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Molecular Endocrinology, doi:10.1210/me.2001-0284
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Molecular Endocrinology 16 (9): 2113-2123
Copyright © 2002 by The Endocrine Society

Angiotensin II Subtype 2 Receptor Activation Inhibits Insulin-Induced Phosphoinositide 3-Kinase and Akt and Induces Apoptosis in PC12W Cells

Tai-Xing Cui, Hironori Nakagami, Clara Nahmias, Tetsuya Shiuchi, Yuko Takeda-Matsubara, Jian-Mei Li, Lan Wu, Masaru Iwai and Masatsugu Horiuchi

Department of Medical Biochemistry (T.-X.C., H.N., T.S., Y.T.-M., J.-M.L., L.W., M.I., M.H.), Ehime University School of Medicine, Ehime 791-0295, Japan; and Centre National de la Recherche Scientifique, UPR0415-Institut Cochin de Genetique Moleculaire (C.N.), Paris 75014, France

Address all correspondence and requests for reprints to: Masatsugu Horiuchi, M.D., Ph.D., Department of Medical Biochemistry, Ehime University School of Medicine, Shigenobu, Onsen-gun, Ehime 791-0295, Japan. E-mail: horiuchi{at}m.ehime-u.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85{alpha} subunit of PI3K, but led to a significant reduction of insulin-induced p85{alpha} phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CROSS-TALK BETWEEN INSULIN and the renin-angiotensin system (RAS) signaling system has drawn great attention because hypertension and insulin resistance often coexist and are leading risk factors for cardiovascular disease (1, 2, 3). Different forms of insulin such as IGF and proinsulin have been shown to participate along with insulin to stimulate vascular growth (4, 5, 6). The growth-promoting effect of insulin may be the most important factor in increased cardiovascular risk (4, 5). It has been demonstrated that insulin activates the vascular RAS via stimulation of angiotensinogen or angiotensin II (AII) production, and up-regulation of AII receptor subtype 1 (AT1) receptor expression, which results in enhanced functional responses such as cell growth of vascular smooth muscle cells (VSMC) on stimulation with AII (7, 8). In addition, AII and IGF have been shown to exert synergistic growth-promoting effects on VSMC through the AT1 and IGF receptors (9).

Molecular cloning and pharmacological studies have defined another subtype of AII receptors, designated the AT2 receptor (10). While both AT1 and AT2 receptors belong to the seven transmembrane, G protein-coupled receptor family and share approximately 30% primary sequence homology, accumulating evidence has revealed that the AT2 receptor functionally antagonizes the AT1 receptor-mediated actions; i.e. the AT2 receptor exerts antigrowth, antihypertrophic, and proapoptotic effects (10, 11). As the AT2 receptor is up-regulated in certain pathological conditions such as vascular injury and inflammation, the effect of AII is considered to be regulated by the balance of expression of the AT1 and AT2 receptors in target tissues, thereby contributing to the pathogenesis of cardiovascular disease and consequent remodeling (10, 12, 13). AT2 receptor stimulation inhibits AT1 receptor- or receptor tyrosine kinase-mediated MAPK via activation of a series of phosphatases including the protein tyrosine phosphatase, SHP-1 [formerly termed protein tyrosine phosphatase (PTP) 1C, SH-PTP-1, SHP, and HCP], a two Src homology 2 (SH2) domain-containing cytosolic tyrosine phosphatase, or the MAPK phosphatase-1 (MKP-1), and the serine/threonine phosphatase 2A (PP2A) (10, 14). These results suggest complex interaction between the AII receptor and receptor tyrosine kinase signaling. A recent report (15) has shown that AT2 receptor stimulation inhibits ERK activation and cell proliferation induced by insulin, through impairing insulin-induced autophosphorylation of the insulin ß-subunit and phosphorylation of IRS-1, suggesting that AT2 receptor stimulation modulates insulin receptor signaling.

One of the growth inhibitory effects of the AT2 receptor is apoptosis (10). The AT2 receptor is highly expressed in fetal tissues, but the expression level rapidly declines after birth and it is re-expressed in remodeling tissues, suggesting that the proapoptotic effect of the AT2 receptor is important in development and regeneration (10). However, the possible cross-talk between the AT2 receptor and insulin receptor signaling in the regulation of apoptosis has not yet been explored.

To examine this possibility, we employed PC12W cells, a subline of the PC12 rat pheochromocytoma cell line, because the cells endogenously express high levels of the AT2 receptor but not AT1 receptor and are widely used to examine the molecular and cellular mechanisms of apoptosis (16), and insulin has been shown to inhibit apoptosis induced by serum-deprivation in PC12 cells (17). We focused on the phosphoinositide 3-kinase (PI3K)/Akt cascade, which plays a critical role in the regulation of apoptosis (17, 18). We have demonstrated that AT2 receptor activation inhibits insulin-mediated IRS-2-associated PI3K activation, Akt phosphorylation, and antiapoptotic effect. We have further shown that stimulation of the AT2 receptor attenuates insulin-induced p85{alpha} phosphorylation, via recruitment of a protein tyrosine phosphatase, SHP-1, to the complex of IRS-2 and PI3K.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Inhibition of Insulin-Induced Activation of Akt by AT2 Receptor Stimulation
Activation of Akt requires the phosphorylation of Thr-308 and Ser-473 in the Akt{alpha} molecule (19, 20). In this study, phosphorylation of Ser-473 was used to evaluate the activation of Akt by immunoblotting using antiphospho Ser-Akt antibody. Activation of Akt in PC12W cells was observed at a minimum concentration of 1 nM insulin and reached a maximum level at approximately 10 nM insulin (data not shown). PC12W cells have been shown to express the AT2 receptor but not the AT1 receptor (16). Consistent with these results, receptor binding assay showed that AT2 receptor binding density was 9.1 ± 0.43 fmol/105 cells, which was up-regulated by serum removal to 21.1 ± 3.3 fmol/105 cells in PC12W cells. In contrast, there was no detectable level of AT1 receptor in the cells. To evaluate whether AT2 receptor stimulation attenuates insulin-induced activation of Akt, PC12W cells were treated with 10 nM insulin with or without 100 nM AII. AII caused a time-dependent decrease in insulin-mediated Akt phosphorylation (Fig. 1AGo), and this inhibitory effect of AII was blocked by an AT2 receptor specific antagonist, PD123329 (Fig. 1BGo). AII alone did not significantly affect Akt phosphorylation (data not shown).



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  		Figure 1. Effect of AII Stimulation on Insulin-Induced Activation of Akt

PC12W cells were treated with 10 nM insulin with or without 100 nM AII for various times (A). PC12W cells were treated for 5 min with 10 nM of insulin alone, or with different concentrations of AII, or with 10-7 M AII and 10-5 M PD123319 (B). The activation of Akt was determined by immunoblotting using an antiphosphoSer-Akt antibody. Results shown are representative immunoblots and densitometric analyses of five (A) or four (B) immunoblots (mean ± SE), respectively. *, P < 0.05 vs. insulin treated. ns, Not significant.

 
Inhibition of Insulin-Induced PI3K Activities by AT2 Receptor Activation
We next investigated whether AT2 receptor-mediated Akt inhibition is the consequence of decreased insulin-induced PI3K activation. As shown in Fig. 2Go, insulin stimulation rapidly and significantly increased p85{alpha} and phosphotyrosine-associated PI3K activity, reaching a peak at 3 min, and declining rapidly. AII significantly inhibited insulin-induced PI3K activity. We observed that insulin-mediated increase in PI3K activity associated with immunoprecipitates by 4G10 was greater than PI3K activity associated with p85{alpha} immunoprecipitated proteins and that inhibitory effect of AT2 receptor on this PI3K activity was weaker than with p85{alpha} immunoprecipitation followed by PI3K assay. These apparent discrepancies might be due to the fact that the immunoprecipitation with p85{alpha} did not specifically immunoprecipitate the activated fraction of PI3K bound to IRS protein, but rather, the total cellular pool of PI3K, which has not been activated by recruitment to tyrosine-phosphorylated proteins such as IRS.



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  		Figure 2. Effect of AT2 Receptor Activation on Insulin-Induced PI3K Activity

Cells were treated with either 10 nM insulin and/or 10-7 M AII for the indicated time periods, and then the p85{alpha} subunit of PI3K-associated PI3K activity (A) or PI3K activity associated with immunoprecipitates by antiphosphotyrosine (pTyr) antibody (B) was measured as described in Materials and Methods. Upper panels show the representative TLC data. Results shown are obtained from five separate experiments (mean ± SE). Values are fold changes in basal p85{alpha}-associated PI3K activity or PI3K activity precipitated with anti-pTyr antibody in nonstimulated PC12W cells. *, P < 0.05 vs. insulin treatment.

 
Furthermore, we examined the effect of AII stimulation on insulin-induced PI3K activation associated with IRS-1 and IRS-2. The expression of IRS-1 and IRS-2 in PC12W cells was confirmed by immunoblotting with anti-IRS-1 and anti-IRS-2 antibodies, and the cell lysate of L6 myoblasts was used as a positive control, which have been shown to express both IRS-1 and IRS-2 (21). In PC12W cells, the expression levels of IRS-1 and IRS-2 were 6.32% and 87.21% of those in L6 myoblasts, respectively (Fig. 3AGo). Insulin stimulation increased both IRS-1- and IRS-2-associated PI3K activity (Fig. 3Go, B and C), whereas IRS-2-associated PI3K activity was inhibited only by AT2 receptor stimulation (Fig. 3CGo). The inhibitory effect of AT2 receptor stimulation on insulin-mediated PI3K activation associated with IRS-2 seemed to be comparable with that in insulin-mediated PI3K activation associated with tyrosine-phosphorylated proteins, whereas inhibitory effect of AT2 receptor on PI3K activity associated with p85{alpha} was weaker, which included both activated and nonactivated fractions of PI3K.



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  		Figure 3. Effect of AT2 Receptor Stimulation on PI3K Activity Associated with IRS-1 or IRS-2

Immunoblots of IRS-1 and IRS-2 were obtained with equal amounts of cell lysates from L6 myoblasts and PC12W cells (A). Cells were treated with 10 nM or/and 10-7 M AII for the indicated time periods, and then IRS-1- (B) or IRS-2- (C) associated PI3K activity was measured as described in Materials and Methods. Results shown are obtained from three separate experiments (mean ± SE). Values are expressed as fold changes in basal IRS-1- or -2-associated PI3K activity in nonstimulated PC12W cells. *, P < 0.05 vs. insulin treatment. IP, Immunoprecipitation.

 
Increases in Association of SHP-1 with Complex of IRS-2 and PI3K and Decreases in Tyrosine Phosphorylation of PI3K by AT2 Receptor Stimulation
Possible implication of tyrosine phosphatases in the negative regulation of IRS-2 phosphorylation has been suggested; however, the specific phosphatases involved have not yet been identified (21). In addition, recent evidence has shown that SHP-1 is involved in the regulation of PI3K activity (22, 23, 24). Thus, to address whether the AT2 receptor-mediated inhibition of IRS-2-associated PI3K activity is a consequence of dephosphorylation of IRS-2 by SHP-1, we first examined whether insulin-induced IRS-2 phosphorylation could be changed by AT2 receptor stimulation. PC12W cells were treated with insulin or/and AII, and immunoprecipitated with anti-IRS-2 antibodies, and IRS-2 phosphorylation was examined by immunoblotting analysis with an antiphosphotyrosine antibody, 4G10. Insulin-induced IRS-2 phosphorylation was not influenced by AII stimulation (Fig. 4Go). This result was confirmed by performing the experiment of immunoprecipitation with 4G10 and immunoblotting with an anti-IRS-2 antibody (Fig. 6AGo). Moreover, the insulin-induced association of IRS-2 with PI3K was also not affected by AII stimulation (Fig. 5Go, upper panel). Interestingly, the association of SHP-1 with IRS-2 was detectable under basal conditions, which was potentiated by AII stimulation, whereas insulin stimulation did not affect the association of SHP-1 with IRS-2 (Fig. 5AGo, middle panel). Immunoprecipitation with anti-p85{alpha} antibody and immunoblotting with an anti-SHP-1 antibody showed no detectable association of SHP-1 with PI3K under either basal conditions or AII stimulation, whereas the association of SHP-1 with PI3K appeared with insulin stimulation, which was further increased by AII stimulation (Fig. 5BGo, middle panel), suggesting that the formation of insulin-mediated a complex of PI3K with IRS-2 is prerequisite for the AT2 receptor-induced further association of IRS-2/PI3K complex with SHP-1. Insulin stimulation increased tyrosine-phosphorylation of PI3K assessed by immunoprecipitation with anti-p85{alpha} antibody and immunoblotting with 4G-10, and this increase was inhibited by AT2 receptor stimulation (Fig. 6BGo).



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  		Figure 4. Effect of AII Stimulation on Insulin-Induced Tyrosine Phosphorylation of IRS-2

PC12W cells were treated with 10 nM insulin or/and 10-7 M AII for the indicated time periods. Lysates were immunoprecipitated (IP) with an anti-IRS-2 antibody and subjected to immunoblotting (IB) with an antiphosphotyrosine antibody (4G10) (A and B) or an anti-IRS-2 antibody (C and D). Results shown are representative immunoblots of three separate experiments.

 


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  		Figure 6. Effects of AII Stimulation on Insulin-Induced Tyrosine Phosphorylation of IRS-2 and p85{alpha} Subunit of PI3K

PC12W cells were treated with 10 nM insulin or/and 10-7 M AII for 3 min. Cell lysates were immunoprecipitated (IP) with an antiphosphotyrosine antibody (4G10) and subjected to immunoblotting (IB) with anti-IRS-2 and anti-p85{alpha} subunit of PI3K antibodies (A). Lysates were IP with anti-p85{alpha} subunit of PI3K antibody and subjected to IB with 4G10 (B). Results shown are representative immunoblots of three separate experiments.

 


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  		Figure 5. Effects of AII Stimulation on Insulin-Induced IRS-2 Association with PI3K or SHP-1

PC12W cells were treated with 10 nM insulin or/and 10-7 M AII for 3 min. Lysates were immunoprecipitated (IP) with an anti-IRS-2 antibody and subjected to immunoblotting (IB) with the anti-p85{alpha} subunit of PI3K, anti-SHP-1 and anti-IRS-2 antibodies (A). Lysates were immunoprecipitated (IP) with an anti-p85{alpha} subunit of PI3K antibody and subjected to immunoblotting (IB) with anti-IRS-2, anti-SHP-1 and anti-p85{alpha} subunit of PI3K antibodies (B). Jurkat T cell lysate was used as a positive control for SHP-1. Results shown are representative immunoblots of three separate experiments.

 
Role of SHP-1 in AT2 Receptor-Mediated Inhibition of PI3K, Akt, and Apoptosis
To examine whether SHP-1 plays a role in the AT2 receptor-mediated down-regulation of the PI3K/Akt cascade, we transfected PC12W cells with a dominant negative (dn) SHP-1 mutant in which the active site cysteine 453 was mutated to serine (C453/S) (25). Overexpression of the dnSHP-1 mutant was confirmed by immunoblotting showing a 3-fold increase in SHP-1 immunoreactivity compared with control vector pcDNA3-transfected cells (Fig. 7AGo). AII stimulation increased SHP-1 activity in pcDNA3-transfected PC12W cells, and no significant increase in SHP-1 activity was observed in response to AII in PC12W cells transfected with the dnSHP-1 mutant (Fig. 7BGo). Insulin-mediated tyrosine-phosphorylation of IRS-1 was inhibited by AII treatment in pcDNA3 or dnSHP-1 transfected PC12W cells (Fig. 8AGo). Moreover, insulin-mediated tyrosine-phosphorylation of IRS-2 was not influenced by AII (data not shown). Insulin-mediated increase in tyrosine-phosphorylation of PI3K assessed by immunoprecipitation with anti-p85{alpha} antibody and immunoblotting with 4G-10 as well as the AII-mediated inhibition of this tyrosine phosphorylation of PI3K was observed in pcDNA3 transfected PC12W cells (Fig. 6BGo). However, AII did not show significant inhibitory effect on insulin-mediated increase in tyrosine-phosphorylation of PI3K in dnSHP-1 transfected cells (Fig. 8BGo). AT2 receptor-mediated inhibition of IRS-2-associated PI3K activity and Akt phosphorylation induced by insulin was also inhibited by dnSHP-1 transfection (Fig. 9Go).



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  		Figure 7. Effect of Overexpression of dnSHP-1 on AT2 Receptor-Mediated SHP-1 Activation

Immunoblots of SHP-1 in control vector (pcDNA) and dnSHP-1 transfected PC12W cells (A). Cells were treated with or without 10-7 M AII for 3 min (B). Lysates were immunoprecipitated with an anti-SHP-1 antibody, and then immunoprecipitates were subjected to PTP activity assay as described in Materials and Methods. Results shown are obtained from four separate experiments (mean ± SE) (B). *, P < 0.05 vs. AII (-).

 


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  		Figure 8. Effect of Overexpression of dnSHP-1 on AT2 Receptor-Mediated Dephosphorylation of PI3K

PC12W cells transfected with control vector pcDNA3 or dnSHP-1 (C453/S) were treated with 10 nM insulin or/and 10-7 M AII for 3 min. Cell lysates were immunoprecipitated (IP) with an anti-IRS-1 antibody and subjected to immunoblotting (IB) with 4G10 and anti-IRS-1 antibodies (A). Lysates were immunoprecipitated (IP) with an anti-p85{alpha} subunit of PI3K antibody and subjected to immunoblotting (IB) with 4G10 and anti-p85{alpha} subunit of PI3K antibodies (B). Results shown are representative immunoblots of three separate experiments.

 


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  		Figure 9. Effect of Overexpression of dnSHP-1 on AT2 Receptor-Mediated Inhibition of IRS-2-Associated PI3K Activity and Akt Activation

Cells were treated with 10 nM insulin or/and 10-7 M AII for 3 min, and then IRS-2-associated PI3K activity was measured as described in Materials and Methods (A). Cells were treated with 10 nM insulin or/and 10-7 M AII for 5 min, and then activation of Akt was determined by immunoblotting using an antiphosphoSer-Akt antibody (B). Results shown are obtained from four separate experiments (mean ± SE). P < 0.05 vs. insulin treatment.

 
To examine the involvement of the AT2 receptor-induced SHP-1/IRS-2/PI3K/Akt signaling cascade in the regulation of insulin receptor-mediated physiological actions, we focused on the interaction of insulin and AII in the regulation of apoptosis. Apoptotic changes were assessed by measurement of caspase-3 activity because caspase-3 plays a central role in serum or trophic factor deprivation-induced apoptosis (26) as well as morphological changes determined by chromatin binding dyes Hoechst 33342. Serum deprivation increased caspase-3 activity and morphological changes of apoptosis time dependently (Fig. 10AGo). Insulin (10 nM) inhibited these apoptotic changes as previously observed in other cell types (16, 27, 28, 29) and wortmannin (0.5 µM) attenuated this insulin’s effect on apoptosis (Fig. 10BGo). We observed that insulin-induced antiapoptotic effect was significantly inhibited by AII (100 nM) stimulation (Fig. 10CGo). This proapoptotic effect of AT2 receptor was diminished in dnSHP-1 transfected PC12W cells (Fig. 11Go).



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  		Figure 10. Effect of AII and Insulin Stimulation on the Regulation of Apoptosis in PC12W Cells

PC12W cells were maintained in serum-free DMEM medium for the indicated time periods (A). PC12W cells maintained in serum-free DMEM were stimulated with 10 nM insulin with vehicle, DMSO or PI3K inhibitor, wortmannin (0.5 µM), and the cells for caspase-3 activity assay and Hoechst 33342 staining were collected 24 h and 48 h after stimulation, respectively (B). PC12W cells were maintained in serum-free DMEM and stimulated with 10 nM insulin with or without 10-7 M AII, and the cells for caspase-3 activity assay and Hoechst 33342 staining were collected 24 h and 48 h after stimulation, respectively (C). Apoptotic changes were assessed by measurement of caspase-3 activity and nuclear chromatin staining with Hoechst 33342 as described in Materials and Methods. Results shown are obtained from six separate experiments (mean ± SE). Values are expressed as fold changes compared with basal level in PC12W cells maintained in DMEM with serum (S). *, P < 0.05 vs. S (serum). §, P < 0.05 vs. SF (serum-free DMEM treatment). P < 0.05 vs. Insulin + AII.

 


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  		Figure 11. Role of SHP-1 in AT2 Receptor-Mediated Proapoptotic Effect in PC12W Cells

PC12W cells transfected with control vector pcDNA3 or dnSHP-1 (C453/S) were maintained in serum-free DMEM and stimulated with 10 nM insulin with or without 10-7 M AII, and the cells for caspase-3 activity assay and Hoechst 33342 staining were collected 24 h and 48 h after stimulation, respectively. Apoptotic changes were assessed by measurement of caspase-3 activity and nuclear chromatin staining with Hoechst 33342 as described in Materials and Methods. Results shown are obtained from six separate experiments (mean ± SE). Values are expressed as fold changes in basal level in PC12W cells maintained in DMEM with serum (S). *, P < 0.05 vs. SF (serum-free DMEM treated). P < 0.05 vs. Insulin + AII.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Insulin not only promotes protein synthesis, glucose uptake, and cellular growth, but also serves as a potent survival factor to inhibit apoptosis via the PI3K cascade (16, 27, 28, 29). In cultured PC12W cells, we also observed that insulin activates PI3K, thereby activating Akt in a time- and dose-dependent manner and preventing apoptosis.

In contrast, AT2 receptor-mediated apoptosis has been demonstrated in PC12W cells, R3T3 mouse fibroblasts, rat neonatal cardiomyocytes, human umbilical venous endothelial cells, and rat fetal VSMC (10, 30). The proapoptotic effect of AT2 receptor stimulation has been shown to be associated with the activation of phosphatases such as SHP-1, MKP-1, and PP2A, the inhibition of ERK activity, and the stimulation of c-Jun NH2-terminal protein kinase (JNK) activity (10, 30, 31). However, little is known about AT2 receptor-mediated regulation of the PI3K/Akt cascade, which has been shown to play a critical role in cell survival (16, 17). In the present study, we observed that AT2 receptor activation dose-dependently inhibited insulin-induced Akt phosphorylation as well as PI3K activation. These results indicate negative cross-talk between AT2 and insulin receptors in regulation of the PI3K/Akt pathway.

IRS proteins coordinate and amplify signals from insulin receptor family members (32). IRS-1 and IRS-2 are the most widely distributed and expressed members of the IRS protein family, which are tyrosine phosphorylated during insulin receptor activation (33, 34). PI3K consists of a catalytic domain and a regulatory domain; the kinase is activated when the regulatory domain binds to tyrosine-phosphorylated motifs in the activated growth factor receptor or docking proteins such as IRS-1 and IRS-2 (32). The binding of p85 regulatory subunits to phosphorylated IRS proteins is the principal mechanism that activates PI3K during insulin stimulation and IGF-I stimulation (35, 36). Therefore, the AT2 receptor-mediated PI3K inhibition might be caused by a reduction of tyrosine phosphorylation of IRS-1 or IRS-2 and their association with PI3K. Indeed, we observed that AT2 receptor activation specifically inhibits IRS-2-associated PI3K activity induced by insulin, whereas insulin-induced IRS-1-associated PI3K activity was not affected by AT2 receptor stimulation. These results suggest the implication of IRS-2 in the AT2 receptor-mediated signaling in PC12W cells. In accordance with our observations, Folli et al. (37) have reported that pretreatment of rat aortic VSMC with AII inhibited insulin-stimulated PI3K activity associated with IRS-1, whereas AII did not impair insulin-stimulated tyrosine phosphorylation of the insulin receptor ß-subunit. Moreover, they observed that AII decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and that AII inhibited the insulin-stimulated association between IRS-1 and the p85 subunit of PI3K, suggesting that activation of the renin-angiotensin system may lead to insulin resistance in the vasculature. The involvement of AII- mediated regulation of IRS-1 and/or IRS-2 would be different in various cell types.

It has been reported that dephosphorylation of IRS-2 is blocked by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, suggesting the involvement of tyrosine phosphatase (21). SHP-1 has been implicated in the negative regulation of a broad spectrum of growth-promoting receptors (38, 39, 40, 41, 42). Interestingly, current data have documented that SHP-1 is also involved in the down-regulation of PI3K/Akt activation (23). Moreover, we previously demonstrated that AT2 receptor-mediated activation of SHP-1 was rapid, peaked at 2–3 min, and returned to basal level at 10–15 min, suggesting that SHP-1 is one of the most proximal effectors in AT2 receptor-mediated apoptosis (25, 30). In this study, we observed that AT2 receptor-mediated inhibition of phosphorylated-Akt was short-lived and was largely reversed by 20–30 min. Consistent with this result, AT2 receptor-mediated inhibition of PI3K was more prominent within 10 min after AII stimulation. These results suggest that SHP-1 might play an important role in the AT2 receptor-mediated inhibition of the PI3-K/Akt cascade, although the involvement of other factors would be possible. Surprisingly, we did not observe that AT2 receptor stimulation inhibits either insulin-induced IRS-2 phosphorylation or the association of IRS-2 and PI3K. However, AT2 receptor stimulation increased the association of SHP-1 with the complex of IRS-2 and PI3K induced by insulin, and insulin-induced PI3K phosphorylation was largely attenuated by AT2 receptor stimulation. These results indicate that AT2 receptor-activated SHP-1 might induce dephosphorylation of PI3K, which decreases PI3K activity. Although the exact mechanism of the interaction of SHP-1 with IRS-2 and/or PI3K remains to be defined, the hypothesis is strongly supported by recent data that SHP-1 dephosphorylates p85 and decreases PI3K activity (23).

It should be mentioned that SHP-1 physically interacts with a number of membrane receptors such as nerve growth factor Trk receptor, epidermal growth factor receptor, c-Kit, IL-3 receptor, erythropoietin receptor, and B cell Fc{gamma}RIIB receptor (38, 39, 40, 41, 42), to terminate their signal. In addition, sst2 somatostatin receptor-mediated activation of SHP-1 has been shown to promote dephosphorylation of tyrosine phosphorylated insulin receptor via association with the insulin receptor (43). However, our data suggest that the functional target of AT2 receptor-activated SHP-1 in the regulation of PI3K seems to be located downstream of the insulin receptor for the following reasons: 1) We could not observe any significant change in insulin-induced phosphorylation of IRS-2 by AT2 receptor stimulation. 2) AT2 receptor activation increased the association of SHP-1 with IRS-2 without insulin stimulation. 3) Recent evidence has shown that SHP-1 is not involved in AT2 receptor-mediated dephosphorylation of the insulin receptor or IRS-1 (15). These observations suggest that IRS-2 serves as an adapter for SHP-1 to inhibit PI3K activity. Taken together, AT2 receptor stimulation increases the association of SHP-1 with IRS-2, and SHP-1 dephosphorylates phosphorylated proteins such as PI3K in the assembly of signaling molecule complexes with IRS-2.

AT2 receptor stimulation inhibited serine-phosphorylaton of Akt almost near to the level without insulin, whereas AT2 receptor-mediated inhibition of PI3K activity associated with IRS-2 was relatively small (approximately one-third decrease compared with the level with insulin). These results suggest that other mechanisms might be involved in AT2 receptor-mediated inhibitory effect on insulin-mediated Akt activation. It has been shown that Akt can be activated via at least two pathways, namely PI3K dependent and independent pathways (44). Activation of protein kinase C (PKC) impairs insulin-mediated Akt activation in 3T3-L1 adipocytes (45) and AT2 receptor stimulation activates PKC in cardiomyocytes (46). Therefore, it is possible that stimulation of AT2 receptor activates PKC, thereby decreasing insulin-mediated Akt activation in PC12W cells. In addition, we have observed that AT2 receptor activation induces accumulation of ceramide in PC12W cells, which was blocked by tyrosine phosphatase inhibitor, orthovanadate (47). The AT2 receptor-mediated generation of ceramide might inhibit Akt, as previously reported (48). These possibilities and more detailed roles of SHP-1 in Akt inactivation have to be addressed for further understanding the mechanism of cross-talk of insulin and AII signaling.

AT2 receptor-mediated inhibition of IRS-2 associated of PI3K, tyrosine-phosphorylation of p85{alpha} subunit of PI3K, and Akt phosphorylation as well as AT2 receptor-mediated proapoptotic effects were abrogated by overexpression of dnSHP-1 mutant. Taken together, these results indicate that AT2 receptor- mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI-3-K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis. Moreover, these results suggest that SHP-1 plays important roles in cross-talk between insulin and AII signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Rabbit antiphosphoSer-Akt and anti-Akt polyclonal antibodies, and the PTP assay kit were purchased from New England Biolabs, Inc. (Beverly, MA). Rabbit anti-IRS-1, anti-IRS-2, and anti-p85{alpha} PI3K polyclonal antibodies and mouse antiphosphotyrosine (4G10) monoclonal antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Mouse anti-PTP1C/SHP-1 monoclonal antibody was purchased from Transduction Laboratories, Inc. (Lexington, KY). All secondary antibodies conjugated with horseradish peroxidase, nitrocellulose membrane Hybond, enhanced chemiluminescence (ECL) reagents and protein G-Sepharose 4 Fast Flow were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). The AT2 receptor antagonist PD123319 was purchased from Research Biochemicals International (Natick, MA) [{gamma}-32P]ATP (3000 Ci/mmol) was obtained from NEN Life Science Products (Boston, MA). LipofectAMINE PLUS and cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD), and all other reagents were from Sigma (St. Louis, MO).

Cell Culture and Transfection
PC12W cells were cultured in DMEM with 10% horse serum, 5% fetal calf serum (FCS), 100 U/ml penicillin, and 1 mg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO2 (16). The SHP-1 (C453/S) mutant inserted in pcDNA3 (25) was transiently transfected in PC12W cells with the LipofectAMINE PLUS reagents according to the manufacturer’s instructions. The transfected cells were fed complete growth medium for 48 h, and were then made quiescent by incubation with serum-free DMEM overnight before appropriate treatment.

Receptor Binding Assay
AT1 and AT2 receptor binding was measured using subconfluent cells grown in 24-well plates. After washing twice PBS containing 0.1% BSA, the cells were incubated for 1 h at 37 C with 0.1 nM 125I-[Sar1, Ile8]AII (NEN Life Science Products) in the absence (for total count) or presence of 1 µM valsartan (provided by Novartis Pharma AG, Basel, Switzerland) or 1 mM PD123319 (Research Biochemicals International). The cells were then washed twice with ice-cold PBS containing 0.1% BSA and lysed in 0.5 N NaOH. The raw radioactivity count of the lysate was measured by {gamma} counter. AT1 receptor binding was calculated as the difference between the total count and the count from samples incubated with valsartan. AT2 receptor binding was determined by subtracting the count of samples incubated with PD123319 from the total count. The net radioactivity count was converted to molar value by using specific activity of the ligand and was normalized by the cell number, which was measured at the same time.

Immunoprecipitation and Immunoblotting
Cells treated under different experimental conditions were washed quickly with ice-cold PBS containing 1 mM Na3VO4, frozen in liquid nitrogen, scraped off and lysed in Nonidet P-40 lysis buffer [1% Nonidet P-40, 25 mM HEPES (pH 7.5), 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 nM okadaic acid, 1 mM sodium orthoavanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 µM aprotinin] for 10–15 min on ice. Insoluble material was removed by centrifugation at 14,000 x g for 20 min at 4 C. Protein concentration was measured in the cleared supernatant using a kit (Bio-Rad Laboratories, Inc., Hercules, CA). Then 50 µl of a 50% slurry of protein G-Sepharose 4 Fast Flow were added to 1 ml of cell lysates with equal amounts of protein, and incubated at 4 C for 1 h with gentle shaking. The precleared lysates were incubated with appropriate antibodies as indicated with constant agitation at 4 C for 2 h and then further incubated with protein G-Sepharose 4 Fast Flow for 1 h. The immunoprecipitates were washed 4 times with Nonidet P-40 lysis buffer. For immunoblotting, the whole cell lysates (20 µg) or immunoprecipitates were subjected to SDS-PAGE and electrotransferred onto Hybond-ECL nitrocellulose membrane. Membranes were incubated with 5% nonfat milk in TBS (Tris-buffered saline)-T [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1% Tween 20] for 1 h at room temperature and incubated with the appropriate primary antibody at 4 C overnight. The membranes were then washed twice with TBS-T and probed with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Membranes were washed several times with TBS-T to remove unbonded secondary antibodies and visualized with an ECL detection kit (Amersham Pharmacia Biotech). Densitometric analysis was performed by using an image scanner (EPSON GT-8000) and NIH image software.

PI3K Assay
The measurement of PI3K activity was performed as previously described with minor modification (49). Briefly, cell lysates (0.5–1 mg protein) were incubated with antibody against the p85{alpha} subunit of PI3K (2 µg/mg of protein), antibody against phospho-tyrosine, antibody against IRS-1 or IRS-2 (2 µg/mg of protein) as described above. The washed pellets were washed once with buffer (0.1 mM NaCl;1 mM EDTA; and 20 mM Tris-HCl, pH 7.5) and then were resuspended in 50 µl kinase reaction buffer (20 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 0.5 mM EGTA) with 10 µg phosphatidylinositol (PI), and incubated at 25 C for 10 min to make micelles of PI. PI dissolved in chloroform was dried with N2 and sonicated at 0.5 µg/µl of kinase reaction buffer in a bath sonicator three times for 5 min each with cooling in between. Assays were initiated with the addition of 5 µl ATP solution (0.4 mM ATP, 100 mM MgCl2, 1 µCi/µl [{gamma}-32P]ATP), and incubated at room temperature for 10 min. The reaction was stopped after the addition of 100 µl chloroform, methanol, and 11.6 N HCl (200:100:2 vol/vol/vol). After centrifugation, the lower organic phase was taken for thin-layer chromatography on a Silica Gel 60 aluminum sheet (Merck, Darmstadt, Germany) and developed in chloroform, methanol, 28% ammonium hydroxide, and water (43:38:4.5:7.5 vol/vol/vol/vol/). The aluminum sheet was exposed to Kodak X-Omat film at -70 C with an intensifying screen, and radiolabeled spots were identified, excised and quantified by scintillation counting.

Measurement of Tyrosine Phosphatase SHP-1 Activity
Activity of SHP-1 was determined as previously described (25). Cell lysates (1 mg protein) were immunoprecipitated with anti-SHP-1 antibody and protein G-Sepharose 4 Fast Flow as described above. The immunoprecipitates were washed three times with lysis buffer lacking Na3VO4 and NaF, and once with phosphatase assay buffer (50 mM Tris-HCl, pH 7.0; 1 mM EDTA; 5 mM dithiothreitol; and 0.01% Brij35). PTP activity was measured as the release of [32P]-orthophosphate from [{gamma}-32P]ATP-radiolabed myelin basic protein.

Caspase-3 Activity Assay
Caspase-3 activity was measured using a previously described protocol (26). Treated cells were rinsed twice with PBS and suspended in 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 10 mM EGTA. After addition of 10 µM digitonin, cells were incubated at 37 C for 10 min. Lysates were cleared by centrifugation at 14,000 x g for 3 min, and the cleared lysates containing 40 µg protein were incubated with 50 µM enzyme substrate Ac-DEVD-MCA at 37 C for 1 h. Levels of released 7-amino-methylcoumarin were measured using a spectrofluorometer (Hitachi F-3000) with excitation at 380 nm and emission at 460 nm. Results were normalized to basal activity in viable cells maintained in serum-containing medium.

Morphological Changes of Apoptois
PC12W cells were seeded into chamber slide. Treated cells were rinsed twice with ice-cold PBS, and fixed with methanol at -20 C for 20 min. After fixation, the cells were washed with PBS, and then stained with Hoechst 33342 (Molecular Probes, Inc., Eugene, OR) at 5 µM for 10 min at room temperature, and were viewed by UV microscopy.

Statistical Analysis
Data are expressed as mean ± SE. One-way analysis of variance with the Newman-Keuls’ test was used to compare differences among means, and P < 0.05 was considered significant.


   ACKNOWLEDGMENTS
 


   FOOTNOTES
 
This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Tokyo Biochemical Research Foundation, the Japan Research Foundation for Clinical Pharmacology, and the Smoking Research Foundation.

Abbreviations: AII, Angiotensin II; AT1 or 2, AII subtype 1 or 2; dn, dominant negative; ECL, enhanced chemiluminescence; IRS, insulin receptor substrate; JNK, c-Jun NH2-terminal protein kinase; MKP-1, MAPK phosphatase-1; PI, phosphatidylinositol; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PP2A, serine/threonine phosphatase 2A; PTP, protein tyrosine phosphatase; RAS, renin-angiotensin system; SH2, Src homology 2; TBS, Tris-buffered saline; VSMC, vascular smooth muscle cells.

Received for publication October 22, 2001. Accepted for publication May 9, 2002.


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TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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