3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors Block Calcium-dependent Tyrosine Kinase Pyk2 Activation by Angiotensin II in Vascular Endothelial Cells

INVOLVEMENT OF GERANYLGERANYLATION OF SMALL G PROTEIN Rap1*

Kumi SatohDagger §, Kazuo Ichihara, Erwin J. Landon, Tadashi InagamiDagger , and Hua TangDagger **

From the Departments of Dagger  Biochemistry and § Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and the  Department of Pharmacology, Hokkaido College of Pharmacy, Otaru 047-0264 Japan

Received for publication, October 6, 2000, and in revised form, January 5, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We recently reported the calcium-dependent activation of tyrosine kinase Pyk2 by angiotensin II (Ang II) in pulmonary vein endothelial cells (PVEC). Since Pyk2 has no calcium binding domain, and neither Ca2+ nor Ca2+/calmodulin directly activates Pyk2, it is not clear how Ca2+ transduces the signal to activate Pyk2, a key tyrosine kinase, in the early events of Ang II signaling. In the present study, we investigated the mechanism of the calcium-dependent activation of Pyk2 in response to Ang II by using 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors and isoprenoid intermediates in PVEC. We have obtained substantial evidence indicating that Ang II activates Pyk2 through calcium-mediated activation of the geranylgeranylated small G protein Rap1 and the Rap1 association with Pyk2. Thus, the small G protein Rap1 is an intermediary signaling molecule linking Ang II-induced calcium signal to Pyk2 activation in PVEC. In addition, our results indicate that 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, a class of cholesterol-lowering drugs, could interrupt Ang II signaling independent of cholesterol lowering in endothelial cells.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The renin-angiotensin system impacts on endothelial function and is involved in cardiovascular remodeling associated with hypertension, atherosclerosis, and heart failure (1). Angiotensin II (Ang II)1 binds to endothelial cell receptors and stimulates the expression of plasminogen activator inhibitor-1 and cell adhesion molecules, which are risk factors for the development of atherosclerosis and myocardial infarction (2, 3). We recently reported that Ang II stimulated a calcium-sensitive tyrosine kinase Pyk2 via the type-1 Ang II (AT1) receptor in pulmonary vein endothelial cells (4). Pyk2 (also called CAKbeta for cell adhesion kinase beta , RAFTK for related adhesion focal tyrosine kinase, CADTK, and FAK2) is related to focal adhesion kinase and is activated by tyrosine phosphorylation in response to various agonists for G protein-coupled receptors such as Ang II that increase intracellular calcium concentration (5-7). Pyk2 is a key tyrosine kinase in the early events of AT1 receptor signaling and is a positive regulator of downstream extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase activation evoked by Ang II in various cell types including endothelial cells (4, 8, 9). We and others have recently shown that Ang II-induced activation of Pyk2 is dependent on intracellular calcium mobilization (4, 6, 7). Since Pyk2 has no calcium binding domain and neither Ca2+ nor Ca2+/calmodulin directly activates Pyk2 (10-12), it is not clear how Ca2+ transduces signal to Pyk2 activation.

Statins, a class of cholesterol-lowering drugs that specifically inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, are now widely used for treatment of patients with hypercholesterolemia (13). HMG-CoA reductase, which converts HMG-CoA to mevalonate, is a rate-limiting enzyme of the mevalonate cascade (14). Mevalonate is a precursor not only in cholesterol synthesis but also in the synthesis of isoprenoid intermediates such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). They are important intermediates for post-translational isoprenylation of a variety of proteins including small G proteins (15). Recent studies have demonstrated that some of the direct effects of HMG-CoA reductase inhibitors on the vascular wall are mediated by inhibition of isoprenoid synthesis but not cholesterol synthesis. For example, HMG-CoA reductase inhibitors attenuate vascular smooth muscle cell migration and proliferation and Ang II-induced cardiac hypertrophy (16-18).

In the present study, we investigated the mechanism of the calcium-dependent activation of Pyk2, a key tyrosine kinase in the early events of AT1 receptor signaling, using pulmonary vein endothelial cells. We found that Ang II- and calcium ionophore-induced activation of Pyk2 were blocked by HMG-CoA reductase inhibitors through the inhibition of geranylgeranylation of the small G protein Rap1 and the inhibition of the subsequent association of Rap1 with Pyk2. Thus, the small G protein Rap1 links the Ang II-induced calcium signal to Pyk2 activation. In addition, our results indicate that the cholesterol-lowering statins could interrupt Ang II signaling independently of their lowering of cholesterol in endothelial cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Ang II was obtained from Peninsula Laboratories. Protein A-Sepharose was purchased from Amersham Pharmacia Biotech. Polyvinylidene difluoride membranes were obtained from Millipore. Monoclonal antibodies against Pyk2, phosphotyrosine (PY20), p130Cas, and paxillin were obtained from Transduction Laboratories. Polyclonal antibodies against Rap1, Rac1, and RhoA were obtained from Santa Cruz Biotechnology. Polyclonal antibody to phospho-specific ERK, alkaline phosphatase-conjugated secondary antibodies, and reagents for chemiluminescense detection were purchased from New England Biolabs. GGTI-286 and botulinum exoenzyme C3 were obtained from Calbiochem. Statins such as simvastatin, lovastatin, atorvastatin, lovastatin, fluvastatin and pravastatin, and RNH-6270, an active form of the AT1 receptor antagonist CS-866 (19), were kindly provided by Sankyo Co. Ltd. All other reagents were from Sigma.

Cell Culture and Stimulation-- Rat pulmonary vein endothelial cells (PVEC) were cultured in RPMI 1640 containing 10% fetal calf serum and grown in a CO2 incubator at 37 °C as described previously (4). Cells were pretreated with HMG-CoA reductase inhibitors, mevalonic acid, FPP, GGPP, GGTI-286, or the exoenzyme C3 in serum-free RPMI 1640 medium as indicated below then washed twice with RPMI 1640 medium and stimulated with 100 nM Ang II for 30 s, 10 µM calcium ionophore A23187 for 3 min, or 100 nM phorbol-12-myristate-13-acetate (PMA) for 5 min.

Subcellular Fractionation-- Cell cytosolic and membrane fractions were prepared as described previously (20). Membranes were suspended in Nonidet P-40 lysis buffer (25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each of leupeptin and aprotinin). The suspension was centrifuged at 100,000 × g for 60 min, and the resulting supernatant was referred to as the solubilized membrane fraction. All procedures were performed at 4 °C.

Radioligand Binding Assay-- PVECs were pretreated with chemicals in serum-free RPMI 1640 medium then washed twice with ice-cold phosphate-buffered saline. The AT1 receptor binding capacity was determined using monoiodinated 125I-[Sar1, Ile8] Ang II (Sar is sarcocine) as described previously (20).

Immunoprecipitation and Immunoblotting-- Cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM Na3VO4 and then lysed on ice in the Nonidet P-40 lysis buffer. The extract was clarified by centrifugation and incubated sequentially (4 h for each incubation at 4 °C) with antibodies as indicated and protein A-Sepharose. The immunoprecipitates were collected and washed four times with the lysis buffer. For immunoblotting, whole cell lysates or immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membrane was probed with various primary antibodies as indicated and detected using the ECL system with alkaline phosphatase-conjugated secondary antibodies according to the manufacturer's protocol.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

AT1 Receptor-mediated Activation of Tyrosine Kinase Pyk2 in PVEC-- We recently reported that type-1 but not type-2 of Ang II receptors were detected in PVEC under our cell culture conditions (4). Pyk2 tyrosine phosphorylation is associated with an increase in its kinase activity (5-7). PVECs were stimulated with 100 nM Ang II for 30 s, which caused a maximal activation of Pyk2 as reported previously (4), then lysates were immunoprecipitated with a Pyk2 antibody, and the immune complexes were subjected to immunoblotting with anti-phosphotyrosine antibody (PY20). As shown in Fig. 1A, Ang II induced a rapid and profound tyrosine phosphorylation of Pyk2, and this effect was blocked by a selective AT1 receptor antagonist, RNH-6270 (19). Alternatively, lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and subjected to immunoblotting with Pyk2 antibody as shown in Fig. 1B. Nearly identical results were obtained by this approach as we and others showed previously (4, 7, 21). These results indicate that Ang II induces a rapid activation of Pyk2 via the AT1 receptor in PVEC.


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Fig. 1.   AT1 receptor-mediated activation of tyrosine kinase Pyk2 in PVEC. PVECs were pretreated with a selective AT1 receptor antagonist RNH-6270 (10 µM) for 20 min then stimulated with 100 nM Ang II for 30 s. Lysates were immunoprecipitated (IP) with Pyk2 antibody and subjected to immunoblotting (IB) with anti-phosphotyrosine antibody PY20 (A) or vice versa (B). Results shown are representative immunoblots of two separate experiments.

Blockade of Ang II-induced Activation of Pyk2 by HMG-CoA Reductase Inhibitors in PVEC-- AT1 receptor activates phospholipase Cbeta through Gq protein to generate inositol trisphosphate and diacylglycerol, which in turn induces intracellular calcium mobilization and activates protein kinase C (PKC), respectively (22). To determine effects and acting sites of HMG-CoA reductase inhibitor on Pyk2 activation by Ang II, PVECs were pretreated for 24 h with simvastatin (5 µM), an HMG-CoA reductase inhibitor, and then stimulated with 100 nM Ang II, 10 µM calcium ionophore A23187, or 100 nM PMA. Cell lysates were immunoprecipitated with Pyk2 antibody, and the immune complexes were subjected to immunoblotting with anti-phosphotyrosine antibody (PY20). As shown in Fig. 2A, the maximal tyrosine phosphorylation of Pyk2 by 30 s stimulation with Ang II (4) and Pyk2 tyrosine phosphorylation by A23187 and PMA were completely inhibited by simvastatin pretreatment. Simvastatin did not affect the protein levels of Pyk2 (Fig. 2A). These results indicate that simvastatin blocks Ang II-induced Pyk2 activation at a site(s) downstream of calcium mobilization and PKC in PVEC.


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Fig. 2.   Blockade of Pyk2 activation by HMG-CoA reductase inhibitors in PVEC. A, PVECs were pretreated with or without 5 µM simvastatin (Sim) for 24 h then stimulated with 100 nM Ang II for 30 s, 10 µM calcium ionophore A23187 for 3 min or 100 nM PMA for 5 min. Lysates were immunoprecipitated (IP) with Pyk2 antibody and subjected to immunoblotting (IB) with anti-phosphotyrosine antibody PY20 (upper panel). The same blot was stripped and reprobed with Pyk2 antibody (lower panel). B-D, PVECs were pretreated for 24 h with various concentrations of simvastatin (B), with 5 µM simvastatin (Sim) for the indicated time periods (C), or with either 10 µM of HMG-CoA reductase inhibitors simvastatin (Sim), lovastatin (Lov), atorvastatin (Ator), fluvastatin (Flu), or pravastatin (Pra) for 24 h (D) then stimulated with 100 nM Ang II for 30 s. Lysates were immnuoprecipitated with anti-phosphotyrosine antibody PY20 and subjected to immunoblotting with Pyk2 antibody (upper panels). Simvastatin did not affect protein levels of Pyk2 (B-D, lower panels). Results shown are representative immunoblots of three independent experiments.

Fig. 2B shows the dose-dependent effect of simvastatin on Ang II-induced Pyk2 tyrosine phosphorylation. Pretreatment of PVEC with 1 µM simvastatin for 24 h inhibited 61% of Pyk2 tyrosine phosphorylation evoked by Ang II. Simvastatin at doses 5-20 µM virtually completely inhibited the Pyk2 tyrosine phsophorylation by Ang II. Fig. 2C showed that simvastatin inhibited Pyk2 tyrosine phosphorylation by Ang II in a time-dependent manner without affecting Pyk2 protein levels. In addition, HMG-CoA reductase inhibitors including simvastatin, lovastatin, atorvastatin, and fluvastatin all blocked Pyk2 tyrosine phosphorylation by Ang II (Fig. 2D). In contrast, pravastatin, a water-soluble HMG-CoA inhibitor, had no effect. This may be related to the hydrophilic nature of the drug, which impairs its diffusion through the plasma membrane (23).

Effect of Simvastatin on AT1 Receptor Expression in PVEC-- To determine the effect of simvastatin on AT1 receptor expression, PVECs were pretreated for 24 h with simvastatin at various concentrations (0-20 µM), and the cell surface AT1 receptors were determined by radioligand binding assay as described previously (20). As shown in Fig. 3, simvastatin (1-20 µM) did not affect the AT1 receptor expression in PVEC. We did observe that pretreatment for 24 h with simvastatin at concentrations higher than 5 µM had a cytotoxic effect, causing some cells to float; however, it did not affect cellular expression of AT1 receptor in remaining attached cells. In addition, we also found that simvastatin did not affect Ang II-induced calcium mobilization detected by Fura-2/AM (24) in PVEC (data not shown). These results plus that of Fig. 2A demonstrate that the HMG-CoA reductase inhibitor simvastatin blocks Ang II-induced Pyk2 activation at a step downstream of calcium mobilization and PKC in PVEC.


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Fig. 3.   Effect of simvastatin on AT1 receptor expression in PVEC. PVECs were pretreated for 24 h with simvastatin at various concentrations (0-20 µM), and the cell surface AT1 receptors were determined by radioligand binding assay using monoiodinated 125I-[Sar1, Ile8] Ang II (Sar is sarcocine). Results shown are representative of three independent experiments (n = 4 per experiment).

Simvastatin Blocks Early Signaling Events of AT1 Receptor through Inhibition of Cellular Geranylgeranylation-- Since statins specifically inhibit the synthesis of mevalonate, which is an isoprenyl precursor for FPP and GGPP (14), we determined the effects of exogenous mevalonic acid and the isoprenoid pyrophosphates on simvastatin-mediated inhibition of endothelial AT1 receptor signaling. As shown in Fig. 4A, pretreatment of PVEC with simvastatin blocked the Ang II-induced tyrosine phosphorylation of Pyk2, and this inhibitory effect was completely eliminated by co-incubation with exogenous mevalonic acid (0.5 mM) and GGPP (5 µM) but not FPP (5 µM) (Fig. 4A). These results indicate that simvastatin blocks Ang II-induced Pyk2 activation by inhibiting synthesis of the mevalonate-derived isoprenoid GGPP. Furthermore, pretreatment of PVEC with GGTI-286 (25), a cell-permeable and selective geranylgeranyltransferase-I inhibitor, suppressed Ang II-induced Pyk2 tyrosine phosphorylation in a dose-dependent manner. GGTI-286 at 50 µM virtually abolished the Pyk2 tyrosine phosphorylation. In contrast, a selective and cell-permeable farnesyltransferase inhibitor (FPT inhibitor III) did not affect Ang II-induced Pyk2 tyrosine phosphorylation (Fig. 4B). In addition, simvastatin and GGTI-286 also blocked Pyk2 tyrosine phosphorylation evoked by calcium ionophore A23187 (Fig. 4C).


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Fig. 4.   Geranylgeranylation-dependent activation of Pyk2 evoked by Ang II and calcium ionophore in PVEC. A, PVECs were pretreated with 5 µM simvastatin (Sim) in the presence or absence of mevalonic acid (Mev, 0.5 mM), FPP (5 µM), or GGPP (5 µM) for 24 h then stimulated with 100 nM Ang II for 30 s. B, PVECs were pretreated with a selective geranylgeranyltransferase-I inhibitor GGTI-286 (15-50 µM) or with 20 µM FPT inhibitor III (FPTI) for 8 h then stimulated with 100 nM Ang II for 30 s. C, PVECs were pretreated with either 5 µM simvastatin for 24 h or 30 µM GGTI-286 for 8 h then stimulated with 10 µM calcium ionophore A23187 for 3 min. Lysates were immnuoprecipitated (IP) with anti-phosphotyrosine antibody PY20 and subjected to immunoblotting (IB) with Pyk2 antibody (A-C, upper panels). Protein levels of Pyk2 were not affected by above pretreatment of PVEC (A-C, lower panels). Results shown are representative immunoblots of three independent experiments.

The focal adhesion proteins paxillin and p130Cas associate constitutively with Pyk2 (26, 27). As shown in Fig. 5, Ang II induced a rapid and profound tyrosine phosphorylation of p130Cas and paxillin, and the effects were blocked by pretreatment of cells with simvastatin. Simvastatin also reduced the basal tyrosine phosphorylation of p130Cas and paxillin (Fig. 5) but did not affect their protein levels (data not shown). Co-incubation of cells with simvastatin and mevalonic acid but not FPP completely prevented simvastatin-mediated inhibitory effects (Fig. 5). We also found that GGPP completely prevented the inhibitory effects of simvastatin on p130Cas and paxillin phosphorylation evoked by Ang II in PVEC (data not shown). Taken together, these results indicate that simvastatin blocks early signaling events of the endothelial AT1 receptor through inhibition of cellular geranylgeranylation independent of its cholesterol-lowering effect in PVEC.


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Fig. 5.   Geranylgeranylation-dependent tyrosine phosphorylation of p130Cas and paxillin in response to Ang II. PVECs were pretreated with 5 µM simvastatin (Sim) in the presence or absence of mevalonic acid (Mev, 0.5 mM) or FPP (5 µM) for 24 h then stimulated with 100 nM Ang II for 30 s. Lysates were immnuoprecipitated (IP) with anti-phosphotyrosine antibody PY20 and subjected to immunoblotting (IB) with antibodies to p130Cas or paxillin as indicated. Results shown are representative immunoblots of three independent experiments.

Geranylgeranylation-dependent Association of Rap1 with Pyk2 in Response to Ang II in PVEC-- The above results indicate that geranylgeranylation of a signal molecule(s) downstream of calcium mobilization is required for Ang II-induced Pyk2 activation in PVEC. Small G proteins including Rap, Ral, Rho, Rac, and Rab are geranylgeranylated (15). Since Pyk2 and Rap1 are both activated by intracellular calcium mobilization and PKC (4-7, 28, 29), we determined whether Rap1 could interact with Pyk2 in response to Ang II. As shown in Fig. 6A, PVEC were stimulated with 100 nM Ang II for 30 s or 10 µM calcium ionophore A23187 for 3 min, then cell lysates were immunoprecipitated with polyclonal anti-Rap1 antibody and subjected to immunoblotting with a monoclonal anti-Pyk2 antibody. Ang II and calcium ionophore A23187 markedly increased the association of Pyk2 with Rap1. Pretreatment of cells with simvastatin blocked the association of Rap1 with Pyk2 evoked by Ang II and calcium ionophore A23187 (Fig. 6A). In an alternative experiment, the amount of Rap1 is also markedly increased in the Pyk2 immunoprecipitates in response to Ang II, and the increased association of Rap1 with Pyk2 was abolished by pretreatment of cells with simvastatin (Fig. 6B). In contrast, Ang II did not induce association of Pyk2 with RhoA or Rac1, although low basal level association of Pyk2 with RhoA or Rac1 was recognizable (Fig. 6C). In addition, pretreatment of PVEC for 24 h with 7 µg/ml exoenzyme C3, a specific inhibitor of RhoA (30), did not affect the Ang II-induced Pyk2 tyrosine phosphorylation (Fig. 6D). Pretreatment of PVEC with a higher dose of exoenzyme C3 (15 µg/ml) did not affect Pyk2 tyrosine phosphorylation by Ang II either (data not shown). Taken together, these results suggest that the calcium-dependent activation of Pyk2 by Ang II requires the geranylgeranylation of Rap1 and the subsequent Rap1 association with Pyk2 in PVEC. Pyk2 may be an effector of Rap1 GTPase.


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Fig. 6.   Geranylgeranylation-dependent association of Rap1 with Pyk2 in response to Ang II in PVEC. A, PVECs were pretreated with or without 5 µM simvastatin (Sim) for 24 h then stimulated with 100 nM Ang II for 30 s or 10 µM calcium ionophore A23187 for 3 min. Lysates were immunoprecipitated (IP) with Rap1 antibody and subjected to immunoblotting (IB) with Pyk2 antibody (upper panel). The same blot was stripped and reprobed with Rap1 antibody (lower panel). B, PVECs were pretreated with or without 5 µM simvastatin (Sim) for 24 h then stimulated with 100 nM Ang II for 30 s. Lysates were immunoprecipitated with Pyk2 antibody and subjected to immunoblotting with Rap1 antibody (upper panel). The same blot was stripped and reprobed with Pyk2 antibody (lower panel). C, PVECs were stimulated with 100 nM Ang II for 30 s, and lysates were immunoprecipitated with antibodies to RhoA or Rac1 and subjected to immunoblotting with Pyk2 antibody (upper panel). The same blot was stripped and reprobed with antibodies to RhoA or Rac1 as indicated (lower panel). D, PVECs were pretreated for 24 h with or without 7 µg/ml exoenzyme C3 then stimulated with 100 nM Ang II for 30 s. Lysates were immunoprecipitated with anti-phosphotyrosine antibody PY20 and subjected to immunoblotting with Pyk2 antibody (upper panel). Exoenzyme C3 treatment did not affect Pyk2 protein levels (lower panel). Results shown are representative immunoblots of three separate experiments.

Geranylgeranylation-dependent Membrane Translocation (Activation) of Rap1 in Response to Ang II in PVEC-- Rap1 is post-translationally modified by geranylgeranylation, which is necessary for its membrane localization and function (15). RhoA has been shown to translocate from cytosol to membrane fraction on activation in response to various agonists (17). As shown in Fig. 7, upper panel, Ang II rapidly (30 s) and markedly increased the level of membrane-bound Rap1. A maximal effect was observed at 30 s of Ang II treatment (data not shown). Pretreatment with simvastatin virtually completely prevented Ang II-induced Rap1 membrane translocation. Furthermore, co-treatment with mevalonate but not FPP reversed the inhibitory effect of simvastatin on Rap1 membrane translocation, suggesting an involvement of Rap1 geranylgeranylation. Indeed, pretreatment with GGTI-286 (25), a cell-permeable and selective geranylgeranyltransferase-I inhibitor, also blocked Ang II-induced Rap1 translocation to membrane (Fig. 7, upper panel). The same blot, when stripped and reprobed with anti-insulin receptor beta  subunit, confirmed that equal amounts of protein were loaded (Fig. 7, lower panel). Thus, geranylgeranylation of Rap1 is required for the Ang II-induced Rap1 membrane translocation (activation), which promotes Rap1 association with Pyk2 and subsequent Pyk2 activation in PVEC.


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Fig. 7.   Geranylgeranylation-dependent membrane translocation of Rap1 in response to Ang II in PVEC. PVECs were pretreated with 5 µM simvastatin (Sim) in the presence of either mevalonic acid (Mev, 0.5 mM) or FPP (5 µM) for 24 h or pretreated with a selective geranylgeranyltransferase-I inhibitor GGTI-286 (30 µM) for 8 h then stimulated with 100 nM Ang II for 30 s. Cellular membranes were directly subjected to immunoblotting (IB) with Rap1 antibody (upper panel). The same blot was stripped and reprobed with insulin receptor beta  subunit (Insulin-Rbeta ) (lower panel). Results shown are representative immunoblots of three separate experiments.

Effects of Simvastatin on ERK Activation by Ang II, Calcium Ionophore, and PMA in PVEC-- It has been shown that Pyk2 is partially involved in Ang II-induced activation of ERK through transactivation of epidermal growth factor receptor (8, 9). The ERK is activated upon phosphorylation of a Thr and a Tyr residue in a TEY motif (31). Specific antibody against phosphorylated ERK detects the activated form of ERK by immunoblotting. As shown in Fig. 8A, simvastatin inhibited Ang II-induced ERK activation by 50%, although it completely inhibited Pyk2 activation at these concentrations in PVEC. The partial inhibitory effect of the HMG-CoA reductase inhibitor lovastatin on ERK activation evoked by serum, epidermal growth factor, and platelet-derived growth factor has been reported recently in mesangial cells (32). Interestingly, the calcium ionophore-induced ERK activation was markedly (85%) inhibited by simvastatin (Fig. 8B). This suggests that activation of Pyk2 may contribute to ERK activation by calcium signal. As expected, simvastatin did not affect PMA-induced activation of ERK in PVEC (Fig. 8B) even though it blocked Pyk2 activation by PMA. This may be due to direct activation of the ERK kinase Raf-1 by PKC (33), leading to ERK activation bypassing Pyk2.


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Fig. 8.   Effects of simvastatin on ERK activation by Ang II, calcium ionophore, and PMA in PVEC. PVECs were pretreated with 5 or 10 µM of simvastatin (Sim) for 24 h then stimulated with 100 nM Ang II for 3 min (A) or 10 µM calcium ionophore A23187 for 3 min or 100 nM PMA for 5 min (B). Lysates were directly subjected to immunoblotting with antibodies against phosphorylated ERK or ERK as indicated. The bar graph shows the ERK1 phosphorylation levels by densitometric analysis. Results shown are representative immunoblots of three separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we investigated the mechanism of calcium-dependent activation of Pyk2 by Ang II in PVEC using HMG-CoA reductase inhibitors and isoprenoid intermediates. We have obtained substantial evidence indicating that Ang II activates Pyk2, a key tyrosine kinase in the early events of the endothelial AT1 receptor signaling, through calcium-mediated activation of geranylgeranylated Rap1 and the Rap1 association with its effector Pyk2.

The first key finding obtained from this study is that the calcium-dependent activation of Pyk2 by Ang II requires geranylgeranylation of the small G protein Rap1 and subsequent Rap1 association with Pyk2 in PVEC. It has been demonstrated that Ang II-induced activation of Pyk2 is dependent on intracellular calcium mobilization (4, 6, 7). However, the mechanism is not clear. Since Pyk2 has no calcium binding domain and neither Ca2+ nor Ca2+/calmodulin directly activates Pyk2 (10-12), it appears that an intermediary protein may link the calcium signal to Pyk2 activation. In the present study, we found that Pyk2 tyrosine phosphorylation evoked by Ang II, calcium ionophore A23187, and PKC activator PMA was blocked by the HMG-CoA reductase inhibitor simvastatin in PVEC. Simvastatin did not affect AT1 receptor expression and the receptor-mediated calcium mobilization in PVEC. Furthermore, exogenous mevalonic acid and GGPP but not FPP completely eliminated the inhibitory effect of simvastatin on Ang II-induced early signaling events in PVEC. Moreover, pretreatment of PVEC with GGTI-286 (25), a cell-permeable and selective geranylgeranyltransferase-I inhibitor, blocked Ang II- and calcium ionophore A23187-induced Pyk2 tyrosine phosphorylation. Thus, geranylgeranylation of a signal molecule(s) downstream of intracellular calcium mobilization and PKC is required for the calcium-dependent activation of Pyk2 by Ang II in PVEC.

Small G proteins including Rap, Ral, Rho, Rac, and Rab are post-translationally modified by geranylgeranylation, which is necessary for their membrane localization and function (15). Geranylgeranylation of RhoA is required for its correct subcellular localization and for interaction with its GDP/GTP cycle regulators, the guanine nucleotide dissociation inhibitor, and guanine nucleotide exchange factor (34, 35). Rab GTPase is known to regulate intracellular vesicular membrane transport and membrane fusion (36). The activation of Rac1 by Ang II (9) and RhoA by Galpha 13 (37) is downstream of Pyk2. Ral is a downstream element in Ras-mediated signaling (38). Rap1 and Pyk2 are both activated rapidly in calcium- and PKC-dependent manners in response to various agonists (4-7, 28, 29). Rap1 is activated when it is translocated to plasma membrane. The translocation is regulated by Rap1 geranylgeranylation (15, 38). Geranylgeranylation of Rap1 could promote Rap1 association with cellular membrane either through lipid-lipid or lipid-protein interactions. We found that Ang II rapidly (30 s) and markedly increased the level of membrane-bound Rap1, and the maximal effect correlated with the maximal activation of Pyk2 by Ang II in PVEC (4). Simvastatin completely prevented Ang II-induced Rap1 membrane translocation. Co-treatment with mevalonate but not FPP reversed the inhibitory effect of simvastatin on Rap1 membrane translocation. Furthermore, GGTI-286 (25), a cell-permeable and selective geranylgeranyltransferase-I inhibitor, also blocked Ang II-induced Rap1 membrane translocation. Thus, our data indicate that Rap1 geranylgeranylation is required for Ang II-induced Rap1 membrane translocation (activation) in PVEC. Small G proteins in an activated GTP-bound form function through binding to effector proteins. For example, Ras binds to and activates its effectors such as serine-threonine kinase Raf-1 and phosphatidylinositol 3-kinase. Interaction of GTP-bound Ras with Raf-1 induces Raf-1 translocation to the plasma membrane and a conformational change, resulting in Raf-1 activation (39). Ras directly activates phosphatidylinositol 3-kinase by binding to the p110 catalytic subunit of phosphatidylinositol 3-kinase (40). In the present study, we found for the first time that Ang II and the calcium ionophore A23187 induced a rapid (30 s) association of Rap1 with Pyk2 in PVEC, and these effects were blocked by the HMG-CoA reductase inhibitor simvastatin. In contrast, Ang II did not induce Pyk2 association with RhoA or Rac1 even though low basal level association of Pyk2 with RhoA or Rac1 was recognizable. In addition, the exoenzyme C3 (30), which specifically inactivates RhoA through ADP-ribosylation of RhoA at Asn41, did not affect Ang II-induced activation of Pyk2 in PVEC. These results strongly indicate that the small G protein Rap1 is an intermediary signaling molecule linking calcium signal to Pyk2 activation by Ang II in PVEC. Pyk2 is related to focal adhesion kinase and is found along actin microfilament-like structures that extended into focal adhesions (41). Subcellular localization of Pyk2 is determined by its carboxyl-terminal domain (42). We recently reported that Pyk2 was detected in membrane and cytosolic fractions in PVEC (4). We found that Ang II did not induce Pyk2 membrane translocation (data not shown). Zheng et al. (42) also report that Ang II did not alter Pyk2 localization. These data suggest that geranylgeranylated Rap1 may only interact with and activate the membrane-associated Pyk2 in response to Ang II. Rap1 may directly interact with Pyk2 when it is geranylgeranylated and translocated to membrane. Alternatively, Rap1 may interact with membrane-associated Pyk2 through its guanine nucleotide exchange factor C3G since Pyk2/p130Cas can form complexes with C3G/Crk in response to various agonists (43, 44)

The second finding obtained from this study is that Rap1 may function in a signaling pathway distinct from Ras through binding to its unique effectors such as Pyk2. Ras and Rap1 are related small GTPases. They are characterized by similarities in the effector domain (38, 45). Previous studies suggest that Rap1 may function as an antagonist of Ras signaling by trapping Ras effectors, in particular Raf-1 (38, 45). However, a different view for the function of Rap1 has been proposed recently. Rap1 may function in a signaling pathway unrelated to Ras despite using similar or different effectors. Rap1 has been shown to be activated by bombesin in the absence of Ras activation and transduces signal to downstream ERK activation in NIH 3T3 cells (29). Rap1 interacts with B-raf, a close relative of Raf-1, and activates the kinase in PC12 cells (46). Our data suggest that Pyk2 may be one of the Rap1-GTPase effectors in PVEC.

Finally, our data indicate that the cholesterol-lowering drugs, statins, could interrupt the renin-angiotensin system by inhibiting AT1 receptor signaling (Pyk2 and ERK) independently of cholesterol lowering in endothelial cells. Hypercholesterolemia, which induces vascular endothelial dysfunction, is one of the major risk factors for the development of atherosclerosis and coronary heart disease (47, 48). Large scale clinical trials have shown that HMG-CoA reductase inhibitors (statins) improve clinical outcomes in patients with atherosclerosis and coronary artery diseases, exceeding beneficial effects expected from plasma cholesterol lowering alone (49-51). However, the mechanisms underlying these beneficial effects are not clear. Ang II binds to endothelial cell receptors and stimulates the expression of plasminogen activator inhibitor-1 and cell adhesion molecules, which are risk factors for the development of atherosclerosis and myocardial infarction (2, 3). We found that statins in clinical use, including simvastatin, lovastatin, atorvastatin, and fluvastatin, all blocked Ang II-induced Pyk2 activation in PVEC. Simvastatin also attenuated downstream ERK activation by Ang II and calcium ionophore in PVEC. Since Pyk2 is a key tyrosine kinase in the early events of endothelial AT1 receptor signaling and is a positive regulator of down-stream ERK and c-Jun NH2-terminal kinase activation evoked by Ang II in various cell types including endothelial cells (4, 8, 9), blockade of the endothelial AT1 receptor signaling pathways by statins could result in an improvement of endothelial function and may, at least in part, explain the beneficial impact of HMG-CoA inhibitors (statins) on clinical outcomes.

In summary, we have obtained substantial evidence indicating that the small G protein Rap1 is an intermediary molecule linking a calcium signal to Pyk2 activation by Ang II in PVEC. Pyk2 may be one of the Rap1-GTPase effectors in PVEC.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HL-58205.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ On leave of absence from the Dept. of Pharmacology, Hokkaido College of Pharmacy, Otaru 047-0264 Japan.

** To whom correspondence should be addressed. Present address: Dept. of Biochemistry, The University of Texas Health Center at Tyler, Tyler, TX 75708. Tel.: 903-877-7938; Fax: 903-877-7558; E-mail: Tang37232@yahoo.com.

Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M009165200

    ABBREVIATIONS

The abbreviations used are: Ang II, angiotensin II; AT1, angiotensin II type-1 receptor; HMG-CoA, 3-hydroxy-3-methyglutaryl-CoA; FPP, farnesylpyrophosphate; GGPP, geranylgeranylpyrophosphate; ERK, extracellular signal-regulated kinase; PVEC, pulmonary vein endothelial cell; PMA, phorbol-12-myristate-13-acetate; PKC, protein kinase C.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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