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• W2091263181 abstract "The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase β. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKα1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKα1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKα1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK → Rac1 → Akt axis in the control of eNOS in endothelial cells. The endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis, is a calcium/calmodulin-dependent phosphoprotein regulated by diverse cell surface receptors. Vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) stimulate eNOS activity through Akt/phosphoinositide 3-kinase and calcium-dependent pathways. AMP-activated protein kinase (AMPK) also activates eNOS in endothelial cells; however, the molecular mechanisms linking agonist-mediated AMPK regulation with eNOS activation remain incompletely understood. We studied the role of AMPK in VEGF- and S1P-mediated eNOS activation and found that both agonists led to a striking increase in AMPK phosphorylation in pathways involving the calcium/calmodulin-dependent protein kinase kinase β. Treatment with tyrosine kinase inhibitors or the phosphoinositide 3-kinase inhibitor wortmannin demonstrated differential effects of VEGF versus S1P. Small interfering RNA (siRNA)-mediated knockdown of AMPKα1or Akt1 impaired the stimulatory effects of both VEGF and S1P on eNOS activation. AMPKα1 knockdown impaired agonist-mediated Akt phosphorylation, whereas Akt1 knockdown did not affect AMPK activation, thus suggesting that AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation. Importantly, we found that siRNA-mediated knockdown of AMPKα1 abrogates agonist-mediated activation of the small GTPase Rac1. Conversely, siRNA-mediated knockdown of Rac1 decreased the agonist-mediated phosphorylation of AMPK substrates without affecting that of AMPK, implicating Rac1 as a molecular link between AMPK and Akt in agonist-mediated eNOS activation. Finally, siRNA-mediated knockdown of caveolin-1 significantly enhanced AMPK phosphorylation, suggesting that AMPK is negatively regulated by caveolin-1. Taken together, these results suggest that VEGF and S1P differentially regulate AMPK and establish a central role for an agonist-modulated AMPK → Rac1 → Akt axis in the control of eNOS in endothelial cells. The AMP-activated protein kinase (AMPK) 2The abbreviations used are: AMPK, 5′-AMP-activated protein kinase; ACC, acetyl-CoA carboxylase; BAEC, bovine aortic endothelial cell(s); eNOS, endothelial nitric-oxide synthase; CaMKKβ, calcium/calmodulin-dependent protein kinase kinase β; siRNA, small interfering RNA; PI3K, phosphoinositide 3-kinase; GSK3-β, glycogen synthase kinase-3-β; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2; S1P, sphingosine 1-phosphate; ERK, extracellular signal-regulated kinase; FOV, field(s) of view; ANOVA, analysis of variance. is an evolutionarily conserved serine/threonine heterotrimeric kinase that was initially characterized as a “fuel gauge” modulating cellular energy flux in eukaryotic cells in response to changes in intracellular AMP levels (for a review, see Ref. 1Hardie D.J. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar). More recent studies have identified a broader role for AMPK in cellular homeostasis and signaling; AMPK is now known to be regulated by a family of upstream AMPK kinases, including the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) (2Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar, 3Hawley S.A. Pan D.A. Mustard K.J. Ross L. Bain J. Edelman A.M. Frenguelli B.G. Hardie D.G. Cell Metab. 2005; 2: 9-19Abstract Full Text Full Text PDF PubMed Scopus (1290) Google Scholar), and the tumor suppressor kinase LKB1 (4Shaw R.J. Kosmatka M. Bardeesy N. Hurley R.L. Witters L.A. DePinho R.A. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3329-3335Crossref PubMed Scopus (1453) Google Scholar). After AMPK undergoes phosphorylation at the threonine 172 site in the activation loop of its catalytic α-subunit, the kinase is activated (1Hardie D.J. Carling D. Eur. J. Biochem. 1997; 246: 259-273Crossref PubMed Scopus (1147) Google Scholar) and can mediate numerous energy-conserving cellular processes, such as promotion of glucose uptake and glycolysis (5Sambandam N. Lopaschuk G.D. Prog. Lipid Res. 2003; 42: 238-256Crossref PubMed Scopus (143) Google Scholar), acceleration of mitochondrial biogenesis (6Reznick R.M. Shulman G.I. J. Physiol. (Lond.). 2006; 574: 33-39Crossref Scopus (291) Google Scholar), and stimulation of fatty acid oxidation with concomitant inhibition of fatty acid synthesis via phosphorylation and inactivation of acetyl-CoA carboxylase (ACC) (7Carling D. Clarke P.R. Zammit V.A. Hardie D.G. Eur. J. Biochem. 1989; 186: 129-136Crossref PubMed Scopus (344) Google Scholar). In addition to phosphorylating ACC, AMPK also has been shown to phosphorylate the endothelial isoform of nitric-oxide synthase (eNOS) on serine 1179 in endothelial cells and cardiac myocytes (8Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (720) Google Scholar). AMPK appears to be involved in the pathways of eNOS activation evoked by a variety of extracellular stimuli that modulate eNOS in endothelial cells, including metformin (9Zou M.H. Kirkpatrick S.S. Davis B.J. Nelson J.S. Wiles 4th, W.G. Schlattner U. Neumann D. Brownlee M. Freeman M.B. Goldman M.H. J. Biol. Chem. 2004; 279: 43940-43951Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar), adiponectin (10Yamauchi T. Kamon J. Minokoshi Y. Ito Y. Waki H. Uchida S. Yamashita S. Noda M. Kita S. Ueki K. Eto K. Akanuma Y. Froguel P. Foufelle F. Ferre P. Carling D. Kimura S. Nagai R. Kahn B.B. Kadowaki T. Nat. Med. 2002; 8: 1288-1295Crossref PubMed Scopus (3483) Google Scholar, 11Chen H. Montagnani M. Funahashi T. Shimomura I. Quon M.J. J. Biol. Chem. 2003; 278: 45021-45026Abstract Full Text Full Text PDF PubMed Scopus (888) Google Scholar), hypoxia (12Nagata D. Mogi M. Walsh K. J. Biol. Chem. 2003; 278: 31000-31006Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and hydroxymethylglutaryl-CoA reductase inhibitors (13Sun W. Lee T.S. Zhu M. Gu C. Wang Y. Zhu Y. Shyy J.Y. Circulation. 2006; 114: 2655-2662Crossref PubMed Scopus (218) Google Scholar). The activation of eNOS by AMPK has been implicated in many of the bioenergetic (14Quintero M. Colombo S.L. Godfrey A. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5379-5384Crossref PubMed Scopus (285) Google Scholar), angiogenic (12Nagata D. Mogi M. Walsh K. J. Biol. Chem. 2003; 278: 31000-31006Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and anti-inflammatory effects of AMPK in endothelial cells. Despite the accumulating evidence linking AMPK with eNOS in the vascular endothelium, the molecular pathways involved in AMPK-mediated eNOS activation remain incompletely characterized. eNOS is also activated by the protein kinase Akt, a phosphoinositide 3-kinase (PI3K)-dependent effector that plays critical roles in numerous cellular responses, including angiogenesis and endothelial cell survival (15Fulton D. Gratton J-P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). A number of recent reports have studied both AMPK and Akt in the context of eNOS activation or nitric oxide release, but the relative role of these protein kinases remains controversial, with evidence for (16Ouchi N. Kobayashi H. Kihara S. Kumada M. Sato K. Inoue T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 1304-1309Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar) and against (17Morrow V.A. Foufelle F. Connell J.M. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) an AMPK/Akt interaction upstream of eNOS. In addition to the uncertain relationship between AMPK and Akt, the eNOS-activating agonists that lead to AMPK phosphorylation are incompletely characterized, as are the pathways that connect these different phosphorylation pathways to eNOS activation and endothelial functional responses, including migration and tube formation. It remains unclear whether vascular endothelial growth factor (VEGF), an angiogenic polypeptide growth factor and potent eNOS agonist, promotes AMPK phosphorylation under normoxic conditions (12Nagata D. Mogi M. Walsh K. J. Biol. Chem. 2003; 278: 31000-31006Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and a recent study (18Reihill J.A. Ewart M. Hardie D.G. Salt I.P. Biochem. Biophys. Res. Commun. 2007; 354: 1084-1088Crossref PubMed Scopus (80) Google Scholar) that implicated AMPK in VEGF-mediated eNOS activation did not define the mechanisms involved in this response. Akt and eNOS are also potently activated by the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) (19Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), but the phosphorylation of AMPK by S1P and the possible involvement of AMPK in S1P-mediated eNOS activation and cell motility have not been previously described. Both VEGF and S1P act in part by stimulating an influx of calcium into the endothelium (19Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 20Dudzinski D.M. Igarashi J. Greif D. Michel T. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 235-276Crossref PubMed Scopus (319) Google Scholar), thereby providing a route for AMPK activation that may depend upon CaMKK. The differential phosphorylation of AMPK by both VEGF and S1P therefore represents a plausible mechanism of eNOS regulation in endothelial cells. Caveolae are plasmalemmal microdomains originally identified on the surface of endothelial and epithelial cells (21Yamada E. J. Biophys. Biochem. Cytol. 1955; 1: 445-458Crossref PubMed Scopus (527) Google Scholar). The scaffolding/regulatory protein of caveolae, caveolin-1 (22Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1873) Google Scholar, 23Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar), is known to interact with and modulate the function of eNOS in endothelial cells (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). We have demonstrated that caveolin-1 negatively regulates the small GTPase Rac1 (25Kinsella B.T. Erdman R.A. Maltese W.A. J. Biol. Chem. 1991; 266: 9786-9794Abstract Full Text PDF PubMed Google Scholar, 26Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar), which in turn modulates the PI3K/Akt/eNOS pathway and regulates migration in endothelial cells (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Gonzalez E. Kou R. Michel T. J. Biol. Chem. 2006; 281: 3210-3216Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). It is therefore possible that AMPK regulates agonist-mediated eNOS activation and endothelial migration by interacting with caveolin-1 or Rac1 in endothelial cells. In the present study, we provide evidence that the eNOS agonists VEGF and S1P differentially promote the phosphorylation of AMPK in vascular endothelial cells in distinct receptor-modulated pathways that involve tyrosine kinases and caveolin-1. Using pharmacological approaches as well as siRNA-mediated protein knockdown methodologies, we elucidate the molecular mechanisms of eNOS activation downstream of AMPK and identify a novel AMPK-Rac1-Akt pathway that functions as a critical determinant of eNOS activity as well as endothelial cell migration and tube formation in the vascular endothelium. Materials—Fetal bovine serum was purchased from Hyclone (Logan, CT); all other cell culture reagents, media, and Lipofectamine 2000 transfection reagent were from Invitrogen. S1P and PP2 were from BioMol (Plymouth Meeting, PA). VEGF, genistein, wortmannin, cyclosporin, SB203580, STO-609, and Compound C were from Calbiochem. Polyclonal antibodies directed against phospho-AMPK (Thr172), AMPK, phospho-ACC (Ser79), ACC, phospho-eNOS (Ser1179), phospho-Akt (Ser473), Akt, phospho-GSK3-β (Ser9), phospho-ERK1/2 (Thr202/Tyr204), and ERK1/2 were from Cell Signaling Technologies (Beverly, MA). Polyclonal Akt1 antibody was from Chemicon. eNOS monoclonal antibody, glycogen synthase kinase-3-β (GSK3-β) monoclonal antibody, and polyclonal caveolin-1 antibody were from BD Transduction Laboratories (Lexington, KY). The monoclonal antibody for VEGFR2 and polyclonal antibody specific for the β isoform of CaMKK were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rac1 monoclonal antibody and Rac activation assay kit were from Upstate Biotechnology, Inc. (Temecula, CA). Super Signal substrate for chemiluminescence detection and secondary antibodies conjugated with horseradish peroxidase were from Pierce. Tris-buffered saline and phosphate-buffered saline were from Boston Bioproducts (Ashland, MA). Other reagents were from Sigma. Cell Culture—Bovine aortic endothelial cells (BAEC) were obtained from Cell Systems (Kirkland, WA) and maintained in culture in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (10%, v/v) as described previously (19Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Cells were plated onto 0.2% gelatin-coated culture dishes and studied prior to cell confluence between passages 5 and 9. siRNA Design and Transfection—Our siRNA duplexes were designed on the basis of established characteristics of siRNA targeting constructs (28Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8186) Google Scholar). All experimental oligonucleotides were purchased from Ambion (Austin, TX). We designed an AMPKα1 siRNA corresponding to bases 234–252 from the open reading frame of bovine AMPKα1: 5′-CCU CAA GCU UUU CAG GCA UdTdT-3′ (Ensembl Transcript ID: ENSBTAT00000000016). We also designed an Akt1 siRNA corresponding to bases 1325–1343 from the open reading frame of bovine Akt1 (5′-GGA CGU GUA CGA GAA GAA GdTdT-3′; Ensembl Transcript ID: ENSBTAG00000017636), a CaMKKβ siRNA from bases 585–603 of the open reading frame of bovine CaMKKβ (5′-GGU GCU GUC CAA AAA GAA AdTdT-3′; Ensembl Transcript ID: ENSBTAG00000010815), and an eNOS siRNA from bases 3948–3966 of the open reading frame of bovine eNOS (5′-CCU GAU CUC UAA AUC AUU CdTdT-3′; Ensembl Transcript ID: ENSBTAT00000007246). siRNA constructs targeting VEGFR2 (32Kou R. SenBanerjee S. Jain M.K. Michel T. Biochemistry. 2005; 44: 15064-15073Crossref PubMed Scopus (27) Google Scholar), Rac1 (27Gonzalez E. Kou R. Michel T. J. Biol. Chem. 2006; 281: 3210-3216Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), and caveolin-1 (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) have been described previously. A nonspecific control siRNA from Dharmacon (Lafayette, CO) was used as a negative control (5′-AUU GUA UGC GAU CGC AGA CdTdT-3′). BAEC were transfected with siRNA as described previously (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and analyzed 48 h after transfection. Drug Treatment and Immunoblotting—12–16 h prior to cell treatments, culture medium was changed to serum-free medium. VEGF and S1P were prepared as previously reported (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Genistein, PP2, cyclosporin A, SB203580, wortmannin, STO-609, and Compound C were solubilized in Me2SO and kept at –20 °C; where indicated, 0.1% (v/v) Me2SO was used as the vehicle control. After drug treatments, BAEC were washed with phosphate-buffered saline and incubated on ice for 20 min in lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA, 2 mm Na3VO4, 1 mm NaF, 2 μg/ml leupeptin, 2 μg/ml antipain, 2 μg/ml soybean trypsin inhibitor, and 2 μg/ml lima trypsin inhibitor). Cells were harvested by scraping and then centrifuged for 5 min at 4 °C. For immunoblot analyses, 20 μg of cellular protein was resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies using protocols provided by the suppliers. Densitometric analyses of the Western blots were performed using a ChemiImager 4000 (Alpha-Innotech). When indicated, for the experiments showing densitometry of Western blots, the ordinate is in arbitrary units. NOS Activity Assay—eNOS activity was quantified as the formation of l-[3H] citrulline from l-[3H] arginine in cultured BAEC, as described previously in detail (19Igarashi J. Bernier S.G. Michel T. J. Biol. Chem. 2001; 276: 12420-12426Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 29Balligand J.L. Kobzik L. Han X. Kaye D.M. Belhassen L. O'Hara D.S. Kelly R.A. Smith T.W. Michel T. J. Biol. Chem. 1995; 270: 14582-14586Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Briefly, reactions were initiated by adding l-[3H]arginine (10 μCi/ml) plus VEGF or S1P, as described below. Each treatment was performed in duplicate cultures, which were then each analyzed in duplicate. The flow-through fraction was analyzed by liquid scintillation counting, and NOS activity was quantitated based on l-[3H]citrulline formation in the cells; the values were expressed as fmol of l-[3H]citrulline produced/well/min. Rac1 Activity Assay—Transfected BAEC in 100-mm dishes were stimulated with VEGF or S1P, and cells were then washed with ice-cold Tris-buffered saline and lysed in lysis buffer (25 mm HEPES, 150 mm NaCl, 1% Nonidet P-40, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, 2 mm Na3VO4, 1 mm NaF). Pull-down of GTP-bound Rac was performed by incubating the cell lysates with glutathione S-transferase fusion protein corresponding to the p21-binding domain of p21-activated kinase-1 bound to glutathione-agarose (Upstate Biotechnology) for 1 h at 4 °C following the instructions provided by the suppliers. The beads were washed three times for 10 min each with lysis buffer, and the protein bound to the beads was eluted with 2× Laemmli buffer and analyzed for the amount of GTP-bound Rac by immunoblotting using a Rac monoclonal antibody. Migration Assay—Cell migration was assayed using a Transwell cell culture chamber containing polycarbonate membrane inserts with an 8-μm pore (Corning Costar Corp.) coated with 0.2% gelatin (24Gonzalez E. Nagiel A. Lin A.J. Golan D.E. Michel T. J. Biol. Chem. 2004; 279: 40659-40669Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). 48 h after transfection in 6-well plates, the cells were trypsinized, and 5 × 104 cells in 100 μl of Dulbecco's modified Eagle's medium, 0.4% fetal bovine serum were added to the upper Transwell chamber. The bottom chamber was filled with 600 μl of media, and the cells were allowed to adhere to the membrane at 37 °C for 1 h. VEGF (10 ng/ml), S1P (100 nm), or vehicle was added to the lower chamber, and the assembly was incubated at 37 °C for 3 h to allow cell migration. After incubation, the membranes were washed with phosphate-buffered saline, and the cells that did not migrate through the membrane were gently removed from the upper surface with a cotton swab. The membranes were then treated with trypsin to detach the migrated cells from the lower surface, and these cells were then counted with a hemocytometer. Tube Formation Assay—250 μl of Matrigel (BD Biosciences) was deposited into wells in a 24-well plate and allowed to solidify for 30 min at 37 °C. 48 h after siRNA transfection, BAEC were trypsinized, and 3 × 104 cells were added to each Matrigel-coated well. Cells were incubated on Matrigel for 9 h at 37 °C and imaged by phase-contrast microscopy (Nikon Eclipse TS100, ×5 objective). Four random fields of view (FOV)/well were examined and photographed by a blinded observer. For quantification purposes, a node was defined as an aggregation of cells from which three or more tubelike structures originated, and a tube referred to a continuous stretch of at least two cells containing no more than two nodes. For each FOV, ImageJ (National Institutes of Health) was used to measure the total tube length and the length per tube in units of pixels. Each experiment was repeated in nine wells. Statistical Analysis—All experiments were performed at least three times. Mean values for individual experiments were expressed as means ± S.E. Statistical differences were assessed by ANOVA or t test when appropriate. A p value of less than 0.05 was considered significant. VEGF- and S1P-mediated AMPK and ACC Phosphorylation—We first studied the effects of VEGF and S1P on the phosphorylation of AMPK and the AMPK substrate ACC in BAEC (Fig. 1). After the addition either of VEGF (10 ng/ml) or S1P (100 nm) to BAEC, AMPK phosphorylation increased within 1 min of agonist addition, reaching a maximum ∼2.5-fold increase by 5 min, with a gradual return to basal levels at ∼30 min following VEGF or S1P addition (Fig. 1, A and B). Immunoblots probed with a phospho-specific ACC antibody revealed a similar time course for ACC phosphorylation, reaching a peak ∼3-fold increase in response to both agonists that gradually returned to base line after ∼30 min following the addition of VEGF or S1P. We next analyzed the dose response to VEGF and S1P for both AMPK and ACC phosphorylation. Fig. 1C shows immunoblots of BAEC lysates from cells treated for 5 min with increasing concentrations of VEGF or S1P and probed with antibodies directed against phospho-AMPK and phospho-ACC; total AMPK and ACC levels serve as the control in this immunoblot analysis. The dose response for VEGF-induced AMPK and ACC phosphorylation demonstrated an EC50 of ∼1 ng/ml, and S1P-induced phosphorylation of both targets showed an EC50 of ∼20 nm; these values fall within the physiological range seen for many other endothelial responses for VEGF and S1P (20Dudzinski D.M. Igarashi J. Greif D. Michel T. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 235-276Crossref PubMed Scopus (319) Google Scholar). Together, these data indicate that both VEGF and S1P induce the reversible receptor-mediated phosphorylation of AMPK and its substrate ACC in endothelial cells. siRNA-mediated Down-regulation of VEGFR2 and Inhibition of Tyrosine Kinases and PI3K in VEGF- and S1P-mediated AMPK and ACC Phosphorylation—Previous work from our laboratory (30Igarashi J. Erwin P.A. Dantas A.P. Chen H. Michel T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10664-10669Crossref PubMed Scopus (173) Google Scholar) and others (31Tanimoto T. Jin Z.G. Berk B.C. J. Biol. Chem. 2002; 277: 42997-43001Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) have observed cross-talk between VEGF and S1P receptors in mediating intracellular endothelial responses. Indeed, it has been previously reported that S1P responses are mediated by VEGF receptor transactivation (31Tanimoto T. Jin Z.G. Berk B.C. J. Biol. Chem. 2002; 277: 42997-43001Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). We explored the possibility of S1P-mediated transactivation of the VEGF receptor 2 (VEGFR2) in promoting AMPK and ACC phosphorylation by using siRNA directed against VEGFR2. BAEC were transfected with duplex siRNAs specific for VEGFR2 (32Kou R. SenBanerjee S. Jain M.K. Michel T. Biochemistry. 2005; 44: 15064-15073Crossref PubMed Scopus (27) Google Scholar), and the effects on siRNA-mediated VEGFR2 knockdown on VEGF- and S1P-induced AMPK and ACC phosphorylation were analyzed (Fig. 2A). VEGFR2 siRNA effectively abrogated AMPK and ACC phosphorylation in response to VEGF, but this siRNA did not affect phosphorylation in response to S1P. These findings indicate that VEGF, but not S1P, acts through the VEGFR2 to activate AMPK and ACC phosphorylation and argue against S1P-mediated VEGFR2 transactivation as an essential component of S1P-mediated AMPK activation. We next used a series of pharmacological inhibitors to assess the role of tyrosine kinases and PI3K in VEGF- and S1P-mediated activation of AMPK and ACC. BAEC were stimulated with agonists following pretreatment with the broad spectrum tyrosine kinase inhibitor genistein, with the Src tyrosine kinase inhibitor PP2, with the PI3K inhibitor wortmannin, with the calcineurin inhibitor cyclosporin, or with the vehicle as a control (Fig. 2, B–D). We found that genistein and PP2 blocked VEGF-induced AMPK and ACC phosphorylation but did not affect S1P-induced AMPK and ACC phosphorylation. In contrast, pretreatment of BAEC with the PI3K inhibitor wortmannin did not affect AMPK or ACC phosphorylation in response to VEGF (data not shown), but wortmannin pretreatment significantly increased phosphorylation of both proteins in response to S1P (42.4 ± 18% increase compared with vehicle-pretreated, n = 3, p < 0.05) (Fig. 2, C and D). We found that the p38 inhibitor SB203580 had no effect in either VEGF- or S1P-induced AMPK or ACC phosphorylation, with positive controls affirming that p38 phosphorylation was partially blocked under these conditions (Fig. 2C) (33Kang Y.J. Seit-Nebi A. Davis R.J. Han J. J. Biol. Chem. 2006; 281: 26225-26234Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Effects of CaMKK Inhibition on VEGF- and S1P-mediated AMPK Phosphorylation Pathways—Both VEGF and S1P increase intracellular calcium in endothelial cells (20Dudzinski D.M. Igarashi J. Greif D. Michel T. Annu. Rev. Pharmacol. Toxicol. 2006; 46: 235-276Crossref PubMed Scopus (319) Google Scholar), and we sought to characterize a possible calcium-dependent upstream kinase responsible for both VEGF- and S1P-induced AMPK phosphorylation. BAEC incubated with the specific CaMKK inhibitor STO-609 (34Tokumitsu H. Inuzuka H. Ishikawa Y. Ikeda M. Saji I. Kobayashi R. J. Biol. Chem. 2002; 277: 15813-15818Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 35Tokumitsu H. Inuzuka H. Ishikawa Y. Kobayashi R. J. Biol. Chem. 2003; 278: 10908-10913Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) did not demonstrate agonist-stimulated AMPK and ACC phosphorylation in response to either VEGF or S1P (Fig. 3A). We next designed and validated siRNA targeting the bovine β isoform of CaMKK; transfection with this siRNA severely impaired both VEGF- and S1P-induced AMPK and ACC phosphorylation (Fig. 3B). These findings suggest that CaMKK may link VEGF- and S1P-activated pathways upstream of AMPK phosphorylation. siRNA-mediated Down-regulation of AMPK Impairs Agonist-mediated eNOS and Akt Activation—We next used siRNA approaches to explore the role of AMPK in VEGF- and S1P-mediated eNOS and Akt activation in BAEC. Pretreatment of endothelial cells with the potent and selective AMPK inhibitor Compound C (36Zhou G. Myers R. Li Y. Chen Y. Shen X. Fenyk-Melody J. Wu M. Ventre J. Doebber T. Fujii N. Musi N. Hirshman M.F. Goodyear L.J. Moller D.E. J. Clin. Invest. 2001; 108: 1167-1174Crossref PubMed Scopus (4472) Google Scholar) inhibited ACC phosphorylation in response to both agonists and also attenuated VEGF- and S1P-induced eNOS phosphorylation at i" @default.
• W2091263181 created "2016-06-24" @default.
• W2091263181 creator A5042325107 @default.
• W2091263181 creator A5069330205 @default.
• W2091263181 creator A5084285813 @default.
• W2091263181 date "2007-07-01" @default.
• W2091263181 modified "2023-10-17" @default.
• W2091263181 title "Agonist-modulated Regulation of AMP-activated Protein Kinase (AMPK) in Endothelial Cells" @default.
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