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INTRODUCTION |
The octapeptide angiotensin II (Ang
II)1 binds to specific high
affinity receptors present in the adrenal cortex, liver epithelial cells, and vascular smooth muscle cells (VSMC), where it elicits a vast
array of biological effects (1). In VSMC, Ang II has been shown to
stimulate proliferative and hypertrophic growth via binding to type 1 receptor (AT1-R), a seven-transmembrane-spanning receptor
coupled to the Gq/11 subtype of heterotrimeric G
proteins (2). Ang II initiates early biochemical events including rapid production of diacylglycerol (an activator of protein kinase C) and
inositol 1,4,5-triphosphate (that induces release of Ca2+
from the sarcoplasmic reticulum) by phospholipase C-mediated hydrolysis
of inositol phospholipids (3-8) and activation of the
mitogen-activated protein kinase/extracellular signal-regulated kinase
(MAPK/ERK) family members ERK1 and ERK2 (9, 10) and the c-Jun
N-terminal kinase (11), which are known to influence c-Jun and
c-Fos transcription. Indeed, some of the intracellular signals mediated
by the AT1 receptor are similar to those activated by
mitogens such as platelet-derived growth factor (PDGF) and epidermal
growth factor (EGF), leading to induction of transcription of several
immediately early growth response genes such as c-fos, c-jun, and c-myc (12-15).
Phosphorylation and dephosphorylation of proteins on tyrosine residues
are now well recognized as important mechanisms for transmitting
extracellular stimuli in cellular events such as cell attachment,
proliferation, differentiation, and migration (16). The phosphorylation
level of the molecules within signaling pathways are regulated by the
activity of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs).
An increase in protein tyrosine phosphorylation has been attributed in
part to the activation of Src (17, 18), the Jak family proteins Jak2
and Tyk2 (10, 19), Pyk-2/CADTK (20), and focal adhesion kinase
pp125FAK (21). A decrease in the level of tyrosine
phosphorylation of cellular proteins is finely controlled by the
activity of PTPs, some containing Src homology-2 (SH2) domains (22).
Two of the SH2 domain-containing PTPs that have been studied are SHP-1
(also known as PTP1C, SHPTP1, HCP, and SHP), which is expressed
predominantly in hematopoietic cells (23-26), and SHP-2 (PTP1D,
SHPTP2, SHPTP3, Syp, or PTP2C), an ubiquitously expressed cytosolic
protein (27). The latter enzyme is known to associate with activated
growth factor receptors such as PDGF receptor (28), EGFR (29),
fibroblast growth factor receptor (30), and insulin receptor (31); with cytokine receptors (32, 33) and adapter molecules with pleckstrin homology domains such as insulin receptor substrate 1 (34) and Grb2-associated binder 1 (35); and with other transmembrane proteins
such as signal regulatory protein (SHPS-1/SIRP/BIT) (36, 37), platelet
endothelial cell adhesion molecule 1 (38), and myelin
P0-related protein PZR (39). Numerous studies suggest that
SHP-2 has a positive role in signaling in mammalian cells. Several
strategies were used to examine the function of SHP-2 in activated
receptor complexes. Microinjection of anti-SHP-2 antibodies or
recombinant fusion protein encoding the SH2 domains of SHP-2 block
insulin, insulin-like growth factor, and EGF-stimulated DNA synthesis
(40). Overexpression of a catalytically inactive cysteine to serine
mutant of SHP-2 (SHP-2 CS) inhibits the activation of MAPK/ERK induced
by insulin (41, 42) or EGF (43, 44) and early gene transcription
and DNA synthesis induced by 
-thrombin (45) and the
proteinase-activated receptor-2 (46). In addition, recent papers report
that SHP-2 plays an important role in the control of cytoskeletal
organization, cell spreading, and migration (47, 48).
The D-type cyclins (cyclin D1, D2, and D3) are thought to be key
regulatory components for progression through G1 phase and for the commitment of mammalian cells to enter S phase and to replicate
their DNA. Cyclin D1 and its catalytic protein partners, the
cyclin-dependent kinases (CDKs), are induced as part of the delayed early response to mitogenic stimulation (49-51). Cyclin D1
preferentially associates with either CDK4 or CDK6, an association that
is prevented by the interaction of CDKs with their inhibitors (52).
Once activated, cyclin D1-CDK4 and/or cyclin D1-CDK6 complexes phosphorylate the 110-kDa retinoblastoma tumor suppressor gene product
(pRb) (53), resulting in the release of E2F transcription factor and
allowing gene transcription required for the progression of the cell
cycle into S phase and DNA synthesis (54).
Early studies demonstrated that p21ras (Ras) induces DNA
synthesis in the nucleus of quiescent cells and that overexpression of
activated Ras is associated with the increased expression of cyclin D1
in NIH 3T3 cells (55) and with the positive regulation of the cyclin D1
promoter in human trophoblasts (JEG-3), in the mink lung epithelial
cell line (Mv1.Lu), and in Chinese hamster ovary fibroblasts (56).
These results suggest that the Ras signaling pathway is directly linked
to the G1/S phase transition of the cell cycle. Indeed,
p21ras activates c-Jun, and members of the c-Jun/AP-1 family
are involved in promoting cellular proliferation (57) and cyclin D1
promoter activity (58), although other transcription factors such as the members of the ETS family proteins participate in this regulation (56).
Cyclin D1 activation by Ras has been attributed to the sequential
activation of Raf kinase, MEK, and MAPK/ERK (59, 60), but recent
findings also reveal that other effectors might contribute to
G1/S phase cell cycle progression (61).
Among the various Ras effectors that interact with the cell cycle
machinery, phosphatidylinositol 3-kinase (PI3K), a heterodimeric protein composed of 85- and 110-kDa subunits that catalyzes the synthesis of 3-phosphorylated phosphoinositides, appears to be a key
intermediate in receptor-stimulated mitogenesis (62, 63). PI3K is
indispensable for G1 to S phase cell cycle progression in
response to a variety of growth factors. Inhibitors of PI3K (LY294002
and wortmannin) have been shown to inhibit S phase entry in a variety
of cell types. In CHO cells, selective activation of PI3K to
physiologically relevant levels was sufficient to stimulate DNA
synthesis and required both p70 S6 kinase and the p21ras/MEK
pathway (64). Inhibitors of PI3K have been shown to block MAPK/ERK
activation by some stimuli, such as insulin or insulin-like growth
factor-1 (65) and lysophosphatidic acid (66) but not others such as EGF
(66) or PDGF (67). Recent studies demonstrate that PI3K is activated in
VSMC after treatment with Ang II and that its activity is crucial for
cell growth (68).
In this study, we investigated the signaling pathway linking the
Ang II receptor to cyclin D1 protein expression and promoter activity
in CHO-AT1A cells. We found that both PI3K and MAPK/ERK are
important components of the mitogenic signal, since they are required
for Ang II-induced S phase cell cycle progression, cyclin D1 protein
expression, and promoter activity. In addition, we show that the
catalytic activity as well as the SH2 domains of SHP-2 and class
IA PI3K are required for Ang II to stimulate cyclin D1
promoter activity, through a pathway that is dependent on MAPK/ERK activation. Although ligand-independent tyrosine phosphorylation (transactivation) of RTKs, such as the EGFR has been suggested to
represent an essential event for MAPK/ERK activation by both Gi- and Gq-coupled receptors (69), we present
evidence that the pathways leading to cyclin D1 promoter activation by
Ang II are independent of EGFR transactivation in CHO-AT1A cells.
This is to our knowledge the first demonstration of the
protein-tyrosine phosphatase SHP-2 acting downstream of a G
protein-coupled receptor triggered to induce cyclin D1 expression
involved in the control of cell cycle progression.
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EXPERIMENTAL PROCEDURES |
Materials--
Acrylamide, salts, and other electrophoresis
materials were purchased from ICN Pharmaceuticals, Inc. (Costa Mesa,
CA) unless otherwise specified. PD098059, PD158780, LY294002, and
wortmannin were obtained from Calbiochem. [
-32P]ATP
was purchased from NEN Life Science Products. Prestained protein
molecular mass markers were from Bio-Rad. Sodium vanadate was from
Fisher. Protein A/G PLUS-agarose was from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). LipofectAMINE-PLUS reagent was from Life
Technologies, Inc. Ang II was purchased from Sigma. Monoclonal
anti-phosphotyrosine antibodies were obtained from Transduction
Laboratories (Lexington, KY) and Upstate Biotechnology, Inc. (Lake
Placid, NY). Anti-SHP-2 antibodies were purchased from Santa Cruz
Biotechnology, Upstate Biotechnology, and Transduction Laboratories.
Monoclonal anti-hemagglutinin (HA) antibody 12CA5 was from Roche
Molecular Biochemicals; monoclonal anti-cyclin D1 antibody (HD11) was
from Santa Cruz Biotechnology; polyclonal anti-p44/p42 ERK1/2 antibody
was from New England Biolabs; and monoclonal phosphospecific MAPK
antibody, which detects only the catalytically active forms of p42/p44
ERK1/ERK2 phosphorylated on Tyr204, was from Santa Cruz
Biotechnology. Rabbit anti-mouse IgG-peroxidase conjugate and goat
anti-rabbit IgG-peroxidase conjugate were from BioSys (Compiègne,
France). Myelin basic protein (MBP) and EGF were from Upstate Biotechnology.
cDNA Plasmids--
HA-tagged p44-ERK1 was a kind gift from
Dr. J. Pouysségur (45). Dr. R. Müller provided the 
973
base pair human cyclin D1 promoter fragment linked to a luciferase
reporter gene (973 CD1/LUC). The different SHP-2 and SHP-1 constructs
were obtained and used as described (44, 70). The dominant negative Ras N17 and constitutive active Ras K12 mutants were provided by Dr. F. Schweighoffer. Constitutive active Raf-1 BXB and dominant negative Raf-1 C4 mutants were gifts from Dr. Z. Luo (71). The plasmid 
p85
(a deletion mutant of the regulatory subunit of PI3K lacking 102 amino
acids from residue 466 to 567 of the inter-SH2 domain that confers
binding to the catalytic subunit p110) was kindly provided by Dr. A. Eder (72). All cDNA plasmids was prepared using the CONCERT high
purity plasmid purification system from Life Technologies.
Cell Culture--
Parental CHO-K1 cells and CHO-AT1A
cells stably expressing a rat vascular AT1A receptor (73)
were maintained in Ham's F-12 medium containing 10% fetal calf serum,
100 µg/ml streptomycin, 100 units/ml penicillin, and 2 mM
L-glutamine. CHO-AT1A cells were supplemented
with 750 µg/ml Geneticin (G418). Cells in 100-mm dishes were grown to
75-85% confluence, washed once with serum-free Ham's F-12 medium,
and growth-arrested in serum-free Ham's F-12 medium supplemented with
0.5 mg/ml bovine serum albumin for 48 h prior to use.
Cell Cycle Analysis--
Cells (0.3 × 106) in
six-well culture dishes were maintained in serum-free medium as
described above, treated with Ang II (10
7
M) or FCS (10%), and collected at various time points
(0-44 h). Then cells were centrifuged at 1000 rpm for 5 min. The
pellet was resuspended in 200 µl of PBS, fixed in 2 ml of ice-cold
70% ethanol, and stored at 4 °C. Cells were centrifuged at 1000 rpm for 5 min and resuspended in 400 µl of PBS containing RNase (100 µg) and propidium iodide (12 µg) for 30 min at 37 °C. The
samples were analyzed by a fluorescence-activated cell sorter
(ELITE, Beckman Coulter).
Measurement of DNA Synthesis--
Incorporation of the thymidine
analogue 5-bromo-2'-deoxyuridine (BrdUrd) was measured to
determine the effect of Ang II on DNA synthesis. CHO-K1 or
CHO-AT1A cells were placed in 96-well cell culture plates
at a concentration of 10,000 cells/well. Twenty-four hours later, cells
were growth-arrested in serum-free Ham's F-12 medium (100 µl/well)
for 72 h before adding either 107
M Ang II, 100 ng/ml EGF, or 10% fetal calf serum. After
18 h (FCS) and 42 h (Ang II and EGF), 10 µM
BrdUrd was added to each well, and cells were incubated for an
additional 6 h and then fixed. When inhibitors were tested, they
were preincubated for 1 h in serum-free Ham's F-12 before adding
Ang II. Quantification of BrdUrd incorporation was performed using a
commercially available detection kit (Roche Molecular Biochemicals).
Transient Transfection and Luciferase Assay--
The 973 base
pair human cyclin D1 promoter fragment linked to the luciferase
reporter gene (973 CD1/LUC) was transiently transfected in CHO-K1 or
CHO-AT1A cells (640 ng/3.106 cells in 100-mm
dishes) together with different amounts of relevant expression vectors
(i.e. 2.5 µg of Ras N17 mutant, 2.5 µg of Raf-1 C4
mutant, 2.5 µg of p85 subunit, 5 µg of SHP-2 CS mutant, 5 µg of the SH2 domains of SHP-2, 5 µg of SHP-1 CS mutant or the corresponding empty vector), using LipofectAMINE-PLUS reagent following
the protocol provided by the supplier. Twenty-four hours following
transfection, cells were trypsinized and aliquoted into 12-well cell
culture dishes. Six hours later, cells were serum-starved for 24 h
in Ham's F-12 medium supplemented with 0.5 mg/ml bovine serum albumin
before preincubation with or without inhibitors for 1 h and then
stimulated for the indicated times with Ang II at the concentrations
indicated. Cells were lysed with 200 µl of passive lysis buffer
(1×), and luciferase activity was assayed in a luminometer (Lumat
LB9507 Berthold) using the kit from Promega (Madison, WI). Relative
luciferase activity is expressed as the ratio of Ang II-stimulated to
unstimulated samples.
Assay of Transfected HA-tagged p44-ERK1--
The activity of
transiently transfected HA-tagged p44-ERK1 was determined by MBP
kinase assay as described (45). HA-tagged p44-ERK1 was transiently
transfected in CHO-K1 or CHO-AT1A cells (0.5 µg/106 growing cells in 60-mm dishes) together with
different amounts of relevant expression vectors (2.7 µg of Ras N17,
Ras K12, Raf1-C4, Raf1-BXB, SHP-2 CS, and SH2 domains of SHP-2 or the
corresponding empty vector). Twenty-four hours following transfection,
cells were serum-starved for 24 h in Ham's F-12 medium
supplemented with 0.5 mg/ml bovine serum albumin before preincubation
with or without inhibitors for 1 h and treatment with
107 M Ang II for the indicated
times. When inhibitors were used, they were pretreated 1 h before
Ang II stimulation. The reactions were terminated by aspirating the
medium, washing the cells with ice-cold PBS (10 mM
Na2HPO4, 1.7 mM
KH2PO4, 136 mM NaCl, 2.6 mM KCl, 2 mM Na3VO4, pH
7.4), and freezing the cells immediately with liquid nitrogen. Each
dish was then treated with 300 µl of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM
Na3VO4, 50 mM NaF, 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml antipain) and placed
on ice for 20 min. The thawed cells were harvested by scraping,
sonicated for 60 s, and then centrifuged at 14,000 × g for 15 min at 4 °C. Protein concentrations were measured with the bicinchoninic acid assay from Pierce. 2 µg of monoclonal anti-HA antibody 12CA5 was added to equal amounts of protein
per sample and allowed to equilibrate overnight at 4 °C. Protein A/G
PLUS-agarose (20 µl) was then added for an additional 2 h.
Immunoprecipitates were washed three times in 1 ml of ice-cold lysis
buffer, once in 1 ml of reaction buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 10 mM MgCl2), and
incubated in 30 µl of reaction buffer containing 25 µM
ATP, 2.5 µCi of [-32P]ATP, and 20 µg of MBP for 30 min at 30 °C. The products of the reaction were resuspended in
SDS-sample buffer, boiled for 5 min at 95 °C, separated on SDS-PAGE
(15% gels), and transferred to nitrocellulose membrane. Blots were
dried, and incorporation of 32P into MBP was analyzed by
exposing to x-ray film and then subjected to quantitation on a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). After
autoradiography, the blots were probed with an anti-HA antibody to
ensure that an equal amount of HA-p44-ERK1 had been immunoprecipitated
in each well.
Western Blotting--
CHO-K1 and CHO-AT1A cells in
100-mm dishes were growth-arrested for 48 h in serum-free Ham's
F-12 and either left untreated or stimulated with
107 M Ang II for the indicated
times. Whole cell extracts and SHP-2 immunoprecipitates were prepared
in ice-cold lysis buffer as described above, resuspended in SDS-sample
buffer, and boiled for 5 min at 95 °C. Reaction mixtures were size
fractionated by SDS-PAGE (8% gels) and transferred to nitrocellulose
membranes. The blot was stained with Ponceau S to confirm equal loading
of proteins and then probed with the indicated antibodies. When cyclin
D1 protein expression was studied, nuclear extracts were prepared according to the method of Andrews and Faller (74), and equal amounts
of proteins were loaded on 10% gels. Immunoblots were developed using
appropriate secondary horseradish peroxidase-coupled antibodies and an
enhanced chemiluminescence (ECL) kit (Pierce).
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RESULTS |
Ang II Stimulates G1 Phase
Progression--
CHO-AT1A cells have been successfully
used to investigate the action of Ang II on cell growth and division
(73), compared with more physiological models such as VSMC or adrenal
cells in culture. To determine whether Ang II stimulates cell cycle
progression in CHO-AT1A cells, flow cytometric analysis was
performed. Cells were treated with 107
M Ang II for 16-44 h, and the proportion of cells in
G0-G1, S, and G2-M was determined
at various time points as indicated in Table
I. The relative proportion of cells in
G0-G1 was reduced from 80.5 to 59.4% at
28 h, concomitant with an increase in cells in S phase (from 5.9 to 29.5%). The proportion of cells in G2-M increased from
13.2 to 27.5% at 44 h. These results indicate that Ang II
decreases the proportion of cells in G1 and increases the proportion of cells in S phase and G2-M and that the effect
persists up to 44 h.
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Table I
Ang II (107 M) and FCS (10%)-treated
CHO-AT1A cells
The percentage of cells within each phase of the cell cycle is shown.
Values are representative of one experiment out of two with identical
results.
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For comparison, cells were also treated with 10% FCS for 12-24 h. The
proportion of cells in S phase increased 68% after 16 h,
concomitant with a decrease in the number of cells in
G0-G1 phase (from 80.5 to 12.9%; Table I). The
proportion of cells in G2-M increased from 13.2 to 48.3 at
20 h.
Ang II-induced DNA Synthesis Is Blocked by both Inhibitors of MEK
and PI3K in CHO-AT1A Cells--
Ang II-stimulated
incorporation of BrdUrd into DNA was examined in quiescent
CHO-AT1A cells and compared with parental CHO-K1 cells,
which do not express AT1A receptors. Whereas
107 M Ang II was unable to
stimulate BrdUrd incorporation in CHO-K1 cells, a 3.5-fold maximal
stimulation was observed for CHO-AT1A cells (Fig.
1). As a control, 10% FCS stimulated
5-fold the incorporation of BrdUrd into CHO-K1 cells. There was
no difference in the kinetics of FCS-dependent BrdUrd
incorporation in CHO-AT1A cells compared with CHO-K1 cells
(data not shown).
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Fig. 1.
Ang II-induced DNA synthesis is blocked by
both inhibitors of MEK and PI3K in CHO-AT1A cells.
Serum-starved cells were stimulated with 107
M Ang II or 100 ng/ml EGF for 48 h and with 10% FCS
for 24 h. Incorporation of BrdUrd was measured using a detection
kit as described under "Experimental Procedures." PD098059 and
LY294002 were preincubated at the indicated concentrations 1 h
before adding Ang II. The results are expressed as the percentage of
stimulation over the basal value established as 100%. Each value is
the mean ± S.E. (bars) from six wells in three
independent experiments.
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A time course of BrdUrd incorporation into DNA was also analyzed after
incubation for various periods of time with EGF (data not shown). Under
these experimental conditions, 100 ng/ml EGF was unable to stimulate
BrdUrd incorporation in CHO-AT1A cells up to 48 h
(Fig. 1).
In order to investigate the pathways connecting Ang II to cell cycle
progression into S phase, we measured the effect of LY294002, an
inhibitor of PI3K, and PD098059, an inhibitor of MEK (the dual specificity kinase that activates p44/p42 MAPK/ERK by phosphorylation), on Ang II-induced BrdUrd incorporation in quiescent
CHO-AT1A cells. Under these conditions, both inhibitors
suppressed BrdUrd incorporation in a concentration-dependent
manner. These results indicate that the proliferation of
CHO-AT1A cells in response to Ang II is dependent on
MAPK/ERK activation and suggest an involvement of PI3K.
Ang II-induced Endogenous Cyclin D1 Protein Expression Is Dependent
upon PI3K and MEK Activities--
Induction of cyclin D1 protein
expression represents one of the earliest effects of growth
factors leading to cell cycle reentry, G1 phase
progression, and the commitment of cells to enter S phase. In order to
examine whether Ang II is able to induce expression of cyclin D1
protein, quiescent CHO-AT1A cells were treated with 107 M Ang II and harvested at
different times. The level of cyclin D1 protein was analyzed by
immunoblotting nuclear extracts with a monoclonal anti-cyclin D1
antibody (HD11). In unstimulated quiescent cells, cyclin D1 protein
expression was low, barely detectable (Fig.
2A). Ang II stimulation led to
an increase of cyclin D1 levels detectable at 16 h poststimulation
and continued to increase until 32 h before declining by 48 h.
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Fig. 2.
Ang II-induced endogenous cyclin D1 protein
expression is dependent upon PI3K and MEK activities in
CHO-AT1A cells. A, serum-starved
CHO-AT1A cells were treated for the indicated times with
107 M Ang II and lysed, and
nuclear extracts were prepared as described under "Experimental
Procedures." B, serum-starved CHO-AT1A cells
were preincubated 1 h with PI3K inhibitor (LY294002 or wortmannin)
or MEK inhibitor (PD098059) at the concentrations indicated, and Ang II
was incubated for an additional 32 h. Cells were lysed, and
nuclear extracts were prepared. Equal amounts of protein were run on
SDS-PAGE (10% gels), transferred onto nitrocellulose membranes, and
hybridized with a monoclonal anti-cyclin D1 antibody (HD11). The blot
was then revealed using a secondary goat anti-mouse horseradish
peroxidase-conjugated antibody before final development with the ECL
system. The Western blots are representative of three independent
experiments.
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To investigate the role of MAPK/ERK and PI3K in this induction, we
tested the effects of PD098059, LY294002, and wortmannin. As shown in
Fig. 2B, PD098059 and LY294002 severely inhibited Ang
II-induced cyclin D1 protein expression in a
concentration-dependent manner. Wortmannin was less
effective probably because the drug is more labile in living cells
(75). These results indicate that PI3K and MAPK/ERK are both required
for up-regulation of cyclin D1 protein expression in response to Ang II
in CHO-AT1A cells.
Ang II Stimulated Cyclin D1 Promoter Activity in a Dose- and
Time-dependent Manner--
To determine whether Ang II was
capable of inducing cyclin D1 promoter activity, we used the 973 base
pair human cyclin D1 promoter fragment linked to the luciferase
reporter gene (973 CD1/LUC). Quiescent CHO-AT1A cells
transiently transfected with 973 CD1/LUC were treated with Ang II at
different concentrations (dose-response; Fig.
3A) and for different times (time course; Fig. 3B) as described. Ang II stimulated cyclin D1
promoter/reporter gene in a concentration-dependent manner,
reaching a maximal increase (5-6-fold above basal level) at
107 M and
106 M for a 24-h stimulation
(Fig. 3A). The specificity of this effect was tested using
CHO-K1 cells in which Ang II at the highest concentration (106 M) was unable to stimulate
cyclin D1 promoter/reporter gene activity (data not shown). The time
course of Ang II-stimulated cyclin D1 promoter/reporter gene activity
was also determined (Fig. 3B) and showed an increase that
was significantly different from control at 8 h (1.8-fold),
reaching a maximum at 32 h (6-7-fold) and remaining at this level
at 48 h. The kinetic in cyclin D1 promoter/reporter gene activity
induced by Ang II was exactly correlated with the increase in cyclin D1
protein expression until 32 h as shown in Fig. 2A. For
longer times (48 h), cyclin D1 promoter/reporter gene activity was
still elevated, but cyclin D1 protein expression decreased, possibly
reflecting a difference in posttranscriptional modifications between
luciferase and cyclin D1 genes.
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Fig. 3.
Ang II stimulated cyclin D1 promoter activity
in a dose- and time-dependent manner. A,
quiescent CHO-AT1A cells transiently transfected with 973
CD1/LUC were treated with the different concentrations of Ang II for
24 h as indicated. Luciferase activity was assayed as described
under "Experimental Procedures." B, quiescent
CHO-AT1A cells transiently transfected with 973 CD1/LUC
were treated with 107 M Ang II
for the time points as indicated. Basal luciferase activity
(unstimulated sample) of the reporter construct was established as
100%. Luciferase activity was shown as the ratio of Ang II-stimulated
to unstimulated samples. The data are shown as the means ± S.E.
(bars) of three independent experiments performed in
triplicate.
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Ang II-stimulated Cyclin D1 Promoter Activity Is Dependent upon
Ras/Raf-1/MEK/ERK and PI3K Activities--
Previous work on the
regulation of cyclin D1 gene transcription has shown that the promoter
is growth factor-regulated and can be activated by oncogenic mutants of
Ras (58, 76, 77). The role of Ras in Ang II-induced cyclin D1
promoter/reporter gene activity was examined in CHO-AT1A
cells transiently transfected with 973 CD1/LUC and an expression
vector for the dominant negative Ras N17 mutant. Overexpression of Ras
N17 reduced Ang II-induced 973 CD1/LUC activity, reaching
approximately 50% inhibition (Fig. 4A). This effect was only
partial, suggesting that additional or parallel pathways might also be
involved. To address this possibility, we tested the effects of
LY294002 and wortmannin on Ang II-induced reporter gene activity.
LY294002 at 50 µM and wortmannin at 200 nM
partially inhibited Ang II-induced promoter activity, reaching approximately 50% inhibition. These results suggest that PI3K is also
involved in Ang II-induced cyclin D1 promoter/reporter gene activity.
To further explore the relationship between PI3K and Ang II-induced
cyclin D1 promoter activity, a dominant-negative form of p85
(p85) was used to inhibit the function of endogenous PI3K. As
expected, Ang II-induced cyclin D1 promoter activity was partially
inhibited (50%) when p85 subunit was coexpressed, therefore
identifying the role of class IA PI3K in this
pathway. We then also tested the effect of Ras N17 overexpression in
the presence of 50 µM LY294002, and under these
conditions, the inhibition was complete (Fig. 4A).
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Fig. 4.
Ang II-stimulated cyclin D1 promoter activity
is dependent upon Ras/Raf-1/MEK/ERK and PI3K activities.
A, quiescent CHO-AT1A cells were transiently
transfected with 973 CD1/LUC and the dominant negative Ras N17, Raf-1
C4, p85 mutants, or empty vector and/or preincubated with the
PI3K inhibitors, LY294002 (LY, 50 µM) and
wortmannin (Wort., 200 nM). Luciferase reporter
gene activity was analyzed 32 h after the addition of Ang II.
B, quiescent CHO-AT1A cells transiently
transfected with 973 CD1/LUC were pretreated or not with the MEK
inhibitor PD098059 at the different concentrations as indicated.
Luciferase reporter gene activity was analyzed 24 h after the
addition of Ang II. The data are presented as means ± S.E.
(bars) from three independent experiments, each performed in
triplicate. *, significantly different compared with empty vector alone
(A) or without inhibitor (B) (p < 0.05; Student's t test).
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The first Ras effectors to be identified in mammalian cells were the
protein kinases of the Raf family. To test the involvement of Raf-1 in
Ang II-induced promoter/reporter gene activity, CHO-AT1A cells were transiently transfected with 973 CD1/LUC and an expression vector for the dominant negative Raf-1 C4 mutant. Interestingly, this
mutant completely inhibited Ang II-induced promoter/reporter gene
activity. These results strongly suggest that the pathway(s) triggered by Ang II and leading to cyclin D1 promoter induction converges toward Raf-1.
It has been largely documented that activated Raf signals through the
phosphorylation of MEK, which in turn activates MAPK/ERK1/2. To test
whether this downstream target of Raf-1 is involved in Ang II-induced
promoter/reporter gene activity, CHO-AT1A cells transiently
transfected with 973 CD1/LUC were treated with various concentrations
of the MEK inhibitor PD098059. Under these conditions, Ang II
stimulation of cyclin D1 promoter/reporter gene activity was completely
inhibited with 50 µM PD98059 (Fig. 4B). Taken
together, these results suggest that the Ras/Raf-1/MEK/ERK pathway
connects Ang II to the induction of cyclin D1 promoter activity and
suggest that class IA PI3K activity also plays a major
role, possibly through an alternate pathway converging toward
Raf-1.
Ang II Induced the Tyrosine Phosphorylation of Intracellular
Proteins--
It is now well known that seven-transmembrane
domain-containing receptors functionally coupled to heterotrimeric
G-proteins are devoid of intrinsic protein tyrosine kinase activity.
However, it has been well documented that some of the intracellular
signaling pathways mediated by the AT1 receptor are
dependent upon stimulation of tyrosine kinase activity. To test our
model, quiescent CHO-AT1A cells were treated for various
times with 107 M Ang II. Whole
cell lysates were prepared, and equal amounts of proteins were
separated on SDS-PAGE, transferred to nitrocellulose membrane, and
analyzed using monoclonal anti-phosphotyrosine antibodies. The results
from Fig. 5A show that Ang II
rapidly stimulated immunoreactive proteins with molecular masses of
120, 90, 75-85, 60-65, and 40-45 kDa. However, a different time
course of induction was observed among the different species. For some
proteins, the induction was seen as early as 2 min (90 kDa) and lasted
up to 16 min. For others (40-45 kDa), the induction was seen at 4 min
and lasted up to 30 min. No such effect was observed when Ang II was
incubated with parental CHO-K1 cells, which do not express
AT1A receptors (data not shown). These results demonstrate
that in CHO-AT1A cells, Ang II is functionally coupled to
intracellular signals similar to those observed in other cell types
i.e. VSMC or adrenal cells.
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Fig. 5.
Implication of the protein-tyrosine
phosphatase SHP-2 in Ang II signaling. A, serum-starved
CHO-AT1A cells were either left untreated (0) or
stimulated for various periods of time (indicated in min) with Ang II.
Whole cell lysates containing equal amounts of total protein were
subjected to SDS-PAGE (8% gels) and analyzed by Western blotting with
monoclonal anti-phosphotyrosine antibodies. Molecular size standards
are shown in kDa on the left. The arrows on the
right denote tyrosine-phosphorylated proteins at 120, 90, 75-85, 60-65, and 40-45 kDa. Data are representative of independent
experiments that were performed at least three times. B,
serum-starved CHO-AT1A cells were either left untreated
(0) or stimulated with 107
M Ang II for the indicated times. SHP-2 was
immunoprecipitated (IP) with a rabbit polyclonal anti-SHP-2
antibody. Bound proteins were separated by SDS-PAGE (8% gels) and
immunoblotted with monoclonal anti-phosphotyrosine
(anti-P-Tyr) antibodies (upper panel).
For loading controls, the blot was stripped and reprobed with a
monoclonal anti-SHP-2 antibody (lower panel).
Data are representative of three independent experiments. C,
serum-starved cells were either left untreated (0) or
stimulated for the indicated times with Ang II
(107 M). Immunoblotting with
antibody specific for active phosphorylated MAPK/ERK
(anti-P-ERK1/2) was performed on whole cell lysates as
described under "Experimental Procedures." The
arrowheads indicate tyrosine-phosphorylated ERK1/2
(upper panel). The amounts of protein were
controlled by probing a duplicate blot with an anti-ERK1/2 antibody
(lower panel). Data are representative of two
independent experiments. D, quiescent CHO-AT1A
cells were transiently transfected with 973 CD1/LUC and either the
catalytically inactive SHP-2 CS mutant, the two SH2 domains of SHP-2,
the catalytically inactive SHP-1 CS mutant, or the empty vector alone.
Cells were stimulated with 107 M
Ang II for 32 h, and luciferase activity was assayed as described
previously. The data are presented as means ± S.E.
(bars) from three independent experiments each performed in
triplicate. *, significantly different compared with empty vector alone
(p < 0.05; Student's t test).
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The Protein-tyrosine Phosphatase SHP-2 is Tyrosine-phosphorylated
following Ang II Treatment--
Ang II-induced tyrosine
phosphorylation is a reversible phenomenon, raising the possibility
that protein-tyrosine phosphatases could also be involved during this
activation. Indeed, in VSMC, Ang II has been shown to stimulate
tyrosine phosphorylation and activation of an ubiquitously expressed
PTP named SHP-2 (78). In order to characterize this protein in our
model, quiescent CHO-AT1A cells were stimulated for
different periods of time with 107
M Ang II, and proteins were immunoprecipitated from whole
cell lysates with an antibody raised against SHP-2. Immune complexes were subjected to SDS-PAGE, and proteins were transferred to
nitrocellulose membrane and revealed using monoclonal
anti-phosphotyrosine antibodies. As shown in Fig. 5B
(upper panel), Ang II stimulated the tyrosine phosphorylation of SHP-2 with a maximum at 8 min and remained at least
up to 30 min. Immunoblotted SHP-2 was stripped and reprobed with a
monoclonal anti-SHP-2 antibody (lower panel) to
confirm identical amounts of protein on the blot. These results
demonstrate that in CHO-AT1A cells, Ang II is functionally
coupled to intracellular protein-tyrosine phosphatase SHP-2 as
observed, for example, in VSMC.
However, SHP-2 was not the only protein whose phosphorylation was
induced by Ang II. Fig. 5C shows that Ang II induced
tyrosine phosphorylation of MAPK/ERK1/2 as tested with an anti-active
phosphospecific antibody. The effect was sustained and lasted up to
8 h poststimulation. These results also confirm the finding that
MAPK/ERK1/2 activation plays a central role during Ang II-induced
cellular signaling.
A Catalytic Inactive Mutant of SHP-2 (SHP-2 CS) and SH2 Domains of
SHP-2 but Not SHP-1 CS Mutant Overexpression Inhibited Ang II-induced
Cyclin D1 Promoter Activity--
To further investigate the role of
SHP-2 in our model, we co-transfected a catalytically inactive SHP-2 CS
mutant or SH2 domains of the phosphatase in CHO-AT1A cells
together with 973 CD1/LUC. Overexpression of both forms resulted in a
strong inhibition of Ang II-stimulated cyclin D1 promoter/reporter gene
activity (Fig. 5D). In contrast, overexpression of wild type
SHP-2 had no significant effect (data not shown). To prove specificity,
we also transfected a catalytically inactive form of the closely
related tyrosine phosphatase SHP-1 (SHP-1 CS) and showed that it does
not affect the Ang II-induced signal.
Thus, it appears that Ang II-induced cyclin D1 promoter activity is
dependent on the intrinsic phosphatase activity and on the SH2 domains
of SHP-2 in CHO-AT1A cells.
Inhibition of EGFR Tyrosine Kinase by PD158780 Does Not Block Ang
II-induced MAPK/ERK and Cyclin D1 Promoter Activities--
The family
of MAPK/ERKs plays a central role in mitogenic signaling in response to
a number of growth-stimulating agents. Tyrosine kinase activity of the
single transmembrane receptor class has been implicated in MAPK/ERK
activation by G protein-coupled receptors by recruiting signaling
complexes containing Shc and GRB2. To clarify the role of EGF receptor
tyrosine kinase in Ang II-induced signal transduction in
CHO-AT1A cells, we tested the effect of the selective
receptor tyrosine kinase inhibitor PD158780 on MAPK activity. As shown
in Fig. 6A, at 100 nM, the compound did not affect Ang II-induced ERK1/2
activation. Increasing the concentration of inhibitor up to 1 µM also did not block (data not shown). On the other
hand, EGF (at 100 ng/ml) had only a modest and transient effect on
MAPK/ERK activation, probably because these cells do not express high
levels of the receptor. These results suggest that EGFR is not involved
in the pathway linking the Ang II receptor to MAPK/ERK activation.
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Fig. 6.
Inhibition of EGFR tyrosine kinase by
PD158780 does not block Ang II-induced MAPK/ERK and cyclin D1 promoter
activities. A, serum-starved CHO-AT1A cells
were either left untreated () or stimulated for various periods of
time with Ang II (107 M) or EGF
(100 ng/ml). When PD158780 was used, the compound was preincubated
1 h before the addition of Ang II. After cell lysis, proteins were
subjected to SDS-PAGE (8% gels) and immunoblotted with antibody
specific for active phosphorylated MAPK/ERK (anti-P-ERK/1/2)
(upper panel). The amounts of protein were
controlled by incubating a duplicate membrane with an anti-ERK1/2
antibody (lower panel). Data are representative
of two independent experiments. B, quiescent
CHO-AT1A cells transiently transfected with 973 CD1/LUC
were treated with Ang II (107 M)
or EGF (100 ng/ml) for 24 h in the presence or absence of the EGFR
tyrosine kinase inhibitor PD158780 at 50 and 100 nM as
indicated. Luciferase reporter gene activity was analyzed as described
previously. The data are presented as means ± S.E.
(bars) from two independent experiments, each performed in
triplicate.
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We then performed similar experiments on Ang II-induced cyclin D1
promoter/reporter gene activity. Fig. 6B shows that EGF (at
100 ng/ml) did not stimulate cyclin D1 promoter activity and that
PD158780 at 50 and 100 nM did not influence the Ang
II-induced effect. Due to all of these results, it is unlikely that Ang
II-induced MAPK/ERK and cyclin D1 promoter activities are dependent on
EGFR transactivation.
Ang II-induced MAPK/ERK Activity Is Inhibited by LY294002; Dominant
Negative Ras N17, Raf-1 C4, SHP-2 CS Mutants and SH2 Domains of
SHP-2--
We have shown that cyclin D1 promoter induction by Ang II
required MAPK/ERK activity. In order to corroborate the
pathway(s) connecting to this activation, we tested the effects
of both PI3K and MEK inhibitor (LY294002 and PD098059, respectively) on
Ang II-stimulated MAPK/ERK activity in CHO-AT1A cells
transiently transfected with HA-tagged p44-ERK1. The kinase was
immunoprecipitated from lysates of cells stimulated with Ang II for
different times (10 min and 2, 5, and 8 h), and enzymatic activity
was determined using the MBP phosphorylation assay. As expected, 50 µM PD098059 completely inhibited Ang II-induced ERK1
activity (data not shown), whereas 50 µM LY294002
partially but significantly blocked this activation (Fig.
7A), confirming an involvement
of PI3K in MAPK/ERK activation in CHO-AT1A cells. Control
experiments performed in CHO-K1 cells transfected with the same amount
of HA-tagged p44-ERK1 and treated with Ang II did not reveal
phosphorylation of MBP (data not shown).
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Fig. 7.
Ang II-induced MAPK/ERK activity is inhibited
by LY294002, dominant negative Ras N17, Raf-1 C4, SHP-2 CS mutants, and
SH2 domains of SHP-2. A, quiescent CHO-AT1A
cells transiently transfected with HA-tagged p44-ERK1 were pretreated
with 50 µM LY294002 (+) or left untreated () and were
stimulated or not with Ang II for different periods of time as
indicated. Cells were lysed, and ERK1 was immunoprecipitated with a
monoclonal anti-HA antibody. Its activity was assessed using the MBP
phosphorylation assay as described under "Experimental Procedures."
B and C, CHO-AT1A cells were
transiently transfected with HA-tagged p44-ERK1 plus (+) either
dominant negative Ras N17 or Raf-1 C4 mutants (B), catalytic
inactive SHP-2 CS mutant, SH2 domains of SHP-2 (C), or empty
vector alone (). Quiescent cells were stimulated or not
(0) with Ang II for the different times indicated and lysed.
ERK1 was immunoprecipitated, and its activity was assayed as described
previously. Results are expressed as -fold increase over basal activity
(unstimulated samples) set at 1. The data shown are representative
autoradiograms from two (A and B) or three
(C) experiments with similar results.
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We further studied the effects of Ras N17, Raf-1 C4, SHP-2 CS, and
SH2 domains of SHP-2 on Ang II-induced MAPK/ERK1 activation after 10 min and after 2 h of stimulation. These times were chosen because
MAPK/ERK activities were elevated compared with 5 and 8 h.
Overexpression of the dominant negative Ras N17 mutant was ineffective
at 10 min but partially inhibited at 2 h, whereas overexpression
of the dominant negative Raf-1 C4 mutant completely inhibited Ang
II-induced ERK1 activation at each time tested (Fig. 7B). As
a positive control, constitutively active Raf-1 BXB or Ras K12 mutants
were each able to activate ERK1 in the absence of Ang II stimulation
(data not shown). In addition, overexpression of dominant negative Ras
N17 and Raf-1 C4 mutants could both inhibit constitutively active Ras
K12-induced ERK1 activity, confirming that both constructs effectively
behave as dominant negative forms in these assays. These results show
that Ang II-induced ERK1 activity is dependent on Raf-1 activity,
independent of Ras at 10 min, and partially dependent on Ras at 2 h. In addition, when dominant negative SHP-2 CS mutant and SH2 domains
were tested, they were both able to partially inhibit Ang II-induced
ERK1 activity at each time tested (Fig. 7C). These results
highlight the importance of the intrinsic phosphatase activity and the
SH2 domains of SHP-2 in Ang II-stimulated MAPK/ERK activity in
CHO-AT1A cells.
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DISCUSSION |
In CHO-AT1A cells, the Ang II receptor is coupled to
more than one G protein (i.e. Gq and possibly
Gi subunits) responsible for inducing an early phase and a
second sustained increase in MAPK/ERK activity (79). The early phase of
MAPK/ERK activation by Ang II is mediated through a pertussis
toxin-insensitive Gq protein via phospholipase C and/or
protein kinase C activation, while the sustained phase of activation
may be mediated by Gq as well as by a pertussis
toxin-sensitive Gi protein. It has been proposed that the
latter phase is an obligatory event for growth factor-induced cell
cycle progression and proliferation (80). In the absence of growth
factors, we found that Ang II was potent at inducing cell cycle
progression of CHO-AT1A (Table I). However, we found a
difference in the initiation and duration of the S phase induced by Ang
II compared with FCS. The delayed and sustained response observed in
Ang II-stimulated DNA synthesis could correlate with the accumulation
of mitogenic factors in the conditioned medium that could function as
autocrine mediators. Among such factors, Wen et al. (79)
showed that in these cells, the Ang II-induced mitogenic response is
associated with sustained MAPK/ERK1/2 activation possibly through
formation of the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic
acid, via activation of Gi protein.
We then identified critical pathways downstream of the AT1A
receptor and found that PI3K and MAPK/ERK activity play a crucial role
in mitogenic signaling of Ang II in CHO-AT1A cells (Fig. 1).
It is well known that cyclin D1 protein expression is regulated by
mitogenic stimuli and that its assembly with
cyclin-dependent kinases is a rate-limiting step in the
G1/S phase progression of the cell cycle, contributing to
cell proliferation. Watanabe et al. (58) previously reported
that Ang II is able to induce cyclin D1 protein expression and promoter
activity in a human adrenal cell line through the AT1
receptor. Activation of the promoter relies on the binding of c-Fos and
c-Jun to an AP-1-responsive element in the cyclin D1-954 promoter
sequence. In our CHO-AT1A cells, we show that Ang II was
able to induce cyclin D1 protein expression (Fig. 2A) and
that both MEK and PI3K inhibitors reduced this induction (Fig.
2B), confirming their important role in cell proliferation.
In order to correlate these results with the activation of cyclin D1
promoter, we tested the effect of Ang II on the 973 base pair
promoter fragment cloned upstream of a luciferase gene (973 CD1/LUC).
CHO-AT1A cells were transiently transfected together with
this construct, and different plasmids encoding mutated forms of Ras,
Raf-1, and SHP-2 or were preincubated with various inhibitors before stimulation with Ang II.
Our results demonstrate that Ang II effectively stimulated 973
CD1/LUC activity (Fig. 3) and show a requirement for MAPK/ERK activity,
since inhibition of MEK by PD098059 was totally inhibitory (Fig.
4B). In addition, we found that p21ras and
also PI3K signaling pathway are linked to the cell cycle machinery
through regulation of cyclin D1 promoter activity, since wortmannin,
LY294002, dominant negative PI3K (p85), and Ras N17 mutants were
partially inhibitory (Fig. 4A). Interestingly, and in
contrast to these partial effects, a dominant negative Raf-1 C4 mutant
completely inhibited Ang II-induced 973 CD1/LUC activity. A complete
inhibition was also observed when Ras N17 and LY294002 were used in
concert. Taken together, these results highlight the central role of
the Raf-1/MEK/ERK pathway during Ang II-induced cyclin D1 promoter
activation and the importance of both Ras and class IA PI3K
upstream of this pathway.
In this study, we also investigated the role of the protein-tyrosine
phosphatase SHP-2 during Ang II-induced cyclin D1 promoter activation.
Little is known about the participation and regulation of SHP-2 in
pathways activated by G protein-coupled receptors that lack intrinsic
tyrosine kinase activity. We found that a catalytic inactive SHP-2 CS
mutant inhibited Ang II-induced cyclin D1 promoter/reporter gene
activity (Fig. 5D), suggesting that the catalytic domain of
SHP-2 plays an important role in pathways leading to cell cycle
progression. In order to act as a positive regulator, SHP-2 must be
able to dephosphorylate phosphotyrosines that negatively regulate key
signaling components such as members of the Src family of
protein-tyrosine kinases, known to activate the Ras/Raf-1/MEK/ERK
pathway (81). The possibility that SHP-2 interacts with
insulin-related substrate-1, resulting in PI3K activation, has also
been suggested in insulin-stimulated Rat-1 fibroblasts (82). However,
additional protein-tyrosine kinases may also be involved in this
regulation. Marrero et al. (83) presented evidence that both
the Jak/STAT and the Ras/MAPK cascades are important components in Ang
II- and PDGF-mediated VSMC proliferation. They also showed that SHP-2
can associate with the Ang II type 1 receptor and serve as a docking
partner for the protein-tyrosine kinase Jak2. These interactions are a
prerequisite for Jak2 phosphorylation and activation (83), leading to
Raf-1 phosphorylation and activation of MAPK/ERK. Therefore, Jak2
represents an additional target for SHP-2 to exert a positive
regulation on Ang II-mediated mitogenic signaling. In this study, we
also found that transfection with the SH2 domains of SHP-2 inhibited
Ang II-induced cyclin D1 promoter/reporter gene activity (Fig.
5D), implying that the adapter function of SHP-2 via
its SH2 domains plays an important role in this induction. But the
participation of SH2 domains interacting with the Ang II type 1 receptor has not been demonstrated in contrast to their association
with activated PDGF and EGF receptors. Indeed, the two SH2 domains
allow SHP-2 to interact with tyrosine-phosphorylated proteins, and it
has been reported that ligation of these domains contributes to
activation of the phosphatase activity (84).
In the case of growth factor receptors, SHP-2 becomes rapidly
tyrosine-phosphorylated upon ligand stimulation, creating a binding
site for the Grb2-Sos complex and therefore facilitating activation of
the Ras/ERK cascade. Moreover, an EGFR-dependent transactivation model has been recently described to mediate Ang II-induced ERK activation (85, 86). However, in our cell system, such a
mechanism is unlikely because Ang II-induced MAPK/ERK and cyclin D1
promoter/reporter gene activities are not affected by PD158780, a
highly specific inhibitor of EGFR tyrosine kinase. Moreover, EGF by
itself was unable to stimulate a sustained MAPK/ERK activation or to
induce cyclin D1 promoter activity and BrdUrd incorporation in
CHO-AT1A cells. Our results show that SHP-2 becomes rapidly
tyrosine-phosphorylated (Fig. 5B) and more precisely that both the SH2 domains and the catalytic activity of the phosphatase are
important for Ang II-induced MAPK/ERK activity (Fig. 6C). We
conclude that SHP-2 plays an important role downstream of the mitogenic
signaling pathway induced by the G protein-coupled Ang II receptor
independently of EGF receptor transactivation.
Depending on the cells and agonists that have been studied, tyrosine
phosphorylation of SHP-2 correlates either with an inhibition (45) or a
stimulation (34, 78) of its phosphatase activity. On the other hand, it
may be difficult to address the influence of tyrosine phosphorylation
on the enzymatic activity, since SHP-2 is subject to an
autodephosphorylation mechanism (87), resulting in a loss of tyrosine
phosphorylation under in vitro phosphatase assay conditions.
Moreover, insulin activates SHP-2 without inducing its tyrosine
phosphorylation (40), suggesting that SHP-2 phosphorylation is not
essential for its activation. Neither the precise mechanisms by
which the phosphatase is regulated in CHO-AT1A cells nor
the mechanisms by which the phosphatase acts to regulate
MAPK/ERK pathway has been investigated in detail. However, our results demonstrate that Ang II-induced MAPK/ERK activation is modulated by
PI3K, Ras, Raf-1, and SHP-2 (Fig. 7) and that it is independent of EGF
receptor transactivation. Further studies are necessary to depict more
precisely the exact mechanism of action of the phosphatase.
Taken together, our results suggest that in CHO-AT1A cells,
SHP-2 plays a crucial role in the Ang II-induced initiation of the
mitotic cyclin D1 promoter activity, leading to cell cycle progression
and proliferation. This is to our knowledge the first demonstration for
an SH2 domain-containing protein-tyrosine phosphatase to regulate a
gene activated by a G-protein-coupled receptor pathway.
,
TOP
ABSTRACT
INTRODUCTION
Supported by a doctoral fellowship from the French Ministère
de l'Education Nationale de l'Enseignement Supérieur et de la Recherche.
