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J Appl Physiol 98: 722-731, 2005. First published October 22, 2004; doi:10.1152/japplphysiol.00715.2004
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HIGHLIGHTED TOPICS
Pulmonary Circulation and Hypoxia

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation

Evgenia V. Gerasimovskaya, Doug A. Tucker, and Kurt R. Stenmark

Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, Denver, Colorado

Submitted 9 July 2004 ; accepted in final form 18 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.

pulmonary hypertension; protein synthesis; environmental stress; oxygen sensing; vascular remodeling

CHRONIC EXPOSURE OF MOST MAMMALIAN SPECIES to reduced oxygen concentrations, either persistent or intermittent, results in the development of pulmonary hypertension. Experiments in several models demonstrate that, in response to moderate levels of global hypoxia (10–13% oxygen), proliferative responses of cells in the pulmonary artery adventitial compartment consistently precede and greatly exceed proliferation of smooth muscle cells (SMC) in the medial compartment (55). In culture, pulmonary artery adventitial fibroblasts have been shown to retain their proliferation ability in response to hypoxia in the absence of any exogenous mitogens (13, 44). The signaling pathways, which ultimately act to cause proliferation in this specific cell population under conditions of moderate hypoxia, remain unclear, although signaling through G proteins, MAPK, and PKC have been shown to be necessary (13, 21, 55). Improved insight into the mechanisms contributing to hypoxia-induced fibroblast proliferation could thus have a significant impact on our understanding of hypoxia-induced pulmonary vascular remodeling.

The phosphatidylinositol 3-kinase (PI3K) pathway has been shown to be important in growth, survival, and proliferation of a wide range of cell types (33, 60). Recent studies have demonstrated that PI3K signaling is involved in vascular SMC and endothelial cell mitogenesis in response to specific growth stimuli (7, 24, 30, 31, 53). Shigematsu et al. (53) have shown that activation of PI3K is essential for the initiation of medial vascular SMC replication after carotid balloon injury in rats. A direct role of PI3K in angiotensin II-induced DNA synthesis and proliferation has been described in cultured porcine carotid artery SMC (49). Importantly, there is indirect evidence that PI3K is involved in hypoxia-induced signaling. The PI3K pathway has been implicated in hypoxic induction of hypoxia-inducible factor-1{alpha} in certain transformed cell lines (29, 39, 62). However, in rat vascular SMC and endothelial cells, hypoxia-induced augmention of growth factor-induced proliferation occurs independent of PI3K (27). Currently, little or no information exists regarding the role of PI3K in regulating hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts, cells capable of serum-independent proliferation under hypoxic conditions (13, 21). Indirect assessment of PI3K activation through Akt phosphorylation is usually performed in vascular cells exposed to hypoxia. However, to our knowledge, there is no direct evidence for PI3K activity in response to hypoxia in these cells.

Several downstream targets of PI3K have been identified, including phosphoinositide-dependent kinase-1, serine/threonine kinase Akt, mammalian target of rapamycin (mTOR), and the p70 ribosomal protein S6 kinase (p70S6K) (15, 22, 49). All may be either dependent on or independent of upstream activation by PI3K in the context of cell proliferation. For example, a recent study showed that mTOR may be activated by hypoxia independent of PI3K and may be critical in mediating the augmented proliferation of SMC and endothelial cells under hypoxic conditions in the presence of growth factors (27). One downstream target of mTOR, p70S6K, increases activation of the ribosomal machinery and induces G1 phase-promoting genes such as cyclin-D. mTOR can also transmit hypoxia-induced signals via 4E-BP1 and other downstream targets. However, the role of mTOR, p70S6K, and 4E-BP1 in cell proliferation may be cell type and/or stimulus specific. For example, angiotensin II-induced proliferation in rat aortic SMC was shown to be dependent on PI3K and Akt signaling pathways, yet independent of mTOR. This suggests that, under certain conditions, mTOR and p70S6K are not downstream targets of PI3K in proliferating cells (18). The role of PI3K, mTOR, and p70S6K activation in mediating hypoxia-induced fibroblast proliferation has not been elucidated.

The purpose of this study was, therefore, to evaluate the role of PI3K and some of its potential downstream targets in hypoxia-induced adventitial fibroblast proliferation. Our approach was to utilize an established model cell system, in which cultured bovine pulmonary artery adventitial fibroblasts were serum starved for a period of 72–84 h and then exposed to hypoxia (3% oxygen) in the absence of exogenous mitogens. In this cell system, the adventitial fibroblasts consistently demonstrate increased growth responses to hypoxic challenge. Pharmacological inhibitors were utilized to evaluate the role of PI3K and its downstream targets in the proliferative response. In addition, kinase assays were performed to evaluate the direct effects of hypoxia on PI3 and p70S6 kinases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture.   Pulmonary artery adventitial fibroblasts were isolated from tissue explants of 120- to 180-day gestational bovine fetuses and cultured as previously described (21). Cells were grown in DMEM, supplemented with 20 mM L-glutamine (Cellgro), nonessential amino acids (1:100, vol/vol), 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma, St. Louis, MO), and 10% FBS (Gemini Bio-Products, Woodland, CA) and used between passages 2 and 6. For all assays, cells were plated at a density of 5 x 105 cells/cm2, cultured to 80% of confluence, growth arrested in DMEM without serum for 72–84 h, and then used for the experiments.

DNA synthesis.   DNA synthesis was determined by [methyl-3H]thymidine incorporation. Cells were plated in 24-well plates at a density of 12 x 10 3 cells/well in DMEM, supplemented with 10% FBS. On the next day, cells were rinsed with PBS and incubated in serum-free DMEM for 72 h. Cells were preincubated either with or without LY-294002 (20 µM, 60 min), wortmannin (100 nM, 2 h), rapamycin (5 nM, 60 min), U0126 (20 µM, 60 min) (Cell Signaling, Beverly, MA), or NL-71-101 (5 µM, 2 h) (Calbiochem, La Jolla, CA), and labeled with 1.0 µCi of [methyl-3H]thymidine (NEN Life Science Products, Boston, MA). Cells were then placed into a Bellco chamber filled with a 3% oxygen-5% CO2-92% N2 gas mixture for 24 h. At the end of incubation, fibroblasts were washed with PBS, incubated with 0.5 ml of 0.2 M perchloric acid, and lysed by 1% SDS/0.1 M NaOH. Samples were mixed with liquid scintillation cocktail (Ecoscint H, National Diagnostics, Atlanta, GA) and counted in a scintillation {beta}-counter (Beckman LS 6500).

Cell extracts and Western blot analysis.   Adventitial fibroblasts were cultured to near confluence and growth arrested in serum-free DMEM for 72–84 h. For the experiments with PI3K, Akt and mTOR inhibitor fibroblasts were preincubated with LY-294002, wortmannin, U0126, NL-71-101, or rapamycin as described in the figure legends. After stimulation with hypoxia (3% oxygen) for various periods of time, cells were washed twice with ice-cold PBS and lysed with Tris·HCl buffer (40 mM, pH 7.5, 4°C) containing 0.1% Triton X-100, 0.25 M sucrose, 3 mM EGTA, 3 mM EDTA, 50 µM {beta}-mercaptoethanol, 1 mM PMSF, and complete protease inhibitors cocktail (Calbiochem). Samples containing 10–25 µg of total cellular protein were subjected to 10% SDS-PAGE, transferred to PVDF membrane, and probed with rabbit polyclonal antibodies against phospho-p70S6K (Thr421/Ser424), phospho-S6 (Ser235/236), phospho-Akt (Ser473), phospho-ERK1/2 (Tyr202/Thr204), and phospho-4E-BP1 (Thr37/40) under conditions recommended by the manufacturer (Cell Signaling Technology, Beverly, MA). After washing with TBS-Tween buffer, membranes were incubated with mouse anti-rabbit peroxidase-conjugated IgG (Amersham; 1:10,000 dilution for 1 h at room temperature). Immunoreactive bands were detected by enhanced chemiluminesence kit (Renaissance, NEN Life Science Product) followed by exposure to Hyperfilm and quantitatively analyzed by Image J densitometry system.

Assay of in vitro PI3K activity.   Adventitial fibroblasts were grown to 80% of confluence in 100-mm2 dishes, growth arrested, and stimulated with hypoxia (3% oxygen). After stimulation, cells were washed two times with ice-cold PBS, containing 0.2 mM activated orthovanadate, and incubated in 750 µl of lysis buffer containing 0.1% Triton X-100, 137 mM NaCl, 20 mM Tris·HCl, pH 7.5, 1 mM MgCl2, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM PMSF, and complete protease inhibitor cocktail (Calbiochem) and incubated for 20 min. After a 20-min incubation at 4°C, cell lysates were centrifuged at 10,000 g for 10 min to sediment insoluble material. Supernatants were normalized for protein content (Bio-Rad assay kit) and were incubated with anti-p85 antibodies or anti-phospho-tyrosine antibodies (clone 4G10) linked to protein A-agarose (1 µg/0.5 mg of total cell protein) (Upstate Biotechnology, Lake Placid, NY). After rocking at 4°C for 1–2 h, agarose beads containing antibody-enzyme complexes were centifugated at 10,000 g for 20 s and washed three times with buffer A: 137 mM NaCl, 20 mM Tris·HCl, pH 7.5, 1 mM MgCl2, 1 mM CaCl2, 1% Nonidet P-40, 0.1 mM sodium orthovanadate; three times with buffer B: 0.1 mM Tris·HCl, pH 7.5, 5 mM LiCl, 0.1 mM sodium orthovanadate; and three times with buffer C: 10 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1 mM sodium orthovanadate. Immunoprecipitates were resuspended in 50 µl of buffer C and combined with 10 µl (20 µg) of sonicated phosphatidyinositol and 10 µl of 100 mM MgCl2. The phosphorylation reaction (final volume of 80 µl) was started by addition of 10 µl of mixture, containing 15 µCi [{gamma}-32P]ATP (10 mM, 3,000 Ci/mmol) and 0.88 µl of 10 mM ATP. The samples were incubated for 15 min at 37°C, and the reaction was stopped by the addition of 20 µl of 6 N HCl. Radiolabeled lipids were extracted with 160 µl of chloroform methanol (1:1, vol:vol). Organic phase was separated by centrifugation for 10 min at 12,000 g at 4°C, and lipids were separated on oxalate-coated silicon TLC plates (Silica Gel 60, Sigma) by chromatography in CH3:MeOH:H2O:NH4OH (60:40:11.3:2, vol:vol) solvent system in parallel with nonradioactive standard. Radioactive spots were revealed by autoradiography and quantitatively analyzed by Image J densitometry system.

Assay of in vitro p70S6K activity.   The p70S6K immunocomplex kinase assay was performed according to the manufacturer's protocol with small modifications (Upstate Biotechnology). Quiescent fibroblasts in 100-mm dishes were growth arrested as indicated above and stimulated with hypoxia (3% oxygen) for the indicated periods of time, washed twice with ice-cold PBS, and solubilized in 700 µl of lysis buffer (50 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 120 mM NaCl, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1% {beta}-mercaptoethanol, 1% Triton X-100, 0.1 mM PMSF, complete protease inhibitors cocktail). Lysates were incubated for 20 min on ice and centrifuged at 12,000 g for 15 min to sediment insoluble material and were precleaned with 40 µl of protein A/G Agarose Plus (Santa Cruz, CA). Equivalent amounts (350 µg) of total cell protein were incubated with 2.5 µg of sheep polyclonal anti-p70S6K antibodies (Upstate Biotechnology), rocking at 4°C for 2 h, and further with 40 µl of protein A-Sepharose for 4°C for 1–2 h. Beads containing antibody-enzyme complexes were centrifuged at 10,000 g for 20 s and washed twice with lysis buffer containing 0.5 M NaCl, twice with lysis buffer alone, and twice with assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM {beta}-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT). Kinase reaction was carried out for 10–30 min at 37°C with S6 ribosomal protein peptide (AKRRRLSSLRA) as a substrate. p70S6K activity was determined as picomoles of phosphate incorporated into substrate per minute.

Data analysis.   Density of bands from Western and Northern blot film images was determined by using National Institutes of Health Image J program. Data are expressed as means ± SE; n equals the number of replicates in one experiment or a number of observations in independent experiments. To evaluate significance of the obtained data, Student-Newman-Keuls test followed by one-way ANOVA was performed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Hypoxia-induced fibroblast proliferation requires PI3K, ERK1/2, and mTOR activation.   Consistent with our previously reported data, exposure of serum-starved fibroblasts (72 h, serum-free DMEM) to hypoxic conditions (3% oxygen, 24 h) caused significant increases in DNA synthesis (13, 21). To assess the importance of the PI3K-Akt pathway in this response, [H3]thymidine incorporation was measured in the presence of pharmacological inhibitors of PI3K (LY-294002 and wortmannin). Both agents inhibited hypoxia-induced DNA synthesis (Fig. 1). LY-294002 was also noted to cause a significant reduction in basal [H3]thymidine incorporation under normoxic conditions. To investigate whether stimulation of Akt is necessary for hypoxia-induced proliferative responses, we examined the effect of the newly developed Akt inhibitor NL-71-101. This compound has been shown to be an ATP competitive inhibitor, which prevents phosphorylation of the Akt substrate GSK3 (46). Our data showed that hypoxia-induced increase in DNA synthesis in adventitial fibroblasts was completely blocked in the presence of NL-71-101.



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  		Fig. 1. Hypoxia induces DNA synthesis in adventitial fibroblasts through phosphatidylinositol 3-kinase (PI3K)-, mammalian target of rapamycin (mTOR)/p70S6K-, and ERK1/2-dependent signaling pathways. Quiescent adventitial fibroblasts (72 h, DMEM without serum) were preincubated with wortmannin (100 nM, 2 h), LY-294002 (20 µM, 60 min), rapamycin (5 nM, 60 min), NL-71-101 (5 µM, 2 h), or U0126 (10 µM, 60 min) followed by stimulation with hypoxia (3% O2) in the presence of 1 µCi of [3H]thymidine per well for 24 h. Incorporated radioactivity was determined in cell lysates as described in MATERIALS AND METHODS. Results are expressed as percentage of basal thymidine incorporation in control cells [1,024 ± 96 counts/million (cpm)/10,000 cells]. Data are means ± SE from 3 independent experiments. Cont, control. *P < 0.05 vs. control.

 
Because in certain cell types PI3K activation leads to mitogen-activated protein kinase kinase 1/2 and ERK1/2 phosphorylation, we evaluated the effect of U0126, a specific mitogen-activated protein kinase kinase 1/2 inhibitor, on hypoxia-induced fibroblast proliferation. We found that U0126 was as effective at blocking hypoxia-induced DNA synthesis in adventitial fibroblasts as the PI3K inhibitors (LY-294002 and wortmannin). This is consistent with our laboratory's previous observations demonstrating that ERK1/2 activation is necessary for hypoxia-induced adventitial fibroblast proliferation (13, 21).

To determine whether mTOR was involved in hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts, we evaluated the effect of rapamycin, a potent pharmacological inhibitor of mTOR. We found that rapamycin completely abolished hypoxia-induced DNA synthesis. It should be noted that the inhibitors used in this study were found not to affect cell viability, as assessed by trypan blue exclusion or lactate dehydrogenase release during the 24-h incubation.

Hypoxia increases PI3K activity and Akt phosphorylation in adventitial fibroblasts.   Although many inhibitor studies have suggested that activation of PI3K is involved in hypoxia-mediated responses, there is little direct evidence for increases in PI3K activity under hypoxic conditions in vascular wall cells. We therefore investigated whether moderate (3% oxygen) hypoxic exposure caused activation of PI3K in adventitial fibroblasts. We found that within 10–30 min of hypoxic exposure, PI3K activity associated with p85 regulatory subunit and with phosphotyrosine immunoprecipitates was increased severalfold (Fig. 2). These data, along with the pharmacological data, support a role for PI3K in hypoxia-induced adventitial fibroblast proliferation.



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  		Fig. 2. Hypoxia induces time-dependent increase in PI3K activity in adventitial fibroblasts. A: growth-arrested cells were exposed to 3% O2 for the indicated times. Equivalent amounts of total cell protein (350–500 µg) were immunoprecipitated with rabbit polyclonal antibodies against the p85{alpha} subunit of PI3K or with anti-phosphotyrosine mouse monoclonal antibodies (4G10). Lipid kinase activity was determined as described in MATERIALS AND METHODS with a L-{alpha}-phosphatidylinositol as a substrate. Ptdins(3)P, product of PI3K activity and L-{alpha}-phosphatidylinositol-3 phosphate; IP, intraperitoneal. B: densitometric analysis of time-dependent formation of PtdIns(3)P. Spots, corresponding to 32P-phospholipids, were verified using unlabeled lipid standards. Data are means ± SE from 4 independent experiments. *P < 0.05 compared with nonstimulated control.

 
Because Akt is a major downstream target of PI3K involved in proliferative responses to mitogenic agonists, we examined the effects of hypoxia on Akt phosphorylation. We found that hypoxia induced a transient phosphorylation of Akt at Ser473 with a maximal effect observed at 30 min (Fig. 3).



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  		Fig. 3. Hypoxia induces Akt phosphorylation in adventitial fibroblasts. Quiescent cells were exposed to hypoxia (3% O2) for the indicated times. Equivalent amounts of total cell protein (40 µg) were subjected to Western blot analysis with antibodies against phosphorylated Akt (top). Phospho-Akt was normalized to total Akt, and quantitative data were expressed as percent of basal level (bottom). Data are means ± SE from 3 independent experiments. *P < 0.05 compared with nonstimulated control.

 
PI3K and ERK1/2 pathways act independently in hypoxia-induced fibroblast proliferation.   Because inhibitors of PI3K and ERK1/2 (LY-294002, wortmannin, and U0126, respectively) reduced hypoxia-stimulated cell responses to nearly the same extent, we examined whether PI3K was required for ERK1/2 activation under hypoxic conditions. We found that hypoxia consistently induced phosphorylation of ERK1/2 by severalfold (Fig. 4). The PI3K inhibitor wortmannin had no significant effect on hypoxia-induced ERK1/2 phosphorylation, indicating that hypoxia initiates independent activation of ERK1/2 and PI3K. We also found that inhibitors of ERK pathway, PD-98059 and U0126, were not effective in blocking hypoxia-induced PI3K activation (not shown). These data are consistent with the findings described for thrombin-, ATP-, and angiotensin II-induced proliferative responses, in which PI3K and ERK1/2 are shown to act as independent signaling pathways, both necessary for cell proliferation (18, 24, 58).



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  		Fig. 4. PI3K is not involved in hypoxia-induced ERK1/2 phosphorylation in adventitial fibroblasts. Growth-arrested adventitial fibroblasts were pretreated with wortmannin (100 nM, 2 h) or vehicle (DMSO, 1:1,000) and stimulated with hypoxia (3% O2) for 10 min. After stimulation, 10 µg of total cell protein were subjected to Western blot analysis with anti-phospho-ERK1/2 (Tyr202/Thr204) antibodies. Data of one typical experiment are shown (top). Phospho-ERK1/2 was normalized to total ERK1/2, and quantitative data are expressed as percent of basal level (bottom). Data are means ± SE from 5 independent experiments. *P < 0.05 compared with nonstimulated control.

 
Hypoxia induces phosphorylation of mTOR, p70S6K, and 4E-BP1 in adventitial fibroblasts.   The data shown in Fig. 1 demonstrate that rapamycin inhibits hypoxia-induced cell proliferation and thus strongly suggest that mTOR is an important regulator of hypoxia-induced cell proliferation. The sensitivity of mTOR to hypoxia was further confirmed by Western blot analyses with phospho-specific mTOR antibodies that recognize the activated state of the protein (Fig. 5A). We found that exposure of adventitial fibroblasts to hypoxia resulted in elevated mTOR phosphorylation at both Ser2448 and Ser2481 in a time-dependent manner, with the maximal responses at 10 and 30 min, respectively.



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  		Fig. 5. Hypoxia activates mTOR, p70S6K, and 4E-BP1 in adventitial fibroblasts. Adventitial fibroblasts were growth arrested as described above and exposed to hypoxia (3% O2) for the indicated time. Forty micrograms of total cell protein were analyzed by Western blotting with anti-phospho-mTOR (Ser2481) and anti-phospho-mTOR (Ser2448) (A), anti-phospho-p70S6K (Thr421/Ser424) (B) or with anti-phospho-4E-BP1 (Thr37/40) antibodies (C). Phospho-mTOR, phospho-p70SK, and phospho-4E-BP1 were normalized to total mTOR, p70S6K, and 4E-BP1, respectively. Quantitative data (bar graphs) are expressed as percent of basal level. Data are means ± SE from 3–6 independent experiments. D: growth-arrested adventitial fibroblasts were preincubated either with rapamycin (5 nM, 60 min) or vehicle (methanol, 1:1,000) and stimulated with hypoxia (3% O2) for indicated times. After stimulation, total cells lysated were prepared, and 350 µg of protein were immunoprecipitated with anti-p70S6K antibodies for kinase assays as described in MATERIALS AND METHODS. p70S6K activity is expressed in relative units; basal activity corresponds to 80.02 pmol Pi incorporated into substrate/min. Data are means ± SE from 5 independent experiments performed on 3 separate cell populations. *P < 0.05 compared with nonstimulated control.

 
Two downstream targets of mTOR believed to be important for cell growth are p70S6K and 4E-BP1. We therefore evaluated the effects of hypoxia on p70S6K and 4E-BP1 phosphorylation and found that hypoxia caused a significant increase in phosphorylation of p70S6K at Thr421/Ser424. This effect was evident at 30 min and was sustained for at least 120 min (Fig. 5B). Similarly, hypoxia caused an increase in 4E-BP1 phosphorylation at Thr37/40, with a maximal effect observed at 30 min (Fig. 5C).

To further investigate the effect of hypoxia on the protein translation pathway, we sought to determine whether an increase in p70S6K phosphorylation was associated with an increase in p70S6K activity. To answer this question, we performed an in vitro kinase assay using the peptide corresponding to S6 ribosomal protein as a substrate. Exposure of adventitial fibroblasts to hypoxia resulted in a time-dependent increase in p70S6K activity (Fig. 5D). The maximal effect of hypoxia on p70S6K activation was observed at 30 min, a finding consistent with the Western blot data demonstrating a time-dependent p70S6K phosphorylation.

Hypoxia induces phosphorylation of p70S6K through mTOR-, PI3K-, and ERK1/2-dependent pathways.   To determine the relationship between PI3K, mTOR, and ERK1/2 pathways in mediating hypoxia-induced activation of p70S6K, we evaluated the effects of the PI3K inhibitor LY-294002, the mTOR inhibitor rapamycin, and the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 on p70S6K phosphorylation. We found that each of these compounds markedly attenuated the hypoxia-induced p70S6K phosphorylation (Fig. 6A). In addition, treatment of cells with the specific Akt inhibitor NL-71-101 resulted in a dramatic decrease in hypoxia-induced p70S6K phosphorylation (Fig. 6B). We also found that LY-294002 and rapamycin both significantly reduced phosphorylation of S6 ribosomal protein (Fig. 7), suggesting that hypoxia-induced activation of the protein translation pathway in adventitial fibroblasts is mediated through mTOR and PI3K.



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  		Fig. 6. PI3K and mTOR are involved in hypoxia-induced p70S6K phosphorylation. Quiescent adventitial fibroblasts were preincubated either with LY-294002 (20 µM, 60 min), rapamycin (5 nM, 60 min), or vechicle (methanol, 1:1,000) (A), or with NL-71-101 (5 µM, 2 h) (B) followed by stimulation with hypoxia (3% O2) or 10% FBS for 30 min. Equivalent amounts of protein (20–40 µg) were subjected to Western blot analysis with anti-phospho-p70S6K (Thr421/Ser424) antibodies. Phospho-p70S6K was normalized to total p70S6K, and values are expressed as percent of the basal level. Quantitative data are means ± SE from 3–6 independent experiments performed on separate cell populations. *P < 0.05 compared with nonstimulated control.

 


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  		Fig. 7. PI3K and mTOR m are involved in hypoxia-induced phosphorylation of S6 ribosomal protein. Quiescent adventitial fibroblasts were preincubated either with LY-294002 (20 µM, 60 min), rapamycin (5 nM, 60 min), or vehicle followed by stimulation with hypoxia (3% O2) or 10% FBS for 30 min. Equivalent amounts of protein (20–40 µg) were analyzed by Western blotting with anti-phospho-S6 (Ser235/236) antibodies (top). Phospho-S6 was normalized to nonphosphorylated S6, and quantitative data are expressed as percentage of the basal level. Data are means ± SE from 3–5 independent experiments. *P < 0.05 compared with nonstimulated control (bottom).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
It is generally assumed that living organisms respond to reduced oxygen concentrations in a cell type- and tissue-specific manner. Pulmonary artery adventitial fibroblasts possess the rather distinct capability, at least for nontransformed cells, to proliferate in response to hypoxia (3% oxygen) independent of exogenous growth factors (44, 51, 55). This capability distinguishes adventitial fibroblasts from many other cell types that often undergo metabolic suppression and inhibition of protein synthesis (hibernation) in response to hypoxia. The goal of this study was to understand the molecular mechanisms that contribute to the autonomous proliferative phenotype of adventitial fibroblasts under hypoxic conditions. Specifically, we were interested in determining the sensitivity of PI3K, Akt, and mTOR to hypoxic stimulation and in understanding the role of these pathways in hypoxia-induced proliferative responses. We provide definitive evidence that hypoxia (3% oxygen) directly activates PI3K signaling in these cells, which ultimately leads to mTOR activation. Activation of the mTOR-p70S6 pathway in fact appears to be a necessary step and a key point of convergence in ERK1/2 and PI3K-Akt pathways that regulate cell cycle progression under hypoxic conditions. These data, when taken in context with our laboratory's previous work demonstrating the importance of G{alpha}i in hypoxia-induced proliferation (13, 21), support a model of hypoxia-induced proliferation of adventitial fibroblasts as shown in Fig. 8.



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  		Fig. 8. Schematic diagram demonstrates intracellular signaling pathways involved in hypoxia-induced adventitial fibroblast proliferation. Hypoxia initiates, potentially through ligand-independent activation of G protein-coupled receptors, PI3K/Akt-, mTOR/p70S6K-, and ERK1/2-dependent signaling pathways that mediate the proliferation of adventitial fibroblasts. PI3K and ERK1/2 pathways are activated by hypoxia independently. PI3K activates Akt, which, in turn, is involved in the cross talk between PI3K and mTOR/p70S6K pathways. ERK1/2 contributes to activation of mTOR/p70S6K pathway by hypoxia, at least on the level of p70S6K. Additional signaling mechanisms are likely involved in control of the translational machinery (box with a question mark). The accumulative effects of PI3K, Akt, mTOR, p70S6K, and ERK1/2 integrate transcriptional and translational pathways that ultimately lead to fibroblast proliferation under hypoxic conditions.

 
The involvement of PI3K-Akt and mTOR pathways in mitogenic responses has been shown in various cell types, including vascular endothelial cells and SMC (32, 33, 49, 54, 61). In many tissues, activation of PI3K, Akt, and mTOR is associated with hypoxia-inducible factor-1{alpha}-dependent VEGF expression, implicating involvement of these kinases in hypoxia-induced angiogenesis (30, 39, 62). However, to our knowledge, no evidence has been presented that demonstrates a role of PI3K in hypoxia-induced cell proliferation. Our studies revealed that hypoxia-induced increases in DNA synthesis in adventitial fibroblasts are highly sensitive to PI3K inhibitors, indicating that PI3K is a critical signaling component in this response (Fig. 1). It is known that several structurally distinct PI3K isoforms are selectively activated in response to various stimuli (33, 60). Class I PI3K{alpha} is activated by receptor tyrosine kinases, whereas PI3K{beta} is synergistically activated by receptor tyrosine kinases and G-protein {beta}{gamma} subunits (38, 43). We previously found that p110{alpha}, p110{beta}, and p85 PI3K subunits are expressed in adventitial fibroblasts (data not shown). The current studies demonstrate that hypoxia-induced PI3K activity can be measured in an in vitro kinase assay after immunoprecipitation with anti-p85 regulatory subunit and phospho-tyrosine antibodies (Fig. 2), suggesting that PI3K{alpha}, as well as PI3K{beta}, are involved in hypoxia-initiated responses in adventitial fibroblasts.

Akt is a downstream target of PI3K known to play a central role in cell growth and survival (14, 31, 54). Consistent with the stimulatory effect of hypoxia on PI3K activity, Akt phosphorylation is also increased under exposure of adventitial fibroblasts to hypoxia. The time course of hypoxia-induced Akt phosphorylation is similar to the early Akt activation observed in response to G protein-coupled receptor stimulation (Refs. 23, 28, 45, and our unpublished data), but it is different from the hypoxia-induced responses observed in rat pheochromocytoma-12 cells or in U373 cells, which showed prolonged and sustained Akt phosphorylation, implicating PI3K-Akt pathways in cell survival (1, 5, 63). The underlying mechanisms through which Akt may be involved in cell proliferation include Akt-dependent phosphorylation, inactivation, and subcellular redistrubution of p21Cip1 and p27kip1 proteins, which are known as inhibitors of cell cycle progression, as well as accumulation of cyclin D1 mediated by GSK-3{beta} (17, 37, 47). In addition to existing data demonstrating serum- and growth factor-induced Akt activation, one recent report clearly demonstrates a role of Akt in angiotensin II-induced proliferation of rat aortic SMC (18). In agreement with these observations, we found that the Akt inhibitor NL-71-101 (5 µM) completely blocked hypoxia-induced DNA synthesis in adventitial fibroblasts. We also found that NL-71-101 at concentrations of ≥10 µM resulted in reduced cell viability under basal conditions, potentially implicating Akt in survival responses in these cells as well. Interestingly, in differentiated SMC, which in contrast to adventitial fibroblasts do not proliferate in response to hypoxia, treatment with the same concentrations of NL-71-101 (≥10 µM) or transfecting a dominant-negative Akt mutant did not affect cell viability under basal conditions (data not shown). Because Akt can participate in both mitogenic and survival pathways, these observations, together with our data showing that PI3K inhibitors decrease the basal rate of DNA synthesis (Fig. 1), are compatible with the idea that hypoxia-proliferative adventitial fibroblasts exhibit a basal or "constitutive" activation of PI3K and Akt and that maintenance of this pathway might be a unique aspect of specific populations of cells capable of exhibiting hypoxia-induced proliferative responses.

mTOR has been shown to play a central role in regulating cell growth, homeostasis, and proliferation (15, 20, 49). Our study demonstrates that mTOR is a critical target of hypoxia, important in mediating hypoxia-induced proliferative responses in adventitial fibroblasts (Fig. 1). We found that hypoxia significantly increases phosphorylation of mTOR as well as its downstream targets p70S6K and 4E-BP1. Moreover, using an in vitro kinase assay (Fig. 5B), we found that hypoxia dramatically induces (up to 9-fold) p70S6K activity. It is remarkable that the magnitude of this response is comparable to the response to 10% serum (up to 11-fold over the basal level, used in our study as a control for maximal stimulation). Although the effect of hypoxia on mTOR phosphorylation is not as dramatic, the sensitivity of hypoxia-induced activation of p70S6K to rapamycin clearly indicates that mTOR is an upstream regulator of p70S6K under hypoxic conditions in these cells. We also found that exposure of adventitial fibroblasts to hypoxia leads to phosphorylation of S6 ribosomal protein, again indicating the importance of protein translational pathways in hypoxia-induced proliferative responses. The sensitivity of mTOR to hypoxic stimulation reported in this study is consistent with the data of Humar et al. (27), demonstrating mTOR-dependent sprout formation and enhanced endothelial cell proliferation in response to hypoxia, as well as with data of Hudson et al. (26), demonstrating a key role of mTOR in hypoxia- and CoCl2-induced hypoxia-inducible factor-1{alpha} expression in pheochromocytoma-3 cells.

The results of the present study show that hypoxia-induced phosphorylation of p70S6K and its downstream target S6 ribosomal protein is significantly decreased by PI3K, ERK1/2, and mTOR inhibitors, indicating the requirement of all these pathways for the response (Figs. 6–8). In addition, the observation that specific Akt inhibitor NL-71-101 completely attenuated hypoxia-induced p70S6K phosphorylation suggests an intermediary role of Akt in PI3K-dependent regulation of 70S6K. A role for Akt in the cross talk between PI3K and mTOR-p70S6K pathways has been shown in several studies in other cell types (8, 41, 52, 59). In addition, there is evidence that mTOR and PI3K pathways may operate in parallel and converge on common downstream targets to support cell growth and proliferation (20). Current knowledge in the field suggests that mTOR may operate as a nutrient and energy sensor to permit mitogens to stimulate cell proliferation. However, specific and coordinated signaling cross talk between mTOR and PI3K pathways is generally believed to occur in a cell type- and stimulus-specific manner, and thus further studies are required for better understanding the pathways specifically involved in linking hypoxia to protein translation in pulmonary artery adventitial fibroblasts. In this regard, it should also be noted that, in our cells, hypoxia induces PI3K-independent activation of ERK1/2 (Fig. 4), a signaling pathway that appears to converge at p70S6K to support the proliferative response. Similar interactions between ERK1/2, PI3K, and p70S6K pathways have been reported in vascular cells and hepatocytes in response to other mitogenic stimuli (12, 35, 58).

In agreement with our studies showing a mitogenic effect of hypoxia (3% oxygen) in adventitial fibroblasts, data by Cogo et al. (10) and Cooper and Beasley (11) have showed that modest hypoxia (5% oxygen) was pro-proliferative for vascular and airway SMC, whereas very low oxygen tension (1% oxygen) resulted in a decrease of SMC proliferation (10). It should also be noted that, in contrast to adventitial fibroblasts, other cell types respond to hypoxia (1–2% oxygen) or anoxia (0% oxygen) by inhibiting protein synthesis and stimulating adaptive cellular pathways (6, 25). For example, in HEK 293 cells, severe hypoxia (1.5% oxygen) attenuated insulin-induced phosphorylation of mTOR and its downstream targets 4E-BP1, p70S6K, S6 ribosomal protein, and eIF4G (2). Although the effect of 1.5% oxygen on HEK 293 cell proliferation was not reported, it is likely that the growth response in these cells would not be increased. Therefore, the effects of hypoxia on HEK 293 cells are more compatible with an adaptive response, whereas adventitial fibroblasts, as shown in this study, exhibit a proliferative response under nearly the same oxygen concentrations (1–3% oxygen) and, not suprisingly, with a different activation profile of signaling intermediates. Basically, because an increase in protein synthesis is necessary for cell proliferation, a positive correlation between these two responses in hypoxia-proliferative cells is expected. The ability of the adventitial fibroblast to proliferate in response to hypoxia and to activate mTOR/p70S6K/S6 protein translational pathways supports this notion. Although not directly evaluated, adventitial fibroblasts might be expected to exhibit metabolic similarities to an oxygen-conforming skeletal muscle cell line (C2C12) in which hypoxic exposure does not result in a supression of protein synthesis (3). Additional data obtained in animal and cell models suggest that hypoxic exposure is associated with enhanced synthesis of extracellular matrix proteins (19, 40, 55, 56) as well as stress-related proteins, including transcription factors and proteins of energy metabolism, all of which are known to be necessary to maintain cell homeostasis (36). Given the above data, it is important to note that, despite the diversity in the hypoxic regulation of the protein translational pathway (mTOR, p70S6K, and S6 protein), a remarkable feature is the capability of these kinases to respond to changes in oxygen concentration.

In conclusion, this study demonstrates that PI3K-Akt, mTOR-p70S6K, and ERK1/2 signaling pathways are key signaling components of hypoxia-induced proliferative responses in pulmonary artery adventitial fibroblasts (Fig. 8). However, intriguing questions remain with regard to exactly how hypoxia initiates activation of individual signaling proteins in cells capable of proliferating under conditions of moderate hypoxia. An essential role for reactive oxygen species as mediators of stimulation of PI3K, Akt, and p70S6K has been proposed in various cell types, including fibroblasts (4, 34, 50, 57). There are data demonstrating redox- and reactive oxygen species-dependent activation of receptor tyrosine kinase and G protein-coupled signaling pathways (9, 16, 42). Because in adventitial fibroblasts both PI3K and p70S6K pathways can be regulated through G proteins, it will be of interest to test the possibility that G proteins can be activated in response to hypoxia in a ligand- and/or receptor-independent manner (Ref. 13 and our unpublished observations). Future understanding of the fundamental mechanisms underlying hypoxic activation of signal transducing proteins and their assemby into signaling networks should have significant implications for therapeutic strategies directed at specific targeting proteins critical for cell proliferation under hypoxic conditions.


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This work was supported by Specialized Center of Research Grant HL-57144 and Program Project Grant 14985 from the National Heart, Lung, and Blood Institute.


   ACKNOWLEDGMENTS
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We thank Dr. Ivan McMurtry, Dr. Raphael Nemenoff, Dr. Maria Frid, Dr. Mark Banks, and Dr. Lynne Bemis for critical reading of this manuscript. We are grateful to Stephen Hofmeister and Victoriya Marusyk for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Stenmark, Professor of Pediatrics, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Box B131, Denver, CO 80262 (E-mail: Kurt.Stenmark{at}UCHSC.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.


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  1. Alvarez-Tejado M, Naranjo-Suarez S, Jimenez C, Carrera AC, Landazuri MO, and del Peso L. Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis. J Biol Chem 276: 22368–22374, 2001.[Abstract/Free Full Text]
  2. Arsham AM, Howell JJ, and Simon MC. A novel hypoxia-inducible factor-independent hypoxic response regulating mammalian target of rapamycin and its targets. J Biol Chem 278: 29655–29660, 2003.[Abstract/Free Full Text]
  3. Arthur PG, Giles JJ, and Wakeford CM. Protein synthesis during oxygen conformance and severe hypoxia in the mouse muscle cell line C2C12. Biochim Biophys Acta 1475: 83–89, 2000.[Medline]
  4. Bae GU, Seo DW, Kwon HK, Lee HY, Hong S, Lee ZW, Ha KS, Lee HW, and Han JW. Hydrogen peroxide activates p70(S6k) signaling pathway. J Biol Chem 274: 32596–32602, 1999.[Abstract/Free Full Text]
  5. Beitner-Johnson D, Rust R, Hsieh TC, and Millhorn DE. Hypoxia activates Akt and induces phosphorylation of GSK-3 in PC12 cells. Cell Signal 13: 23–27, 2001.[CrossRef][ISI][Medline]
  6. Boutilier RG and St-Pierre J. Surviving hypoxia without really dying. Comp Biochem Physiol A Mol Integr Physiol 126: 481–490, 2000.[CrossRef][Medline]
  7. Braun-Dullaeus RC, Mann MJ, Seay U, Zhang L, von Der Leyen HE, Morris RE, and Dzau VJ. Cell cycle protein expression in vascular smooth muscle cells in vitro and in vivo is regulated through phosphatidylinositol 3-kinase and mammalian target of rapamycin. Arterioscler Thromb Vasc Biol 21: 1152–1158, 2001.[Abstract/Free Full Text]
  8. Cheatham L, Monfar M, Chou MM, and Blenis J. Structural and functional analysis of pp70S6k. Proc Natl Acad Sci USA 92: 11696–11700, 1995.[Abstract/Free Full Text]
  9. Chen EY, Mazure NM, Cooper JA, and Giaccia AJ. Hypoxia activates a platelet-derived growth factor receptor/phosphatidylinositol 3-kinase/Akt pathway that results in glycogen synthase kinase-3 inactivation. Cancer Res 61: 2429–2433, 2001.[Abstract/Free Full Text]
  10. Cogo A, Napolitano G, Michoud MC, Barbon DR, Ward M, and Martin JG. Effect of hypoxia on rat airway smooth muscle cell proliferation. J Appl Physiol 94: 1403–1409, 2003.[Abstract/Free Full Text]
  11. Cooper AL and Beasley D. Hypoxia stimulates proliferation and interleukin-1{alpha} production in human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 277: H1326–H1337, 1999.[Abstract/Free Full Text]
  12. Coutant A, Rescan C, Gilot D, Loyer P, Guguen-Guillouzo C, and Baffet G. PI3K-FRAP/mTOR pathway is critical for hepatocyte proliferation whereas MEK/ERK supports both proliferation and survival. Hepatology 36: 1079–1088, 2002.[CrossRef][ISI][Medline]
  13. Das M, Bouchey DM, Moore MJ, Hopkins DC, Nemenoff RA, and Stenmark RK. Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on G protein-mediated activation of mitogen-activated protein kinases. J Biol Chem 276: 15631–15640, 2001.[Abstract/Free Full Text]
  14. Datta SR, Brunet A, and Greenberg ME. Cellular survival: a play in three Akts. Genes Dev 13: 2905–2927, 1999.[Free Full Text]
  15. Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, and Thomas G. Mammalian TOR: a homeostatic ATP sensor. Science 294: 1102–1105, 2001.[Abstract/Free Full Text]
  16. Deora AA, Win T, Vanhaesebroeck B, and Lander HM. A redox-triggered ras-effector interaction. Recruitment of phosphatidylinositol 3'-kinase to Ras by redox stress. J Biol Chem 273: 29923–2998, 1998.[Abstract/Free Full Text]
  17. Diehl JA, Cheng M, Roussel MF, and Sherr CJ. Glycogen synthase kinase-3{beta} regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12: 3499–3511, 1998.[Abstract/Free Full Text]
  18. Dugourd C, Gervais M, Corvol P, and Monnot C. Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation. Hypertension 41: 882–890, 2003.[Abstract/Free Full Text]
  19. Falanga V, Zhou L, and Yufit T. Low oxygen tension stimulates collagen synthesis and COL1A1 transcription through the action of TGF-{beta}1. J Cell Physiol 191: 42–50, 2002.[CrossRef][ISI][Medline]
  20. Fingar DC and Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23: 3151–3171, 2004.[CrossRef][ISI][Medline]
  21. Gerasimovskaya EV, Ahmad S, White CW, Jones PL, Carpenter TC, and Stenmark KR. Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J Biol Chem 277: 44638–44650, 2002.[Abstract/Free Full Text]
  22. Gingras AC, Raught B, and Sonenberg N. Regulation of translation initiation by FRAP/mTOR. Genes Dev 15: 807–826, 2001.[Free Full Text]
  23. Goel R and Baldassare JJ. {beta}-Arrestin 1 couples thrombin to the rapid activation of the Akt pathway. Ann NY Acad Sci 973: 138–141, 2002.[Abstract/Free Full Text]
  24. Goncharova EA, Ammit AJ, Irani C, Carroll RG, Eszterhas AJ, Panettieri RA, and Krymskaya VP. PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 283: L354–L363, 2002.[Abstract/Free Full Text]
  25. Hochachka PW, Buck LT, Doll CJ, and Land SC. Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack. Proc Natl Acad Sci USA 93: 9493–9498, 1996.[Abstract/Free Full Text]
  26. Hudson CC, Liu M, Chiang GG, Otterness DM, Loomis DC, Kaper F, Giaccia AJ, and Abraham RT. Regulation of hypoxia-inducible factor 1{alpha} expression and function by the mammalian target of rapamycin. Mol Cell Biol 20: 7004–7014, 2002.
  27. Humar R, Kiefer FN, Berns H, Resink TJ, and Battegay EJ. Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling. FASEB J 16: 771–780, 2002.[Abstract/Free Full Text]
  28. Huwiler A, Rolz W, Dorsch S, Ren S, and Pfeilschifter J. Extracellular ATP and UTP activate the protein kinase B/Akt cascade via the P2Y(2) purinoceptor in renal mesangial cells. Br J Pharmacol 136: 520–529, 2002.[CrossRef][ISI][Medline]
  29. Jiang BH, Jiang G, Zheng JZ, Lu Z, Hunter T, and Vogt PK. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ 12: 363–369, 2001.[Abstract/Free Full Text]
  30. Jiang BH, Zheng JZ, Aoki M, and Vogt PK. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells. Proc Natl Acad Sci USA 97: 1749–1753, 2000.[Abstract/Free Full Text]
  31. Jung F, Haendeler J, Goebel C, Zeiher AM, and Dimmeler S. Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death. Cardiovasc Res 48: 148–157, 2000.[Abstract/Free Full Text]
  32. Kanda S, Hodgkin MN, Woodfield RJ, Wakelam MJ, Thomas G, and Claesson-Welsh L. Phosphatidylinositol 3'-kinase-independent p70 S6 kinase activation by fibroblast growth factor receptor-1 is important for proliferation but not differentiation of endothelial cells. J Biol Chem 272: 23347–23353, 1997.[Abstract/Free Full Text]
  33. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, and Waterfield MD. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17: 615–675, 2001.[CrossRef][ISI][Medline]
  34. Kosmidou I, Xagorari A, Roussos C, and Papapetropoulos A. Reactive oxygen species stimulate VEGF production from C2C12 skeletal myotubes through a PI3K/Akt pathway. Am J Physiol Lung Cell Mol Physiol 280: L585–L592, 2001.[Abstract/Free Full Text]
  35. Krymskaya VP, Penn RB, Orsini MJ, Scott PH, Plevin RJ, Walker TR, Eszterhas AJ, Amrani Y, Chilvers ER, and Panettieri RA Jr. Phosphatidylinositol 3-kinase mediates mitogen-induced human airway smooth muscle cell proliferation. Am J Physiol Lung Cell Mol Physiol 277: L65–L78, 1999.[Abstract/Free Full Text]
  36. Kumar GK and Klein JB. Analysis of expression and posttranslational modification of proteins during hypoxia. J Appl Physiol 96: 1178–1186, 2004.[Abstract/Free Full Text]
  37. Liang J, Zubovitz J, Petrocelli T, Kotchetkov R, Connor MK, Han K, Lee JH, Ciarallo S, Catzavelos C, Beniston R, Franssen E, and Slingerland JM. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest. Nat Med 10: 1153–1160, 2002.
  38. Maier U, Babich A, and Nurnberg B. Roles of non-catalytic subunits in gbetagamma-induced activation of class I phosphoinositide 3-kinase isoforms beta and gamma. J Biol Chem 274: 29311–2917, 1999.[Abstract/Free Full Text]
  39. Mazure NM, Chen EY, Laderoute KR, and Giaccia AJ. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 90: 3322–3331, 1997.[Abstract/Free Full Text]
  40. McKenzie JC, Clancy J Jr, and Klein RM. Autoradiographic analysis of cell proliferation and protein synthesis in the pulmonary trunk of rats during the early development of hypoxia-induced pulmonary hypertension. Blood Vessels 21: 80–89, 1984.[ISI][Medline]
  41. Nave BT, Ouwens M, Withers DJ, Alessi DR, and Shepherd PR. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427–431, 1999.[CrossRef][ISI][Medline]
  42. Nishida M, Schey KL, Takagahara S, Kontani K, Katada T, Urano Y, Nagano T, Nagao T, and Kurose H. Activation mechanism of Gi and Go by reactive oxygen species. J Biol Chem 277: 9036–9042, 2002.[Abstract/Free Full Text]
  43. Okada T, Hazeki O, Ui M, and Katada T. Synergistic activation of PtdIns 3-kinase by tyrosine-phosphorylated peptide and beta gamma-subunits of GTP-binding proteins. Biochem J 317: 475–480, 1996.[Medline]
  44. Peacock AJ, Scott P, Plevin R, Wadsworth R, and Welsh D. Hypoxia enhances proliferation and generation of IP3 in pulmonary artery fibroblasts but not in those from the mesenteric circulation. Chest 114: 24, 1998.
  45. Polakiewicz RD, Schieferl SM, Gingras AC, Sonenberg N, and Comb MJ. µ-Opioid receptor activates signaling pathways implicated in cell survival and translational control. J Biol Chem 273: 23534–23541, 1998.[Abstract/Free Full Text]
  46. Reuveni H, Livnah N, Geiger T, Klein S, Ohne O, Cohen I, Benhar M, Gellerman G, and Levitzki A. Toward a PKB inhibitor: modification of a selective PKA inhibitor by rational design. Biochemistry 41: 10304–10314, 2002.[CrossRef][Medline]
  47. Rossig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, and Dimmeler S. Akt-dependent phosphorylation of p21(Cip1) regulates PCNA binding and proliferation of endothelial cells. Mol Cell Biol 16: 5644–5657, 2001.
  48. Saward L and Zahradka P. Angiotensin II activates phosphatidylinositol 3-kinase in vascular smooth muscle cells. Circ Res 81: 249–257, 1997.[Abstract/Free Full Text]
  49. Schmelzle T and Hall MN. mTOR, a central controller of cell growth. Cell 103: 253–262, 2000.[CrossRef][ISI][Medline]
  50. Schumacker PT. Current paradigms in cellular oxygen sensing. Adv Exp Med Biol 543: 57–71, 2003.[ISI][Medline]
  51. Scott PH, Belham CM, Peacock AJ, and Plevin R. Intracellular signaling pathways that regulate vascular cell proliferation: effect of hypoxia. Exp Physiol 82: 317–326, 1997.[ISI][Medline]
  52. Sekulic A, Hudson CC, Homme JL, Yin P, Otterness DM, Karnitz LM, and Abraham RT. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells. Cancer Res 60: 3504–3513, 2000.[Abstract/Free Full Text]
  53. Shigematsu K, Koyama H, Olson NE, Cho A, and Reidy MA. Phosphatidylinositol 3-kinase signaling is important for smooth muscle cell replication after arterial injury. Arterioscler Thromb Vasc Biol 20: 2373–2378, 2000.[Abstract/Free Full Text]
  54. Shiojima I and Walsh K. Role of Akt signaling in vascular homeostasis and angiogenesis. Circ Res 90: 1243–1250, 2002.[Abstract/Free Full Text]
  55. Stenmark KR, Gerasimovskaya E, Nemenoff RA, and Das M. Hypoxic activation of adventitial fibroblasts: role in vascular remodeling. Chest 122: 326–334, 2002.
  56. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89–144, 1997.[CrossRef][ISI][Medline]
  57. Tu VC, Bahl JJ, and Chen QM. Signals of oxidant-induced cardiomyocyte hypertrophy: key activation of p70 S6 kinase-1 and phosphoinositide 3-kinase. J Pharmacol Exp Ther 300: 1101–1110, 2002.[Abstract/Free Full Text]
  58. Wilden PA, Agazie YM, Kaufman R, and Halenda SP. ATP-stimulated smooth muscle cell proliferation requires independent ERK and PI3K signaling pathways. Am J Physiol Heart Circ Physiol 275: H1209–H1215, 1998.[Abstract/Free Full Text]
  59. Wilson M, Burt AR, Milligan G, and Anderson NG. Wortmannin-sensitive activation of p70s6k by endogenous and heterologously expressed Gi-coupled receptors. J Biol Chem 271: 8537–8540, 1996.[Abstract/Free Full Text]
  60. Wymann MP and Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1436: 127–150, 1998.[Medline]
  61. Yu Y and Sato JD. MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor. J Cell Physiol 178: 235–246, 1999.[CrossRef][ISI][Medline]
  62. Zhong H, Chiles K, Feldser D, Laughner E, Hanrahan C, Georgescu MM, Simons JW, and Semenza GL. Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res 60: 1541–1545, 2000.[Abstract/Free Full Text]
  63. Zundel W, Schindler C, Haas-Kogan D, Koong A, Kaper F, Chen E, Gottschalk AR, Ryan HE, Johnson RS, Jefferson AB, Stokoe D, and Giaccia AJ. Loss of PTEN facilitates HIF1-mediated gene expression. Genes Dev 14: 391–396, 2000.[Abstract/Free Full Text]



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