Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation

Yong D. Li1, Edward R. Block1,2, and Jawaharlal M. Patel1,2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha /Akt (PKB-alpha ) 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-alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow mTOR right-arrow 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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha , and p70S6K; 3) to determine the phosphorylation status of ERK1/2, PKB-alpha , and p70S6K proteins; and 4) to monitor DNA synthesis.

Although not shown, treatment of cells with wortmannin, LY-294002, PD-98059, and rapamycin had no cytotoxic effects as determined by lactate dehydrogenase release and morphological observations (26).

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-alpha -phosphatidylinositol (Avanti, Alabaster, AL) in kinase buffer, and 5 µM [gamma 32P]ATP (25 Ci/mmol). The reactions were performed for 15 min at room temperature and terminated by the addition of 15 µl of 4 N HCl. Lipids were extracted with 160 µl of methanol/chloroform (1:1 vol/vol). After centrifugation (14,000 g, 10 min), the chloroform-containing lipid phase was reextracted with 150 µl of an equal volume mixture of 0.5 N HCl-methanol followed by vortex and centrifugation (14,000 g, 10 min). The chloroform extracts (10 µl) were then resolved by thin-layer chromatography (1% potassium oxalate-treated plates; Analteck, Newark, DE) in a chloroform-methanol-ammonium hydroxide (75:58:17 vol/vol/vol) solvent system as previously described (25). Detection of phosphorylated PI was performed by autoradiography on XAR film (Eastman Kodak). The bands were scanned using the Fluor-S MultImager system (Bio-Rad) to quantify PI content.

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 [gamma 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-alpha 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-alpha was immunoprecipitated by incubation at 4°C with 5 µg of anti PKB-alpha 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 [gamma 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-alpha 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 [gamma 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-alpha 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   ANG IV stimulation of phosphatidylinositol 3-kinase (PI3K) activity is inhibited by wortmannin (Wort) but not by rapamycin (Rap). Serum-starved cells were pretreated with or without 15 nM Rap or 0.1 µM Wort for 45 min before stimulation with 1 µM ANG IV or vehicle (control) for 15 min. PI3K activity was determined by a lipid kinase assay described in EXPERIMENTAL PROCEDURES. The reaction product phosphatidylinositol phosphate (PIP) was separated by thin-layer chromatography and visualized by autoradiography. A: representative autoradiogram; B: densitometric analysis of 4 separate autoradiograms shown in A. Values are means ± SE (n = 4). *P < 0.05 vs. control; #P < 0.05 vs. ANG IV or Rap + ANG IV.

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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Fig. 2.   ANG IV concentration- and time-dependent activation of PI-dependent kinase (PDK)-1. Cell monolayers were incubated with or without (control) ANG IV (1 µM) for 15-120 min (A) or with 0 (control)-5 µM ANG IV (B) at 37°C or were pretreated for 30 min with 100 nM Wort or 50 µM LY-294002 (LY) followed by ANG IV (1 µM) stimulation (C). After incubation, PDK-1 activity was determined as described in EXPERIMENTAL PROCEDURES. Values are means ± SE (n = 4 in A and B, n = 8 in C). *P < 0.05 vs. control.

Increased phosphorylation of PKB-alpha is associated with ANG IV-mediated activation of PKB-alpha . 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-alpha as early as 5 min (P < 0.01), and the increase persisted for 60 min (P < 0.05). ANG IV-mediated activation of PKB-alpha 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-alpha (Fig. 3).


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Fig. 3.   ANG IV-mediated increased phosphorylation and activation of protein kinase B-alpha /Akt (PKB-alpha ). Cell monolayers with or without [32P]orthophosphate labeling and with or without pretreatment with 50 µM LY or 100 nM Wort were stimulated with 1 µM ANG IV for 0 (control) to 60 min at 37°C. A: representative images of 3 separate immunoblots of PKB-alpha using PKB-alpha and Ser473 phospho-specific PKB-alpha antibodies in [32P]orthophosphate-unlabeled cells. B: the effects of LY and Wort on phosphorylation of PKB-alpha in [32P]orthophosphate-labeled cells. C: the effect of ANG IV on the catalytic activity of PKB-alpha . Data in C represent means ± SE (n = 4) for each time point. *P < 0.01 and **P < 0.05 vs. control.

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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Fig. 4.   ANG IV increases phosphorylation and activity of p70 ribosomal S6 kinase (p70S6K). Cell monolayers were prelabeled with [32P]orthophosphate (5 µCi/ml) for 2 h and then incubated with 1 µM ANG IV at 37°C for 15-60 min. After incubation, phosphorylated p70S6K was analyzed as described in EXPERIMENTAL PROCEDURES. A: representative autoradiograph of phosphorylated p70S6K. B: densitometric analysis of 4 separate autoradiographs shown in A. *P < 0.05 and **P < 0.001 vs. control. C: time-dependent effect of ANG IV stimulation on the catalytic activity of p70S6K measured by monitoring S6 peptide phosphorylation. *P < 0.05 vs. control. D: effects of the PI3K inhibitor Wort and the mammalian target of rapamycin kinase inhibitor Rap on ANG IV-mediated activation of p70S6K. *P < 0.05 vs. control and **P < 0.05 vs. ANG IV. Data in C and D represent means ± SE (n = 4) for each time point and/or treatment.



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Fig. 5.   Rap blocks ANG IV-mediated phosphorylation of p70S6K. Cell monolayers were metabolically labeled with 32P]orthophosphate (5 µCi/ml) for 2 h followed by incubation with or without Rap (15 nM) for 15 min and then stimulation by 1 µM ANG IV for 30 min at 37°C. After final incubation, phosphorylated p70S6K was analyzed as described in EXPERIMENTAL PROCEDURES. A: representative autoradiograph of phosphorylated p70S6K. B: densitometric analysis of 3 separate autoradiographs shown in A. *P < 0.05 vs. control and #P < 0.05 vs. ANG IV.

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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Fig. 6.   ANG IV-mediated phosphorylation and expression of extracellular signal-related kinase 1/2 (ERK1/2). Cell monolayers were pretreated with or without 10 µM PD-98059 or 15 nM Rap for 15 min followed by stimulation with 1 µM ANG IV for 60 min at 37°C. Cells were then processed for immunodetection using phospho-specific ERK1/2 (P-ERK-1/2) antibody or ERK1/2 antibody as described in EXPERIMENTAL PROCEDURES. Representative immunoblot of P-ERK1/2 (A) or ERK1/2 (B). C: densitometric analysis of 4 separate P-ERK1/2 immunoblots shown in A. Densitometric analysis of ERK1/2 expression from 4 separate blots shown in B was comparable with control irrespective of treatments (not shown). *P < 0.01 vs. respective controls; **P < 0.01 vs. control or ANG IV; #P < 0.01 vs. control or Rap.

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 right-arrow 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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Fig. 7.   Time- and ANG IV concentration-dependent cell proliferation. Subconfluent cell monolayers were incubated with or without (control) the presence of 5 µM ANG IV for 2-72 h (A) or with or without (0 time) the presence of varying concentrations (5 nM-50 µM) of ANG IV for 24 h (B) at 37°C. Incorporation of 5-bromo-2'-deoxy-uridine (BrdU) was monitored as described in EXPERIMENTAL PROCEDURES. Values are means ± SE; n = 4 for each ANG IV concentration or time point. *P < 0.05 and **P < 0.01 vs. control or 0 time.



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Fig. 8.   Pretreatment with Wort, Rap, or PD-98059 alone or Rap and PD-98059 in combination blocked ANG IV-induced cell proliferation. Serum-starved (24 h) subconfluent cells were incubated with or without (control) the presence of ANG IV (5 µM) for 12 h at 37°C. In some experiments: 1) serum-starved cells were pretreated with Wort (1 µM) or PD-98059 (PD, 10 µM) or Rap (15 nM) for 45 min followed by incubation with 1 µM ANG IV for 12 h at 37°C; 2) serum-starved cells were incubated with ANG IV for 4 h and then treated with Rap (15 nM) for 12 h (ANG IV + Rap); 3) serum-starved cells were pretreated with PD (10 µM) and Rap (15 nM) for 45 min followed by incubation with ANG IV (1 µM) for 12 h at 37°C (Rap + PD + ANG IV). After incubation, BrdU incorporation was monitored as described in EXPERIMENTAL PROCEDURES. Values are means ± SE; n = 6. *P < 0.01 vs. control; #P < 0.05 vs. ANG IV; **P < 0.01 vs. ANG IV.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow PDK-1/PKB right-arrow mTOR right-arrow p70S6K and Ras right-arrow MEK right-arrow 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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Fig. 9.   Critical signaling modules in ANG IV-induced lung endothelial cell proliferation. MEK-1/2 Raf-mitogen-activated protein kinase kinase 1/2; 4E-BP1, intuition factor 4E binding protein; mTOR, mammalian target of rapamycin; eIF-4E, eukaryotic translation initiation factor 4E.

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 right-arrow PDK-1/PKB right-arrow 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 right-arrow PDK-1/PKB right-arrow 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.


    ACKNOWLEDGEMENTS

We thank Bert Herrera for tissue culture assistance and Weihong Han and Dihua He for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 283(4):L707-L716