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B and Monocyte Chemoattractant Protein-1 by Angiotensin II Type 2 Receptor-Activated Src Homology Protein Tyrosine Phosphatase-1 in Fetal Vascular Smooth Muscle Cells
Department of Medical Biochemistry, Ehime University School of Medicine (L.W., M.I., Z.L., T.S., L.-J.M., T.-X.C., J.-M.L., M.O., M.H.), Shigenobu, Onsen-gun, Ehime 791-0295, Japan; and Centre National de la Recherche Scientifique, UPR0415-Institut Cochin de Genetique Moleculaire (C.N.), Paris, France
Address all correspondence and requests for reprints to: Dr. Masatsugu Horiuchi, 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.
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ABSTRACT |
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B (NF-B) binding to the corresponding cis DNA element of the MCP-1 promoter region and a decrease in the cytosolic inhibitory protein-B (IB) protein level via AT1 receptor stimulation, whereas stimulation of the AT2 receptor decreased Ang II-induced MCP-1 expression, NF-B DNA binding, and IB degradation, suggesting that activation of the AT2 receptor attenuated AT1 receptor-mediated MCP-1 expression via a decrease in NF-B DNA binding and an increase in IB stability. Moreover, we demonstrated that AT2 receptor stimulation attenuated TNF
-mediated NF-B activation and MCP-1 expression. A tyrosine phosphatase inhibitor, orthovanadate, attenuated the AT2 receptor-mediated increase in IB protein. Moreover, we observed that two IB subunits (IB and IBß) were tyrosine-phosphorylated after Ang II stimulation. Transfection of a dominant-negative Src homology protein tyrosine phosphatase-1 mutant into vascular smooth muscle cells inhibited the AT2 receptor-mediated increase in IB, leading to a significant increase in AT1 receptor-induced NF-B activation and MCP-1 expression. Taken together, our results demonstrated that AT2 receptor stimulation attenuated MCP-1 expression via IB stabilization, and Src homology protein tyrosine phosphatase-1 might play a critical role in the transcriptional regulation of MCP-1 expression through the control of IB protein stability. |
INTRODUCTION |
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Like many other proinflammatory genes, activation of the MCP-1 gene is controlled by the transcription factor, nuclear factor-B (NF-B). NF-B is present in the cytosol of unstimulated cells in an inactive form bound to the inhibitory protein-B (IB) (5). The ankyrin repeat domains of IB proteins prevent nuclear translocation of NF-B by masking the nuclear localization sequences of NF-B. In response to inflammatory stimuli, IB was phosphorylated and targeted for ubiquitination and degradation. Dissociation and degradation of IB result in activation of NF-B, which may be defined as translocation of the NF-B complex from the cytoplasm to the nucleus. After translocation to the nucleus, NF-B binds specific promoter elements of DNA and induces transcription of relevant genes, such as MCP-1.
The AT2 receptor subtype is a member of the G protein-coupled receptor superfamily with relatively low sequence homology with the AT1 receptor subtype. Accumulating evidence has revealed opposite intracellular effects of the AT1 and AT2 receptors, particularly on the regulation of protein kinases and phosphatases (6, 7). It is postulated that the final response to Ang II stimulation in cells coexpressing AT1 and AT2 receptors is dependent on the relative expression levels of these two receptors. The growth inhibitory effects of the AT2 receptor have been shown to be associated with the activation and/or induction of a series of phosphatases (1), including the protein tyrosine phosphatase Src homology protein tyrosine phosphatase-1 (SHP-1) (8), MAPK phosphatase-1 (9), and serine/threonine phosphatase 2A (10), which results in the inactivation of AT1 receptor- and/or growth factor-activated ERK. Therefore, we postulated that the AT2 receptor-activated specific phosphatase(s) antagonizes the signaling pathway involved in AT1 receptor-mediated MCP-1 production. To explore the roles of signaling cross-talk of the AT1 and AT2 receptors in the regulation of MCP-1 expression, we focused on NF-B/IB in fetal VSMC, which endogenously express both AT1 and AT2 receptors.
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). Stimulation with Ang II in the presence of an AT2 receptor-specific antagonist, PD123319, further increased MCP-1 expression, whereas addition of an AT1 receptor blocker, valsartan, decreased Ang II-mediated MCP-1 expression (Fig. 1B). We did not observe any significant difference in MCP-1 expression between control VSMC and VSMC treated with Ang II and valsartan. Neither valsartan nor PD123319 alone influenced MCP-1 expression. These results suggest that AT2 receptor stimulation antagonized AT1 receptor-mediated MCP-1 mRNA expression. To further clarify the possibility that AT2 receptor stimulation inhibits MCP-1 expression, we examined the effect of CGP42112A, an AT2 receptor agonist, on TNF-induced MCP-1 expression, as it has been reported that TNF stimulation enhances MCP-1 expression via enhancement of NF-B binding and inhibition of IB protein degradation in VSMC (12, 13, 14). We observed that CGP42112A decreased TNF-induced MCP-1 expression (Fig. 1B).
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B Binding and Degradation of Cytosolic IB via Cross-Talk of AT1 and AT2 ReceptorsB binding, we performed electrophoretic mobility shift assay (EMSA) using nuclear extracts prepared from fetal VSMC stimulated with Ang II for 30, 60, 90, and 120 min and a 32P-labeled NF-B-binding oligonucleotide probe and observed a time-dependent increase in the complex formation (Fig. 2A) that reached a peak after 90 min of Ang II stimulation. As shown in Fig. 2B, addition of a 50-fold excess of unlabeled NF-B-binding oligonucleotide as a competitor abolished Ang II-induced NF-B binding, but the addition of a mutant NF-B-binding oligonucleotide did not influence this binding. Incubation of nuclear extract with anti-NF-B antibodies decreased this binding. Stimulation with Ang II in the presence of PD123319 further increased NF-B binding, whereas addition of valsartan decreased Ang II-induced MCP-1 expression (Fig. 2C). These results suggest that AT2 receptor stimulation attenuated the AT1 receptor-mediated increase in NF-B binding. We also examined the effect of CGP42112A, an AT2 receptor agonist, on TNF-induced NF-B binding and observed that CGP42112A effectively inhibited TNF-induced NF-B binding (Fig. 2C).
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B activation is regulated by IB proteins; that is, the dissociation and consequent degradation of IB result in an increase in NF-B nuclear translocation and its binding to the corresponding cis DNA element of the MCP-1 promoter region. Therefore, we first examined the protein levels of cytosolic IB after stimulation with Ang II (Fig. 3). Cytosolic extracts were isolated from VSMC, and the levels of IB and IBß were determined by Western blot. Upon Ang II stimulation, cytosolic IB and IBß levels were decreased, reaching a nadir after 60 min of Ang II stimulation. Simultaneous addition of valsartan attenuated the Ang II-mediated decrease in cytosolic IB and IBß levels, whereas addition of PD123319 exaggerated the decrease in IB in the cytosol, suggesting that AT2 receptor stimulation attenuated the AT1 receptor-mediated decrease in cytosolic IB. TNF decreased cytosolic IB and IBß levels and CGP42112A attenuated this effect of TNF (Fig. 3B).
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B Protein Levels and Attenuation of MCP-1 Expression, pretreatment with orthovanadate or okadaic acid further enhanced the Ang II-induced increase in MCP-1 mRNA expression, and these effects were comparable with the effect of AT2 receptor blockade by PD123319. Pretreatment with orthovanadate or okadaic acid also attenuated the inhibitory effect of AT2 receptor stimulation by CGP42112A on TNF-induced MCP-1 expression (Fig. 4). Neither orthovanadate nor okadaic acid affected basal MCP-1 expression. Next, we examined the effects of these phosphatase inhibitors on Ang II-regulated NF-B nuclear translocation (Fig. 5). Consistent with the results obtained by EMSA (Fig. 2
), nuclear translocation of NF-B protein subunits p65 and p50 was enhanced by specific AT1 receptor stimulation (Ang II plus PD123319), and Ang II-induced nuclear translocation of NF-B was inhibited by selective AT2 receptor stimulation (Ang II plus valsartan; Fig. 5). Moreover, we observed that both tyrosine and serine/threonine phosphatase inhibitors further increased the Ang II-induced enhancement of NF-B protein levels in the nucleus. Addition of these phosphatase inhibitors also enhanced the Ang II-mediated decrease in levels of cytosolic IB and IBß (Fig. 6). The inhibitory effects of CGP42112A on TNF-mediated nuclear translocation of NF-B and the decrease in levels of cytosolic IB were inhibited by orthovanadate or okadaic acid (Figs. 5 and 6).
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B and consequently may stabilize IB. VSMC were treated with Ang II and immunoprecipitated with anti-IB antibodies, and tyrosine phosphorylation of IB was examined by immunoblotting analysis with an antiphosphotyrosine antibody, 4G10 (Fig. 7). Tyrosine phosphorylation of IB and IBß was enhanced by Ang II and peaked after 10 min of Ang II stimulation (Fig. 7A). Simultaneous addition of valsartan attenuated the Ang II-mediated increase in phosphorylation of IB and IBß, whereas addition of PD123319 exaggerated the Ang II-induced tyrosine phosphorylation of IB, suggesting that AT2 receptor stimulation attenuated the AT1 receptor-mediated tyrosine phosphorylation of IB (Fig. 7B). We also examined the effect of CGP42112A on TNF-induced IB phosphorylation and observed that CGP42112A effectively inhibited TNF-induced phosphorylation of IB and IBß (Fig. 7B). To investigate whether SHP-1 plays a role in AT2 receptor-mediated inhibition of IB and IBß degradation, NF-B DNA binding activity, and MCP-1 expression, we transfected rat fetal VSMC with a dominant-negative (dn) SHP-1 mutant in which the active cysteine 453 was mutated to serine (C453/S) (16). Transfection of the dnSHP-1 mutant was confirmed by immunoblotting, showing a 6-fold increase in SHP-1 immunoreactivity compared with the control vector pcDNA3-transfected VSMC and the decrease in SHP-1 activity (Fig. 8A). We first examined the functional significance of SHP-1 activation in the AT2 receptor-mediated inhibition of IB and IBß. Basal levels of IB and IBß were not changed in either pcDNA3-transfected or dnSHP-1-transfected VSMC. The Ang II-mediated decrease in IB and IBß protein levels was more marked in dnSHP-1-transfected VSMC than in pcDNA3-transfected VSMC (Fig. 8B). The Ang II-induced decrease in IB and IBß was not significantly influenced by PD123319 in dnSHP-1-transfected VSMC (Fig. 8B). Next we compared the effects of Ang II treatment on NF-B binding activity determined by EMSA and MCP-1 mRNA expression in pcDNA3- or dnSHP-1-transfected fetal rat VSMC. In dnSHP-1-transfected VSMC, the Ang II-mediated increases in NF-B binding activity and MCP-1 expression were further exaggerated compared with those in pcDNA3-transfected VSMC (Fig. 8, C and D). We also compared the effect of CGP42112A on the TNF-induced decrease in IB and IBß, NF-B binding activity, and MCP-1 expression in both cells and found that the effect of CGP42112A was attenuated in dnSHP-1-transfected VSMC (Fig. 8, B–D).
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DISCUSSION |
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B, which coordinates the up-regulation of proinflammatory genes (18). This proinflammatory action of Ang II on the vessel wall interacts synergistically with those of other cardiovascular risk factors, such as dyslipidemia and diabetes. One of the major Ang II-mediated activated proinflammatory genes in monocytes and VSMC is MCP-1, which is inhibited by coadministration of an intracellular antioxidant (19). These Ang II-induced proinflammatory actions in the vessel wall are mediated via AT1 receptor stimulation. Despite increasing evidence suggesting possible important functions of the AT2 receptor in vascular remodeling, the specific effect of the AT2 receptor on vascular inflammation is a total enigma. Consistent with our recent in vivo study demonstrating that MCP-1 expression in the injured artery was exaggerated in AT2 receptor-null mice (4), we observed in this study that AT2 receptor stimulation antagonized AT1 receptor-mediated activation of NF-B, thereby inhibiting MCP-1 expression in cultured fetal VSMC. Moreover, the AT2 receptor is highly expressed in certain pathological conditions, such as inflammation and vascular injury (1), and we demonstrated that AT2 receptor stimulation significantly inhibited TNF-induced phosphorylation and degradation of IB, nuclear translocation of NF-B, NF-B binding, and MCP-1 expression, suggesting that these inhibitory effects of AT2 receptor stimulation would contribute to its antiinflammatory actions against other proinflammatory factors as well as AT1 receptor stimulation. In contrast, Ruiz-Ortega et al. (12) demonstrated that Ang II activates NF-B via both AT1 and AT2 receptors in adult rat VSMC, and Wolf et al. (20) reported that AT2 receptor stimulation could induce NF-B activation in AT2 receptor-expressed COS-7 cells, glomerular endothelial cells, and PC12 cells. These apparent discrepancies could occur because the signaling of these cells may be different from that of the embryonic rat VSMC used in the present study, as the cells used in previous studies were transfected and overexpressed with AT1 and AT2 receptors, whereas embryonic VSMC in our study have endogenous AT1 and AT2 receptors, and/or could be due to different experimental conditions, such as the concentration of Ang II or the schedule of the reaction.
NF-B is a critical transcription factor for Ang II-induced MCP-1 gene expression (2, 3, 21). NF-B is a heterodimer of p50 and p65 subunits present in an inactive form in the cytoplasm complexed with its inhibitor, IB. NF-B activation consists of the dissociation of IB and nuclear translocation. In this study we observed that Ang II increased nuclear translocation of NF-B and increased NF-B DNA binding with a decrease in cytosolic IB and IBß levels in fetal VSMC, and PD123319 exaggerated these effects of Ang II, suggesting that AT2 receptor stimulation attenuated AT1 receptor-mediated NF-B activation by decreasing IB levels. Phosphorylation of IB and IBß dissociates these proteins from NF-B and determines their fate through degradation. We observed that the addition of orthovanadate and okadaic acid further exaggerated not only the Ang II-mediated increase in MCP-1 expression and NF-B binding, but also the Ang II-mediated decrease in cytosolic IB levels to a similar extent when PD123319 was added. Furthermore, we found that CGP42112A-mediated inhibitory effects on TNF-induced degradation of IB, nuclear translocation of NF-B, and MCP-1 expression were attenuated by the addition of orthovanadate or okadaic acid. These results implied that the inhibitory effect of AT2 receptor stimulation on AT1 receptor- or TNF-mediated NF-B/MCP-1 activation is at least partly due to the phosphatases.
Our results suggest that both tyrosine phosphatase and serine/threonine phosphatase are involved in the AT2 receptor-mediated inhibition of IB and IBß degradation. Consistent with our observation, specific serine phosphorylation sites of IB mediated by IB kinases (IKKs) and other kinases have been located at two serine residues within their amino-terminal regulatory domains (S32 and S36 in IB, and S19 and S23 in IBß) (22). It has also been reported that phosphorylation at the serine of IB and IBß by serine/threonine kinase triggers polyubiquitination of the IB proteins (23) and, as with other proteins (24), targets them for rapid degradation by the 26S proteasome. Degradation of IB is also mediated by phosphorylation of IB at tyrosine residue 42 or by mechanisms involving unidentified protein tyrosine phosphatases (12, 25). It has also been reported that AT1 receptor-mediated MCP-1 expression was decreased by a tyrosine kinase inhibitor and a MAPK inhibitor (2). Imbert et al. (25) reported that stimulation of Jurkat T cells with a protein tyrosine phosphatase inhibitor and the T cell activator, pervanadate, led to NF-B activation through tyrosine phosphorylation, but not degradation of IB, and that pervanadate-induced IB phosphorylation and NF-B activation required expression of the T cell tyrosine kinase, p56ick. In addition, inhibition of protein tyrosine phosphatase activities by pervanadate or various vanadate-based compounds has been reported to specifically increase the activation of NF-B and downstream responsive genes (26). In this study we showed that both IB and IBß were tyrosine-phosphorylated by Ang II stimulation, and this Ang II-induced tyrosine phosphorylation of IB and IBß was specifically inhibited by an AT1 receptor blocker, but was enhanced by an AT2 receptor blocker, supporting the idea that AT1 receptor stimulation tyrosine-phosphorylates both IB and IBß, whereas AT2 receptor stimulation antagonizes this effect of AT1 receptor stimulation. Moreover, our results suggest that tyrosine phosphorylation of IB and IBß is closely associated with degradation of IB and IBß, and the consequent increase in nuclear translocation of NF-B is associated with the enhancement of MCP-1 expression in VSMC.
To examine whether the AT2 receptor-mediated dephosphorylation of the tyrosine residue is actually involved in Ang II-regulated MCP-expression in VSMC, we focused on SHP-1, a soluble tyrosine phosphatase that participates in negative regulation of the receptor tyrosine kinase pathway (27). However, the role of SHP-1 in regulating proinflammatory signaling is incompletely understood. Previously, we demonstrated that SHP-1 is pivotal in the antigrowth effect of the AT2 receptor (8, 11, 15). Moreover, involvement of SHP-1 in AT2 receptor signaling has been suggested (28). To establish the functional link between AT2 receptor-mediated activation of SHP-1 and inhibition of the degradation of IB, consequent inhibition of NF-B binding, and suppression of MCP-1 expression, we employed dnSHP-1. We demonstrated that preventing SHP-1 activation abrogated AT2 receptor-induced protection of IB degradation in fetal VSMC, with a decrease in NF-B binding and MCP-1 expression against AT1 receptor stimulation or TNF stimulation. Consistent with these results, Massa et al. (29) reported increased sensitivity of astrocytes lacking SHP-1 to various inducers of NF-B. These studies suggest an additional role of SHP-1 in controlling specific and nonspecific immune responses where induction of NF-B is involved.
It seems possible that SHP-1 could directly dephosphorylate the tyrosine residues of IB; however, this possibility requires more extensive examination. On the other hand, there is evidence suggesting that IKKs are themselves phosphorylated and activated by the MAPK cascade (30). Hoshi et al. (31) also reported that PD98059, a MAPK kinase (MEK) inhibitor, suppressed NF-B transcriptional activity. MEK kinase-1 has been shown to be copurified with IKK activity (30), and MEK kinase-1 enhances the ability of IKKs to phosphorylate IB and activate NF-B (32). It is possible that IKKs may be regulated at several levels by SHP-1, as our previous results suggested that SHP-1 is involved in AT2 receptor-mediated inactivation of ERK (8, 11). Moreover, Shibasaki et al. (28) reported that the AT2 receptor inhibits epidermal growth factor receptor trans-activation by increasing the association with SHP-1 tyrosine phosphatase and decreases ERK activity. In addition to SHP-1, AT2 receptor-mediated inhibition of ERK might be dependent on other phosphatases, such as MAPK phosphatase-1 and serine/threonine phosphatase 2A, which results in the inactivation of AT1- and/or growth factor-activated ERK (1). Consistent with these observations, we demonstrated in this study that inhibition of serine/threonine phosphatases as well as tyrosine phosphatases effectively abrogated the inhibitory effect of the AT2 receptor on IB phosphorylation, suggesting that the AT2 receptor-mediated inhibitory pathway of ERK is also involved in this receptor-mediated inhibition of NF-B activation.
Ozes et al. (33) reported that the Akt serine-threonine kinase is involved in the activation of NF-B by TNF by activating NF-B-inducing kinase, and that Akt mediates IKK phosphorylation at threonine 23. These findings indicate that Akt is part of a signaling pathway that is necessary for inducing key immune and inflammatory responses. Moreover, Madrid et al. (34) demonstrated that activated phosphoinositide 3-kinase or Akt stimulates NF-B-dependent transcription by stimulating trans-activation domain 1 of the p65 subunit rather than by inducing NF-B nuclear translocation via IB degradation. It has also been reported that upon platelet-derived growth factor stimulation, Akt transiently associates in vivo with IKK and induces IKK activation (35). Very recently, we demonstrated that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition of IRS-2-associated PI-3-K activity contribute at least partly to the inhibition of Akt phosphorylation in PC12W cells (15). These results suggest that AT2 receptor-activated Akt could be involved in the protective effect of the AT2 receptor on IB degradation and the inhibition of NF-B activation and MCP-1 expression.
Impairment of the capacity of a vessel to dilate in the presence of endothelial dysfunction reflects, at least in part, increased oxidative stress due to enhanced catabolism of nitric oxide (NO) caused by increased generation of the superoxide anion. In addition to being a vasodilator, NO is an endogenous inhibitor of VSMC growth and migration (36), of the activity of the transcription factor NF-B, and of the expression of proinflammatory molecules (37). With an imbalance between NO and reactive oxygen species (ROS), there is a propensity for vasospasm, VSMC proliferation, prothrombosis, and proinflammatory and prooxidant states. AT2 receptor-mediated NO production is a well established phenomenon in various tissues and cells (38), and it can be assumed that this NO production might be involved in AT2 receptor-mediated NF-B inactivation. In addition, the possible interesting roles of the AT2 receptor in NADPH oxidase and production of ROS need to be addressed in the near future to fully understand the molecular and cellular mechanisms of the antiinflammatory effect of AT2 receptor stimulation.
Taken together, these results suggest that AT2 receptor stimulation antagonizes the effect of AT1 receptor-mediated MCP-1 expression by dephosphorylation of IB and consequent inhibition of nuclear translocation of NF-B, and that SHP-1 is a critical phosphatase that mediates the antiinflammatory action of the AT2 receptor. AT1 receptor-activated NF-B also induced proinflammatory genes, such as adhesion molecules, including vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, and inflammatory cytokines, such as IL-1, TNF, and IL-6, in addition to MCP-1 (39). The potential inhibitory effects of AT2 receptor stimulation on these proinflammatory genes via NF-B inactivation need to be further clarified, and detailed analysis will contribute to further understanding of the pathogenesis of vascular inflammation, atherosclerosis, and vascular remodeling.
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MATERIALS AND METHODS |
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-smooth muscle (-SM) actin (clone 1A4; Sigma-Aldrich Corp., St. Louis, MO) (41). Cells at passage 2–3 were used for the experiments. AT1 and AT2 receptor binding was measured as previously described (40).
Northern Blot Analysis
Rat fetal VSMC were grown in DMEM with 10% fetal bovine serum to a subconfluent state in 10-cm dishes and were incubated for an additional 24 h in serum-free DMEM to reach a quiescent state. Cells were then treated with vehicle or Ang II (10-7 M) with or without valsartan (10-5 M; provided by Novartis Pharma AG, Basel, Switzerland) or PD123319 (10-5 M; Sigma-Aldrich Corp.). To examine the involvement of phosphatase, cells were incubated with sodium orthovanadate (10-5 M vanadate) or okadaic acid (10-5 M) for 16 h, then stimulated with Ang II. Recombinant rat TNF- was purchased from Genzyme (Minneapolis, MN). An AT2 receptor agonist, CGP42112A, was purchased from Sigma-Aldrich Corp. RNA was prepared from the cells using TRIzol reagent (Life Technologies, Inc.). To prepare the hybridization probe for MCP-1, RT-PCR was performed as previously described (4). PCR primers for MCP-1 were 5'-ACT GAA GCC AGC TCT CTC TTC CTC-3' (forward) and 5'-TTC CTT CTT GGG TCA GCA CAG AC-3' (reverse). To verify the identities of the PCR products, we sequenced the PCR products and confirmed that the sequences of PCR products matched the predicted sequences. PCR-amplified DNA of MCP-1 was subcloned into pGEM-T Easy Vector (Promega Corp., Madison, WI). After size fractionation on a denaturing agarose-formaldehyde gel, total RNA (25 µg) was transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Little Chalfont, UK). Hybridization was carried out with 32P-labeled probes of rat MCP-1 cDNA and/or the 0.78-kb PstI-XbaI fragment of human glyceraldehyde-3-phosphate dehydrogenase in Rapid-Hyb buffer (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis was performed using an image scanner (GT-8000, Epson, Tokyo, Japan) and NIH image software.
EMSA
NF-B DNA binding activity was determined using EMSA as previously described (42). Briefly, after stimulation, the cells were collected with a cell scraper and suspended in buffer A [10 mM HEPES (pH 7.9), 0.5 mM phenylmethylsulfonyfluoride (PMSF), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM dithiothreitol (DTT)] and homogenized with a Dounce homogenizer (Kontes Co., Vineland, NJ). Nuclear and cytosolic fractions were separated by centrifugation at 1,000 x g for 15 min at 4 C, and the nuclear fraction was washed twice in buffer A and resuspended in buffer C [20 mM HEPES (pH 7.9), 0.5 mM PMSF, 1.5 mM MgCl2, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, and 25% (vol/vol) glycerol]. After incubation for 1 h at 4 C, the nuclear fraction was centrifuged at 10,000 x g for 30 min. The supernatant was dialyzed in buffer D [20 mM HEPES (pH 7.9), 0.5 mM PMSF, 0.1 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, and 20% (vol/vol) glycerol] and then cleared by centrifugation and stored at -80 C until use. Specific oligonucleotide probes of NF-B rat MCP-1 (5'-GTC TGG GAA CTT CCA ATG C-3') carrying the rat MCP-1 NF-B consensus sequence or its mutation (mutant NF-B oligonucleotide, 5'-GTC TGG GAA CTC GGA ATG C-3'; mutated part is underlined) were prepared as previously reported (43), labeled with [
-32P]deoxy-ATP (3000 Ci/mmol; Amersham Pharmacia Biotech) and T4 polynucleotide kinase (Promega Corp.), and employed to examine the specificity of NF-B binding to the rat MCP-1 gene. Nuclear extracts (10 µg) were equilibrated for 10 min in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 µg/ml of poly(dI-dC) (Pharmacia Biotech, Uppsala, Sweden)], then the labeled probe (30,000 cpm) was added, and the mixture was incubated for 30 min at room temperature. Competition assays with a 50-fold excess of unlabeled NF-B and mutant NF-B oligonucleotides were performed to establish the specificity of the reaction. To examine the specificity of binding, 1 µg anti-p65 or anti-p50 NF-B antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added and incubated with nuclear extracts for 1 h before addition of labeled probe. The reaction was stopped by adding gel loading buffer (250 mM Tris-HCl, 0.2% xylene cyanol, and 40% glycerol) and was run on 4% acrylamide gel in Tris-borate-EDTA buffer. The gel was dried and exposed for autoradiography.
Immunoprecipitation and Western Blot Analysis
After stimulation with Ang II, with or without valsartan or PD123319, or stimulation with TNF, with or without CGP42112A, the cells were quickly washed twice with HEPES-buffered saline and frozen in liquid nitrogen. The cells were lysed in lysis buffer [20 mM HEPES (pH 7.8), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml aprotinin, 1 mM Na3VO4, and 1 mM NaF], and the supernatant fraction was obtained as the cell lysate by centrifugation at 12,000 rpm for 25 min at 4 C. The cell lysate (300–500 µg protein) was incubated with 1 µg anti-IB (Santa Cruz Biotechnology, Inc.) or anti-IBß (Santa Cruz Biotechnology, Inc.) at 4 C for 12 h and precipitated by the addition of 25 µl protein A/G-agarose (Amersham Pharmacia Biotech). The immunoprecipitate was resuspended in 1x Laemmli sample buffer and run on 10% SDS-PAGE. The proteins were then transferred to nitrocellulose membrane (Amersham Pharmacia Biotech), blotted with antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY) or anti-IB, IBß, p50, p65 (Santa Cruz Biotechnology, Inc.), or -SM actin antibodies, and detected by an enhanced chemiluminescence method (Amersham Pharmacia Biotech). Densitometric analysis was performed using an image scanner (GT-8000, Epson) and NIH image software.
Plasmid Constructs and Transfection
The SHP-1 (C453/S) mutant cDNA was inserted into the pcDNA3 vector (16). Transient transfection was performed with 0.5 µg plasmid DNA/10-cm dish and Lipofectamine Plus (Life Technologies, Inc.) according to the manufacturer’s instructions (DNA:Lipofectamine:Plus ratio, 1:10:10 µl). After transfection, the transfected cells were fed with complete growth medium for 48 h to allow protein expression. Transfected cells were identified by ß-galactosidase staining. The transfection efficiency determined using only the strongly stained cells was 26 ± 5%, similar to that previously reported (11). Overexpression of the SHP-1 (C453/S) mutant was confirmed by immunoblotting with anti-SHP-1 antibody (Santa Cruz Biotechnology, Inc.).
Measurement of Tyrosine Phosphatase SHP-1 Activity
SHP-1 activity was determined as previously described (8). Cell lysates (1 mg protein) were immunoprecipitated with anti-SHP-1 antibody and protein G-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech, Buckinghamshire, UK). 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 DTT, and 0.01% Brij35 solution]. Protein tyrosine phosphatase activity was measured as the release of [32P]orthophosphate from [-32P]ATP-radiolabeled myelin basic protein.
Statistical Analysis
Results are expressed as the mean ± SE in the text and figures. Data were analyzed using ANOVA. If a statistically significant effect was found, the data were further analyzed by Dunnett’s test to detect the difference between the groups. Statistical significance was assumed for P < 0.05.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: Ang II, Angiotensin II; AT2, angiotensin II type 2; dn, dominant negative; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; IB, inhibitory protein-B; IKK, inhibitory protein-B kinase; MCP-1, monocyte chemoattractant protein-1; MEK, MAPK kinase; NF-B, nuclear factor-B; NO, nitric oxide; PMSF, phenylmethylsulfonyfluoride; SHP-1, Src homology protein tyrosine phosphatase-1; -SM, -smooth muscle; VSMC, vascular smooth muscle cell.
Received for publication February 19, 2003. Accepted for publication December 10, 2003.
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, P < 0.05 vs. Ang II.
, P < 0.05 vs. control, pcDNA3; 
, P < 0.05 vs. Ang II, pcDNA3; *, P < 0.05 vs. control;