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Dual Role of Src Homology Domain 2-Containing Inositol Phosphatase 2 in the Regulation of Platelet-Derived Growth Factor and Insulin-Like Growth Factor I Signaling in Rat Vascular Smooth Muscle Cells

Department of Clinical Pharmacology (T.S., K.K., R.A., I.K.) and First Department of Internal Medicine (T.W., A.S., H.H., S.M., K.F.), Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan; and Sainou Hospital (H.I.), Toyama 930-0887, Japan

Address all correspondence and requests for reprints to: Toshiyasu Sasaoka, M.D., Ph.D., Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: tsasaoka-tym{at}umin.ac.jp.

	Abstract

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Src homology domain 2 (SH2)-containing inositol phosphatase 2 (SHIP2) possesses 5-phosphatase activity and an SH2 domain. The role of SHIP2 in platelet-derived growth factor (PDGF) and IGF-I signaling was studied by expressing wild-type (WT-) and a catalytically defective (IP-) SHIP2 into rat aortic smooth muscle cells by adenovirus-mediated gene transfer. PDGF- and IGF-I-induced tyrosine phosphorylation of their respective receptors and phosphatidylinositol 3-kinase (PI3-kinase) activity were not affected by the expression of either WT- or IP-SHIP2. SHIP2 possessed 5'-phosphatase activity to hydrolyze the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate in vivo. Akt and glycogen synthase kinase 3ß are known to be downstream molecules of PI3-kinase, leading to the antiapoptotic effect. Overexpression of WT-SHIP2 inhibited PDGF- and IGF-I-induced phosphorylation of these molecules and the protective effect of poly(ADP-ribose) polymerase degradation, whereas these phosphorylations and the protective effect were enhanced by the expression of IP-SHIP2, which functions in a dominant negative fashion. Regarding the Ras-MAPK pathway, PDGF- and IGF-I-induced tyrosine phosphorylation of Shc was not affected by the expression of either WT- or IP-SHIP2, whereas both expressed SHIP2 associated with Shc. Importantly, PDGF and IGF-I stimulation of Shc/Grb2 binding, MAPK activation, and 5-bromo-2'-deoxyuridine incorporation were all decreased in both WT- and IP-SHIP2 expression. These results indicate that SHIP2 plays a negative regulatory role in PDGF and IGF-I signaling in vascular smooth muscle cells. As the bifunctional role, our results suggest that SHIP2 regulates PDGF- and IGF-I-mediated signaling downstream of PI3-kinase, leading to the antiapoptotic effect via 5-phosphatase activity, and that SHIP2 regulates the growth factor-induced Ras-MAPK pathway mainly via the SH2 domain.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE PROLIFERATION AND apoptosis of vascular smooth muscle cells (VSMCs) play a key role in the development of atherosclerosis (1, 2, 3, 4). Platelet-derived growth factor (PDGF) and IGF-I are important regulators of the cell growth and death of VSMCs (3, 4, 5, 6, 7). Therefore, elucidation of the regulatory mechanism of PDGF and IGF-I signaling in the proliferation and apoptosis of VSMCs is important for understanding the pathogenesis of atherosclerosis. PDGF and IGF-I bind to cognitive receptors, leading to the phosphorylation of multiple tyrosine residues (8, 9). The activated PDGF receptor directly binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) and the phosphorylated IGF-I receptor transmits the signal to the p85 subunit via insulin receptor substrates, resulting in activation of the p110 catalytic subunit of PI3-kinase (8, 9). PI3-kinase activation is important for exerting subsequent signaling to Akt and glycogen synthase kinase 3ß (GSK3ß), leading to the antiapoptotic effect of PDGF and IGF-I (10, 11). On the other hand, Shc is known to be tyrosine-phosphorylated upon PDGF and IGF-I stimulation (8, 9). The tyrosine-phosphorylated Shc binds to Grb2, which is important for the growth factor-induced Ras-MAPK activation resulting in DNA synthesis (11, 12, 13, 14).

We and others have recently cloned SH2-containing inositol 5'-phosphatase 2 (SHIP2), which possesses 5'-phosphatase activity and an SH2 domain (15, 16). SHIP2 is known to hydrolyze the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] via 5'-phosphatase activity (17, 18). There is evidence to suggest that downstream target molecules of PI3-kinase are negatively regulated by SHIP2 (17, 18, 19). In addition, SHIP2, via the SH2 domain, is able to associate with the Tyr³¹⁷ residue of Shc, which is a binding site of Grb2 (16). Because the association of Shc with Grb2 is important for PDGF- and IGF-I-induced Ras-MAPK activation resulting in DNA synthesis, SHIP2 also appears to regulate Ras-MAPK signaling by interfering with the binding of Shc to Grb2 (11, 12, 13, 14). Thus, although the role of SHIP2 has been mainly clarified in insulin signaling (18, 19, 20), the impact of SHIP2 on PDGF and IGF-I signaling is largely unknown. As PDGF and IGF-I are critical growth factors for DNA synthesis and antiapoptosis in VSMCs in the process of the development and progression of atherosclerosis (10, 11, 12, 13, 14), clarification of the involvement of SHIP2 in the regulation of PDGF and IGF-I signaling is important for understanding the novel control mechanism of these signalings. To clarify the role of SHIP2 and the molecular mechanisms of the involvement in PDGF and IGF-I signaling, wild-type (WT-) and a catalytically defective (IP-) SHIP2 were expressed in rat aortic smooth muscle cells by means of adenovirus-mediated gene transfer. PDGF and IGF-I signaling leading to DNA synthesis and antiapoptosis was compared in WT- and IP-SHIP2-expressing cells. Furthermore, the pathological meaning of SHIP2 in VSMCs was investigated by examining the change in SHIP2 expression after chronic treatment with insulin.

	Materials and Methods

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Materials
Human recombinant IGF-I and insulin were provided by Fujisawa Pharmaceutical Co. (Osaka, Japan) and Novo Nordisk Pharma Ltd. (Copenhagen, Denmark), respectively. Human recombinant PDGF-BB was purchased from Life Technologies, Inc. (Grand Island, NY). [-³²P]ATP (111 TBq/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA). Two polyclonal anti-SHIP2 antibodies were described previously (16). A monoclonal antiphosphotyrosine antibody (PY20) was obtained from Transduction Laboratories (Lexington, KY). A polyclonal anti-PDGF ß-receptor antibody, a polyclonal anti-GSK3 antibody, a polyclonal anti-Akt antibody, a monoclonal anti-poly (ADP-ribose) polymerase (anti-PARP) antibody, and a monoclonal tetramethyl rhodamine isothiocyanate-conjugated mouse anti-HA antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A polyclonal anti-Thr³⁰⁸ phosphospecific Akt antibody, a polyclonal anti-Ser⁴⁷³ phosphospecific Akt antibody, and a polyclonal anti-Ser^21/9phosphospecific GSK3 antibody were purchased from New England Biolabs, Inc. (Beverly, MA). Enhanced chemiluminescence reagents were obtained from Amersham Pharmacia Biotech Corp. (Uppsala, Sweden). DMEM, MEM vitamin mixtures, and MEM amino acid solutions were purchased from Life Technologies, Inc. Japan (Tokyo, Japan). All other reagents were of analytical grade and purchased from Sigma-Aldrich Corp. (St. Louis, MO) or Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Construction of adenovirus vectors
cDNAs encoding rat wild-type SHIP2 (WT-SHIP2) and a phosphatidylinositol 5'-phosphatase-defective mutant of SHIP2 (IP-SHIP2) were described previously (16, 18). WT-SHIP2 and IP-SHIP2 were subcloned into the vector pAxCAwt and transferred to recombinant adenovirus by homologous recombination using an Adenovirus Expression Vector Kit (Takara Biomedicals, Tokyo, Japan). An adenovirus vector encoding the hemagglutinin-tagged pleckstrin homology (PH) domain of Grp1(Grp1-PH-HA) was provided by Dr. T. Asano and H. Katagiri (University of Tokyo, Japan).

Cell culture and infection of adenovirus
Aortic smooth muscle cells were prepared from thoracic aortas of 8- to 9-wk-old male Sprague Dawley rats. In brief, rats were anesthetized, and the thoracic aorta was removed and transferred to a sterile petri dish containing Hanks’ Balanced Salt Solution. Fat and connective tissues were removed, and the vessel was washed free of blood. A longitudinal incision was made, and the endothelial layer was removed. The denuded vessel was minced into small pieces and placed in a digestion medium containing 0.6 mg/ml collagenase and 15 U/ml elastase in DMEM. The tissue was digested for 90 min at 37 C with shaking. The dispersed VSMCs were centrifuged and resuspended in DMEM supplemented with 10% fetal calf serum (21). The growth medium was changed every 2 d. Subcultures of VSMCs from passages 3–5 were used in all experiments. Cells were identified as VSMCs by immunostaining for smooth muscle -actin. WT-SHIP2, IP-SHIP2, and Grp1-PH were transiently expressed in VSMCs by adenovirus-mediated gene transfer (18). A multiplicity of infection (m.o.i.) of 10 plaque-forming units (pfu)/cell was used to infect VSMCs in DMEM containing 2% fetal calf serum, with the virus being left on the cells for 16 h before removal. Subsequent experiments were conducted 24–48 h after the initial addition of the virus. The efficiency of adenovirus-mediated gene transfer of WT-SHIP2, IP-SHIP2, and Grp1-PH as measured by immunostaining was approximately 95%.

Immunoprecipitation and Western blotting
Aortic smooth muscle cells grown in six-well plates were serum-starved for 16 h in DMEM. The cells were treated with various concentrations of PDGF, IGF-I, or insulin at 37 C for specific periods. They were then lysed in a buffer containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium deoxycholate, 1 mM ß-glycerophosphate, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride, 1 mM Na₃VO₄, 50 mM sodium fluoride, 10 µg/ml aprotinin, and 10 µM leupeptin (pH 7.4) for 15 min at 4 C. Lysates obtained from the same number of cells were centrifuged to remove insoluble materials. The supernatants (100 µg protein) were immunoprecipitated with the antibodies or precipitated with glutathione-Sepharose beads for 2 h at 4 C. The precipitates or whole cell lysates were then separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Transblot apparatus (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA or 5% nonfat milk (pH 7.5) for 2 h at 20 C. The membranes were then probed with the specified antibodies for 2 h at 20 C or for 16 h at 4 C. After the membranes had been washed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5) blots were incubated with horseradish peroxidase-linked secondary antibody, followed by enhanced chemiluminescence detection using the ECL reagent according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Arlington Heights, IL) (18). Densitometric analysis was conducted directly from the blotted membrane using a Bio-Rad Molecular Imager system. The same amounts of protein were confirmed to be loaded among the samples, and appropriate amounts of WT-SHIP2 and IP-SHIP2 were assured to be expressed in all Western blotting experiments.

Measurement of PI3-kinase activity
Serum-starved aortic smooth muscle cells grown in 10-cm dishes were stimulated with 1 nM PDGF or 14 nM IGF-I at 37 C for 5 min. The cells were lysed in a buffer containing 20 mM Tris, 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1 mM Na₃VO₄, 1% Nonidet P-40, 10% glycerol, 2 mM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin, pH 7.6. The cell lysates were centrifuged to remove insoluble materials. The supernatants were immunoprecipitated with anti-PY20 antibody for 2 h at 4 C. The precipitates were washed twice with buffer A [Tris-buffered saline, 1% Nonidet P-40, 0.1 mM Na₃VO₄, and 1 mM dithiothreitol (DTT), pH 7.6], twice with buffer B (100 mM Tris, 500 mM LiCl, 0.1 mM Na₃VO₄, and 1 mM DTT, pH 7.6), and twice with buffer C (10 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.6). The phosphorylation reaction was started by adding 20 µl PI solution containing 0.5 mg/ml PI, 50 mM HEPES, 1 mM NaH₂PO₄, and 1 mM EGTA (pH 7.6) at 20 C, followed by 10 µl of the reaction mixture containing 250 µM [-³²P]ATP (0.37 MBq/tube), 100 mM HEPES, and 50 mM MgCl₂ (pH 7.6) for 5 min. The reaction was stopped by the addition of 15 µl 8 M HCl. The products were extracted by adding 130 mM chloroform/methanol (1:1), followed by centrifugation. The organic phase was removed and spotted on a Silica Gel thin layer chromatography plate (Merck & Co., Rahway, NJ). The plates were developed and dried (18). The phosphorylated inositol was visualized by autoradiography and quantitated using a Fuji BAS 2000 image analyzer (Fuji Film, Tokyo, Japan).

Confocal laser microscopy for measurement of PI(3,4,5)P3 formation
Serum-starved rat aortic smooth muscle cells grown on coverslips were treated with 1 nM PDGF or 14 nM IGF-I at 37 C for 10 min. The cells were fixed with in 3.7% formaldehyde in PBS for at 23 C for 10 min. After being washed, the cells were permeabilized with 0.1% Tween 20 in PBS for 3 min and blocked with 10% BSA in PBS for 20 min. The cells were then incubated with tetramethyl rhodamine isothiocyanate-conjugated mouse antihemagglutinin antibody. After the coverslips were mounted, the cells were analyzed with a confocal laser fluorescence inverted microscope (LSM510, Carl Zeiss, Inc., Oberkochen, Germany) and evaluated for the presence of cell surface Grp1-PH (18). In all cell countings, the observer was blind to the experimental condition of each coverslip.

DNA synthesis assay
Serum-starved aortic smooth muscle cells grown in 96-well plates were incubated with various concentrations of PDGF or IGF-I. Then, the cells were treated with 10 mM 5-bromo-2'-deoxyuridine (BrdU) for 3 h. BrdU incorporation into DNA was measured by a colorimetric reaction with peroxidase-linked anti-BrdU antibody using a Cell Proliferation ELISA kit according to the manufacturer’s instructions (Roche, Mannheim, Germany).

Statistical analysis
The data are represented as the mean ± SE.P values were determined by Bonferroni test with ANOVA, and P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Structures of SHIP2 constructs and expression in rat aortic smooth muscle cells
SHIP2 is a 140-kDa protein composed of an SH2 domain at the N terminus, a central 5'-phosphatase catalytic domain, and a proline-rich region including a PTB domain binding consensus at the C terminus (Fig. 1A) (16). Three amino acids located within the catalytic domain of SHIP2, which are highly conserved among known 5'-phosphatases, were mutated to generate a 5'-phosphatase-defective SHIP2 (IP-SHIP2). WT-SHIP2 and IP-SHIP2 were transiently expressed in aortic smooth muscle cells by adenovirus-mediated gene transfer (Fig. 1B). Endogenous SHIP2 was observed in control aortic smooth muscle cells transfected with lacZ alone. By transfecting cells with either WT-SHIP2 or IP-SHIP2 at an m.o.i. of 10 pfu/cell, we observed similar levels of expression of WT-SHIP2 and IP-SHIP2 that were 10-fold greater than the levels of endogenous SHIP2. Treatment with PDGF and IGF-I did not affect the expression of WT-SHIP2 and IP-SHIP2 (Fig. 1B).

View larger version (57K): [in this window] [in a new window]   FIG. 1. Structures of SHIP2 constructs and expression in rat aortic smooth muscle cells. A, Structures of WT-SHIP2 and IP-SHIP2 containing Pro687 to Ala, Asp701 to Ala, and Arg702 to Gly are shown. The three domains of SHIP2 are an SH2 domain, a 5'-phosphatase domain, and a carboxyl-terminal proline-rich domain containing a tyrosine phosphorylation site. B, Aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. After the infection, the cells were lysed and subjected to immunoblot analysis with anti-SHIP2 antibody. Results are representative of three separate experiments.

View larger version (49K): [in this window] [in a new window]   FIG. 2. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced tyrosine phosphorylation of their receptors and PI3-kinase activation. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and subsequently treated with 1 nM PDGF or 14 nM IGF-I at 37 C for the periods indicated. For the measurement of receptor tyrosine phosphorylation, the cell lysates were immunoprecipitated with anti-PDGF receptor antibody (A) or anti-IGF-I receptor antibody (B). The precipitates were separated by 7.5% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. For the study with PDGF-induced (C) and IGF-I-induced (D) PI3-kinase activation, the cell lysates were immunoprecipitated with antiphosphotyrosine antibody. The washed immunoprecipitates were assayed for PI3-kinase activity with phosphatidylinositol as a substrate, and the labeled phosphatidylinositol (3 ) phosphate product (PI3P) was resolved by thin layer chromatography and visualized by autoradiography. Results are the mean ± SE of four separate experiments.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced formation of PI(3,4,5)P3. Rat aortic smooth muscle cells were transfected with Grp1-PH at an m.o.i. of 10 pfu/cell. The cells were cotransfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF or 14 nM IGF-I at 37 C for 10 min. The cells were then fixed in 3.7% formaldehyde and visualized by confocal laser fluorescence microscopy. The percentage of cells displaying cell surface fluorescence of Grp1-PH was determined by counting at least 100 cells at each point. Results are the mean ± SE of three separate experiments. *, P < 0.05 vs. percentage of cells displaying cell surface fluorescence of Grp1-PH in PDGF- (A) or IGF-I (B)-stimulated lacZ-transfected control cells.

View larger version (50K): [in this window] [in a new window]   FIG. 4. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced phosphorylation of Akt. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A, C, E, and G) or 14 nM IGF-I (B, D, F, and H) at 37 C for 10 min. The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with antiphosphospecific Akt antibody (A and B), anti-Akt antibody (E and F), or anti-SHIP2 antibody (G and H). The amount of phosphorylated Akt was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. Akt phosphorylation in PDGF- (C) or IGF-I (D)-stimulated lacZ-transfected control cells.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced phosphorylation of GSK3ß. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A) or 14 nM IGF-I (B) at 37 C for 10 min. The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with antiphosphospecific GSK3ß antibody. The amount of phosphorylated GSK3ß was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. GSK3ß phosphorylation in PDGF- (C) or IGF-I (D)-stimulated lacZ-transfected control cells.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Effect of SHIP2 overexpression on PDGF- and IGF-I-mediated protection of PARP fragmentation induced by H2O2. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with H2O2 in the absence or presence of 1 nM PDGF or 14 nM IGF-I at 37 C. The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-PARP antibody. The relative amount of PARP of high molecular weight vs. that of low molecular weight was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. the relative amount of PARP of high molecular weight vs. that of low molecular weight in PDGF- (A) or IGF-I (B)-stimulated lacZ-transfected control cells.

View larger version (32K): [in this window] [in a new window]   FIG. 7. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced tyrosine phosphorylation of Shc. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A) or 14 nM IGF-I (B) at 37 C for the periods indicated. The cell lysates were immunoprecipitated with anti-Shc antibody at 20 C for 2 h. The precipitates were separated by 7.5% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody. The amount of phosphorylated Shc was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. Shc phosphorylation at respective concentrations of PDGF (C) or IGF-I (D) in lacZ-transfected control cells.

View larger version (29K): [in this window] [in a new window]   FIG. 8. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced SHIP2 association with Shc. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A) or 14 nM IGF-I (B) at 37 C for 10 min. The cell lysates were immunoprecipitated with anti-Shc antibody at 20 C for 2 h. The precipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-SHIP2 antibody. The amount of SHIP2 associated with Shc was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. SHIP2 associated with Shc in PDGF- (C) or IGF-I (D)-stimulated lacZ-transfected control cells.

View larger version (31K): [in this window] [in a new window]   FIG. 9. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced Shc association with Grb2. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A) or 14 nM IGF-I (B) at 37 C for 10 min. The cell lysates were immunoprecipitated with anti-Shc antibody at 20 C for 2 h. The precipitates were separated by 7.5% SDS-PAGE and immunoblotted with anti-Grb2 antibody. The amount of Grb2 associated with Shc was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. Grb2 associated with Shc in PDGF- (C) or IGF-I (D)-stimulated lacZ-transfected control cells.

View larger version (28K): [in this window] [in a new window]   FIG. 10. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced MAPK activation. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without 1 nM PDGF (A) or 14 nM IGF-I (B) at 37 C for 10 min. The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with antiphosphospecific MAPK antibody. The amount of phosphorylated p42 MAPK was quantitated by densitometry. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. MAPK phosphorylation in PDGF- (C) or IGF-I (D)-stimulated lacZ-transfected control cells.

View larger version (23K): [in this window] [in a new window]   FIG. 11. Effect of SHIP2 overexpression on PDGF- and IGF-I-induced BrdU incorporation. Rat aortic smooth muscle cells were transfected with lacZ, WT-SHIP2, or IP-SHIP2 at an m.o.i. of 10 pfu/cell. The cells were serum-starved for 16 h and treated with or without the indicated concentrations PDGF or IGF-I at 37 C for 20 h. BrdU incorporation for the next 3 h was assayed. Dose-response curves for PDGF- (A) and IGF-I (B)-induced BrdU incorporation are shown. Results are the mean ± SE of four separate experiments. *, P < 0.05 vs. BrdU incorporation at respective concentrations in lacZ-transfected control cells.

View larger version (29K): [in this window] [in a new window]   FIG. 12. The changes in SHIP2 expression caused by chronic insulin treatment in VSMC. Rat aortic smooth muscle cells were treated with insulin at 37 C for the indicated times. The cell lysates were separated by 7.5% SDS-PAGE and immunoblotted with anti-SHIP2 antibody (A) or anti-Akt antibody (B). The amount of SHIP2 was quantitated by densitometry (C). Results are the mean ± SE of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The expression of both WT-SHIP2 and IP-SHIP2 did not affect PDGF- and IGF-I-induced tyrosine phosphorylation of the cognitive receptor and activation of PI3-kinase. In contrast, PDGF- and IGF-I-induced phosphorylation of Akt was inhibited by the expression of WT-SHIP2, whereas phosphorylation was enhanced by the expression of IP-SHIP2 in VSMCs. These results are consistent with previous reports that SHIP2 negatively regulates downstream molecules of PI3-kinase via 5'-phosphatase activity in the insulin signaling system (18). The present study further supports the idea that SHIP2 negatively regulates PDGF- and IGF-I-induced phosphorylation of Akt via the 5'-phosphatase of SHIP2 in VSMCs. GSK3ß is located downstream of Akt and is implicated in the antiapoptotic signaling of PDGF and IGF-I (10, 11). In accordance with the decreased phosphorylation of Akt caused by overexpression of WT-SHIP2, PDGF- and IGF-I-induced phosphorylation of GSK3ß was reduced by overexpression of WT, whereas these effects were augmented by the expression of IP-SHIP2. Furthermore, H₂O₂ is an important mediator of apoptotic signaling, and its introduction resulted in the cleavage of PARP, a substrate of caspase-3 located downstream of Akt in VSMCs (24). The PDGF- and IGF-I-induced inhibitory effect on H₂O₂-mediated degradation of PARP was attenuated by the expression of WT-SHIP2, whereas it was again apparently enhanced by the expression of IP-SHIP2. These results indicate that PDGF- and IGF-I-induced antiapoptotic signaling mediated by the Akt/GSK3ß pathway was negatively regulated by the 5'-phosphatase activity of SHIP2.

It is of note that IGF-I also possesses a function to maintain the quiescence and contractile phenotype, whereas PDGF is one of the most powerful stimuli of vascular smooth muscle cell proliferation (7, 9, 25, 28, 34). In this regard, this IGF-I action may be regulated in a different manner than the growth proliferation and antiapoptotic actions. As IGF-I-induced maintenance of the quiescence and contractile phenotype also appears to be regulated at least in part by the MAPK and/or PI3-kinase cascades (7, 9, 34), the possible involvement of SHIP2 in this IGF-I action remains to be elucidated in VSMCs.

PDGF- and IGF-I-induced phosphorylations of Akt and GSK3ß were only partly inhibited by the expression of WT-SHIP2. Although the reason why WT-SHIP2 overexpression only partially inhibited activation of the downstream effectors of PI3-kinase is unclear, it is possible that the expression of WT-SHIP2 is not high enough to completely inhibit the effects of PDGF and IGF-I. However, this is unlikely, because a higher expression level of WT-SHIP2 resulted in a similar degree of inhibition (data not shown). Rather, PI(3,4)P2 is possibly involved, although it has a lesser role than PI(3,4,5)P3, in the activation of Akt. Along this line, it is known that both PI(3,4,5)P3 and PI(3,4)P2 bind with high affinity to the PH domain of Akt, leading to the recruitment of Akt to be phosphorylated on the plasma membrane (18, 19). Another possible explanation is that there is a redundant pathway that regulates the PI3-kinase product PI(3,4,5)P3 in the control of the antiapoptotic effect of PDGF and IGF-I. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a possible candidate factor for mediating the alternative pathway for the hydrolysis of PI(3,4,5)P3 to PI(4,5)P2 (35, 36). Interestingly, it is reported that overexpression of PTEN also resulted in an inhibition of PDGF-stimulated Akt activation in VSMCs (37). It would be interesting to further clarify how SHIP2 and PTEN may cooperatively or solely participate in the regulation of the PI3-kinase product PI(3,4,5)P3, resulting in an antiapoptotic effect by PDGF and IGF-I. We also cannot rule out the possibility of the existence of another 5'-lipid phosphatase responsible for PDGF and IGF-I signaling in VSMCs, because SHIP2 is considered the 5'-lipid phosphatase relatively specific to insulin signaling based on studies with knockout mice lacking SHIP2 (20). Along this line, skeletal muscle and kidney enriched inositol phosphatase (SKIP) is identified as another 5'-lipid phosphatase that relatively specifically hydrolyzes PI(3,4,5)P3 (38).

In summary, we clarified the role of SHIP2 in PDGF and IGF-I signaling in VSMCs. Via 5'-phosphatase activity to hydrolyze PI3-kinase products, SHIP2 appears to negatively regulate downstream molecules of PI3-kinase including the Akt-GSK3ß pathway, leading to the antiapoptotic effect of PDGF and IGF-I. Via the SH2 domain, the association of SHIP2 with Shc competed for Shc binding to Grb2. Thus, SHIP2 also appears to be involved in regulation of the Ras-MAPK pathway, leading to PDGF- and IGF-I-induced DNA synthesis. As SHIP2 regulates PDGF and IGF-I signaling by dual mechanisms, alteration of the expression and/or change in the enzymatic activity of SHIP2 might result in inadequate cell growth and/or apoptosis. This might be a novel cause of the development and progression of atherosclerosis, especially in type 2 diabetes, because the expression of SHIP2 is altered in the state of insulin resistance (26, 27). Along this line, chronic treatment with insulin decreased the expression levels of SHIP2 in VSMCs as shown in Fig. 12, whereas it enhanced the expression in 3T3-L1 adipocytes (Fukui, K., T. Sasaoka, and M. Kobayashi, unpublished observations). Further studies will be required to investigate the involvement of SHIP2 in the pathological state of atherosclerosis.

	Acknowledgments

 
We thank Drs. Tomoichiro Asano and Hideki Katagiri for kindly providing an adenovirus vector encoding HA-tagged PH-domain of Grp1, and Drs. Hiroshi Tsuneki, Yoshitaka Sugihara, and Takefumi Korenaga for their technical assistance.

	Footnotes

 
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science and Japan Growth Foundation (to T.S.).

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; DTT, dithiothreitol; GSK, glycogen synthase kinase; m.o.i., multiplicity of infection; PARP, poly(ADP-ribose) polymerase; PDGF, platelet-derived growth factor; pfu, plaque-forming units; PH, pleckstrin homology; PI3-kinase, phosphatidylinositol 3-kinase; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate; PI(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog deleted on chromosome 10; Src, Src homology domain; SHIP2, Src homology domain 2-containing inositol phosphatase 2; VSMC, vascular smooth muscle cell; WT, wild-type.

Received February 10, 2003.

Accepted for publication May 27, 2003.

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