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Endocrinology, doi:10.1210/en.2007-0285
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Endocrinology Vol. 148, No. 8 4023-4031
Copyright © 2007 by The Endocrine Society

Differential Regulation of Hypoxia-Inducible Factor-1 through Receptor Tyrosine Kinase Transactivation in Vascular Smooth Muscle Cells

Marie-Claude Lauzier1, Elisabeth L. Pagé1, Maude D. Michaud and Darren E. Richard

Centre de Recherche de L’Hôtel-Dieu de Québec and the Department of Medicine, Université Laval, Québec, Québec, Canada G1R 2J6

Address all correspondence and requests for reprints to: Darren E. Richard, Centre de Recherche de L’Hôtel-Dieu de Québec, 10 Rue McMahon, Québec, Canada G1R 2J6. E-mail: darren.richard{at}crhdq.ulaval.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of many genes expressed in hypoxic conditions. In vascular smooth muscle cells, the vasoactive hormone angiotensin II (Ang II) is a very potent inducer and activator of HIF-1. As opposed to hypoxia, which induces HIF-1{alpha} by protein stabilization, Ang II induced HIF-1{alpha} through transcriptional and translational mechanisms. Interestingly, a number of intracellular signaling events triggered by Ang II are mediated by the transactivation of receptor tyrosine kinases. The major receptor tyrosine kinases shown to be transactivated by Ang II in vascular smooth muscle cells are the epidermal growth factor receptor and the IGF-I receptor. In this study, we demonstrate that the transactivation of both these receptor tyrosine kinases is involved in HIF-1 complex activation by Ang II. More interestingly, this modulation of HIF-1 is at different degrees and through different pathways. Our results show that transactivation of IGF-I receptor is essential for HIF-1{alpha} protein translation through phosphatidylinositol 3-kinase/p70S6 kinase pathway activation, and epidermal growth factor receptor transactivation is implicated in HIF-1 complex activation through the stimulation of the p42/p44 MAPK pathway. Our results therefore show that Ang II-induced receptor tyrosine kinase transactivation is essential in both the induction and activation of HIF-1. These findings identify novel and intricate signaling mechanisms involved in HIF-1 complex activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPOXIA ACTIVATES GENES that are essential for the cellular adaptation to changes in oxygen levels. Hypoxic gene regulation is mainly mediated through a ubiquitous transcription factor, hypoxia-inducible factor-1 (HIF-1) (1). HIF-1 regulates gene expression by binding the hypoxia response element (HRE) found on the promoters of target genes. HIF-1 is composed of two subunits, HIF-1{alpha} and HIF-1ß (2). Whereas HIF-1ß is found in the nucleus of all cells, HIF-1{alpha} is highly labile and rapidly degraded by the ubiquitin-proteasome system under normal oxygen tension (3, 4). The hydroxylation of specific HIF-1{alpha} proline residues permits the recruitment of pVHL (product of von Hippel-Lindau tumor-suppressor gene), a recognition component of the ubiquitin ligase complex, targeting HIF-1{alpha} for proteasome-mediated degradation (5, 6, 7). In most, if not all, mammalian cells, the lack of oxygen quickly blocks hydroxylation and permits the accumulation of HIF-1{alpha} protein and the formation of the HIF-1 transcription complex. Maximal activation of HIF-1 complex transactivation capacity is enhanced by the coactivation of the p42/p44 MAPK pathway (8, 9, 10).

Vascular smooth muscle cells (VSMCs) respond in a similar manner to low oxygen conditions (11). However, additional mechanisms also induce and activate HIF-1 in nonhypoxic conditions. In VSMCs, vasoactive factors such as angiotensin II (Ang II) and thrombin strongly activate HIF-1 through high-level induction of the HIF-1{alpha} protein (11, 12). HIF-1{alpha} induction by Ang II in VSMCs is markedly different from hypoxic induction. Ang II activates two separate pathways that are necessary for HIF-1{alpha} protein induction in VSMCs under normoxic conditions (13). First, activation of diacylglycerol-sensitive protein kinase C plays an important part in the increase of HIF-1{alpha} transcription. Second, Ang II increases HIF-1{alpha} translation by reactive oxygen species (ROS)-dependent activation of the phosphatidylinositol 3-kinase (PI3K)/p70S6 kinase (p70S6K) pathway and through the 5'-untranslated region of HIF-1{alpha} mRNA.

Elegant studies have demonstrated that in many cell lines, activation of G protein-coupled receptors (GPCRs) stimulate different signaling pathways through the transactivation of receptor tyrosine kinases (RTK) (14, 15, 16). In Ang II-stimulated VSMCs, the Ang II type 1 receptor (AT1) has been shown to transactivate the epidermal growth factor receptor (EGFR) and IGF-I receptor (IGF-IR) (17, 18, 19). Interestingly, growth factors such as epidermal growth factor (EGF) and IGF-I are clear activators of HIF-1 in a number of different cellular models, including VSMCs (11, 20, 21). The objective of the current study was to determine whether IGF-IR and EGFR transactivation could be implicated in HIF-1 modulation by GPCR stimulation. We demonstrate that RTK transactivation is implicated in both HIF-1 induction and activation after Ang II stimulation. Transactivation of IGF-IR by Ang II plays a crucial role in increasing HIF-1{alpha} protein translation through the activation of the PI3K/p70S6K pathway. On the other hand, transactivation of EGFR by Ang II increases HIF-1{alpha} protein levels and HIF-1 complex activation through p42/p44 MAPK pathway activation. These results identify the intricate signaling mechanisms involved in HIF-1{alpha} protein regulation and activation after GPCR activation of VSMCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Ang II, thrombin, EGF, IGF-I, PD98059, and diphenyleneiodonium (DPI) were from Sigma (St. Louis, MO). AG1024 and AG1478 were from Calbiochem (La Jolla, CA). All cell culture reagents were from Invitrogen (Carlsbad, CA). Anti-HIF-1 antiserum was raised by our laboratory in rabbits immunized against the last 20 amino acid of the C termini of human HIF-1{alpha} (8). Monoclonal antiphospho-p42/p44 MAPK and {alpha}-tubulin antibodies were from Sigma. Total polyclonal p42/p44 MAPK antibody was from Upstate Biotechnology (Lake Placid, NY). Antiphospho-p70S6K (Thr389), antiphospho-EGFR (Tyr1068), antiphospho-IGF-IR (Tyr1131), anti-EGFR, and anti-IGF-IRß antibodies were from Cell Signaling (Beverly, MA). Horseradish peroxidase-coupled antimouse and antirabbit antibodies were from Promega (Madison, WI). The PRE-tk-LUC vector was a kind gift from Dr. Steven McKnight (University of Texas Southwestern Medical Center, Dallas, TX).

Cell culture
VSMCs were isolated from thoracic aorta of 6-wk-old male Wistar rats by enzymatic dissociation (22). Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), 50 U/ml penicillin, and 50 U/ml streptomycin in a humid atmosphere (5% CO2-95% air). Cells were passaged upon reaching confluence and all experiments were performed between passages 4 and 12. Cells were serum deprived for 16 h before treatment. Hypoxic conditions were obtained by placing cells in a sealed hypoxic workstation (Biotrace International PLC, Bridgend, UK). The oxygen level in this workstation was maintained to 1% with the residual gas mixture containing 94% nitrogen and 5% carbon dioxide.

Western blot analysis
Confluent cells were lysed in 2x Laemmli sample buffer. Protein concentration was determined by Lowry assay. Samples were resolved in sodium dodecyl sulfate (SDS)-polyacrylamide gel (8%) and electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp., Bedford, MA). Proteins were revealed with specific antibodies as indicated and visualized with an enhanced chemiluminescence system (GE Healthcare Life Sciences, Piscataway, NJ) or with the Odyssey infrared imaging system (LI-COR, Lincoln, NE). Western blots were quantified using Odyssey quantification software or Scion Image (http://www.scioncorp.com).

35S-methionine metabolic cell labeling and immunoprecipitation
Confluent cells were rendered quiescent 16 h before a 3-h stimulation with Ang II in methionine-free DMEM. Easy Tag Express 35S protein labeling mix (PerkinElmer Life Sciences, Norwalk, CT) was then added at a final concentration of 0.3 mCi/ml. Cells were labeled for 30 min and harvested in a lysis buffer [50 mM Tris (pH 8.0), 0.1% SDS, 1% Nonidet P-40, 5 mM deoxycholic acid, 150 mM NaCl, 2 mM dithiothreitol]. Cell extracts (1 mg) were precleared with 50 µl protein A-Sepharose (GE Healthcare Life Sciences) followed by the addition of 3 µl anti-HIF-1{alpha} antiserum. Extracts were left 2 h at 4 C before the addition of 50 µl protein A-Sepharose for 2 h at 4 C. Sepharose beads were then washed three times with lysis buffer and resuspended in 2x Laemmli sample buffer. Samples were resolved in SDS-polyacrylamide gels (8%) and electrophoretically transferred onto a polyvinylidene difluoride membrane. Membranes were exposed to BioMax MS film (Kodak, Rochester, NY) followed by Western blotting as described above.

Luciferase assay
VSMCs, seeded in six-well plates, were transfected with a pGL3 (R2.2) 3HRE-TK-LUC luciferase reporter vector (2 µg/well). This construct was generated by inserting the 3HRE-TK promoter from the PRE-tk-LUC construct (23) into the pGL3(R2.2)-basic Rapid Response luciferase reporter vector (Promega). Renilla reniformis luciferase expression vector (250 ng/well) was used as a control for transfection efficiency. Transfection was performed on 45% confluent cells with Superfect transfection reagent (QIAGEN, Valencia, CA) at a 1:5 DNA to reagent ratio. Three hours after transfection, fresh medium was added to cells. Twelve hours after transfection, cells were deprived of FBS for 16 h. Cells pretreated or not for 15 min with indicated compounds were stimulated for 6 h with Ang II. Cells were then washed with cold PBS, and luciferase assays were performed with the dual-luciferase reporter assay system (Promega). Results were quantified with a Luminoskan Ascent microplate reader with integrated injectors (Thermo Electron, San Jose, CA). Results are expressed as a ratio of beetle luciferase activity over R. reniformis luciferase activity. Experiments are an average ± SD of triplicate data.

HIF-1{alpha} dephosphorylation assay
HIF-1{alpha} dephosphorylation assay was performed on VSMC nuclear extracts using endogenous HIF-1{alpha} as substrate. Preparation of nuclear extracts was performed as described previously (24). Phosphatase buffer supplied by the manufacturer was added to lysates and incubated with {lambda}-phosphatase (New England Biolabs, Beverly, MA) for 1 h at 30 C. Samples were resolved in SDS-polyacrylamide gel (8%) and proteins were revealed by Western blotting.

Northern blot
Confluent cells were lysed and RNA was isolated with TRIzol reagent (Invitrogen). RNA resolved on a 1% agarose / 6% formaldehyde gel was transferred to Hybond N+ nylon membrane (GE Healthcare Life Sciences) and hybridized with a radioactive cDNA probe comprising the first 900-bp coding sequence of the human HIF-1{alpha} gene. An 18S oligonucleotide probe was labeled with {gamma}32P-ATP using polynucleotide kinase (GE Healthcare Life Sciences) and used for a loading control. Northern blots were quantified using a STORM phosphor imaging system equipped with ImageQuant software (GE Healthcare Life Sciences).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RTK transactivation is implicated in HIF-1{alpha} induction after Ang II stimulation of VSMCs
Ang II induces HIF-1{alpha} in VSMCs through both transcriptional and translational mechanisms (13). The Ang II AT1 receptor can transactivate different RTKs in VSMCs to mediate several intracellular signaling events (17, 19, 25). To study the implication of RTK transactivation on HIF-1{alpha} induction by Ang II, we pretreated VSMCs with AG1478 and AG1024, specific inhibitors of EGFR and IGF-IR kinases, respectively. These compounds are derivatives of the tyrphostin family of tyrosine kinase inhibitors and their strong specificity against the activity of these RTKs is well documented (19, 26, 27). As seen in Fig. 1AGo (middle panel), stimulation with Ang II transactivated the IGF-IR in VSMCs. The transactivation of IGF-IR by Ang II, as evaluated with a phospho-specific IGF-IR antibody, was completely blocked by pretreatment with AG1024. More interestingly, the inhibition of IGF-IR transactivation with AG1024 strongly decreased HIF-1{alpha} protein induction by Ang II (Fig. 1AGo, upper panel). As seen in Fig. 1BGo (middle panel), stimulation with Ang II also potently transactivated the EGFR in VSMCs. The transactivation of EGFR by Ang II, as evaluated with a phospho-specific EGFR antibody, was completely blocked by pretreatment with AG1478. The inhibition of EGFR transactivation with AG1478 also decreased HIF-1{alpha} protein induction by Ang II (Fig. 1BGo, upper panel). Quantification of immunoreactive HIF-1{alpha} demonstrated that during Ang II stimulation, IGF-IR inhibition with AG1024 decreased HIF-1{alpha} levels by 84.5%, whereas EGFR inhibition with AG1478 decreased HIF-1{alpha} levels by 36.1%. As expected, the Ang II receptor AT1 subtype was implicated in EGFR and IGF-IR transactivation. Losartan, a specific AT1 inhibitor, completely blocked the induction of HIF-1{alpha} and the phosphorylation of both EGFR and IGF-IR. PD123319, an AT2-specific inhibitor, had no effect on either HIF-1{alpha} induction or RTK transactivation (Fig. 2AGo). The effects of AG1478 and AG1024 are specific to HIF-1{alpha} induction by GPCRs because AG1478 and AG1024 also inhibited HIF-1{alpha} induction by thrombin in a similar manner (Fig. 2BGo) without having any effect on HIF-1{alpha} induction by hypoxia (Fig. 2CGo). These results demonstrate that in VSMCs, transactivation of IGF-IR, and to a minor degree EGFR, are implicated in HIF-1{alpha} protein induction after stimulation of GPCRs.


Figure 1
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  		FIG. 1. Activation of tyrosine kinase receptors by Ang II induces HIF-1{alpha} protein. Quiescent VSMCs were maintained under control conditions or in the presence of Ang II (100 nM) for 4 h. Pretreatment of cells with AG1024 (5 µM, A) or AG1478 (0.5 µM, B) was performed for 15 min before stimulation. Total cell extracts (25 µg) were resolved by SDS-PAGE (6 or 8%) and immunoblotted using anti-HIF-1{alpha} (A and B), antiphospho- and total IGF-IR antibodies (A), and antiphospho- and total EGFR (B). Western blots are representative of at least three experiments performed on different cell cultures. C, HIF-1{alpha} Western blots were quantified with Scion Image or an Odyssey infrared imaging system using {alpha}-tubulin as an internal loading control. Results are expressed as the percentage of immunoreactive HIF-1{alpha}, compared with cells treated only with Ang II. Data expressed are an average ± SD of at least three independent experiments.

 

Figure 2
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  		FIG. 2. Activation of tyrosine kinase receptors by GPCRs induces HIF-1{alpha} protein. Quiescent VSMCs were maintained under control conditions in the presence of Ang II (100 nM), thrombin (5 U/ml), or in hypoxic conditions (1% O2) for 4 h. Pretreatment of cells with AG1478 (0.5 µM), AG1024 (5 µM), Losartan (100 µM), or PD123319 (1 µM) was performed for 15 min before stimulation. Total cell extracts (25 µg) were resolved by SDS-PAGE (6 or 8%) and immunoblotted using anti-HIF-1{alpha} (A–C), antiphospho-EGFR and antiphospho-IGF-IR (A and B), and {alpha}-tubulin antibodies (C). Western blots are representative of at least three experiments performed on different cell cultures.

 
RTK transactivation is implicated in HIF-1 activation after Ang II stimulation of VSMCs
To determine whether RTK transactivation plays a role in the stimulation of a transcriptionally active HIF-1 complex, we evaluated Ang II-induced stimulation of a HIF-1-sensitive reporter. VSMCs were transiently transfected with the pGL3 (R2.2) 3HRE-TK reporter vector followed by treatment with AG1024 or AG1478 and stimulation with Ang II. We used this specific reporter vector to minimize the duration of exposure of the cells to the AG compounds. Using the pGL3 (R2.2) 3HRE-TK reporter vector, we obtained maximal luciferase expression in VSMCs after a 6-h stimulation with Ang II or hypoxia (results not shown). Under these conditions, stimulation of VSMC for 6 h with Ang II alone increased reporter activity by 2.6-fold over basal levels (Fig. 3Go). As expected, pretreatment with the IGF-IR inhibitor AG1024 completely blocked Ang II induced HIF-1 reporter activity, which was compatible with the inhibition of HIF-1{alpha} protein induction. However, pretreatment of cells with the EGFR inhibitor AG1478 also blocked Ang II-induced HIF-1 reporter activity by nearly 80%. Pretreatment with AG1024 or AG1478 alone had no effect on basal luciferase expression (results not shown). These results indicate that although having only partial effects on HIF-1{alpha} induction by Ang II, the EGFR can strongly regulate HIF-1 transcriptional activity.


Figure 3
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  		FIG. 3. Activation of tyrosine kinase receptors by Ang II regulates HIF-1 complex activity. VSMCs (six-well plate) were transfected with 2 µg of pGL3 (R2.2) 3HRE-TK reporter plasmid and 250 ng of an expression vector coding for R. reniformis luciferase was used to normalize transfection efficiency. Twelve hours after transfection, cells were deprived of FBS for 16 h. Cells were pretreated with AG1478 (0.5 µM) or AG1024 (5 µM) for 15 min and then maintained under control conditions or stimulated with Ang II (100 nM) for 6 h. VSMCs were lysed and luciferase activity was measured using the dual-luciferase reporter assay. Results are expressed as a ratio of beetle luciferase activity to R. reniformis luciferase activity. Data expressed are an average of at least three independent experiments performed in triplicate.

 
RTK transactivation is implicated in increasing HIF-1{alpha} translation after Ang II stimulation of VSMCs
Our previous studies have shown that Ang II induces HIF-1{alpha} protein levels under normoxic conditions by increasing HIF-1{alpha} mRNA transcription and up-regulating HIF-1{alpha} translation (13). Therefore, we investigated whether RTK transactivation by Ang II was implicated in either of these two events. To determine whether RTK transactivation could increase HIF-1{alpha} transcription, we pretreated VSMCs with AG1478 and AG1024 and measured Ang II-induced HIF-1{alpha} mRNA levels by Northern blot. As seen in Fig. 4AGo, inhibition of RTK transactivation with AG1024 or AG1478 did not significantly modify HIF-1{alpha} mRNA expression after Ang II stimulation. To determine whether IGF-IR or EGFR transactivation by Ang II could be implicated in regulating HIF-1{alpha} protein translation, pulse-labeling experiments were performed. VSMCs were stimulated or not for 3 h with Ang II followed by a pulse-labeling period with 35S-methionine/cysteine for 30 min. In nonstimulated cells, very little labeled HIF-1{alpha} was immunoprecipitated from VSMCs (Fig. 4BGo, upper panel). In cells treated with Ang II, a strong labeling of HIF-1{alpha} protein was observed. When cells were treated with the IGF-IR inhibitor, AG1024, 10 min before the pulse-labeling period, HIF-1{alpha} labeling was nearly completely blocked. When cells were treated with the EGFR inhibitor, AG1478, only 19.7% of HIF-1{alpha} labeling was blocked. The effect of AG1478 is consistent with its modest inhibition of HIF-1{alpha} protein levels seen in Fig. 1BGo. It is important to note that these short-term treatment periods with the AG compounds did not affect total HIF-1{alpha} protein levels induced for 3 h with Ang II (Fig. 4BGo, lower panel). These results demonstrate that HIF-1{alpha} protein synthesis is increased through RTK transactivation after Ang II stimulation.


Figure 4
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  		FIG. 4. Activation of IGF-IR by Ang II increases HIF-1{alpha} mRNA translation. A, Quiescent VSMCs were maintained under control conditions or in the presence of Ang II (100 nM) for 4 h. Pretreatment of cells with AG1478 (0.5 µM) or AG1024 (5 µM) was performed for 15 min before stimulation. Total RNA was extracted and resolved in formaldehyde/agarose gels. Northern blots were performed using a specific radiolabeled HIF-1{alpha} probe (upper panel). An 18S RNA probe was used as a control for gel loading. Northern blots were quantified by phosphor imaging using 18S RNA as a control for gel loading (lower panel). Results are expressed as the percentage of HIF-1{alpha} mRNA, compared with cells treated only with Ang II. Data expressed are an average ± SD of three independent experiments performed on different cell cultures. B, Quiescent VSMCs were maintained under control conditions or in the presence of Ang II (100 nM) for 3 h. AG1478 (0.5 µM) or AG1024 (5 µM) was then added for 10 min before the addition of 35S-methionine/cystein labeling mix (0.3 mCi/ml) for 30 min. Whole-cell extracts (1 mg) were immunoprecipitated using an anti-HIF-1{alpha} antiserum. The samples were then analyzed by SDS-PAGE (8%) followed by autoradiography (upper panel) or immunoblotted (lower panel) using an anti-HIF-1{alpha} antiserum. Results were quantified by phosphor imaging. Results are normalized to total HIF-1{alpha} protein levels (quantified with Scion Image or an Odyssey infrared imaging system) and expressed as the percentage of 35S-labeled HIF-1{alpha}, compared with cells treated only with Ang II. Data expressed are an average ± SD of three independent experiments performed on different cell cultures.

 
HIF-1 activation through IGF-IR transactivation: role of the PI3K/p70S6K pathway and ROS
We have shown that Ang II activates the PI3K/p70S6K pathway and that the activation of this pathway leads to increased HIF-1{alpha} translation (13). IGF-I is a potent activator of the PI3K/AKT/p70S6K pathway and has been shown to induce HIF-1{alpha} protein levels through translational mechanisms (20). As demonstrated in Fig. 5BGo, IGF-I induced HIF-1{alpha} protein levels and increased PI3K/p70S6K pathway activation in VSMCs. More interestingly, activation of p70S6K by Ang II was completely blocked by the IGF-IR inhibitor, AG1024 (Fig. 5AGo). In comparison, inhibition of Ang II-induced EGFR transactivation with AG1478 had only minor effects on the activation of the PI3K/p70S6K pathway by Ang II (results not shown). Therefore, our results demonstrate that IGF-IR transactivation plays a pivotal role in HIF-1{alpha} induction by Ang II in VSMCs. Transactivation of the IGF-IR by Ang II leads to robust PI3K/p70S6K pathway activation, a key player in increasing HIF-1{alpha} translation.


Figure 5
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  		FIG. 5. HIF-1 activation through IGF-IR transactivation; implication of the PI3K/p70S6K pathway and ROS. A and B, Quiescent VSMCs were pretreated with AG1024 (5 µM) for 15 min and then maintained under control conditions or in the presence of Ang II (100 nM) or IGF-I (50 ng/ml) for 4 h. C and D, Quiescent VSMCs were pretreated with DPI (10 µM) for 15 min and then maintained under control conditions or in the presence of Ang II (100 nM, A) or IGF-I (50 ng/ml, B) for 4 h. Total-cell extracts (25 µg) were resolved by SDS-PAGE (6 or 8%) and immunoblotted using anti-HIF-1{alpha}, antiphospho-p70S6K, and antiphospho-IGF-IR antibodies. Western blots are representative of at least three experiments performed on different cell cultures.

 
Our previous studies have shown that ROS are involved in PI3K/p70S6K pathway activation and HIF-1{alpha} translation after Ang II stimulation. Therefore, we wanted to determine whether ROS were upstream or downstream of IGF-IR transactivation by Ang II. Cells were pretreated with DPI and catalase to inhibit Ang II-stimulated ROS production in VSMCs. These two agents potently inhibit HIF-1{alpha} protein induction by Ang II (11). When cells were pretreated with DPI (Fig. 5CGo) or catalase (results not shown) before stimulation with Ang II, transactivation of IGF-IR by Ang II was reduced to basal levels. These results suggest that ROS activity on the PI3K/p70S6K pathway is upstream of IGF-IR transactivation by Ang II. However, when cells were pretreated with DPI before direct IGF-IR stimulation with IGF-I, an inhibition of HIF-1{alpha} protein levels and the p70S6K pathway were also observed (Fig. 5DGo). DPI did not affect IGF-IR phosphorylation after IGF-I stimulation (results not shown). Taken together, these results suggest that Ang II transactivation of IGF-IR requires ROS generation and that IGR-IR activation further produces ROS leading to elevated HIF-1{alpha} levels through PI3K/p70S6K pathway activation.

HIF-1 activation through EGFR transactivation: role of the p42/p44 MAPK pathway
Our results show that AT1-mediated EGFR transactivation is involved in both HIF-1 induction and activation after Ang II stimulation. Studies have shown that the activation of the p42/p44 MAPK pathway permits the phosphorylation of HIF-1{alpha} and cofactors leading to increased transcriptional activation (8, 9, 10, 28, 29). Our previous studies demonstrated that in VSMCs, the p42/p44 MAPK pathway is partially involved in HIF-1{alpha} protein induction by Ang II (11). In VSMCs, stimulation of the EGFR is a main activator of p42/p44 MAPK (30). We attempted to investigate whether the p42/p44 MAPK pathway activation through EGFR transactivation could be implicated in HIF-1 induction and activation after Ang II stimulation. In VSMCs, Ang II activated the p42/p44 MAPK pathway rapidly, maximal induction seen at 5 min followed by a signal that is persistent for up to 6 h (Fig. 6Go). EGFR inhibition by AG1478 treatment repressed p42/p44 MAPK pathway activation after Ang II stimulation at all time points evaluated (Fig. 6AGo). On the other hand, IGF-IR transactivation did not participate in p42/p44 MAPK activation in VSMCs after Ang II treatment because AG1024 had no effect on p42/p44 MAPK activation by Ang II (Fig. 6BGo). Additionally, no significant p42/p44 MAPK activation was observed after direct stimulation of VSMC with IGF-I (see Fig. 8Go, center panel).


Figure 6
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  		FIG. 6. Activation of p42/p44 MAPK pathway by Ang II is dependent on EGFR transactivation. Quiescent VSMCs were pretreated with AG1478 (0.5 µM, A) or AG1024 (5 µM, B) for 15 min and then maintained under control conditions or in the presence of Ang II (100 nM) for different periods. A, Total cell extracts (25 µg) were resolved by SDS-PAGE (8%) and immunoblotted using antiphospho-p42/p44 MAPK and anti-p42/p44 MAPK antibodies. Western blots are representative of at least three experiments performed on different cell cultures.

 

Figure 8
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  		FIG. 8. Importance of the p42/p44 MAPK pathway for HIF-1{alpha} induction in VSMCs. Quiescent VSMCs were maintained under control conditions in the presence of Ang II (100 nM), IGF-I (50 ng/ml), or EGF (100 ng/ml) for 4 h. Pretreatment of cells with PD98059 (20 µM) was performed for 15 min before stimulation. Total cell extracts (25 µg) were resolved by SDS-PAGE (6 or 8%) and immunoblotted using anti-HIF-1{alpha}, antiphospho-p42/p44 MAPK, or anti-p42/p44 MAPK antibodies. Western blots are representative of at least three experiments performed on different cell cultures.

 
HIF-1{alpha} phosphorylation can be observed through an electrophoretic mobility shift of HIF-1{alpha} protein (8). In VSMCs stimulated with Ang II, this mobility shift of HIF-1{alpha} was apparent by the diffuse pattern of the protein band observed in Western blots. This diffuse migration pattern was indeed caused by phosphorylation because the treatment of VSMC extracts with {lambda}-phosphatase eliminated the diffuse pattern of HIF-1{alpha} migration (Fig. 7Go). These results demonstrate that HIF-1{alpha} phosphorylation is present in VSMC treated with Ang II.


Figure 7
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  		FIG. 7. Phosphorylation of HIF-1{alpha} induced by Ang II in VSMCs. Quiescent VSMCs were maintained under control conditions or in the presence of Ang II (100 nM) for 4 h. Nuclear extracts were treated with {lambda}-phosphatase (10 U/µg of protein extract). Protein extracts (10 µg) were resolved by SDS-PAGE (8%) and immunoblotted using anti-HIF-1{alpha}, antiphospho-p42/p44 MAPK, and anti-p42/p44 MAPK antibodies. Western blots are representative of at least three experiments performed on different cell cultures.

 
The upward shift in HIF-1{alpha} migration can be induced by p42/p44 MAPK activation (8, 9, 10). Interestingly, the treatment of VSMCs with PD98059, an inhibitor of p42/p44 MAPK activation, caused a change in HIF-1{alpha}’s migration pattern (Fig. 8Go, upper panel) (11). These results suggest that EGFR transactivation by Ang II leads to HIF-1 phosphorylation by p42/p44 MAPK and increased HIF-1 complex activity. Similar to the effect of PD98059, the inhibition of EGFR transactivation by AG1478 also reduced the diffuse pattern of HIF-1{alpha} migration (Fig. 1BGo, upper panel). These results suggest that during Ang II stimulation of VSMCs, HIF-1{alpha} is phosphorylated through EGFR-transactivation-induced p42/p44 MAPK activation and that this phosphorylation could be responsible, at least in part, for increased HIF-1 transcriptional activity (Fig. 3Go).

As observed during EGFR inhibition with AG1478 (Fig. 1BGo), p42/p44 MAPK pathway inhibition with PD98059 also causes a decrease in HIF-1{alpha} protein levels (Fig. 8Go, upper panel) (11). After Ang II stimulation, the p42/p44 MAPK pathway is a known activator of protein translation in VSMCs (31). Decreased HIF-1{alpha} protein translation seen during AG1478 treatment would explain lower HIF-1{alpha} protein levels during p42/p44 MAPK pathway inhibition (Fig. 4BGo). Interestingly, induction of HIF-1{alpha} by EGF alone was completely inhibited after p42/p44 MAPK pathway inhibition with PD98059, whereas IGF-I mediated HIF-1{alpha} induction was not significantly affected (Fig. 8Go, upper panel). Taken together, these results suggest that EGFR transactivation plays a central role in HIF-1 complex activity by increasing both HIF-1{alpha} protein induction and its level of phosphorylation through p42/p44 MAPK activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The HIF-1 transcription factor is highly induced by hypoxia in all mammalian cell types. We have shown that in VSMCs, nonhypoxic stimuli also increased HIF-1 complex levels by inducing HIF-1{alpha} (11). HIF-1{alpha} induction by Ang II is mediated through transcriptional activation of the HIF-1{alpha} gene, via activation of protein kinase C, and translational induction of the HIF-1{alpha} protein, via ROS production and activation of the PI3K/p70S6K pathway (13). It is now well accepted that the stimulation of many GPCRs with their respective agonists lead to the transactivation of tyrosine kinase receptors in a number of different cell types. In VSMCs, Ang II, endothelin-1, and thrombin have been shown to transactivate the EGF (18, 32, 33, 34, 35, 36, 37, 38, 39) and IGF-I receptors (19, 40, 41). Interestingly, both these growth factors are described as important activators of the HIF-1 complex (20, 21). In this work, we investigated whether RTK transactivation is implicated in HIF-1{alpha} induction by GPCRs in VSMCs. Our studies demonstrate that both EGF and IGF receptor transactivation are involved in HIF-1 activation by Ang II through the action of two different signaling pathways. Transactivation of IGF-IR promotes the activation of the PI3K pathway, which increases HIF-1{alpha} translation. EGFR transactivation permits the increase of HIF-1 complex activity via activation of the p42/p44 MAPK pathway. These two pathways cooperate together to strongly increase and activate the HIF-1 complex and stimulate transcription of target genes.

IGF-I is a potent inducer of HIF-1. Fukuda et al. (20) demonstrated that IGF-I induces HIF-1 by increasing HIF-1{alpha} protein synthesis through the activation of the PI3K pathway. The PI3K pathway is strongly activated by Ang II in VSMCs (42). Our previous studies demonstrated that the PI3K pathway, activated through the Ang II-AT1 receptor, mediated this increase in HIF-1{alpha} protein translation. The present studies now route this effect through AT1’s ability to transactivate the IGF-IR. Through the transactivation of the IGF-IR, Ang II/AT1 mediates the majority of its ability to activate the PI3K pathway (19). This is demonstrated by the potency of AG1024 to block PI3K pathway activation after Ang II stimulation. EGFR transactivation by AT1 also leads to PI3K pathway activation (25, 43). However, we found that in VSMCs, EGF is far less potent than IGF-I to induce HIF-1{alpha} and AG1478 had only minor inhibitory effects on PI3K pathway activation after Ang II stimulation.

Stimulation of VSMCs by Ang II leads to elevated levels of ROS (34). These increased ROS levels activate the PI3K pathway and stimulate HIF-1{alpha} protein translation (13, 34). We investigated the implication of ROS in IGF-IR transactivation by Ang II. Our results suggest that ROS interact with IGF-IR transactivation at two levels leading to PI3K pathway activation. First, ROS are implicated in the transactivation of IGF-IR by Ang II/AT1 because agents that decrease intracellular ROS levels in VSMCs, such as DPI and catalase, block IGF-IR phosphorylation induced by Ang II. Second, after IGF-IR activation, ROS are needed to mediate the activation of the PI3K pathway and downstream targets, such as p70S6K. At this time, the mechanisms involved in ROS-mediated activation of the IGF-IR are not known. However, studies have shown that H2O2 is a potent activator of the IGF-IR in VSMCs (44). Upstream and downstream of the IGF-IR, it is possible that activation by ROS may be due to changes in the balance of protein phosphorylation-dephosphorylation. ROS-mediated increases of IGF-IR transactivation and PI3K pathway activation in VSMCs could be mediated through the oxidative inactivation of specific phosphatases such as protein tyrosine phosphatase-1B, Src homology phosphatase-2, and phosphatase and tensin homolog deleted from chromosome 10 as demonstrated in a number of different cell systems (45, 46, 47, 48, 49). Finally, it is interesting to note that ROS are not implicated in EGFR transactivation by Ang II in VSMCs (Lauzier, M. C., and D. E. Richard, unpublished results). This is reflected in the inability of DPI and catalase to inhibit the p42/p44 MAPK pathway in VSMCs (11). These findings are a possible explanation for the low potency of PI3K pathway activation through Ang II-mediated EGFR transactivation.

Our studies demonstrate that EGFR transactivation is indeed important in HIF-1 activation by Ang II. Even though AG1478 has a minor effect on HIF-1{alpha} protein induction, the potent effect of EGFR inhibition on HIF-1 complex activity more than makes up for this lack in effectiveness. In VSMCs, the full and long-term activation of p42/p44 MAPK is dependent on EGFR transactivation because this activation is effectively inhibited by AG1478. Different groups, including ours, have shown that p42/p44 MAPK pathway stimulation is essential for the activation of HIF-1 complex and enhances its transcriptional activity (8, 9, 28, 29). However, the mechanism leading to HIF-1 complex activation by p42/p44 MAPK has not been completely elucidated. We have shown that HIF-1{alpha} is strongly phosphorylated and that p42/p44 MAPK can directly phosphorylate HIF-1{alpha} in vitro (8). Potential p42/p44 MAPK phosphorylation sites on HIF-1{alpha} have recently been identified. Mylonis et al. (10) have shown that phosphorylation on serine residues 641 and 643 of human HIF-1{alpha} permits its nuclear translocation and accumulation, leading to increased transcriptional activity of the HIF-1 complex. It has also been suggested that the implication of p42/p44 MAPK in HIF-1 activation is mediated via the phosphorylation of p300/cAMP response element-binding protein (CBP), which is the major cofactor of the HIF-1 complex. Sang et al. (9) have shown that inhibition of p42/p44 MAPK activation blocks the interaction between p300/CBP and the C-terminal end of HIF-1{alpha}, leading to a decrease in the transcriptional activity of HIF-1. Our studies suggest that p42/p44 MAPK activation through EGFR transactivation by Ang II/AT1 is responsible for enhancing HIF-1-dependent gene transcription by promoting the phosphorylation of HIF-1{alpha} and possibly p300/CBP.

Overall, our work demonstrates the importance of RTK transactivation by GPCR agonists in the induction and activation of the HIF-1 transcription factor. EGFR and IGF-IR activation after an Ang II treatment both lead to increased HIF-1 activation by different mechanisms and signaling pathways. These two mechanisms cooperate together, leading to a strong effect on HIF-1-dependent gene induction. In cardiovascular research, cross talk between RTK receptors and GPCRs has many implications. In VSMCs, Ang II stimulation leads to a general activation of survival, proliferation and intracellular homeostasis pathways. Interestingly, HIF-1 induces the expression of many genes involved in these processes. Whereas most studies in this area have delineated the signaling pathways involved in this transactivation, our study is the first to describe how an important transcription factor and its downstream genes are modulated through these elegant signaling pathways. Ang II is also involved in many pathological processes such as hypertension (50), cardiovascular hypertrophy (51, 52, 53, 54), and atherosclerosis (55). Given the scope of genes induced by HIF-1, it will be interesting to see how the activation of this transcription factor through RTK transactivation fits into the regulation of these pathologies.

In conclusion, our study identifies novel signaling intermediates implicated in the regulation of the HIF-1 complex by Ang II in VSMCs (Fig. 9Go). The binding of Ang II to the AT1 receptor provokes the transactivation of two different RTKs, EGFR and IGF-IR. The transactivation of IGF-IR by Ang II plays a crucial role in increasing HIF-1{alpha} protein translation through the activation of the PI3K/p70S6K pathway. The transactivation of EGFR by Ang II also increases HIF-1{alpha} protein levels but plays a more crucial role in the activation of the HIF-1 complex through p42/p44 MAPK pathway activation. Together, these mechanisms permit a maximal induction and activation of the HIF-1 complex by Ang II to activate the expression of HIF-1 target genes. Given the importance of HIF-1-activated genes in vascular biology, we believe that determining of the pathways responsible for regulating the HIF-1 complex will have a very strong impact in vascular biology.


Figure 9
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  		FIG. 9. Schematic representation of signaling mechanisms involved in HIF-1 activation after Ang II stimulation of VSMCs. Stimulation of the AT1 receptor by Ang II leads to the transactivation of the IGR-IR and the EGFR. Transactivation of the IGF-IR and activation of the downstream PI3K pathway involves the production of ROS leading to the activation of p70S6K. PI3K/p70S6K pathway activation increases HIF-1{alpha} subunit translation. Increased HIF-1{alpha} leads to the formation of the HIF-1 transcription complex and activation of target genes. Transactivation of the EGFR leads to the activation of p42/p44 MAPK. p42/p44 MAPK causes increased HIF-1{alpha} subunit translation and also phosphorylates HIF-1{alpha}. Increased phosphorylated HIF-1 leads to increased transcription of target genes.

 


   Acknowledgments
 
We thank Guylaine Soucy and Geneviève Robitaille for their excellent technical assistance, Marc-André Déry for generation of the pGL3 (R2.2) 3HRE-TK-LUC vector, and Jacques Huot and Jean-Philippe Gratton for critical review of the manuscript.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR; MOP-49609) and the Heart and Stroke Foundations of Québec and Canada. D.E.R. is the recipient of a CIHR New Investigator Award. M.-C.L., E.L.P., and M.D.M. are recipients of doctoral scholarships from the CIHR.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 17, 2007

1 M.-C.L. and E.L.P. contributed equally to this work. Back

Abbreviations: Ang II, Angiotensin II; AT1, Ang II type 1 receptor; CBP, cAMP response element-binding protein; DPI, diphenyleneiodonium; EGF, epidermal growth factor; EGFR, EGF receptor; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; HIF, hypoxia-inducible factor; HRE, hypoxic response element; IGF-IR, IGF-I receptor; PI3K, phosphatidylinositol 3-kinase; p70S6K, p70S6 kinase; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SDS, sodium dodecyl sulfate; VSMC, vascular smooth muscle cell.

Received March 1, 2007.

Accepted for publication May 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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