D1 and cdk4 kinase activity
(Figure 5A and 5B), although the
combination of PD98059 and calphostin C tended to inhibit ET-1-induced
cyclin D1 expression and cdk4 kinase activity. We used up to 100
µmol/L of PD98059 and 200 ng/mL of calphostin C to study their
effects. However, neither the ET-1–induced increase in cyclin D1
expression nor the ET-1–induced increase in cdk4 kinase activity was
significantly inhibited by them. In marked contrast, PD98059 and/or
calphostin C significantly inhibited ET-1–induced activation of cdk2
kinase (Figure 5C). In contrast to the effects of PD98059 and
calphostin C, LY294002 significantly inhibited ET-1–induced cyclin D1
expression and cdk4 kinase activity (Figure 6A and 6B). The ET-1–induced cyclin D1
expression and cdk4 kinase activity were inhibited by 73±20%
(P<0.05) and by 72±15% (P<0.05) in the
presence of 50 µmol/L LY294002, respectively. LY294002 also
significantly inhibited ET-1–induced cdk2 kinase activity to the basal
level (Figure 6C). To confirm that PD98059 indeed inhibited
ET-1–induced activation of ERK1/2 kinase, we measured ERK1 kinase
activity in the presence and the absence of PD98059. ET-1–induced ERK1
kinase activity was indeed inhibited by PD98059 pretreatment when MBP
was used as the substrate (Figure 7A, left panels). We also used the antibody that recognizes only
phosphorylated, catalytically active ERK1/2.
ET-1–induced phosphorylation of ERK1/2 was completely
blocked by PD98059 (Figure 7B, left panels). We therefore
concluded that PD98059 inhibited ET-1–induced activation of ERK1/2 in
our system. It is well-known that PKC activates ERK in some
cases.26 27 Thus, we examined whether PKC was
involved in ET-1–induced ERK activation. ET-1–induced ERK1/2
activation was significantly inhibited by calphostin C when ERK1/2
activity was assessed by its capacity to phosphorylate MBP
(Figure 7A, right panels). ET-1–induced
phosphorylation of ERK1/2 was also significantly
inhibited by calphostin C (Figure 7B, right panels).
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Effects of the MEK1, PKC, and PI-3-K Inhibitors on
ET-1–Induced Entry Into the S Phase
The results described above suggested that ET-1 induced
activation of the cell-cycle regulatory machinery and that
ET-1–induced activation depended on the ERK-, PKC-, and
PI-3-K-mediated pathways. To further study the mechanisms of action of
ET-1, we examined whether ET-1–induced S-phase entry depended on the
ERK-, PKC-, and PI-3-K–mediated pathways by measuring
3H-thymidine incorporation. As shown in Figure 8A, ET-1 increased
3H-thymidine incorporation in a time-dependent
manner. ET-1–induced 3H-thymidine uptake started
to increase around 16 hours after stimulation and remained at high
levels until 24 hours after the stimulation, although the increase was
not so striking. Thus, we examined the effects of PD98059, calphostin
C, and LY294002 on ET-1–induced 3H-thymidine
uptake 16 hours after stimulation (Figure 8B). ET-1 induced a
significant increase in 3H-thymidine uptake, and
this increase was significantly inhibited by PD98059, calphostin C, and
LY294002. These reagents did not significantly affect the basal
3H-thymidine uptake (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we have demonstrated that ET-1 induced cyclin D1 protein expression, cdk4 kinase activity, and cdk2 kinase activity in time- and dose-dependent manners. Although the molecular mechanisms of cell-cycle regulation have been intensively studied, serum mitogen was the major mitogen used to induce cell-cycle progression in those studies. Other mitogens, including thrombin, insulin, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor (PDGF), were also used to induce cell-cycle progression. However, little is known about the molecular mechanisms of cell-cycle progression mediated by heterotrimeric G-protein–coupled receptors. A recent study demonstrated that ATII induced cyclin D1 protein expression and cyclin D1 promoter activity and that the ATII-induced increase in cyclin D1 promoter activity depended on Ras and ERK, with dominant negative mutants of Ras and ERK, although the effect of the mutants on cyclin D1 expression at protein level was not examined in that study.31 However, little is known about the molecular mechanisms of ET-1–induced cell-cycle progression. Although ET-1 induced cyclin D1 protein expression, we did not observe significant upregulation of cyclin A or downregulation of p27kip1, which was reportedly observed when serum mitogen or epidermal growth factor was used to induce progression of the cell cycle.17 18 It is possible that serum mitogens and receptor tyrosine kinase-mediated signaling pathways follow other pathways to affect cell-cycle progression.
Our results showed that ET-1–induced upregulation of cyclin D1 and cdk4 kinase activity was not inhibited by the MEK1/2 inhibitor PD98059 or the PKC inhibitor calphostin C, whereas ET-1–induced activation of cdk2 kinase activity was inhibited by PD98059 and/or calphostin C. These results demonstrate that ET-1–induced activation of the ERK and PKC pathways may be involved in the progression of late G1/S phase. Several reports have suggested that mitogen-induced cyclin D1 upregulation depended on the ERK pathway.32 33 Although we do not know the reason for the difference, it is noteworthy that in several reports potent mitogens, such as serum mitogen, PDGF and thrombin, induced sustained activation of ERK for more than 9 hours.32 33 Weber et al32 showed that inhibition of the sustained phase, but not of the initial phase of ERK activation, was sufficient to inhibit cell-cycle progression. In contrast, ET-1–induced activation of ERK lasted for a shorter period (<1 hour) in this study and others.8 10 Foschi et al8 demonstrated that ET-1–induced ERK activation in turn phosphorylated SosI, which deactivated Ras and ERK. It is thus possible that ERK-mediated activation of cyclin D1 and cdk4 kinase activity requires sustained activation of ERK and that other signaling pathways may be used for the activation of cyclin D1 and cdk4 kinase unless sustained ERK activation is ensured. Thus, it is plausible to speculate that ERK1/2 is not the only pathway for inducing cyclin D1 expression and cdk4 kinase activity and that signaling molecules found further upstream, such as Ras, may follow an alternate pathway to induce cyclin D1 and cdk4 kinase activity. We therefore examined whether PI-3-K–mediated pathways, which locate downstream of Ras, were involved in ET-1–induced upregulation of cyclin D1 and cdk4 activity. Surprisingly, LY294002 significantly inhibited ET-1–induced cyclin D1 expression and cdk4 kinase activity. LY294002 also significantly inhibited ET-1–induced cdk2 kinase activity, although it is not clear whether the suppression of cdk2 kinase activity was a direct effect of PI-3-K inhibition by LY294002 or was mediated by the suppression of cdk4 kinase activity. PI-3-K–mediated pathways seem to play roles in glycogen synthesis, antiapoptotic actions, and cell growth.34 35 It is reported that a retrovirus-encoded PI-3-K could transform fibroblasts.28 However, little is known of the mechanisms by which PI-3-K–mediated pathways are linked to the cell-cycle regulatory machinery, although Akt/PKB, some PKC isoforms, Rac, and p70S6K are downstream candidate targets of PI-3-K.36 37 38 39 Future studies, especially those with a dominant-negative PI-3-K mutant, will elucidate the role of PI-3-K–mediated pathways in ET-1–induced cell-cycle progression.
Several reports have suggested that PKC activation is involved in cell-cycle progression or cell-cycle arrest, which depends on the timing of PKC activation during the cell cycle, PKC isozymes, and cell type.22 23 24 25 Several PKC isozymes seem to play roles in the progression of the cell cycle from G1 to the S phase or from G2 to the M phase.23 25 On the other hand, PKC-induced cell-cycle arrest correlates with the upregulation of p21waf1/cip1 and p27kip1 or downregulation of cdk activating kinase.22 24 Furthermore, some PKC isozymes can activate ERK in Ras-dependent or Ras-independent manners,26 27 which in turn modulate the cell-cycle regulatory machinery. Collectively, PKC-mediated pathways seem to be involved in the modulation of the cell-cycle regulatory machinery at multiple points. Our results suggested that ET-1–induced activation of PKC might be involved in modulating cdk2 kinase activity rather than cdk4 kinase activity, although we did not directly examine whether calphostin C inhibited PKC activity in our system. Previous reports demonstrated that the IC50 of calphostin C was 50 nmol/L and that the dosage was sufficient to inhibit PKC-mediated signaling pathways.40 We used up to 200 ng/mL (254 nmol/L) of calphostin C in our experiments and did not observe any effects of calphostin C on ET-1–induced upregulation of cyclin D1 or cdk4 kinase activity. Thus, it is reasonable to speculate that a major target of ET-1–induced PKC activation in cell-cycle progression may be cdk2 rather than cdk4 and that PKC may be involved in the regulation of late G1/S-phase progression. It is noteworthy that calphostin C inhibited ET-1–induced ERK activation in our system. Thus, it is possible that ET-1–induced PKC activation might modulate the cell-cycle regulatory machinery partly by activating ERK.
ET-1–induced increase in 3H-thymidine uptake was
completely inhibited by PD98059, calphostin C, and LY294002, which
suggested that ET-1–induced S-phase entry was, at least in part,
mediated by ERK1/2, PKC, and PI-3-K. However, ET-1–induced
3H-thymidine incorporation was not so potent. It
was not simply because ET-1 did not fully activate
intracellular signaling pathways in NIH3T3 cells, because ET-1 induced
ERK1 activation almost to the same extent as that observed with serum
mitogen (Figure 4
). Furthermore, previous reports suggested that
the 3T3 fibroblasts expressed sufficient amounts of ET-1
receptors.41 42 Although we do not know the mechanisms, it
is possible that a sustained ERK activity might be required for
adequate progression of G1 phase as discussed above or that serum
mitogen and potent mitogens such as PDGF follow additional pathways to
fully activate the cell-cycle regulatory machinery. While we
were preparing this article for publication, Pedram et
al43 reported that ET-3 induced the biosynthesis of cyclin
D1 as well as cyclin E and cyclin A in astrocytes. In some ways their
results seem different from ours; they measured the transcript level
and newly synthesized protein level, whereas we measured the level of
the total amount of protein by Western blot analysis. It is
possible that the discrepancy may be due to the difference in the
methodology or cell type.
In summary, ET-1 induced upregulation of cyclin D1 protein, cdk4 kinase activity, and cdk2 kinase activity. ET-1–induced activation of cdk2 depended, at least in part, on the ERK-, PKC-, and PI-3-K–mediated pathways, whereas ET-1–induced increase in cyclin D1 protein and cdk4 kinase activity depended partly on PI-3-K–mediated pathways, which suggested that these pathways may be involved in the progression of the cell cycle at distinct points in ET-1–stimulated NIH3T3 cells. Additional studies are required to elucidate the mechanisms by which PI-3-K–mediated pathways are linked to the cell-cycle regulatory machinery.
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Acknowledgments |
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This work was supported in part by grants-in-aid 07557055 and 09281206 from the Ministry of Education, Culture and Science of Japan, awarded to Dr Yasunobu Hirata. We also thank Etsuko Taira and Marie Morita for their technical assistance.
Received October 27, 1998; accepted January 6, 1999.
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