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Am J Physiol Cell Physiol 292: C2185-C2196, 2007. First published February 28, 2007; doi:10.1152/ajpcell.00204.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G{alpha}i3/Ras/Raf/MAPK pathway

Jianyu Li,1 Xiangmin Zhao,1 Xinmei Li,1 Kenneth M. Lerea,2 and Susan C. Olson1

Departments of 1Biochemistry and Molecular Biology and 2Cell Biology and Anatomy, New York Medical College, Valhalla, New York

Submitted 25 April 2006 ; accepted in final form 16 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G{alpha}i3 dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT2 antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G{alpha}i3, Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT2-dependent increase in eNOS expression in PAECs.

Src; mitogen-activated protein kinase

ANGIOTENSIN II (ANG II), a key regulator of fluid homeostasis, vascular tone, and cell growth (7), exerts its physiological affects by binding to two major G protein-coupled receptor subtypes, type 1 (AT1) and type 2 (AT2). The AT1 receptor, which mediates vasoconstriction, water and sodium intake, and induction of cell growth, is linked to the activation of many signaling molecules (7, 18) including GTP-binding proteins, PLC, PKC, phosphatidylinositol 3-kinase, ERK1/2, and tyrosine kinases. While the AT2 receptor is linked to neuronal differentiation, apoptosis, inhibition of cell growth, and regulation of blood pressure (6), there is much controversy in the literature regarding the signaling pathways downstream of the AT2 receptor. Investigators have demonstrated that the AT2 receptor is linked to both the activation and inhibition of protein phosphatases (15, 16, 38) and as well as ERK1/2 phosphorylation (11, 37). Originally, the AT2 receptor was thought to be expressed mainly in fetal tissue; however, numerous studies have now reported the expression of the AT2 receptor in adult adrenal tissue and the brain, kidney, heart (6, 18), and lung (27). Nevertheless, the physiological role of the AT2 receptor remains to be determined. Many studies have suggested that the AT2 receptor functions in a protective role (10, 11). For example, the AT2 receptor plays a protective role in ischemic neuropathy (33), myocardial infarction (24, 39), congestive heart failure (25), and during states of ANG II excess (39). Although the pathways mediated via the AT2 receptor have not been clearly delineated, it appears that activation of AT2 receptor opposes the effects of the AT1 receptor (6, 18).

Several recent studies (2, 4, 14, 27, 30, 39) have demonstrated that ANG II stimulates endothelial cells to produce nitric oxide (NO), a key regulator of blood pressure, vascular tone, and angiogenesis. NO is synthesized from L-arginine by a family of NADPH-dependent NO synthases (NOSs) (1): two constitutively expressed enzymes, neuronal and endothelial NOS (eNOS), and a cytokine-inducible isoform. The activity of the major isoform present in the cardiovascular system, eNOS, can be regulated by association with calcium/calmodulin, heat shock protein 90, and caveoli as well as by posttranslational phosphorylation (1). In addition, several stimuli can induce the expression of eNOS mRNA, ultimately leading to an increase in NO production (10). To date, there are many studies (6, 38, 40) linking ANG II and NO production; however, the mechanism by which ANG II leads to an increase in NO depends not only on the experimental conditions but also appears to depend on the cell type and signaling pathways present in that cell.

We (20, 26, 27) have recently reported that ANG II stimulates Src tyrosine kinase via a pertussis toxin-sensitive AT2 receptor, which, in turn, activates the MAPK pathway, resulting in increased NOS protein expression in bovine pulmonary artery endothelial cells (PAECs). ANG II activation of ERK has been previously described in several cell types (7, 36) and has been shown to involve the Gbeta{gamma}-subunits of a pertussis toxin-sensitive G protein (19) and the EGF receptor (EGFR) (8) as well as the adapter proteins sequence homology of collagen (Shc) and growth factor receptor-bound protein 2 (Grb2) (31). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and to an increase in eNOS protein expression in PAECs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. DMEM, fungizone, penicillin-streptomycin solution, FBS, and HBSS were purchased from GIBCO-Invitrogen (Grand Island, NY). The Ras Activation Assay Kit, H-Ras cDNA [Dominant Negative (DN)] Expression Kit, Src cDNA (DN) Expression Kit, Shc antibody, Grb2 antibody (Western), son of sevenless (Sos) antibody, Src antibody (phosphorylated and total Src), Ras antibody, and the phosphotyrosine antibody 4G10 were from Upstate Chemicon. Erk1/2 antibody, actin antibody, lactate dehydrogenase (LDH) antibody, protein A/G agarose, ANG II, PD-123319, and protease inhibitor cocktail were from Sigma Chemical (St. Louis, MO). Caveolin-1 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Acrylamide, N,N,N',N'-tetramethylethylene diamine (TEMED), ammonium persulfate, nitrocellulose membranes, and the protein assay kit were purchased from Bio-Rad (Hercules, CA). Horseradish peroxidase-conjugated secondary antibodies and normal IgG were from Transduction Laboratories (San Diego, CA). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine (PP2), FTI-277, and Raf-1 inhibitor were from Calbiochem (Los Angeles, CA). Anti-phospho-ERK1/2 was purchased from Cell Signaling (Beverly, MA). For the immunoprecipitation experiments, anti-Grb2 antibody was from Serotec (Raleigh, NC) and anti-Src antibody was from Bioscience (Camarillo, CA). Candesartan was a gift from Dr. Peter Morsing (AstraZeneca). DN mutant cDNAs of G{alpha}i2 (G203T) and G{alpha}i3 (G202T) were purchased from University of Missouri, Rolla cDNA Resource Center (Rolla, MO).

Cell culture. Calf lungs, obtained immediately after slaughter, were transported to the laboratory in ice-cold buffered HBSS (pH 7.4). PAECs were isolated from the secondary branches of the main intrapulmonary artery of calf lungs as previously described (26). Subculture cells were maintained in DMEM supplemented with 15% FBS, penicillin-streptomycin (100 U/ml and 100 µg/ml), and fungizone (2.5 µg/ml). Unless otherwise stated, cells were grown to 90% confluence and then made quiescent for 24 h before experiments were conducted. Control and treated cells were matched in each experiment for cell line, passage number (passages 3–6), and time to monolayer confluence.

Western blot analysis. PAECs were harvested in Laemmli sample buffer containing 62.5 mM Tris·HCl (pH 6.8), 2% SDS, and 10% glycerol. Cell lysate proteins (30 µg), as determined by the Bradford method, were separated by SDS-PAGE and transferred to nitrocellulose membranes using a Bio-Rad electrophoretic transfer cell. Membranes were probed with various primary antibodies as indicated, followed by their respective horseradish peroxidase-conjugated secondary antibodies. Peroxidase activity was determined using enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) and by exposing membranes to X-ray film. Relative amounts of proteins were quantitated using the AlphaImager Tm2000 documentation and analysis system. Results are expressed relative to untreated cells.

Coimmunoprecipitation. PAECs were harvested with modified RIPA buffer [50 mM Tris·HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, and protease inhibitory cocktail]. After centrifugation, protein lysates (200–600 µg) were incubated with 2–4 µg of the desired antibody overnight and then precipitated with protein A/G-agarose for 2 h. Immunoprecipitations were also performed using an equivalent amount of nonimmune IgG (mouse or rabbit) or in the absence of the immunoprecipitating antibody. Precipitates were washed three times in ice-cold modified RIPA buffer and resuspended in reduced Laemmli sample buffer. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with the desired antibodies.

Transient transfection of PAECs with DN mutant cDNAs. PAECs were plated at 40–60% confluent in six-well tissue culture plates. One microgram of Src DN cDNA (K296R/Y528F) in pUSEamp was mixed with 6 µl PLUS reagent in 100 µl serum-free DMEM for 15 min at room temperature. Serum-free DMEM was then mixed with LipofectAMINE Reagent (4 µl, Invitrogen), which was then added to the DNA mixture and incubated at room temperature for 15 min. LipofectAMINE-PLUS reagent-DNA complexes were added to each well of the cell culture plates and incubated for 3 h at 37°C in a CO2 humidified incubator. The DNA-containing medium was removed from the cells, and fresh medum was added. Cells were incubated for an additional 48–72 h. Src expression was monitored by Western blot analysis using an anti-Src antibody, and Src kinase activity was determined as previously described (20). Successful transfection was monitored by overexpression of Src as assessed by Western blot analysis. As a control, PAECs were transfected with an empty vector (1 µg). Transfected PAECs were then treated with 100 nM ANG II for the desired lengths of time.

PAECs were transiently transfected with Ras DN mutant cDNA (S19N) following the same protocol except that LipofectinAMINE was used as the transfection vehicle. Successful transfection was evaluated by overexpression of Ras in transfected cells compared with nontransfected cells.

PAECs were transfected (LipofectAMINE) with the DN mutant cDNAs of G{alpha}i2 (Q205L) and G{alpha}i3 (Q204L) for 24 h prior to being stimulated with 100 nM ANG II for 1 min, 15 min, or 8 h. Successful transfection was evaluated by overexpression of the respective G protein {alpha}-subunits in transfected cells compared with nontransfected cells.

Ras activity assay. Ras activity was measured using the affinity precipitation assay (Ras Activation Kit, Upstate Biotechnology) in which only Ras bound to GTP binds to Raf. Briefly, the recombinant Ras GTP-binding domain of c-Raf (RBD; residues 51–131 of c-Raf), fused to glutathione-S-transferase, was precoupled to glutathione-Sepharose beads. PAEC lysates were mixed with 15 µl of the RBD-Sepharose beads and incubated overnight at 4°C. Beads were washed three times with lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 25 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail], and bound proteins were separated by SDS-PAGE. Ras-GTP was determined by immunoblot analysis. Total Ras expression in PAEC lysates was analyzed by Western blot analysis.

Preparation of PAEC membrane and cytosolic fractions. PAECs were treated with 100 nM ANG II for the desired times, washed with ice-cold PBS three times, and scraped into homogenization buffer containing 20 mM Tris·HCl (pH 7.5), 0.33 M sucrose, 2 mM EDTA, 0.5 mM EGTA, and protease inhibitory cocktail (27). Cells were then homogenized, and nuclei and unbroken cells were removed by centrifugation at 1,000 g for 10 min. The supernatant was collected and centrifuged at 100,000 g for 30 min. The resulting supernatant contained cytosolic proteins. The pellet was then placed in ice-cold homogenization buffer containing 1% Triton X-100 for 60 min and then centrifuged at 100,000 g for 30 min. The supernatant, which contained solubilized membrane proteins, was then collected. To assess the effectiveness of subcellular fractionation, Western blot analysis of proteins from the membrane and cytosolic fractions was performed using anti-caveolin antibody, a marker for the membrane fraction, and anti-LDH antibody, a marker for the cytosolic fraction.

Assay of NOS activity by quantitation of nitrite production. Confluent PAECs in six-well plates were cultured in DMEM containing 2% FBS without phenol red and antibiotics. Cells were treated with buffer, PP2, FTI-277, or Raf-1 inhibitor prior to being stimulated with ANG II. After 8 h (9), nitrate reductase was added to the culture media to convert nitrate into nitrite (NO2–). NO production was then measured as the amount of its stable metabolite, NO2–, using the Colorimetric NO Assay Kit (Calbiochem). Absorbance was measured at 540 nm, and the NO2– concentration was determined using sodium nitrite as a standard. The amount of NO2– formed in the media was normalized to the protein content in the respective dishes.

Statistical analysis. Data are expressed as means ± SE; n refers to the number of experiments performed in a minimum of three different cell preparations. Data from at least three independent experiments were averaged, and statistical significance was evaluated by Student's t-test (with paired control and conditions) or one-way ANOVA with the Bonferroni post test. Statistical significance was identified at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We (20, 27) have previously reported that ANG II stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which in turn, activates the MAPK pathway, resulting in an increase in NOS expression in PAECs. The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK phosphorylation and an increase in eNOS protein expression in PAECs.

ANG II-dependent increases in Src phosphorylation and eNOS protein expression are mediated via G{alpha}i3. Since PAECs express pertussis toxin-sensitive G proteins [G{alpha}i2 and G{alpha}i3 (20)], we used DN cDNAs of these G protein {alpha}-subunits to investigate whether either subunit linked ANG II to Src activation (21) and/or the increase in eNOS protein expression. Expression of the respective DN G protein {alpha}-subunits was monitored by Western blot analysis (Fig. 1A, top: autoradiograms). Transfection of PAECs with DN cDNA of G{alpha}i3 blocked the ANG II induction of Src phosphorylation (Tyr416) at 1 min (Fig. 1A, bottom autoradiograms and graph) and eNOS protein expression at 8 h (Fig. 1B), whereas transfection with the DN mutant cDNA of G{alpha}i2 had no effect. Transfection of PAECs with G protein {alpha}-subunit DN cDNAs had no effect on the expression of the AT2 receptor (Fig. 1S-A, online supplement).1


Figure 1
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  		Fig. 1. ANG II-induced Src phosphorylation and increases in endothelial nitric oxide (NO) synthase (eNOS) protein expression are mediated via G{alpha}i3. Pulmonary artery endothelial cells (PAECs) were transfected with control vector, G{alpha}i2 dominant negative (DN) cDNA, or G{alpha}i3 DN cDNA for 24 h before being stimulated with buffer or 100 nM ANG II for 1 min (A) or 8 h (B). A, top: Western blot (WB) demonstrating overexpression of G{alpha}i2 or G{alpha}i3 DN mutants. Src was immunoprecipitated with a mouse anti-Src antibody (Ab), and the activation of Src was determined by immunoblot analysis with a rabbit anti-phosphorylated (p)-Src antibody (Tyr416). The total amount of Src in the immunoprecipitate complex was determined by reprobing the same blot with a mouse anti-Src antibody. Bottom: representative autoradiograms of p-Src, Src, and IgG bands. Blots were scanned and relative levels of p-Src and Src were determined by laser densitometry. The graph represents pooled data of 6 separate experiments. Results were normalized by arbitrarily setting the densitometry of control cells (vector) to 100%. B, top: representative autoradiogram of eNOS and actin protein expression. Blots were scanned and relative levels of eNOS expression were quantified by laser densitometry. Bottom: graph representing pooled data from 6 separate experiments. Values are means ± SE. *P < 0.05 vs. untreated control; #P < 0.05 vs. ANG II-treated cells.

 
ANG II-induced Src-dependent tyrosine phosphorylation of Shc and the association of Shc with Grb2. All three isoforms of the adaptor protein Shc (46, 52, and 66 kDa), which arise from alternative RNA splicing or alternative translational initiation of the same message (28), are expressed in PAECs (Fig. 2A). Stimulation of PAECs with 100 nM ANG II for 0–30 min had no effect on the expression of Shc isoforms (Fig. 2A). To determine whether ANG II stimulated an association between Src tyrosine kinase and Shc, PAECs were treated with 100 nM ANG II for 0–5 min. PAEC lysates were immunoprecipitated with an anti-Src antibody, and the immunocomplexes were subjected to SDS-PAGE followed by immunoblot analysis with an anti-Shc antibody. ANG II stimulated a rapid (1 min, 2-fold increase), but transient, association between Src and the 66-kDa isoform of Shc (Fig. 2B, top). Equal amounts of Src were immunoprecipitated at each time point (Fig. 2B, bottom).


Figure 2
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  		Fig. 2. ANG II stimulates a Src-dependent increase in sequence homology of collagen (Shc) tyrosine phosphorylation. A: PAECs were treated with 100 nM ANG II for the times indicated. The representative immunoblot shows the expression of the three isoforms of Shc (46, 52, and 66 kDa) in PAECs. B: PAECs were treated with 100 nM ANG II for the times indicated. Src immunocomplexes were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with an anti-Shc and anti-Src antibodies. Immunoprecipitation (IP) was performed in the absence of anti-Src antibody (no Ab). The autoradiogram is representative of 3 separate experiments. C: PAECs were treated with 100 nM ANG II for the times indicated. Tyrosine-phosphorylated proteins were immunoprecipitated, and Shc tyrosine phosphorylation was determined by immunoblot analysis with an anti-Shc antibody (n = 3). D: PAECs were pretreated with 100 µM 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine (PP2) for 30 min and then treated with 100 nM ANG II for 15 min. Shc was immunoprecipitated, and tyrosine phosphorylation was determined by immunoblot analysis with the anti-phosphotyrosine antibody 4G10 (top autoradiogram). Blots were stripped and reprobed with an anti-Shc antibody. IP was performed in the absence of anti-Shc antibody (no Ab). Bottom: bar graph showing the quantitative analysis of protein tyrosine phosphorylation for Shc. Results were normalized by arbitrarily setting the densitometry of untreated cells to 1.0. Values are means ± SE; n = 6. *P < 0.05 vs. untreated control; #P < 0.05 vs. ANG II-treated cells.

 
To investigate whether ANG II stimulates an increase in Shc tyrosine phosphorylation, PAECs were stimulated with 100 nM ANG II for 0–30 min. Tyrosine-phosphorylated proteins were immunoprecipitated with the anti-phosphotyrosine antibody 4G10, and the immunocomplexes were subjected to Western blot analysis with an anti-Shc antibody. ANG II stimulated an increase in tyrosine phosphorylation of the 66-kDa isoform of Shc that reached a maximum at 15 min (2.02 ± 0.23-fold increase; Fig. 2C). To determine whether this Shc tyrosine phosphorylation was dependent on Src, PAECs were treated with 100 µM PP2, a Src family tyrosine kinase inhibitor, for 30 min before being stimulated with 100 nM ANG II. After 15 min, Shc was immunoprecipitated from PAEC lysates, and tyrosine phosphorylation determined by Western blot analysis with the anti-phosphotyrosine antibody 4G10. As shown in Fig. 2D, the ANG II-dependent increase in tyrosine phosphorylation of Shc (66 kDa) was prevented (1.1 ± 0.09-fold increase) when PAECs were pretreated with PP2. Equal amounts of Shc were immunoprecipitated at each time point (Fig. 2D).

Since a previous report (28) has demonstrated that tyrosine phosphorylation of Shc leads to its association with another adaptor protein, Grb2, we examined whether ANG II induced an association between Shc and Grb2. PAECs were treated with 100 nM ANG II for 0–30 min. PAEC lysates were immunoprecipitated with an anti-Shc antibody, and the immunoprecipitates were separated by SDS-PAGE followed by immunoblot analysis with anti-Grb2. As shown in Fig. 3A, top, ANG II stimulated a significant increase in Grb2 association with Shc (66 kDa) between 10 and 15 min (2.20 ± 0.27-fold increase compared with time 0). As shown in Fig. 3A, bottom, equal amounts of Shc (66 kDa) were immunoprecipitated at each time point. Pretreatment of PAECs with the Src inhibitor PP2 prevented this ANG II-dependent Grb2-Shc association at 15 min (Fig. 3B). Even though PAECs also express the 46- and 52-kDa Shc isoforms, the only isoform that consistently was tyrosine phosphorylated and associated with Grb2 in an ANG II-dependent manner was the 66-kDa Shc isoform.


Figure 3
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  		Fig. 3. ANG II induced an association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos). A: PAECs were treated with 100 nM ANG II for the times indicated. Shc was immunoprecipitated, and the presence of Grb2 was determined by immunoblot analysis. Detection of Shc in the immunocomplex was assessed by WB analysis. IP was performed in the absence of anti-Shc antibody (no Ab). The association of Grb2 and Shc is expressed as a ratio relative to basal levels. Values are means ± SE; n = 4. *P < 0.05 vs. time 0. B: the association of Grb2 and Shc is mediated via Src. PAECs were left untreated or pretreated with 100 µM PP2 for 30 min before being stimulated with ANG II for 15 min. Grb2 was immunoprecipitated, and the presence of Shc was determined by WB analysis. Blots were stripped and reprobed with anti-Grb2 antibody. Shown is a representative autoradiogram of 3 different experiments. C: PAECs were treated with 100 nM ANG II for the times indicated. Membrane and cytosolic fractions were isolated by differential centrifugation as described in MATERIALS AND METHODS. Grb2 and Shc were detected by immunoblot analysis. Shown is a representative autoradiogram of 3 different experiments. D: Grb2 was immunoprecipitated, and the presence of Shc and Sos was determined by immunoblot analysis. The total amount of Grb2 in immunoprecipitates was determined by reprobing the same blots with anti-Grb2 antibody. IP was performed in the absence of anti-Srb-2 antibody (no Ab). Shown is a representative autoradiogram of 3 different experiments. E: the association of Grb2 and Sos is mediated via Src. PAECs were left untreated or pretreated with 100 µM PP2 for 30 min before being stimulated with ANG II for 15 min. Grb2 was immunoprecipitated, and the presence of Sos was determined by WB analysis. Blots were stripped and reprobed with anti-Grb2 antibody. Shown is a representative autoradiogram of 3 different experiments.

 
Activation of Grb2 involves translocation from the cytosol to the plasma membrane (35); thus, the effect of ANG II on the subcellular location of Grb2 was investigated. ANG II stimulated an increase in Grb2 associated with the membrane fraction at 5 min (Fig. 3C, top). In addition, ANG II stimulated an increase in the membrane localization of all three isoforms of Shc (46, 52, and 66 kDa) that was seen as early as 1 min and was maintained up to 30 min (Fig. 3C, bottom).

ANG II induced an association between Grb2 and Sos. To determine whether Grb2 associates with the guanine nucleotide exchange protein Sos in an ANG II-dependent manner, PAECs were stimulated with ANG II (100 nM) for 0–30 min. Cell lysates were immunoprecipitated with anti-Grb2 antibody and subjected to immunoblot analysis with an anti-Sos antibody. Coimmunoprecipitation data revealed that ANG II induced an association between Grb2 and Sos, which was seen as early as 5 min and was maximal by 15 min (Fig. 3D, top). The same Grb2 immunocomplex was probed for the presence of Shc. ANG II stimulated an association of Grb2 with Shc that was seen as early as 5 min and was maximal up to 15 min (Fig. 3D, middle). The presence of Grb2 in the immunocomplex is shown in Fig. 3D, bottom. Pretreatment of PAECs with PP2 demonstrated that Src activation was also required for the Grb2-Sos association (Fig. 3E). These data suggest that Grb2, Shc, and Sos associate with each other in an ANG II-dependent manner.

ANG II stimulated an increase in Ras GTP binding via a pertussis toxin-sensitive AT2 receptor-linked Src pathway. Translocation of Grb2/Shc/Sos to the membrane leads to an increase in Ras GTP binding (22); thus, we investigated whether Ras mediates the ANG II-dependent increase in MAPK activation. ANG II (1 µM) stimulated a time-dependent increase in Ras GTP binding with maximal activation occurring at 15 min (3.19 ± 0.28-fold vs. basal; Fig. 4A). During the time frame of this experiment, ANG II had no effect on total Ras protein levels.


Figure 4
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  		Fig. 4. ANG II activation of Ras is mediated via a pertussis toxin (PTX)-sensitive ANG II type 2 (AT2) receptor, Src-dependent pathway. A: PAECs were treated with 1 µM ANG II for the times indicated. Top: representative immunoblot of GTP-bound Ras and total Ras. 100x GDP prevented Ras from binding GTP. Bottom: graph representing the densitometric analysis of immunoblots from 3 experiments. B: PAECs were pretreated with either the AT1 receptor antagonist candesartan (Cand; 10 µM) or the AT2 receptor antagonist PD-123319 (10 µM) for 15 min before being stimulated with 1 µM ANG II for 15 min. Top: representative immunoblot of GTP-bound Ras and total Ras. Bottom: densitometric analysis of immunoblots from 3 experiments. C: PAECs were pretreated with 50 ng/ml PTX for 6 h before being stimulated with 1 µM ANG II for 15 min. Top: representative immunoblot of GTP-bound Ras and total Ras. Bottom: densitometric analysis of immunoblots from 3 experiments. D: PAECs were transiently transfected with either Src DN mutant cDNA or treated with 100 µM PP2 for 30 min before being stimulated with 1 µM ANG II for 15 min. Ras-GTP was isolated using the Ras Activation Kit and assessed by WB analysis. Top: immunoblot showing the expression of total Ras and overexpression of DN Src. Bottom: graph representing pooled data from 6–9 experiments. Values are means ± SE. *P < 0.05 vs. untreated controls; #P < 0.05 vs. ANG II-treated cells.

 
Since we (2) have previously shown that ANG II-induced Src activation is mediated via the AT2 receptor in PAECs, we investigated which ANG II receptor subtype is linked to this increase in Ras GTP binding. PAECs were pretreated with either 10 µM candesartan, an AT1 antagonist, or 10 µM PD-123319, an AT2 receptor antagonist, for 15 min before being stimulated with ANG II. Pretreatment of PAECs with PD-123319 had no effect on basal Ras GTP binding but completely blocked the ANG II-dependent increase in Ras GTP binding at 15 min (1.11 ± 0.31-fold increase), whereas the AT1 receptor antagonist had no effect (Fig. 4B). Treatment of PAECs with either receptor antagonist had no effect on total Ras protein levels or on the expression of the AT2 receptor (Fig. 1S-B, online supplemental data).

Given that the ANG II-dependent increase in eNOS is blocked by pertussis toxin (27), we examined the effect of pertussis toxin on Ras GTP binding activity. PAECs were pretreated with pertussis toxin for 6 h and then treated with ANG II for 15 min. Pertussis toxin treatment did not effect cell viability as over 90% of cells were viable as determined by a trypan blue dye exclusion assay. In addition, ANG II-stimulated STAT3 phosphorylation at 30 min was not affected by pertussis toxin (Fig. 2S, online supplemental data), demonstrating that pertussis toxin treatment was not lethal to PAECs. The ANG II-dependent increase in Ras GTP binding was significantly attenuated by pertussis toxin (1.42 ± 0.32-fold increase; Fig. 4C). Treatment of PAECs with pertussis toxin did not alter the expression of Ras. Collectively, these data suggest that the ANG II-dependent increase in Ras activation is mediated via a pertussis toxin-sensitive AT2 receptor in PAECs.

Since Src tyrosine kinase mediates the ANG II-dependent increase in the expression of eNOS (20), we examined the role of Src in regulating Ras GTP binding in PAECs. For this experiment, PAECs were either transfected with Src DN mutant cDNA or pretreated with 100 µM PP2 for 30 min before being stimulated with ANG II. The ANG II-dependent increase in Ras GTP binding at 15 min (Fig. 4D) was completely blocked by Src DN mutant cDNA transfection (0.8 ± 0.18-fold increase) or by pretreatment with PP2 (0.92 ± 0.08-fold increase). Neither AT2 receptor (Fig. 1S-C, online supplement) nor total Ras protein levels were changed as a result of inhibition of Src activity (Fig. 4D). Overexpression of DN Src is shown in Fig. 4D.

These data, taken together with our previously published results (20, 27), suggest that AT2 receptor-dependent activation of Src leads to an increase in Ras GTP binding in PAECs.

ANG II activates Raf-1 kinase via AT2 receptor activation of Src and Ras. Because Ras GTP binding can lead to activation of the serine/threonine kinase kinase Raf-1 (3), the next set of experiments was directed at investigating ANG II-dependent Raf-1 activation. ANG II stimulated a time-dependent increase in Raf-1 phosphorylation (Ser338) that was found to be significant starting at 15 min and was maintained for up to 2 h (Fig. 5A). In addition, ANG II stimulated an increase in Raf-1 translocation from the cytosolic to membrane fraction at 15 min (Fig. 5B). Markers identifying membrane (caveolin) and cytosolic fractions (LDH) are shown in Fig. 5B.


Figure 5
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  		Fig. 5. ANG II-induced Raf-1 serine phosphorylation is mediated via the AT2 receptor and is downstream of Src tyrosine kinase. A: PAECs were treated with 100 nM ANG II for the times indicated. Phosphorylated Raf-1 was identified by WB analysis with an anti-phosphoserine Raf-1 antibody (Ser338) (n = 3). B: cells were treated with 100 nM ANG II for 15 min. Membrane and cytosolic fractions were isolated by differential centrifugation, and Raf-1 was identified by WB analysis. Shown is a representative blot of 2 separate experiments. The cytosol and membrane fractions were identified using an anti-lactate dehydrogenase antibody or anti-caveolin antibody, respectively. Bands were visualized using chemiluminescence. C: cells were pretreated with the AT1 receptor antagonist Cand (1 µM, 15 min), the AT2 receptor antagonist PD-123319 (1 µM, 15 min), or the Src tyrosine kinase inhibitor PP2 (10 µM, 30 min) before being stimulated with 100 nM ANG II for 15 min. Raf-1 activation was determined by WB analysis with an anti-phosphoserine Raf-1 antibody (Ser338). Values are means ± SE; n = 7. *P < 0.05 vs. basal; **P < 0.01 vs. untreated controls; #P < 0.05 vs. ANG II; ##P < 0.01 vs. ANG II.

 
Pretreatment of PAECs with the AT1 receptor antagonist candesartan did not have any effect on ANG II-dependent Raf-1 phosphorylation at 30 min, whereas the AT2 receptor antagonist PD-123319 blocked the ANG II-dependent increase in Raf-1 phosphorylation (Fig. 5C). Furthermore, the Src tyrosine kinase inhibitor PP2 inhibited both basal and ANG II-stimulated Raf-1 phosphorylation (Fig. 5C). Collectively, these data suggest that the ANG II-dependent activation of Raf-1 is via an AT2 receptor/Src-linked pathway.

Ras mediates ANG II-dependent increases in ERK1/2 phosphorylation. To determine whether G{alpha}i3 mediates the ANG II-dependent activation of MAPK, PAECs were transfected with control vector, G{alpha}i2 DN cDNA, or G{alpha}i3 DN cDNA for 24 h before being stimulated with buffer or with 100 nM ANG II. ERK1/2 phosphorylation at 15 min was determined using an anti-phospho-p44/42 MAPK antibody. Transfection with G{alpha}i2 DN cDNA partially blocked the ANG II-dependent increase in ERK1/2 phosphorylation, whereas transfection with G{alpha}i3 DN cDNA completely blocked the ANG II-dependent increase in ERK1/2 phosphorylation (Fig. 6A). Overexpression of G protein {alpha}-subunits is shown in Fig. 6A.


Figure 6
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  		Fig. 6. Ras mediates ANG II-dependent MAPK activation. A: PAECs were transfected with control vector, G{alpha}i2 DN cDNA, or G{alpha}i3 DN cDNA for 24 h before being stimulated with buffer or 100 nM ANG II. The overexpression of G protein {alpha}-subunits is shown. ERK1/2 phosphorylation at 15 min was determined using an anti-p-p44/42 MAPK antibody. On a parallel blot, total ERK1/2 expression was assessed by immunoblot analysis. B and C: PAECs were transiently transfected with Ras DN mutant cDNA (B) or treated with FTI-277 (FTI; C) for 24 h before being stimulated with 100 nM ANG II. ANG II-dependent ERK1/2 phosphorylation at 15 min was determined using an anti-p-p44/42 MAPK antibody. B: representative immunoblots of p-ERK1/2 and total ERK1/2 and overexpression of DN Ras. C: representative immunoblots of p-ERK1/2 and total ERK1/2. D: graphed data representing p-ERK2 normalized against total ERK2. Values are means ± SE; n = 4. *P < 0.05 vs. untreated control; #P < 0.05 vs. ANG II-treated cells.

 
To investigate the role of Ras in the ANG II induction of MAPK, PAECs were either transfected with Ras DN mutant cDNA or treated with 1 µM FTI-277, a farnesyl transferase inhibitor, before being stimulated with ANG II for 15 min. The ANG II-dependent increase in ERK2 phosphorylation (2.65 ± 0.41-fold increase, P < 0.05) was blocked by transfection of PAECs with Ras DN mutant cDNA (1.54 ± 0.45-fold increase; Fig. 6, B and D) and by pretreatment with FTI-277 (0.92 ± 0.25-fold increase; Fig. 6, C and D), demonstrating that Ras mediates the ANG II-dependent activation of MAPK. Overexpression of the Ras DN mutant was monitored by Western blot analysis with an anti-Ras antibody (Fig. 6B). Blockade of Ras activation with FTI-277 had no effect on the ANG II-dependent tyrosine phosphorylation of Shc (Fig. 3S, online supplemental data).

ANG II-dependent increase in eNOS protein expression is mediated via Ras and Raf. The ANG II induction of eNOS protein expression at 8 h (1.74 ± 0.25-fold increase) was blocked by transfection of PAECs with Ras DN mutant cDNA (0.71 ± 0.09-fold increase; Fig. 7, A and C) or by pretreatment of PAECs with FTI-277 (0.81 ± 0.14-fold increase; Fig. 7, B and C). Furthermore, treatment of PAECs with a Raf-1 selective inhibitor prevented the ANG II-dependent increase in eNOS protein expression (0.75 ± 0.17-fold increase; Fig. 8). Taken together, these data demonstrate that Ras and Raf-1 mediate the ANG II-dependent increase in eNOS protein expression in PAECs.


Figure 7
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  		Fig. 7. ANG II-dependent increases in eNOS protein expression are dependent on Ras. A: PAECs were transfected with Ras DN mutant cDNA before being stimulated with 100 nM ANG II for 8 h (n = 5). Shown are representative immunoblot of eNOS and actin and overexpression of DN Ras. B: PAECs were pretreated with 1 µM FTI, a farnesyl transferase inhibitor, for 24 h before being stimulated with ANG II for 8 h (n = 6). Shown are representative immunoblots of eNOS and actin. C: blots were scanned and relative levels of eNOS expression were quantified by laser densitometry and normalized to actin levels. Values are means ± SE. *P < 0.05 vs. untreated control cells; #P < 0.05 vs. ANG II-treated cells.

 

Figure 8
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  		Fig. 8. ANG II-dependent increases in eNOS protein expression are dependent on Raf-1 kinase. PAECs were pretreated with 1 µM Raf-1 inhibitor for 1 h before being stimulated with 100 nM ANG II for 8 h. eNOS and actin protein expression were determined by immunoblot analysis. Blots were scanned and relative levels of eNOS expression were quantified by laser densitometry. Values are means ± SE; n = 6. *P < 0.05 vs. untreated control cells; #P < 0.05 vs. ANG II-treated cells.

 
Since the Shc/Grb2/Sos-Ras/Raf/ERK cascade is a well-recognized growth-promoting cascade activated by many growth factors (3, 28, 35), the effect of ANG II on PAEC growth was tested. Cell proliferation was assessed using two methods: reduction of MTS tetrazolium (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega) and incorporation of bromodeoxyuridine (BrdU; BrdU Cell Proliferation Assay Kit; Upstate Chemicon). PAECs were plated at 1 x 103 cells/well in a 96-well plate and allowed to attach overnight. When the cells reached 50% confluence, media were replaced with serum-free media to induce quiescence, and then either 100 nM or 1 µM ANG II was added to the cells. Cell growth was assessed at 12 and 24 h by monitoring the MTS reduction (absorbance: 490 nm). ANG II did not stimulate PAEC proliferation. However, when these cells were placed in complete media (containing serum), they resumed growth until cells reached 100% confluence. Results were the same when we monitored ANG II-stimulated BrdU incorporation in PAECs.

ANG II stimulated NO production via a Src-, Ras-, and Raf-dependent pathway. To show that this increase in eNOS protein expression leads to an increase in NO production, PAECs were pretreated with 100 µM PP2, 1 µM FTI-277, or 1 µM Raf-1 inhibitor before being stimulated with 100 nM ANG II. ANG II stimulated a 40% increase in NO2– production (74.40 ± 4.88 nmol/mg, n = 8, P < 0.05) compared with control cells (55.84 ± 4.24 nmol/mg) that was blocked when PAECs were pretreated with PP2 (60.3 ± 4.88 nmol/mg), FTI-277 (55.44 ± 4.56 nmol/mg), or the Raf-1 kinase inhibitor (55.04 ± 5.80 nmol/mg). These compounds had no effect on basal NO2– production.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
We (20, 26, 27) have previously reported that the ANG II-dependent increase in eNOS protein expression in PAECs is mediated via AT2 receptor pertussis toxin-sensitive Src-dependent MAPK activation. In the present study, we provide experimental evidence that AT2 receptor induction of eNOS protein expression is via a G{alpha}i3 Src-mediated pathway involving recruitment of the adapter proteins Shc and Grb2, the guanine nucleotide exchange protein Sos, Ras GTP-binding protein, and the serine threonine kinase Raf-1 (Fig. 9).


Figure 9
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  		Fig. 9. A hypothetical scheme whereby AT2 receptor-dependent Src activation of MAPK and the increase in eNOS expression is mediated via a G{alpha}i3/Src/Shc/Grb2/Sos/Ras/Raf pathway in PAECs. ANG II, via G{alpha}i3, stimulates Src-dependent phosphorylation of the adaptor protein Shc, which then associates with the adapter protein Grb2 and the guanine nucleotide exchange protein Sos. Sos then stimulates Ras GTP binding, which results in sequential activation from Ras to Raf-1 to MEK and ultimately to MAPK and to an increase in NO production. In vascular smooth muscle cells, NO activates soluble guanylyl cyclase (sGC), which induces an increase of cGMP, leading to vasodilation. Our hypothesis is that, in the pulmonary system, ANG II increases NO, via an endothelial AT2 receptor, to counterbalance its AT1 receptor-dependent vasoconstriction in smooth muscle cells.

 
Even though the physiological effects of the AT2 receptor have not been fully elucidated, an emerging role of the AT2 receptor is to mediate vasodilation and to counteract the vasoconstrictive effect of the AT1 receptor (6, 7, 18). Our previous results (27) demonstrated that activation of the AT2 receptor leads to an increase in eNOS mRNA, protein expression, and NO production in PAECs. In agreement with our studies, Ritter and colleagues (29) found that ANG II increases eNOS protein expression via an AT2 receptor-dependent calcineurin-nuclear factor of activated T cells pathway in cardiomyocytes. Other investigators (4, 34) have also demonstrated that the AT2 receptor is linked to an increase in NO synthesis, yet found that the mechanism involves acute activation of NOS via the bradykinin pathway. More recently, Hiyoshi and colleagues (14) demonstrated that an AT2 receptor-dependent increase in eNOS phosphorylation leads to an increase in NO production independent of Akt in aortas of mice with two-kidney, one-clip hypertension.

In contrast, other studies have demonstrated that ANG II increases NO production either by activating the eNOS enzyme (30) or increasing eNOS protein expression (32) via the AT1 receptor. Nevertheless, published results from our laboratory (9) have demonstrated that treatment of PAECs with the AT1 receptor antagonist losartan leads to an increase in eNOS mRNA and protein and NO production, suggesting that the function of the AT1 receptor in PAECs is to inhibit NO production in PAECs. Furthermore, recent data from our laboratory (41) have suggested that an AT1/PLC/Ca2+/PKC-{alpha}-dependent pathway downregulates eNOS protein expression in PAECs. The reason for the discrepancies between these studies are not entirely clear but could be explained either by differences in the duration of exposure and the concentration of ANG II, the type of ANG II receptor subtypes expressed on the cells, or by differences in the source of endothelial cells.

Even though the AT2 receptor has the seven-transmembrane protein topology typical of the G protein-coupled family of receptors (6, 7, 18), whether it couples to GTP-binding proteins remains controversial. Results from our previous study (27) and data from this study (Fig. 4) indicated that a pertussis toxin-sensitive G protein is involved in the ANG II stimulation of Ras GTP binding, ERK1/2 activation, and the increase in eNOS protein expression in PAECs. In this study, we also demonstrated that transfection of PAECs with the DN mutant cDNA of G{alpha}i3 blocks the ANG II-dependent increase in Src phosphorylation, ERK1/2 phosphorylation, and eNOS protein expression. And, finally, using coimmunoprecipitation experiments, we have demonstrated that G{alpha}i3 associates with both the AT2 receptor (S. C. Olson, unpublished observation) and Src (20) in PAECs.

Similarly, in preglomerular smooth muscle cells, AT2 receptor generation of NO is blocked by pertussis toxin, suggesting that it is mediated via a member of the G{alpha}i family of GTP-binding proteins (2). Furthermore, in neuronal cells, AT2 receptor activation of PP2A is sensitive to pertussis toxin (16), and, in renal proximal tubule cells, AT2 receptor-dependent activation of PLA2 is G protein mediated (13). Conversely, in NIE-115 neuroblastoma cells, AT2 receptor inhibition of ERK occurs via a G protein-independent mechanism (5). The results of this study as well as well as previously published reports (6, 11, 18) clearly demonstrate that the AT2 receptor activates diverse signaling pathways in numerous cell lines and demonstrates the importance of identifying ANG II-dependent cell-specific responses.

The results from this study demonstrate that ANG II stimulates an increase in Src-dependent phosphorylation of the adaptor protein Shc (Fig. 2) and an increase in the membrane localization of Shc (Fig. 3). There was also an increase in Shc coassociation with the adapter protein Grb2 and an increase in Grb2 associated with the membrane fraction. Grb2 then associates with and results in the activation of the guanine nucleotide exchange protein Sos, followed by an increase in Ras GTP binding. This ANG II-dependent increase in Ras GTP binding is mediated via a pertussis toxin-sensitive AT2 receptor and is dependent on Src activation. A linkage between Ras and the AT2 receptor has been reported previously by Douglas and colleagues (17), who demonstrated that ANG II-dependent activation of Ras was blocked by PD-123319 in renal proximal tubular cells.

In PAECs, this AT2 receptor-dependent increase in Ras GTP binding leads to an increase in Raf-1 serine phosphorylation and an increase in Raf-1 associated with the membrane. Additionally, this Raf-1 activation is dependent on Src as pretreatment with the Src tyrosine kinase inhibitor PP2 blocked Raf phosphorylation. Supporting our studies demonstrating a linkage between the AT2 receptor and Raf serine-threonine kinase is the study by Gendron and colleagues (12), who demonstrated that in the neuronal NG108-15 cell line, the ANG II-induced sustained increase in p42/p44 MAPK activity was dependent on AT2 receptor activation of the neuronal-specific B-Raf kinase.

Investigators have demonstrated that the AT2 receptor is linked to both inhibition (15, 37) and activation (11, 37) of the MAPK cascade. In several cell lines, including vascular smooth muscle cells, cardiomyocytes, rat pheochromocytoma (PC12W) cells, and fibroblasts undergoing apoptosis, Horiuchu and colleagues (15) showed that AT2 receptor activation of MAPK phosphatase-1 leads to inactivation of ERK1/2. Similarly, Stroth et al. (37) found that the AT2 receptor is linked to inhibition of MAPK in NGF-stimulated PC12W cells. Conversely, other studies (11, 17, 37), including our own (20), have demonstrated that the AT2 receptor is linked to MAPK activation. Douglas and colleagues demonstrated that ANG II-dependent activation of Ras (17) and MAPK (13) in renal proximal tubular cells was mediated via the AT2 receptor. Gendron et al. (11) demonstrated in NG108-15 cells that the AT2 receptor-dependent increase in ERK1/2 mediates ANG II-dependent increases in neurite outgrowth. Finally, in quiescent PC12W cells, Stroth and colleagues (37) demonstrated that ERK activation by ANG II could be inhibited by the AT2 receptor antagonist PD-123177.

In conclusion, we propose that the following signaling pathway links ANG II-dependent Src activation to the MAPK cascade and to the increase in eNOS protein expression in pulmonary endothelium: ANG II via G{alpha}i3 stimulates Src-dependent phosphorylation of Shc and its association with Grb2, which then recruits Sos. Sos then stimulates Ras GTP binding, which results in the sequential activation from Ras to Raf-1 to MEK and ultimately to MAPK (Fig. 9). Our hypothesis is that, in the pulmonary system, ANG II increases NO, via an endothelial AT2 receptor, to counterbalance its AT1 receptor-dependent vasoconstriction in smooth muscle cells (Fig. 9). Since recent studies (9, 23) with NOS knockout mice have demonstrated that eNOS is the primary source of NO in the lung, characterization of the proteins/enzymes that regulate NO production in the pulmonary endothelium is essential for the understanding of the control of pulmonary vascular tone both under normal and pathological conditions. We propose that this AT2 receptor-dependent increase in eNOS may provide a protective mechanism in the pulmonary circulation when it is challenged by elevated levels of ANG II, such as those seen during hypoxic conditions and in renin-dependent systemic hypertension.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grant HL-63182.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. C. Olson, Dept. of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595 (e-mail: susan_olson{at}nymc.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.

1 Supplemental material for this article is available online at the American Journal of Physiology-Cell Physiology website. Back


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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