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Am J Physiol Heart Circ Physiol 286: H1954-H1962, 2004. First published December 23, 2003; doi:10.1152/ajpheart.00134.2003
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SAM68: a downstream target of angiotensin II signaling in vascular smooth muscle cells in genetic hypertension

Mohammed El Mabrouk, Quy N. Diep, Karim Benkirane, Rhian M. Touyz, and Ernesto L. Schiffrin

Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec, Canada H2W 1R7

Submitted 26 January 2003 ; accepted in final form 19 December 2003


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated whether phosphatidylinositol 3-kinase (PI3K) and 68-kDa Src associated during mitosis (SAM68) are involved in angiotensin II (ANG II) growth signaling in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). PI3K activity was assessed by measuring the phosphorylation of the regulatory subunit p85{alpha} and kinase activity of the catalytic 110-kDa subunit of PI3K. The PI3K-SAM68 interaction was assessed by coimmunoprecipitation, and SAM68 activity was evaluated by poly(U) binding. SAM68 expression was manipulated by SAM68 antisense oligonucleotide transfection. VSMC growth was evaluated by measuring [3H]leucine and [3H]thymidine incorporation as indexes of protein and DNA synthesis, respectively. ANG II increased the phosphorylation of p85{alpha} and kinase activity of the 110-kDa PI3K subunit in VSMCs from SHR and transiently increased p85{alpha}-SAM68 association. In Wistar-Kyoto (WKY) rat cells, ANG II increased SAM68 phosphorylation without influencing poly(U) binding. In SHR, ANG II did not influence SAM68 phosphorylation but increased SAM68 binding to poly(U). ANG II stimulated phosphoinositol phosphate synthesis by PI3K in SAM68 immunoprecipitates in both groups, with significantly enhanced effects in SHR. Inhibition of PI3K, using the selective inhibitor LY-294002, and downregulation of SAM68, by antisense oligonucleotides, significantly decreased ANG II-stimulated incorporation of [3H]leucine and [3H]thymidine in VSMCs, showing the functional significance of PI3K and SAM68. Our data demonstrate that PI3K and SAM68 are involved in ANG II signaling and that SAM68 is differentially regulated in VSMCs from SHR. These processes may contribute to the enhanced ANG II signaling and altered VSMC growth in SHR.

growth; renin-angiotensin system; signal transduction; spontaneously hypertensive rat

VASCULAR SMOOTH MUSCLE CELL (VSMC) growth and extracellular matrix deposition are processes required for vascular remodeling in hypertension. Angiotensin II (ANG II) plays a major role in the development of hypertension (16, 25, 32). VSMC growth signaling through ANG II receptors involves several distinct intracellular signaling pathways including tyrosine kinases, MAPKs (ERK1/2, JNK, and p38 kinase), Janus kinase (JAK)/signal transducer and activator of transduction (STAT), NF-{kappa}B, and phosphatidylinositol 3-kinase (PI3K) (5, 31, 37). PI3Ks are ubiquitous lipid kinases. The predominant form is a heterodimer enzyme consisting of a p110 catalytic subunit and p85{alpha} regulatory subunit. PI3K phosphorylates phosphatidylinositols at the D-3 inositol ring. PKB/Akt, which is downstream of PI3K, is activated by binding of 3-phosphoinositides, arising from action of PI3K. These kinases are implicated in many biological functions such as cell growth, differentiation, survival, cytoskeletal organization, and cell motility (33).

PI3K interacts via its SH2 and SH3 domains with various proteins that contain a tyrosine phosphorylation-specific motif and proline-rich domains (30). One of these proteins is 68-kDa Src associated during mitosis (SAM68), a member of the signal transduction and activation RNA (STAR) proteins. SAM68 interacts with several signaling proteins, such as Grb2, phospholipase C-{gamma}, p85{alpha}, p47phox, JAK3, Cbl, SHP-1, p120GAP, and Nck, and with RNA via its KH, QUA1, and QUA2 domains. It is a target of several tyrosine kinases such as Src, Fyn, Lck, Tec, Jak3, Brk, Zap70, and Btk (8, 11, 18, 19, 30, 38). Thus SAM68 plays a role in signal transduction and RNA metabolism. SAM68 is tyrosine phosphorylated downstream of Src, Fyn, Lck, Btk, Tec, Zap70, and Sik in mitosis in tumor cell lines (6, 11, 14, 18, 26). However, some studies have reported that SAM68 is tyrosine phosphorylated upon stimulation with insulin, CD3, CD4, CD16, or CD32 (12, 17, 18, 30). Furthermore, SAM68 has been implicated in cell cycle control and cell differentiation (24).

We (10) previously demonstrated that PI3K plays a role in ANG II-stimulated ERK1/2 activation in spontaneously hypertensive rat (SHR)-derived mesenteric artery SMCs. Likewise, PI3K influences heightened norepinephrine neuromodulatory actions of ANG II in SHR-derived neurons (41). Thus the question arises as to whether PI3K and SAM68 are involved in perturbed signaling by ANG II in SHR. In the present study, we investigated the relationships between PI3K and SAM68 in ANG II-stimulated VSMCs and determined whether these interactions are altered in SHR. Moreover, we assessed the functional role of PI3K in ANG II-mediated VSMC growth responses in SHR.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Chemicals. Losartan (AT1 receptor antagonist) was a gift from Dr. R. D. Smith (Merck; Whitehouse Station, NJ). PD-123319 (AT2 receptor antagonist) was a gift of Dr. J. Keiser (Parke-Davis; Ann Arbor, MI). The anti-PI3K p85{alpha} antibody and horseradish peroxidase-conjugated 4G10 anti-phospho-tyrosine (p-Tyr) antibody were from Upstate Biotechnology (Lake Placid, NY). Antibodies against SAM68, p-Tyr (PY99) AC, anti-rabbit IgG-FITC, and protein A/G plus agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). LY-294002 (specific PI3K inhibitor) was from Calbiochem (La Jolla, CA). Phospho-specific antibodies against STAT3 and PKB/Akt were from Cell signaling (Mississauga, Ontario, Canada). AGPOLY(U) type 6 was from Amersham Pharmacia Biotech (Quebec, Canada). NE-PER nuclear and cytoplasmic extraction reagents were from Pierce (Rockford, IL).

Cell culture. The present study was conducted according to recommendations from the Animal Care Committee of the Clinical Research Institute of Montreal and the Canadian Council of Animal Care. Wistar-Kyoto (WKY) and SHR (17 wk old) were from Taconic Farms (Germantown, NY). VSMCs from mesenteric arteries were obtained by enzymatic digestion and cultured as described (10). VSMCs from passages 3 to 6 were used for all experiments. VSMCs at 80–90% confluence were maintained in serum-free DMEM for 48 h to render cells quiescent.

[3H]thymidine incorporation. VSMCs were stimulated with ANG II for 24 h in the absence or presence of the PI3K inhibitor LY-294002 (10–5 mol/l). [3H]thymidine (1 µCi/ml) was added for the last 4 h of incubation. To stop the reaction, cells were washed with cold PBS. Thereafter, cells were trypsinized (0.25 U/ml) and collected with an automatic LKB Wallac cell harvester on to glass fiber filtermats. Filters were dried and placed in a plastic bag, to which scintillation cocktail (10 ml) was added. Radioactivity incorporated into DNA was counted in a LKB Wallac 1205 BETAPLATE counter.

[3H]leucine incorporation. VSMCs were stimulated with ANG II for 24 h in the absence or presence of LY-294002 (10–5 mol/l). [3H]leucine (1 µCi/ml) was added at the same time as ANG II. Thereafter, cells were washed with PBS and incubated with trichloracetic acid (TCA; 5%) for 30 min at 24° C. Cells were washed twice with 5% TCA and three times with tap water and then solubilized in 0.2 mol/l NaOH for 30 min at 37° C. Radioactivity incorporated into proteins was determined as for measurement of [3H]thymidine incorporation.

Immunoprecipitation and immunoblotting. VSMCs were stimulated with ANG II for the indicated times, washed, and lysed on ice in lysis buffer [25 mmol/l HEPES (pH 7.5), 50 mmol/l NaCl, 50 mmol/l NaF, 5 mmol/l EDTA, 1 mmol/l Na3VO4, 1% Nonidet P-40, 1 µg/ml aprotinin, 0.7 µg/ml pepstatin, and 1 mmol/l PMSF]. Cells were scraped and centrifuged at 16,000 g for 20 min. Proteins (500 µg) were incubated with p85{alpha} antibody, PY99 antibody against the p-Tyr residue conjugated to agarose, or SAM68 antibody overnight at 4° C. Thereafter, immunocomplexes were adsorbed to protein A/G-agarose, washed, and eluted by boiling in sample buffer [125 mmol/l Tris·HCl (pH 6.8), 2% SDS, 2% glycerol, and 1 mmol/l {beta}-mercaptoethanol]. Proteins were separated by 10% SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked in PBS containing 3% (wt/vol) nonfat dry milk, and incubated for 20 min at 37° C for p85{alpha} and p-Tyr antibodies or 5% Tris-buffered saline-0.1% Tween 20 for SAM68, PKB, ERK1/2, and STAT3 antibodies. Membranes were probed with anti-p85{alpha} (1/8,000), anti-p-Tyr (AG-410, 1/1,000), or anti-SAM68, anti-PKB, anti-ERK1/2, and anti-STAT3 antibodies (1/1,000) overnight at 4° C. Detection was carried out using anti-rabbit IgG-horseradish peroxidase. Blots were developed by enhanced chemiluminescence. Band intensity was analysed using Image Quant software 5.0 (Molecular Dynamics; Sunnyvale, CA).

PI3K assay. PI3K activity was quantified by an in vitro assay using phosphatidylinositol (PtdIns) as the substrate (3). Briefly, p85{alpha} and SAM68 immunocomplexes were washed with lysis buffer; three times with 100 mmol/l Tris·HCl (pH 7.5), 5 mmol/l LiCl, and 0.1 mmol/l sodium orthovanadate; two times with washing buffer (20 mmol/l Tris·HCl, 100 mmol/L NaCl, 1 mmol/L EDTA, and 0.1 mmol/l Na3VO4); and two times with reaction buffer [20 mmol/l Tris·HCl (pH 7.5), 10 mmol/l NaCl, 0.5 mmol/l EGTA, and 0.2 mg/ml PtdIns]. The immunoprecipitates were resuspended in 50 µl of reaction buffer, and the assay was started by the addition of 5 µl of [{gamma}-32P]ATP (1 µC/sample), 100 mmol/l MgCl2, and 0.4 µmol/l ATP. After 30 min, the reaction was stopped by the addition of 150 µl of chloroform-methanol-HCl (100:200:2). The 32P-labeled lipid products of the kinase reaction were separated by thin layer chromatography and analysed by Image Quant software 5.0.

Poly(U) binding assay. The poly(U) binding assay was performed as described by Taylor and Shalloway (36). Cell lysates (500 µg/ml) were incubated with poly(U)-agarose (20 µl) for 30 min at 4° C. Thereafter, beads were washed with lysis buffer. Beads were resuspended in 25 µl of SDS-PAGE sample buffer. Proteins were separated by 10% SDS-PAGE, transferred to PVDF membranes, and blotted with anti-SAM68 antibody.

Liposomal transfection with SAM68 antisense oligonucleotide. VSMC derived from WKY were transfected with SAM68 antisense phosphorothioated oligodeoxynucleotides (ODN) (5'-CTCAGCCATGAGTTCAGG-3'). The corresponding sense ODN (5'-CCTGAACTCATGGCTGAG-3') was used as a control. Transfection was performed with 50 nmol/l ODN performed in serum-free, antibiotic-free DMEM with 5 µg/ml lipofectamine reagent (GIBCO-BRL) for 24 h at 37° C in 5% CO2-95% air, as recommended by the manufacturer. Culture medium was replaced with lipofectamine-free DMEM. VSMCs were cultured for a further 24 h.

SAM68 immunofluorescence. VSMCs grown on glass coverslips were fixed in PBS containing 3.7% formaldehyde and 0.2% Triton X-100 for 10 min at 25° C. Fixed cells were incubated with PBS blocking solution (10% goat serum and 0.2% Triton X-100; 20 min, 25° C). Thereafter, cells were probed with anti-SAM68 antibody in blocking solution (1/50) for 1 h at 25° C. Cells were washed three times for 5 min, incubated with anti-rabbit IgG-FITC, and diluted in blocking solution (1/50) for1hat25° C. Coverslips were washed with PBS-BSA, mounted with mounting media (Molecular Probes; Eugene, OR), and visualized by Zeiss LSM 510 confocal microscopy.

Nuclear and cytoplasmic fractions. VSMC were stimulated with ANG II at the indicated times as already described. After stimulation, cells were washed with cold PBS, scraped, and transferred to Eppendorf tubes. Methodology of the nuclear and cytoplasmic preparation was based on the manufacturer's instructions (NE-PER, Pierce).

Statistical analysis. Results are reported as means ± SE and were compared by Student's t-test or ANOVA, followed by a post hoc Bonferroni multiple-comparisons test. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ANG II-induced PI3K activity in WKY and SHR. ANG II effects on PI3K activity in VSMCs was assessed by measuring the phosphorylation of the regulatory subunit p85{alpha} and the lipid kinase activity of the catalytic subunit p110 in VSMCs. Cells were stimulated with ANG II (10–7 mol/l) for the indicated times (Fig. 1, A and B). ANG II caused a time-dependent increase of p85{alpha} phosphorylation and of the lipid kinase activity at as early as 2.5 min, reaching a maximal effect at 5 min. Thereafter, responses plateaued for 30 min. Despite the similar kinetics of ANG II-induced PI3K activity in WKY and SHR, the response to ANG II was significantly higher in VSMCs from SHR than WKY.



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  		Fig. 1. Time course of ANG II-induced p85{alpha} subunit phosphorylation (p-p85{alpha}) and phosphatidylinositol 3-kinase (PI3K) activity. Serum-deprived vascular smooth muscle cells (VSMCs) were stimulated with ANG II (10–7 mol/l), lysed, and immunoprecipitated (IP) with an antibody against the p85{alpha} subunit. The immunocomplex formed was assayed for p85{alpha} phosphorylation (A) and PI3K activity (B). A, top: representative immunoblot (IB) of ANG II-induced phosphorylation of p85{alpha}. B, top: representative autoradiogram of thin-layer chromatography. A and B, bottom: means ± SE of 3 different experiments. Pty, phospho-tyrosine; JkT, Jurkat cells; SHR, spontaneously hypertensive rats; WKY, Wistar-Kyoto rats; PIP, phosphatidylinositol 4,5-bisphosphate. *P < 0.01 vs. WKY counterpart.

 

AT1 receptor-mediated p85{alpha} phosphorylation. To investigate the receptor subtype mediating the ANG II-stimulated PI3K activation, VSMCs were pretreated with 10–5 mol/l losartan (AT1 receptor blocker) or PD-123319 (AT2 receptor blocker) (Fig. 2). Losartan blocked the ANG II effect on p85{alpha} phosphorylation, whereas PD-123319 had no effect, indicating that ANG II activates PI3K exclusively via AT1 receptors. ANG II receptor antagonists did not have any effect on basal p85{alpha} phosphorylation. Thus AT1 receptors mediated ANG II-stimulated PI3K activation in VSMCs.



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  		Fig. 2. AT1 receptor-mediated p85{alpha} subunit phosphorylation. VSMCs were stimulated with ANG II in the presence or absence of losartan (Los; 10–5 mol/l) or PD-123319 (PD123; 10–5 mol/l) for 5 min. A: representative Western blots. B and C: means ± SE of 4 different experiments in WKY and SHR, respectively. *P < 0.05 vs. other groups.

 

Effect of PI3K inhibition on ANG II-induced VSMC growth. Similar to our previous findings (10), our data here demonstrate that, whereas ANG II induces hypertrophy of WKY VSMCs, it induced proliferation of SHR VSMCs (Fig. 3). To assess whether ANG II-induced growth effects are mediated through PI3K-dependent pathways, we examined the effects of LY-294002. As shown in Fig. 3, LY-294002 attenuated the increase of [3H]leucine incorporation induced by ANG II in VSMCs from WKY and SHR and [3H]thymidine incorporation induced by ANG II in SHR. It did not have any significant effect on basal [3H]leucine or [3H]thymidine incorporation and did not influence [3H]leucine incorporation in VSMCs from WKY.



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  		Fig. 3. Effect of LY-294002 (LY) on ANG II-induced protein and DNA synthesis in VSMCs from WKY and SHR. VSMCs were pretreated with LY (PI3K inhibitor; 10–5mol/l) for 30 min, incubated in DMEM supplemented with [3H]leucine and [3H]thymidine, and stimulated with ANG II in the presence or absence LY for 24 h. A and B: effect of LY on [3H]leucine and [3H]thymidine incorporation in VSMCs from WKY. C and D: effects of LY on [3H]thymidine and [3H]leucine incorporation in VSMCs from SHR. Data are means ± SE of 4 experiments (each in triplicate). *P < 0.05 vs. other groups.

 

ANG II stimulated interaction of PI3K and SAM68 and their localization in VSMCs from WKY and SHR. SAM68 coprecipitated with p85{alpha} under basal conditions (Fig. 4A). The association between p85{alpha} and SAM68 was transient with a peak at 2.5 min. p85{alpha} and SAM68 did not coprecipitate with anti-p85{alpha} antibody, suggesting that p85{alpha}-SAM68 complex formation induces a conformational change in p85{alpha} that is no longer recognized by the p85{alpha} antibody (data not shown). This interaction was not different between VSMCs derived from WYK and SHR. Localization of SAM68 was investigated in ANG II-stimulated VSMCs by immunoblotting and immunocytochemistry (Fig. 4, B and C). SAM68 was localized in the nucleus before and after stimulation with ANG II. We also investigated the PI3K translocation induced by ANG II. Under basal conditions, the p85{alpha} subunit of PI3K was localized in the cytoplasm and nucleus, and this was not affected by ANG II stimulation in either WKY or SHR. Thus the ANG II-induced interaction of PI3K and SAM68 appears to be primarily intranuclear.



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  		Fig. 4. ANG II-induced 68-kDa Src associated during mitosis (SAM68) association with p85{alpha} and their localization in VSMCs. VSMCs were exposed to ANG II for the indicated times. A, top: after immunoprecipitation with anti-SAM68, proteins were subjected to electrophoresis followed by Western blot using antibody against p85{alpha}. A, bottom: means ± SE of 8 experiments. *P < 0.05 vs. control by ANOVA, followed by a post hoc Bonferroni test; #P < 0.05 vs. control by Student's t-test. B and C: localization of SAM68 and p85{alpha} in VSMCs from WKY and SHR. B: SAM68 and PI3K localization induced by ANG II. Protein (15 µg) from the cytoplasm (c) or nucleus (n) from VSMCs were subjected to SDS-PAGE, transferred, and probed with antibodies against SAM68 and p85{alpha}. C: immunostaining of VSMCs from WKY and SHR with SAM68 antibody. Quiescent VSMCs were stimulated with ANG II, fixed with 3.7% formaldehyde, and permeabilized with 0.2% Triton X-100. Cells were exposed to SAM68 antibody and visualized with a second antibody conjugated with FITC. VSMCs were imaged by a laser scanning confocal microscope (x600 magnification). Results are representative of 3 experiments.

 

ANG II-induced SAM68 phosphorylation in VSMCs. SAM68 was slightly tyrosine phosphorylated under basal conditions (Fig. 5). SAM68 was further tyrosine phosphorylated upon ANG II stimulation. In WKY, ANG II induced a slow and sustained increase in SAM68 phosphorylation with a maximal effect at 10 min. In contrast, in SHR, ANG II induced a weak phosphorylation of SAM68, which was lower (P < 0.01) than that in WKY.



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  		Fig. 5. Effect of ANG II on SAM68 phosphorylation. Tyrosine-phosphorylated SAM68 (p-SAM68) was immunoprecipitated using an antibody against phospho-tyrosine (pTyr) residues. Immunoprecipites were analyzed by Western blot with SAM68 antibody. A: representative Western blot. B: means ± SE of 3 different experiments. *P < 0.01 vs. WKY counterpart.

 

Phosphorylation of SAM68-induced by ANG II decreased the interaction of SAM68 with poly(U). Several studies have shown that SAM68 binds RNA with a UAAA motif and ribonucleotide homopolymers with a preference for poly(U) (23, 39). Only the dephosphorylated form of SAM68 has the ability to interact with RNA. The ANG II-induced interaction of SAM68 with poly(U) was time dependent, with significantly greater (P < 0.05) responses in SHR than WKY (Fig. 6). In SHR, ANG II induced a transient binding of SAM68 to poly(U). It increased the interaction of SAM68 and poly(U) with a peak at 5 min, followed by a decrease after 10 min. In contrast, ANG II did not have significant effects on the binding of SAM68 to poly(U) in WKY, whereas it significantly and persistently tyrosine phosphorylated SAM68. In SHR, the poly(U) binding assay demonstrated that the interaction was transient and lasted for first 10 min. This response suggested that other mechanisms could control the SAM68-RNA interaction after 10 min of stimulation with ANG II. One of the mechanisms that could modify SAM68 is threonine phosphorylation. An independent regulatory mechanisms of tyrosine phosphorylation of SAM68 and poly(U) binding properties could be another possibility in VSMCs from SHR.



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  		Fig. 6. Time course of the nucleic acid binding ability of dephosphorylated SAM68 induced by ANG II. Results demonstrated the differential inability of dephosphorylated SAM68 induced by ANG II to bind poly(U) in WKY and SHR. A: representative Western blot of dephosphorylated SAM68 in WKY and SHR. B: means ± SE of 3 different experiments. *P < 0.05 vs. WKY counterpart.

 

To examine the ANG II receptor subtypes whereby ANG II induced SAM68 phosphorylation, we measured ANG II-induced SAM68 phosphorylation and its binding to poly(U) in WKY and SHR, respectively, in the absence and presence of losartan or PD-123319 (Fig. 7, A and B). Treatment of VSMCs with losartan or PD-123319 did not influence basal levels of SAM68 phosphorylation or its interaction with poly(U). However, losartan and PD-123319 decreased the effect of ANG II on phosphorylation of SAM68 (20.5% and 27.5%, respectively) in VSMCs from WKY (Fig. 7A). In SHR, losartan and PD-123319 decreased SAM68 poly(U) binding (21% and 31%, respectively) in ANG II-stimulated VSMCs (Fig. 7B). Pretreatment of VSMCs with losartan and PD-123319 abrogated the effect of ANG II on SAM68 tyrosine phosphorylation and poly(U) binding.



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  		Fig. 7. Effects of AT1 and AT2 receptor blockers on ANG II-stimulated tyrosine phosphorylation of SAM68 in WKY (A) and nucleic acid binding ability of SAM68 in SHR (B). VSMCs from WKY and SHR, pretreated with Los and PD1233, were stimulated with ANG II for 10 or 2.5 min, respectively. Top: representative immunoblots of p-SAM68 in WKY and nucleic acid binding of SAM68 in SHR. Bottom: means ± SE of at least 5 experiments. *P < 0.05; #P < 0.01.

 

Effects of SAM68 antisense ODN on ANG II signaling pathways and cell growth. To investigate the role of SAM68 in intracellular ANG II signaling, we examined the effects of SAM68 antisense ODN on ERK1/2, STAT3, and PKB/Akt pathways stimulated by ANG II (Fig. 8). SAM68 antisense ODN, but not sense ODN, downregulated SAM68 expression (~60% reduction). Downregulation of SAM68 did not affect ANG II-stimulated ERK1/2, STAT3, and PKB/Akt in VSMCs. To investigate other possible functions of SAM68, we assessed phosphatidylinositol 4,5-bisphosphate (PIP) production in SAM68 precipitates from ANG II-stimulated cells. ANG II stimulation increased PIP synthesis associated with SAM68, as demonstrated in SAM68 immunoprecipitates, with significantly greater responses in SHR than WKY (Fig. 8B).



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  		Fig. 8. Effects of SAM68 antisense oligonucleotide on ANG II-stimulated ERK1/2, signal transducer and activator of transcription (STAT)3, and PKB/Akt phosphorylation in WKY VSMCs and PI3K activity in anti-SAM68 immunoprecipitates. A: VSMCs were stimulated with ANG II for 5 min for ERK1/2 and PKB/Akt and for 30 min for STAT3. {alpha}-Actin was used as a control protein. B, top: time course of PI3K activity in SAM68 immunoprecipitates in VSMCs derived from WKY and SHR. B, bottom: means ± SE of 3 experiments. *P < 0.01 vs. WKY counterpart.

 

To assess whether SAM68 influences VSMC function, ANG II growth-inducing actions were assessed in SAM68 antisense ODN-transfected cells. As demonstrated in Fig. 9, ANG II-induced incorporation of [3H]leucine was significantly attenuated in antisense ODN-transfected but not sense ODN-transfected cells.



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  		Fig. 9. Effects of SAM68 antisense and sense oligonucleotides on ANG II-stimulated protein synthesis in VSMCs from WKY (A) and SHR (B). VSMCs were transfected with oligonucleotides as described inMETHODS. Cells were stimulated with ANG II for 24 h and incubated in DMEM supplemented with [3H]leucine. Data are means ± SEM of 3 experiments. *P < 0.05 vs. ANG II-treated groups; #P < 0.01 vs. control.

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The involvement of PI3K in the regulation of cell growth, differentiation, survival, motility, and metabolic actions of many hormones is well established (33). However, its implication in vascular changes associated with hypertension is unclear. The present study characterizes for the first time the role of PI3K and the Src-associated protein SAM68 in ANG II-induced VSMC growth in SHR. Our findings indicate that ANG II mediates part of its growth effects via PI3K-dependent pathways in WKY and SHR. ANG II stimulates the interaction of p85{alpha} with SAM68 in the nucleus. SAM68 is tyrosine phosphorylated upon cell stimulation by ANG II in VSMCs and is differentially regulated in SHR. AT1 and AT2 receptors mediate tyrosine phosphorylation of SAM68 in WKY and its RNA binding properties in SHR. In the present study, ANG II increased PIP synthesis in SAM68 immunoprecipites significantly more in VSMCs from SHR than WKY.

VSMCs from small arteries were studied because these are the vessels that contribute physiologically to blood pressure regulation and pathophysiologically to the development of hypertension. Abnormalities in primary cultured VSMCs from SHR are maintained in serially passaged cells. Alterations in signaling events induced by ANG II in SHR may indeed be due to genetic differences with normotensive rats and not the result of culture conditions.

AT1 receptors have been linked to vascular remodeling because of their implication in SMC hypertrophy and/or hyperplasia, extracellular matrix deposition, and inflammatory responses (28, 37). In our study, ANG II mediated p85{alpha} phosphorylation via AT1 receptors in VSMCs from WKY and SHR. ANG II increased the phosphorylation of p85{alpha} and the lipid kinase activity of PI3K, which was significantly higher in VSMCs from SHR than WKY. This finding is in agreement with and extends previous studies (31). Consistent with our results, the activity of PI3K is impaired in SHR neurons (41) and skeletal muscle of fructose-hypertensive rats (4). The higher activity of PI3K in SHR VSMCs could be due to a decrease of p85{alpha} dephosphorylation. Regulator of ubiquitous kinase (Ruk), an adapter protein, interacts with p85{alpha} and decreases lipid kinase activity of PI3K (13). Downregulation of this adapter protein could play a role in enhanced PI3K activity, but this remains to be demonstrated.

The role of PI3K in cell growth has been investigated in VSMCs stimulated by different growth factors such as IGF-1 and PDGF (9,15) and by vasoconstrictors such as ANG II (31) and ATP (40). PI3K mediated the expression of cyclin D1 and the subsequent proliferation of airway SMCs stimulated by PDGF (26). PI3K also plays a role in VSMC proliferation in neointima after balloon injury (32). Furthermore, expression of constitutively active or dominant negative mutant PI3K in the heart of transgenic mice showed an increase or decrease in heart size, respectively, associated with parallel change in the size of myocytes (35). The antiapoptotic role of PI3K has also been demonstrated in VSMCs. It contributes to cell growth, as cell growth depends on the balance between proliferative and apoptotic pathways (1). We demonstrate here that ANG II induced hypertrophy of VSMCs from WKY and hypertrophy/proliferation in VSMCs from SHR. Indeed, our previous studies suggest that the PI3K pathway plays an important role in ERK activation in VSMCs from SHR but not in WKY (10). Therefore, we examined the effect of a selective PI3K inhibitor (LY-294002) in VSMC growth induced by ANG II. PI3K is required for ANG II signaling pathways leading to hypertrophy in WKY and hypertrophy/proliferation in SHR. A higher and persistent stimulation of PI3K in response to ANG II in SHR may be sufficient to shift the signaling equilibrium toward cell proliferation (10).

In addition to binding to the catalytic p110 subunit of PI3K, the regulatory p85{alpha} subunit of PI3K, which is the best characterized isoform, interacts with many tyrosine kinase receptors and several signaling molecules, such as p130Cas and SAM68 (21, 30, 42). In quiescent VSMCs, ANG II induced a transient association of SAM68 and p85{alpha} in SHR. SAM68 localized in the nucleus, whereas PI3K is localized in both the cytoplasm and nucleus. Thus the interaction between SAM68 and p85{alpha} probably occurs primarily in the nucleus, where it could control PIP synthesis. Indeed, ANG II stimulates PI3K activity, as assessed by PIP formation in the anti-SAM68 immunoprecipitate, which was higher in SHR than WKY VSMCs. p85{alpha} may also act as a signal transducer protein. The involvement of p85{alpha} in p53-mediated apoptosis induced by oxidative stress seemed to be independent of the catalytic subunit p110 and its lipid kinase (42).

Increasing evidence suggests that SAM68 plays a role in signal transduction of TCR-CD4, CD32, and insulin receptors in nonproliferative cells (12, 17). ANG II stimulated tyrosine phosphorylation of SAM68, and the response was sustained for up to 30 min in VSMCs from WKY but not SHR. The inability of ANG II to stimulate SAM68 phosphorylation in SHR may be due to an abnormality of the SAM68 signaling pathway in this model of genetic hypertension. The kinase(s) upstream of SAM68 in the ANG II signaling pathway in VSMC is unknown.

SAM68, member of the family of STAR proteins, contains a KH domain and binds to RNA (38). The ability of SAM68 to bind to RNA with a UAAA motif (23) is negatively regulated by tyrosine phosphorylation (39). ANG II induced an increase of SAM68 binding to poly(U) in VSMCs from SHR. This is consistent with the inability of ANG II to increase the tyrosine phosphorylation of SAM68 in SHR. In contrast, ANG II did not increase the binding of SAM68 to poly(U) in WKY, whereas it did significantly and persistently tyrosine phosphorylate SAM68. The specific role of SAM68 in RNA metabolism is presently not well understood. However, it has been implicated in RNA stability. A new role has been attributed to SAM68 in cell cycle control. Liu et al. (24) reported that NIH3T3 murine fibroblasts deficient in SAM68 undergo irreversible tumorigenesis. In contrast, Barlat et al. (2) demonstrated that SAM68 controlled cell proliferation by promoting the S phase entry in NIH3T3 cells.

We investigated the functional role of SAM68 in VSMCs by using ODN antisense technology. Downregulation of SAM68 did not alter phosphorylation of ERK1/2, PKB/Akt, or STAT3, but significantly attenuated ANG II-induced incorporation of [3H]leucine in WKY and SHR. These findings suggest that ANG II influences VSMC growth through SAM68-mediated pathways that are independent of ERK1/2, PKB/Akt, and STAT3. The exact mechanisms whereby SAM68 influences cell growth are unclear and require further investigation.

Effects of ANG II are mediated by AT1 and AT2 receptors (7). In VSMCs, the role of the AT2 receptor in VSMC growth is controversial (20, 22, 29). We investigated which ANG II receptor subtypes mediate SAM68 phosphorylation and its ability to bind poly(U) in WKY and SHR. Our findings demonstrate that AT1 and AT2 receptors mediated SAM68 phosphorylation in WKY and its interaction with poly(U) in SHR, suggesting that both receptors could play a role in SAM68 signaling in VSMCs. The direct implication of AT2 receptors in the transcription machinery has been shown by Ruiz-Ortega et al. (28), who demonstrated that AT1 and AT2 receptors mediate NF-{kappa}B activation in VSMCs. Peng et al. (27) demonstrated that AT1 and AT2 receptors act synergistically to induce the expression of FGF-2 in bovine adrenal medullary chromaffin cells.

In conclusion, findings from our study demonstrate that PI3K and the Src-associated protein SAM68 are involved in ANG II signaling and that they play a role in VSMC growth. ANG II stimulation increases phosphorylation of SAM68 and modulates the interaction of SAM68 with p85{alpha}, primarily in the nucleus. Furthermore, PIP generation by PI3K appears to be regulated by SAM68 in the nucleus. In SHR, PI3K and SAM68 signaling pathways are upregulated in ANG II-stimulated cells. These processes may contribute, at least in part, to enhanced VSMC growth in SHR.


   ACKNOWLEDGMENTS
 
GRANTS

This study was supported by Canadian Institutes of Health Research (CIHR) Grants 13570 and 44018 and by a CIHR Group Grant to the Multidisciplinary Research Group on Hypertension. M. El Mabrouk was the recipient of a studentship from the Heart and Stroke Foundation of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. L. Schiffrin, Clinical Research Institute of Montreal, 110 Pine Ave. West, Montreal, Quebec, Canada H2W 1R7 (E-mail: ernesto.schiffrin{at}ircm.qc.ca).

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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