1 Department of Medicine, University of Florida College of Medicine; and 2 Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608-1197
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ABSTRACT |
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Signaling events
involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial
cell (PAEC) proliferation were examined. ANG IV significantly increased
upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent
kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and
protein kinase B-/Akt (PKB-
) activities, as well as downstream
p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of
these proteins. ANG IV also significantly increased
5-bromo-2'-deoxy-uridine incorporation into newly synthesized DNA in a
concentration- and time-dependent manner. Pretreatment of cells with
wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an
inhibitor of the mammalian target of rapamycin kinase and p70S6K,
diminished the ANG IV-mediated activation of PDK-1 and PKB-
as well
as phosphorylation of p70S6K. Although an inhibitor of
mitogen-activated protein kinase kinase, PD-98059, but not rapamycin,
blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and
rapamycin independently caused partial reduction in ANG IV-mediated
cell proliferation. However, simultaneous treatment with PD-98059 and
rapamycin resulted in total inhibition of ANG IV-induced cell
proliferation. These results demonstrate that ANG IV-induced DNA
synthesis is regulated in a coordinated fashion involving multiple
signaling modules in PAEC.
p70 S6 kinase; protein kinase B; phosphatidylinositol 3-kinase
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INTRODUCTION |
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THE VASCULAR ENDOTHELIUM processes a variety of biologically active substances in the circulation including angiotensin IV (ANG IV), a metabolic product of ANG II, in animals and humans (4, 40, 41, 54, 55). The presence of ANG IV-specific binding sites has been identified in various mammalian tissues and cells, including the brain, kidney, and heart, as well as in lung endothelial cells (8, 14, 15, 28, 35). Although the functional role of ANG IV is not fully defined, it has been reported that ANG IV increases blood flow in the brain and kidney and causes endothelium-dependent vasodilation of rabbit cerebral arterioles (22, 23). We previously reported that ANG IV stimulation increases the catalytic activity of the constitutively expressed lung endothelial cell isoform of nitric oxide synthase (eNOS), leading to increases in nitric oxide (NO) release, production of cGMP, and NO-cGMP-mediated porcine pulmonary artery vasorelaxation (8, 28). This ANG IV-mediated vasodilation of the pulmonary artery is endothelium dependent and is regulated by intracellular calcium release through receptor-coupled G protein-phospholipase C and phosphatidylinositol 3 kinase (PI3K) signaling mechanisms (8). The activation of PI3K plays a critical role in downstream signaling in the regulation of multiple cellular processes, including protein synthesis and cell proliferation. In fact, an essential step in the signaling events that trigger cellular proliferation is the induction of high-level protein synthesis (19, 36). ANG IV is known to increase protein and DNA synthesis in mammalian cells (29, 50) including vascular endothelial cells (18, 27). However, the critical signaling events linked to ANG IV-mediated increased DNA synthesis remain elusive.
Agonist- and/or growth factor-mediated activation of PI3K is known to be associated with the regulation of downstream signaling processes involving activation of PI-dependent kinase-1 (PDK-1), protein kinase B (PKB or Akt), the mammalian target of rapamycin (mTOR) kinase, and p70 ribosomal S6 kinase (p70S6K), leading to enhanced cell growth and proliferation in diverse systems (1, 2, 17, 21, 49). mTOR is an upstream effector of at least two distinct translational regulators: p70S6K and initiation factor 4E binding protein (4E-BP1) (10, 33). Rapamycin, a lipophilic macrolide, is a potent and selective inhibitor of TOR proteins and signaling to downstream targets including p70S6K and 4E-BP1 (10, 33). Thus TOR signaling plays a critical role in the regulation of cell growth. In addition, several agonists and/or growth factors have also been shown to activate mitogen-activated protein kinases (MAPK) including the Raf-MAPK kinase 1/2 (MEK1/2)-extracellular signal-related kinase 1/2 (ERK1/2) module. In fact, a number of mitogenic stimuli are known to increase MEK1/2-mediated phosphorylation of MAPK ERK1 and 2, identified as 44- and 42-kDa MAPK, respectively, leading to modulation of vascular endothelial cell function including protein synthesis and cell growth (20, 44, 46). The role of MEK/ERK1/2 in endothelial cell translational signaling has not been described. However, insulin has been reported to activate mRNA translation by increasing the phosphorylation of the eukaryotic translation initiation factor 4E (eIF-4E) (11), which was blocked by the MEK1/2 inhibitor PD-98059 in Chinese hamster ovary (CHO) cells (12). This indicates a potential role for ERK1/2 in translational signaling and the regulation of cell growth.
The objective of the present study was to identify selective signaling
pathways involving ERK1/2 or PI3K/p70S6K linked to ANG IV-mediated
increased DNA synthesis in lung endothelial cells. The results identify
critical roles for PI3K mTOR
p70S6K as well as for ERK1/2
pathways in ANG IV-mediated enhanced DNA synthesis in pulmonary artery
endothelial cells (PAEC). Rapamycin inhibition of mTOR or its
downstream effector p70S6K also suggests that translational signaling
plays a major role in ANG IV-induced cell proliferation PAEC.
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EXPERIMENTAL PROCEDURES |
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Cell culture and treatment. Endothelial cells were isolated from the main pulmonary artery of 6- to 7-mo-old pigs and were propagated in monolayers as previously described (26). Third- to fifth-passage cells in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) were used in all experiments. In each experiment, cell monolayers were studied 1 or 2 days after confluence and were matched for cell line, passage, and days after confluence.
To determine the potential effects of ANG IV on PI3K, PDK-1, PKB, ERK1/2, and p70S6K activity and/or phosphorylation as well as on DNA synthesis, we incubated cell monolayers in RPMI 1640 with or without (control) ANG IV (5 nM-5 µM) for 15 min-24 h at 37°C or as indicated in selective experiments. In some experiments, cell monolayers were preincubated for 15 min with or without the presence of the PI3K inhibitors wortmannin (100 nM) (47) and LY-294002 (50 µM) (48), the MEK1/2 inhibitor PD-98059 (10 µM), which inhibits phosphorylation of ERK1/2 (9), or the mTOR/p70S6K inhibitor rapamycin (15 nM) (32) or were subjected to metabolic labeling with [32P]orthophosphate (5 µCi/ml) for 2 h followed by incubation with or without the presence of ANG IV as described above. After treatment, cells were used 1) to determine the potential cytotoxic effects of the various modulators of the signaling pathways; 2) to measure the catalytic activities of PI3K, PDK-1, PKB-PI3K activity. Immediately after treatment, cells were lysed at 4°C with lysis buffer containing 20 mM Tris · HCl (pH 8.1), 1 mM MgCl2, 1 mM CaCl2, 137 mM NaCl, 10% glycerol, 1% Nonidet (NP)-40, 150 µM vanadate, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Equal amounts of cell lysates (400 µg of protein) were incubated with 2 µg of anti-PI3K (p85) antibody (Upstate Biotechnology, Lake Placid, NY) at 1:10 dilution with lysis buffer for overnight at 4°C. The immune complexes were precipitated with 50 µl of protein A-Sepharose, and the pellets were washed three times with isotonic phosphate-buffered saline containing 1% NP-40 followed by two washes with 100 mM Tris · HCl, pH 7.4, containing 0.5 M LiCl and two washes with 100 mM Tris · HCl, pH 7.4, containing 100 mM NaCl and 1 mM each of EDTA and DTT. The pellets were resuspended in PI3K assay buffer (20 mM Tris · HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EGTA).
We initiated the lipid kinase assay by adding 50 µl of immunoprecipitate, 60 µl (10 µg) of sonicated L-PDK-1 activity.
PDK-1 activity was determined using a PDK-1 assay kit (Upstate
Biotechnology) through activation of glucocorticoid-regulated kinase
(SGK) by PDK-1 following the manufacturer's suggested procedures. Equal amounts of cell lysate (50 µg of protein) were used to
phosphorylate the substrate (RPRAATF) in the presence of activated or
inactive SGK and [32P]ATP (500 counts · min
1 · pmol
1) for
30 min at 30°C. Phosphorylated substrate was collected on a
phosphocellulose P81 filter and quantified by scintillation spectroscopy. We determined specific activity by subtracting
nonspecific phosphorylation of substrate using appropriate blanks such
as inactive SGK or active PDK-1 without inactive SGK. One unit of PDK-1
activity equals 1 nmol of phosphate transferred to
substrate · 30 min
1 · mg
protein
1.
PKB- activity.
After treatment, cells were washed and suspended in lysis buffer
containing 50 mM Tris · HCl, pH 7.5, 0.1% Triton X-100, 1 mM
each of EDTA and EGTA, 50 mM NaF, 10 mM sodium glycerophosphate, 5 mM
sodium pyrophosphate, 1 mM sodium orthovanadate, 0.1%
2-mercaptoethanol, 0.27 M sucrose, 1 µM microcystin-LR, and protease
inhibitor cocktail (Boehringer Mannheim). After insoluble material was
removed by centrifugation, PKB-
was immunoprecipitated by incubation
at 4°C with 5 µg of anti PKB-
antibody conjugated to 5 µl of
protein G-Sepharose. Immunoprecipitates were washed three times with 50 mM Tris · HCl buffer, pH 7.5, containing 0.1 mM each of EDTA
and EGTA, 0.1% 2-mercaptoethanol, and 0.5 M NaCl and twice with same the buffer without NaCl. Kinase activity was measured by incubation of
equal amounts of immunoprecipitated protein with peptide substrate (GRPRTSSFAEG, termed "Crosstide") and [
32P]ATP
(0.5 µCi) for 10 min at 30°C. Phosphorylated substrate was collected on a phosphocellulose P81 filter and quantified by
scintillation spectroscopy.
p70S6K activity.
Immediately after treatment, cell lysates were prepared as described in
PKB- activity. Equal amounts of lysate protein (750 µg)
were incubated with 5 µg of polyclonal rabbit p70S6K antibody (Santa
Cruz Biotechnology) for 2 h at 4°C. The antigen-antibody complex
was then precipitated with protein A-Sepharose beads with gentle
agitation for 1 h at 4°C. The immunoprecipitates were washed three times with ice-cold phosphate buffer, and kinase activity was
determined using a p70S6K assay kit (Upstate Biotechnology). In brief,
10 µg substrate peptide (AKRRRLSLAR); 10 µl inhibitor cocktail
targeted to PKC, PKA, and calmodulin-dependent kinases; 10 µl
immunoprecipitate; 500 µM ATP; 75 mM MgCl2; and
[
32P]ATP (0.5 µCi) were incubated at 30°C for 30 min. Phosphorylated substrate was collected on a P81 phosphocellulose
filter and quantified by scintillation spectroscopy.
Western blot analysis and autoradiography.
After treatment, cells with or without
[32P]orthophosphate metabolic labeling were lysed and
equal amounts of cell lysate protein (50 µg) were separated on
SDS-PAGE. Proteins were transferred from gel to polyvinylidene
difluoride membranes and immunodetected using polyclonal
anti-phospho-specific ERK1/2 antibody, anti-phospho-specific PKB-
antibody, or anti-p70S6K antibody (Upstate Biotechnology) as previously
described. Phosphorylated p70S6K was detected by immunoprecipitation
and autoradiography on XAR film (Eastman Kodak). Densitometric analysis
of the blots was performed using a Bio-Rad Fluor-S MultImager system.
Cell proliferation assay. We assessed cell proliferation by monitoring incorporation of 5-bromo-2'-deoxy-uridine (BrdU) into newly synthesized DNA of replicating cells. Subconfluent cells were seeded into 96-well plates (5 × 103 cells/well) coated with 1.5% gelatin in 12% fetal calf serum for 24 h followed by additional incubation in serum-free medium for 24 h. Cells were then stimulated with varying concentrations of ANG IV (2 nM-50 µM) for 2-72 h at 37°C. After incubation, cells were washed, and we measured BrdU incorporation by monitoring absorbance at 405 nm with a BioTek F600 microplate reader.
Statistical analysis. Statistical significance for the effect of ANG IV on PI3K, PDK-1, PKB, and p70S6K activities, as well as PKB, p70S6K, and ERK1/2 phosphorylation and DNA synthesis were determined using ANOVA and Student's paired t-test (53). Values are means ± SE for n experiments. Differences between treatments and/or groups were considered significant if P < 0.05.
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RESULTS |
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ANG IV stimulation increases catalytic activity of PI3K.
Because a number of agonists are known to activate PI3K through a G
protein-coupled signaling mechanism, we first examined the effects of
ANG IV on PI3K activity and on its potential effects on downstream
effector kinases. As illustrated in Fig.
1, ANG IV significantly
(P < 0.05) increased the catalytic activity of PI3K in
PAEC. Pretreatment of cells with the PI3K inhibitor wortmannin, but not
with the mTOR/p70S6K selective inhibitor rapamycin, significantly (P < 0.05) diminished ANG IV-mediated activation of
PI3K.
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ANG IV stimulation of PDK-1 activity.
To determine whether ANG IV also activates PDK-1 and whether such
activation is dependent or independent of PI3K, we next examined ANG
IV's effect on PDK-1 activity with and without the presence of PI3K
and mTOR/p70S6K inhibitors. As shown in Fig. 2, ANG IV caused concentration- and
time-dependent stimulation of PDK-1 activity in PAEC. PDK-1
activity was significantly (P < 0.05) increased in the
presence of 0.5 µM ANG IV and remained elevated with increasing
concentrations up to 5 µM ANG IV. A time-dependent increase in PDK-1
activity was observed in PAEC incubated with 1 µM ANG IV. Significant
increases were observed as early as 15 min. Similarly, pretreatment of
cells with wortmannin and LY-294002 slightly but not significantly
reduced ANG IV-stimulated activation of PDK-1. Pretreatment of cells
with rapamycin had no effect on PDK-1 activity in control and ANG
IV-stimulated cells (not shown). This indicates that ANG IV-induced
activation of PDK-1 is independent of PI3K.
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Increased phosphorylation of PKB- is associated with ANG
IV-mediated activation of PKB-
.
Several reports have suggested that PKB could be activated in a
PI3K-dependent manner. To examine this, we determined ANG IV's effect
on PKB activity, expression, and phosphorylation with or without the
presence of PI3K-specific inhibitors. ANG IV-stimulation caused a
significant increase in the catalytic activity of PKB-
as early as 5 min (P < 0.01), and the increase persisted for 60 min
(P < 0.05). ANG IV-mediated activation of PKB-
was
associated with increased phosphorylation, but not expression, of this
protein. Pretreatment of cells with the PI3K-selective inhibitors
LY-294002 and wortmannin blocked ANG IV-mediated phosphorylation of
PKB-
(Fig. 3).
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ANG IV-stimulated activation of p70S6K is mediated through
increased phosphorylation.
To examine whether p70S6K, a critical component of the translational
signaling pathway, is activated upon stimulation with ANG IV, we
performed a kinase assay using S6 peptide as a substrate in parallel
with Western blotting to determine phosphorylation and expression state
of this enzyme (Fig. 4, A and
B). ANG IV stimulation caused time-dependent activation of
p70S6K up to 2 h (P < 0.05 for all time points)
(Fig. 4C). Pretreatment of cells with the PI3K inhibitor
wortmannin and the mTOR kinase-selective inhibitor rapamycin abolished
ANG IV-induced activation of p70S6K (Fig. 4D). ANG
IV-induced activation of p70S6K was associated with increased
phosphorylation of this protein and was blocked by pretreatment with
the PI3K inhibitor wortmannin and the mTOR kinase-selective inhibitor
rapamycin (Figs. 4 and 5). Collectively, these results indicate that activation of PI3K and/or mTOR signaling are required for p70S6K activation.
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Rapamycin failed to block ANG IV-mediated phosphorylation of
ERK1/2.
Because pathways involving PI3K and MEK or ERK1/2 have been implicated
in the regulation of cell growth and proliferation, we examined whether
ANG IV increases phosphorylation of ERK1/2. As shown in Fig.
6, ANG IV stimulation significantly
increased phosphorylation of ERK1/2 (P < 0.05).
Pretreatment with the ERK1/2 selective inhibitor PD-98059, but not with
the mTOR/p70S6K-selective inhibitor rapamycin, blocked ANG IV-mediated
phosphorylation of ERK1/2. However, ERK1/2 expression was comparable
with control irrespective of treatments with ANG IV alone or with or
without PD-98059 and rapamycin.
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ANG IV-induced cell proliferation was blocked by wortmannin,
PD-98059, and rapamycin.
To determine whether ANG IV stimulation causes increased DNA synthesis
and whether such a response is regulated through PI3K and p70S6K- or
ERK1/2-mediated signaling, we measured ANG IV-induced cell
proliferation by monitoring BrdU incorporation into newly synthesized
DNA. As shown in Fig. 7, ANG IV
stimulation resulted in increased DNA synthesis in a concentration- and
time-dependent manner. The time-dependent response revealed that
stimulation with 5 µM ANG IV resulted in a gradual increase in DNA
synthesis with maximal cell proliferation observed at 24 h of
incubation (P < 0.01 at 24 h). The
concentration-dependent study revealed a significant (P < 0.05) increase in DNA synthesis after stimulation with as little as
50 nM ANG IV for 24 h. To determine the selective roles of PI3K
mTOR/p70S6K and MAPK/ERK1/2 signaling on ANG IV-induced DNA
synthesis, we examined the effects of inhibitors of PI3K, mTOR, and
ERK1/2 alone or in combination. As shown in Fig.
8, ANG IV significantly
(P < 0.01) increased DNA synthesis. Pretreatment with
the PI3K inhibitor wortmannin, with the MEK1/2 inhibitor PD-98059, or
with the mTOR inhibitor rapamycin alone partially, but significantly,
reduced ANG IV-induced DNA synthesis compared with control
(P < 0.05). However, when cells were first incubated with ANG IV for 4 h, rapamycin treatment caused a slight, but not
significant, reduction in DNA synthesis. Simultaneous pretreatment with
rapamycin and PD-98059 completely abolished ANG IV-stimulated DNA
synthesis (P < 0.01).
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DISCUSSION |
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The present study provides experimental evidence for the first
time that ANG IV plays a role in proliferation of lung vascular endothelium in culture through activation of at least two distinct signaling modules: PI3K PDK-1/PKB
mTOR
p70S6K and Ras
MEK
ERK1/2 (Fig. 9). Our results
demonstrate that ANG IV-stimulated activation of PI3K and ERK1/2 in
upstream signaling and of mTOR/p70S6K in downstream signaling plays a
critical role in the regulation of cell proliferation in lung
endothelial cells. The ANG IV-induced overall DNA synthesis appears to
be regulated in a coordinated fashion by both the MEK/ERK1/2 and the
PI3K/p70S6K signaling pathways as simultaneous inhibition of both
pathways is required to block total DNA synthesis. These results also
suggests that mTOR signaling plays a major role in ANG IV-mediated
control of cell growth, as delayed inhibition of mTOR signaling by
rapamycin failed to block ANG IV-induced DNA synthesis. This is
most likely due to delayed blocking of a critical time-dependent event
in G1 to S phase transition in cell cycle progression.
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We previously reported that ANG IV receptor-mediated vasorelaxation of pulmonary artery is endothelium dependent and regulated by intracellular calcium release through a receptor-coupled G protein-, phospholipase C-, and PI3K-signaling mechanism (8). The results of this study are consistent with the well-established role of agonist- and mitogen-induced activation of PI3K and its key signaling role in the regulation of diverse processes, including proliferation, apoptosis, secretion, inflammation, and organization of the actin cytoskeleton in a variety of cell types (5, 16). Although activation of PI3K is generally known to be associated with modulation of multiple downstream effector kinases, our results indicate that ANG IV-mediated activation of PDK-1 is independent of PI3K activation in lung endothelial cells. PDK-1 is believed to be constitutively active in a variety of cell types (1, 6). However, our results demonstrate for the first time that ANG IV can enhance PDK-1 activity above basal level. This is unique in the context that no other agonist, growth factor, or mitogen has been shown to increase PDK-1 activity in any cell type. Although the mechanism responsible for ANG IV-mediated activation of PDK-1 remains elusive, several potential possibilities exist. For example, phosphorylation of Ser241 was reported to be essential for PDK-1 activity possibly through autophosphorylation by PDK-1 (6). In addition, PDK-1 expressed in unstimulated 293T cells was shown to be phosphorylated at Ser25, Ser241, Ser393, Ser396, and Ser410. The level of phosphorylation of each serine site and PDK-1 activity was unaffected by stimulation with insulin or insulin-like growth factor (6). ANG IV may mediate PDK-1 activation through phosphorylation of a critical serine site. Alternatively, a recent study suggested that a mutation at Ala280 or deletion of the pleckstrin homology domain can increase PDK-1 autophosphorylation (52). PDK-1 has been shown to be activated by phosphorylation of tyrosine residues after stimulation with a tyrosine phosphatase inhibitor peroxyvanadate (13, 31). However, insulin stimulation of 293T cells failed to detect tyrosine phosphorylation of PDK-1 (13, 31). Another recent report suggested that ANG IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells (7). Therefore, it is possible that ANG IV-mediated activation of PDK-1 may be linked with increased tyrosine phosphorylation in lung endothelial cells. Alternatively, ANG IV may directly or indirectly activate PDK-1 through autophosphorylation by triggering conformational changes. The precise mechanism involved in ANG IV-mediated activation of PDK-1 remains to be determined.
Phosphorylation of PKB at Thr308 and Ser473 is known to be essential for activation of this kinase (1, 24). Recent studies have shown that the phosphorylation of PKB at Thr308 by PDK-1 (42) and at the Ser473 hydrophobic site is possibly associated with PDK-2-mediated autophosphorylation (45). The current model of PKB activation involving PDK-1 and PDK-2 suggests that the binding of PI3K products, namely PI-3,4 phosphate (PIP2) and PI-3,4,5 phosphate (PIP3), with the enzyme is critical. This binding is believed to lead to a conformational change that exposes Thr308 and Ser473 to the membrane-associated PDK-1 and PDK-2 resulting in enhanced phosphorylation (52). Because the PI3K inhibitors LY-294002 and wortmannin blocked ANG IV-mediated phosphorylation of PKB despite enhanced activation of PDK-1, our data support the notion that: 1) ANG IV-induced phosphorylation of PKB is independent of PDK-1 activation and 2) PI3K- catalyzed PIP2 and PIP3 generation is critical for increased phosphorylation and activation of PKB.
Our results also demonstrate that ANG IV caused increased phosphorylation and activity of p70S6K as well as increased phosphorylation of ERK1/2. Phosphorylation and/or activation of p70S6K were blocked by the PI3K inhibitors LY-294002 and wortmannin as well as by the mTOR inhibitor rapamycin, whereas phosphorylation of ERK1/2 was blocked by PD-98059 but not by rapamycin. Therefore, activation of p70S6K appears to be linked with upstream PI3K rather than ERK1/2 signaling in lung endothelial cells. Whether PKB, a downstream effector of PI3K, plays a direct or indirect role in ANG IV-mediated activation of p70S6K is unknown. However, studies with overexpression of PKB suggest that in mammalian cells mTOR kinase activity is not altered (30, 39).
Very little is known about the role of the PI3K PDK-1/PKB
p70S6K module in the control of vascular endothelial cell growth. It is
known that mTOR lies upstream of p70S6K and 4E-BP1 and that the TOR
proteins are members of the PI3K superfamily that regulates cell growth
and differentiation (1, 10, 33). The TOR proteins exhibit
protein kinase activity that is selectively inhibited by rapamycin
without modulation of other kinases, including PI3K and ERK1/2
(10, 33, 43). A number of studies have reported that
insulin, insulin-like growth factor, platelet-derived growth factor, or
shear stress activate ERK1/2 or p70S6K or both in diverse cell types
including vascular endothelium (20, 38, 40, 46). Despite
activation of both ERK1/2 and p70S6K by these stimuli, ultimate protein
synthesis or DNA synthesis is regulated by selective pathways in
specific cells. For example, shear stress-induced DNA synthesis in
human umbilical vein endothelial cells is exclusively regulated by
p70S6K-mediated signaling irrespective of the activation of both ERK1/2
and p70S6K (20), whereas in 3T3-L1 adipocytes, insulin-induced protein synthesis is regulated by both the ERK1/2 and
p70S6K pathways (38). Our results demonstrate equal
involvement of ERK1/2 and mTOR/p70S6K signaling in ANG IV-induced DNA
synthesis in lung endothelial cells, as PD-98059 or rapamycin alone
resulted in only partial inhibition of DNA synthesis, but the
combination of PD-98059 and rapamycin caused near total inhibition of
DNA synthesis. The coordinated role of MEK/ERK1/2 and
PI3K/PDK-1-PKB/p70S6K modules in ANG IV-mediated, increased DNA
synthesis is associated with the induction of a high level of protein
synthesis, which is an essential step in the pathway by which growth
factors trigger cell proliferation. If so, the function of p70S6K is
critical in the regulation of a class of mRNA transcripts that contain an oligopyrimidine tract at their transcriptional start site (10, 33). This class of mRNAs encodes for many of the components of
the protein synthetic apparatus and represents ~30% of the total
mRNA. Its translation into protein is regulated by p70S6K (10,
33). Thus p70S6K function is essential to the regulation of the
translational pathway required for G1 to S phase transition in cell
cycle progression (10, 33). Our data support the notion that timely regulation of the mTOR downstream effector is critical to
the regulation of G1 to S phase transition and the proliferative response, as rapamycin failed to reverse ANG IV-mediated activated signaling events and DNA synthesis. Although downstream effectors of
mTOR, i.e., p70S6K and 4E-BP1, are known to be involved in translational regulation of protein synthesis as well as in G1 to S
phase transition in cell cycle progression, ERK1/2 has also been
suggested to play a role in cell growth and proliferation through
transcriptional and translational pathways in a variety of cells,
including vascular endothelial cells (38, 49). For example, bradykinin-stimulated protein synthesis was reported to be
dependent on the MAPK/MEK-mediated activation of p70S6K in rat
ventricular cardiomyocytes (34). A recent report indicates that G protein-coupled receptor agonists such as phenylephrine, endothelin-1, and insulin activate p70S6K isoform S6K2 by ERK signaling
in rat ventricular cardiomyocytes, which was completely blocked by
rapamycin (51). Whether ANG IV stimulation also results in
activation of S6K2 through ERK1/2 signaling in lung endothelial cells
remains to be determined. In addition, MAPK/MEK and ERK1/2 have been
suggested to enhance mRNA translation through increased phosphorylation
of eIF-4E in CHO cells overexpressing the insulin receptor (11,
12). Although 4E-BP1 has been shown to bind to eIF-4E, leading
to inhibition of mRNA translation (10, 33, 43), the
precise role of 4E-BP1 and/or eIF-4E proteins and their mechanistic
link in the regulation of ANG IV-induced DNA synthesis remain to be determined.
In summary, the integration of multiple signaling modules involving the
PI3K PDK-1/PKB
mTOR/p70S6K pathway and the MEK/ERK1/2 pathway
is essential to ANG IV-stimulated endothelial cell proliferation. These
findings also illustrate that coordinated control of transcriptional and translational signaling in ANG IV-induced cell proliferation is
dependent on the cell type as well as the nature of the external stimulus, e.g., growth factors, agonists, or shear stress (20, 34, 38, 49). Identification of two distinct signaling pathways in the regulation of ANG IV-induced cell growth is physiologically significant, as increased rates of protein synthesis and cell cycle
progression represent key events in vascular proliferative diseases.
Targeting the transcriptional and translational signaling by
therapeutic intervention may prevent uncontrolled upregulation of cell
cycle proteins and may interrupt other potential pathophysiological events, such as cellular migration or hypertrophy, during the development of vascular diseases (3, 37). For example,
immunosuppressive drugs such as rapamycin have the potential to control
cell cycle progression via the p70S6K-dependent translational pathway
without affecting protein synthesis regulated through transcriptional signaling. In contrast, inhibition of MEK/ERK1/2 might alter the level
of gene expression without affecting the synthesis of proteins regulated through translational signaling as well as the processes of
G1 to S phase transition in cell cycle progression.
The physiological and pathophysiological significance of the observations reported here is important in understanding the potential role of ANG IV in the regulation of endothelial cell proliferation in lung vasculature. This is particularly relevant since ANG IV stimulation was previously reported to cause early and sustained increases of the catalytic activity of eNOS in lung endothelial cells as well as of NO-cGMP-mediated pulmonary artery vasorelaxation (8, 27, 28). Although NO production has been suggested to mediate multiple effects including enhanced cell proliferation, it remains to be determined whether ANG IV-mediated lung endothelial cell NO production and cell proliferation are causally linked.
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ACKNOWLEDGEMENTS |
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We thank Bert Herrera for tissue culture assistance and Weihong Han and Dihua He for technical assistance.
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FOOTNOTES |
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This work was supported by Medical Research Service of the Department of Veterans Affairs and by National Heart, Lung, and Blood Institute Grant HL-58679 (J. M. Patel).
Address for reprint requests and other correspondence: J. M. Patel, Research Service (151), VA Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: Pateljm{at}medicine.ufl.edu).
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.
May 17, 2002;10.1152/ajplung.00024.2002
Received 18 January 2002; accepted in final form 1 May 2002.
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