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. Author manuscript; available in PMC: 2012 Jun 26.
Published in final edited form as: J Biol Chem. 2007 Jun 6;282(32):23679–23686. doi: 10.1074/jbc.M700563200

The Effects of mTOR-Akt Interactions on Anti-apoptotic Signaling in Vascular Endothelial Cells*

Olivier Dormond ‡,§,1, Joren C Madsen §, David M Briscoe ‡,2
PMCID: PMC3383050  NIHMSID: NIHMS312680  PMID: 17553806

Abstract

Recent studies have determined that mTOR mediates the activation of the protein kinase Akt in several cell types, but little is known about the association between mTOR and Akt in vascular endothelial cells. Furthermore, the functional significance of mTOR/Akt signaling has not been characterized in the endothelium. In these studies we treated endothelial cells with the mTOR inhibitor rapamycin, and we found that it decreases Akt phosphorylation and activity, as determined by phosphorylation of its substrate glycogen synthase kinase-3. This effect of rapamycin on Akt phosphorylation could not be demonstrated in endothelial cells transfected with a rapamycin-resistant mTOR construct. Also, in the presence of rapamycin, vascular endothelial growth factor, tumor necrosis factor, and insulin failed to phosphorylate Akt, further indicating that mTOR regulates Akt activation in endothelial cells. The activation of Akt is well established to mediate pro-survival signals. In part this is mediated via the phosphorylation and inactivation of the proapoptotic Akt substrates Foxo1 and Foxo3a. We find that rapamycin totally blocks vascular endothelial growth factor and Akt-inducible phosophorylation of these transcription factors in endothelial cells. Furthermore, inhibition of Akt activity by rapamycin increased the number of endothelial cells undergoing apoptosis after serum withdrawal as well as after stimulation by vascular endothelial growth factor or tumor necrosis factor. Taken together these observations demonstrate first, that mTOR regulates the phosphorylation and activation of Akt in endothelial cells and, second, that a major effect of mTOR inhibition in endothelial cells is to suppress Akt-inducible pro-survival signals.

Growth factors that are essential for angiogenesis, defined as the formation of new blood vessels from preexisting ones, induce protective genes in endothelial cells (EC)3 (1). Analysis of the signaling pathways that facilitate growth factor-mediated EC survival have demonstrated a critical function for the serine/threonine kinase Akt in this response (2). For example, vascular endothelial growth factor (VEGF), insulin, and ligation of the Tie2 receptor by angiopoietin-1, which all promote angiogenesis, induce Akt-dependent signals (3–5). Akt is evolutionarily conserved and in mammalian cells consists of three highly homologous isoforms that share more than 80% of their amino acid sequence. Moreover, intracellular signals leading to Akt activation are conserved across species (6, 7). The binding of cytokines and growth factors to vascular EC increases phosphatidylinositol 3-kinase activity, resulting in the production of phosphatidylinositol 3,4,5-triphosphates and the recruitment and activation of phosphoinositide-dependent kinase 1 within the cell membrane. Akt is phosphorylated by phosphoinositide-dependent kinase 1 (PDK1) at a threonine residue (Thr-308) in the activation loop and by another putative PDK2 kinase at a serine residue (Ser-473) in the carboxyl-terminal domain. Although several candidates have been proposed to function as the putative phosphoinositide-dependent kinase 2 (PDK2) kinase, including PDK1, integrin-linked kinase 1, ataxia telangiectasia mutant, and DNA-dependent protein kinase, recent studies have demonstrated that the rapamycin-insensitive Sin-1·Rictor·mTOR complex functions as PDK2 (8–10).

Upon activation Akt mediates pro-survival and anti-apoptosis in part via the phosphorylation and inhibition of the Bcl-2 homolog BAD, the phosphorylation and inactivation of the FoxO subfamily of forkhead transcription factors, and the transcriptional co-activator Yes-associated protein (11–13). In addition, Akt mediates cell proliferation and migration in part via the phosphorylation and activation of the endothelial nitric-oxide synthase and glycogen synthase kinase-3β known to be functional in cell cycle progression as well as via the inactivation of p21 and p27 (14–18).

Rapamycin is a well established immunosuppressive agent that has recently been found to possess anti-angiogenic activities (19, 20). Studies of its mechanism of action have shown that when bound to its intracellular receptor, the immunophilin FK506-binding protein-12, rapamycin interacts and inhibits the formation of a complex composed of mTOR, raptor, and mLST8 (called mTORC1) (21). Inhibition of mTORC1 results in the hypophosphorylation of p70 S6 kinase and 4E-BP1, which are involved in the control of the translation initiation, ribosome biogenesis, and other growth and proliferation events (22, 23). Another mTOR complex consisting of mTOR, rictor, Sin1, and mLST8 (called mTORC2) has been found to regulate Akt activity and actin polymerization (8, 10, 24, 25). Although mTORC2 is rapamycin-insensitive, in several studies it has been found that prolonged exposure to rapamycin may inhibit mTORC2 function by blocking the assembly of the newly synthesized complex within the cell (26). Thus, depletion of available intracellular mTOR by rapamycin may biologically result in an inhibition of mTORC2 and, thus, inhibition of phosphorylation and activation of Akt.

In this study we have evaluated mTOR-Akt interactions and signaling pathways in EC using rapamycin to regulate mTOR activity. We find that a major biological effect of rapamycin is to inhibit Akt phosphorylation and Akt-induced anti-apoptotic signaling. Moreover, we find that the ability of rapamycin to inhibit the migration and proliferation of EC is independent of its effects on mTORC2 activity.

MATERIALS AND METHODS

Reagents and Plasmids

Rapamycin was gifted to the laboratory by Wyeth-Ayerst Research (Princeton, NJ). Antibodies used for Western blot were anti-phospho-Ser-473 Akt, anti-phospho-Thr-308 Akt, anti-phospho-Ser-256 Foxo1, anti-phospho-Ser-253 Foxo3a, anti-phospho 4E-BP1 (Ser-65), anti Foxo1, anti Foxo3a, anti-4E-BP1, and anti-mTOR all purchased from Cell Signaling Technologies (Beverly, MA). Anti-Rictor antibody was purchased from Bethyl Laboratories (Montgomery, TX), and anti-Akt was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rapamycin-resistant mTOR plasmid (RRmTOR) was kindly gifted to the laboratory by Robert Abraham (27), and the wild type myr-Akt plasmid (WT Akt) was obtained from D. Mukhopadhyay (Mayo Clinic, Rochester, MN). The dominant active mutant of Akt (S473D/T308D) (2DART) was obtained by directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with Akt as a backbone (William Sellers, Addgene, plasmid 9021, Cambridge, MA) (28). rhuVEGF was purchased from R & D (Minneapolis, MN), insulin was purchased from Sigma (St. Louis, MO), and TNF was a gift from Biogen (Cambridge, MA).

Cell Culture

Human umbilical vein endothelial cells were isolated from umbilical cords as previously described (29) and cultured in M199 (Cambrex, Walkersville, MD) supplemented with 20% fetal bovine serum (Invitrogen), 1% EC growth factor, 100 units/ml penicillin, 100 μg streptomycin, 2 mm l-glutamine. EC were used for the experiments between passages 2 and 5.

Transfection

EC were transfected with the WT Akt, a constitutively active form of the kinase, 2DAkt, and RRmTOR constructs using the Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions and as previously described (30), Transfected cells were rested for an additional 12 h before initiation of the experiment. In all assays we used an empty vector (pcDNA3) as a control for the Akt or mTOR constructs.

Western Blot Analysis

EC were washed one time with phosphate-buffered saline and lysed with ice-cold radioimmune precipitation assay buffer (Boston Bioproducts, Worcester, MA) containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl , 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptine, 10 μg/ml aprotinin, 1 mm sodium orthovanadate, 50 mm NaF, and 10 mm pyrophosphate. Lysates were centrifuged at 4 °C for 15 min, and samples containing 30–50 μg of proteins were separated on polyacrylamide gel with Tris-glycine-SDS running buffer (Bio-Rad) and transferred onto a polyvinylidene difluoride membrane (PerkinElmer Life Sciences) for 1 h. Membranes were blocked with 5% milk in Tris-buffered saline-Tween 20 for 1 h and incubated overnight with the primary antibody. Membranes were washed and incubated with a secondary peroxidase-linked antibody, and the reactive bands were detected by chemiluminescence (Pierce).

Immunoprecipitation

EC were grown in 10-cm cell culture plates, treated with rapamycin or vehicle for 12 h, rinsed in cold phosphate-buffered saline, and lysed in ice-cold lysis buffer (containing 0.3% CHAPS, 40 mm HEPES, pH 7.5, 120 mm NaCl, 1 mm EDTA, 10 μg/ml leupeptine, 10 μg/ml aprotinin, 1 mm sodium orthovanadate). Lysates were centrifuged at 4 °C for 15 min, and supernatants were collected. Immunoprecipitations were performed with 500 μg of total protein incubated with 3 μg of anti-mTOR antibody for 90 min at 4 °C. Immunocomplexes were captured with protein A-Sepharose beads (Amersham Biosciences) and washed four times in CHAPS-containing buffer, resuspended in protein loading buffer containing SDS (Boston Bioproducts), and boiled for 5 min. Finally, immunoprecipitated proteins were separated on a polyacrylamide gel and analyzed by Western blot.

Apoptosis Assay

EC were serum-starved overnight by reducing the concentration of fetal bovine serum to 5%. Subsequently, the cells were treated with rapamycin, TNF or VEGF as indicated. After treatment, floating cells and attached EC were collected and fixed in 70% ethanol overnight. The cells were washed twice in phosphate-buffered saline before incubation in phosphate-buffered saline containing propidium iodide (50 μg/ml) and RNase A (100 μg/ml). Propidium iodide staining was analyzed by fluorescence-activated cell sorter using Cellquest software (BD Biosciences). In some experiments, EC were transfected with an empty vector (pcDNA3), WT Akt, or with 2DAkt as indicated.

Akt Kinase Assay

Akt kinase activity was assessed using an in vitro Akt kinase assay kit (Cell Signaling Technology, Beverly, MA). Briefly, EC were treated with rapamycin for the indicated time period, and cells were collected in lysis buffer according to the manufacturer’s instructions. 200 μg of protein lysate was incubated overnight with the anti-Akt antibody provided in the kit. Captured Akt was incubated with 1 μg of recombinant glycogen synthase kinase-3β in the reaction buffer and run on a polyacrylamide gel, and phosphorylation of glycogen synthase kinase-3 was analyzed by Western blot.

Migration Assay

Migration assays were performed as previously described (31). Briefly, the lower surface of a Transwell filter (8-μm pores, Corning Costar, Nagog Park, MA) was coated with fibronectin (10 mg/ml) and blocked with 1% bovine serum albumin. EC were used untransfected or transfected with an empty vector (pcDNA3) or the 2DAkt construct and were rested in cell culture medium for 12 h (as above). The cells were subsequently untreated or treated with rapamycin for an additional 24 h before the migration assay. 4 × 104 EC were added to the upper chamber of the Transwell, and after 3 h the filters were fixed in 2% paraformaldehyde and stained in 0.5% crystal violet. Migrated cells on the lower side of the filter were counted by light microscopy in three high-power fields (count/ high-power field).

Proliferation Assay

EC were transfected with an empty vector or 2DAkt as above. Subsequently, 5 × 103 cells were plated in 96-well cell culture plates in serum starvation medium (5% serum) for 36 h in the absence or presence of rapamycin. Proliferation of EC was assessed by [3H]thymidine (1 μCi/well) incorporation in the final 12 h of cell culture. Radioactivity (cpm) was measured using a Tomtec automated cell harvester (Hamden, CT).

Statistics

Data were analyzed by Student’s t test. Values of p < 0.05 were considered statistically significant.

RESULTS

Rapamycin Reduces Akt Phosphorylation and Activity in EC

Accumulating evidence suggests that rapamycin can decrease Akt activity by inhibiting mTORC2-mediated Akt phosphorylation of the Ser-473 site (9, 26, 32). However, little is known about the association between mTOR and Akt in vascular EC. Moreover, the functional significance of mTOR/Akt signaling has not been characterized in EC. To evaluate the function of this signaling pathway, we first treated serum-starved EC with rapamycin (0.1–10 ng/ml), and Akt phosphorylation was determined by Western blot. We observed that rapamycin significantly blocked Akt phosphorylation at both 1 and 10 ng/ml (Fig. 1A). This decrease in Akt (Ser-473) phosphorylation started at 4 h and was maximal 12 h after exposure of EC to rapamycin (Fig. 1B), suggesting that rapamycin targets mTORC2 in EC. Furthermore, these observations suggest that mTORC2 inhibition by rapamycin requires prolonged exposure to the drug, as described in other cell types (26). We also found that treatment of EC with rapamycin for shorter times (20, 40, and 60 min) resulted in an increase in Akt(Ser-473) phosphorylation, similar to that previously reported in non-EC (26, 33, 34). Nevertheless, as expected, this brief exposure to rapamycin (20–60 min) inhibited mTORC1 and resulted in hypophosphorylation of 4E-BP1 in EC (Fig. 1C).

FIGURE 1. Inhibition of Akt phosphorylation and Akt activity by rapamycin.

FIGURE 1

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A, EC were serum-starved for 6 h and treated with increased concentrations of rapamycin for 12 h as indicated. Cell lysates were examined for pAkt(Ser-473) and total Akt by Western blot. B and C, serum-starved EC were treated with rapamycin (10 ng/ml) for the indicated times. Cell lysates were analyzed for pAkt (Thr-308 and Ser-473) and total Akt (panel B) or p4E-BP1(Ser-65), total 4E-BP1, pAkt(Ser-473), and total Akt (panel C) by Western blot. D, serum-starved EC were treated with rapamycin (10 ng/ml) for the indicated times. Immunoprecipitated Akt was assessed for its activity using glycogen synthase kinase-3 as a substrate. Relative Akt activity was evaluated by the expression of phosphorylated glycogen synthase kinase-3/total Akt by Western blot. The bar graph illustrates the -fold change in phosphorylated glycogen synthase kinase/Akt as assessed by densitometry (*, p < 0.005 versus untreated cells, n = 3). E, EC were transfected with an empty vector (pcDNA3) or a rapamycin-resistant mTOR construct (RRmTOR). Subsequently, the cells were serum-starved for 6 h, treated with rapamycin for 12 h, and analyzed for the expression of pAkt and total Akt by Western blot. F, EC were serum-starved for 6 h and treated with rapamycin (10 ng/ml) for 12 h as indicated. Immunoprecipitations (IP) were performed using saturating concentrations of an anti-mTOR antibody, and immunocomplexes were captured with protein A-Sepharose beads as described under “Materials and Methods.” Complexes were boiled in SDS buffer and resolved by gel electrophoresis. Western blot (WB) was performed with an anti-rictor and anti-mTOR antibody on immunoprecipitated complexes or whole cell lysates as indicated. The illustrated blots are representative of three with similar findings.

In these studies rapamycin also decreased Akt(Thr-308) phosphorylation (Fig. 1B), which we interpret to indicate that Ser-473 phosphorylation is required for Thr-308 phosphorylation and, thus, for full Akt kinase activity (as suggested by others (35, 36)). Because the phosphorylation of both Akt(Ser-473) and Akt(Thr-308) is necessary for the activation of Akt-induced signaling (37), we next wished to examine if prolonged exposure of EC to rapamycin would decrease Akt kinase activity. EC were treated with rapamycin (10 ng/ml) for 1–12 h, and Akt activity was examined using a commercial kinase activity assay. As illustrated in Fig. 1D, we found that brief exposure to rapamycin (1 h) slightly increased Akt activity, but longer treatment for greater than 4 h markedly inhibited Akt activity (p < 0.005). These findings are consistent with our observations by Western blot in Fig. 1, A and B.

To further confirm that mTOR mediates Akt phosphorylation in EC, we transfected EC with a plasmid encoding a rapamycin-resistant form of mTOR (RRmTOR) (27), and we treated these transfected cells with rapamycin (as above). As illustrated in Fig. 1E, we found that overexpression of RRmTOR in EC prevented the inhibitory effect of rapamycin on the phosphorylation of Akt. Finally, to evaluate whether rapamycin targets mTORC2 in EC, by immunoprecipitation we assessed the co-expression and association between mTOR and rictor in untreated or rapamycin-treated EC (Fig. 1F). As illustrated, we found that mTOR formed a complex with rictor in untreated EC and that rapamycin inhibited co-precipitation of both proteins. Taken together these findings show that prolonged exposure of EC to rapamycin reduces Akt phosphorylation and its kinase activity and that this effect is mediated through the inhibition of mTORC2.

Rapamycin Prevents VEGF-, TNF- and Insulin-mediated Akt Phosphorylation

Proinflammatory cytokines such as TNF and growth factors such as VEGF and insulin are well established to induce Akt activity and its associated downstream signaling (3, 4, 38). We next wished to examine if rapamycin could inhibit cytokine- and growth factor-mediated phosphorylation of Akt. Serum-starved EC were treated for 12 h with rapamycin before exposure to VEGF, TNF, or insulin, and Akt phosphorylation was analyzed by Western blot. As expected, the phosphorylation of the Akt(Ser-473) and Akt-(Thr-308) was induced when EC were exposed to all three agonists (Fig. 2). However, we found that this effect was totally blocked by pretreatment of the cells with rapamycin. These findings are suggestive that rapamycin has significant effects to inhibit cytokine- and growth factor-mediated biological effects in EC.

FIGURE 2. Rapamycin inhibits VEGF-, TNF-, and insulin-mediated activation of Akt.

FIGURE 2

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EC were serum-starved for 6 h and treated for 12 h with rapamycin (Rapa, 10 ng/ml) or vehicle (C) as a control. Subsequently, VEGF (20 ng/ml), TNF (100units/ml), or insulin (10−7 m) was added into cultures for 30 min. Cell lysates were examined for the expression of pAkt (Thr-308 and Ser-473) or total Akt by Western blot. The illustrated blots are representative of 3–5 similar experiments.

Rapamycin Inhibits Akt-mediated Prosurvival Signaling in EC

Nextwe analyzed whether mTOR-regulated activation of EC results in down-stream effects on known Akt substrates such as those mediating its classical pro-survival function. VEGF is well established to protect EC from undergoing apoptosis, and this function is reported to be mediated by Akt (3). Downstream of Akt, the FoxO subfamily of transcription factors Foxo1 and Foxo3a are known to regulate apoptosis (12). As expected, we found that VEGF induced the phosphorylation of Foxo1 and Foxo3a in EC; however, pretreatment of cells with rapamycin decreased base-line as well as inducible Foxo1 and Foxo3a phosphorylation (Fig. 3, A and B). We interpret these findings to suggest that the ability of rapamycin to inhibit Akt phosphorylation in EC has biological effects to inhibit the pro-survival function of growth factors such as VEGF.

FIGURE 3. Effect of rapamycin on the FoxO subfamily of forkhead transcription factors.

FIGURE 3

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EC were serum-starved for 6 h and treated with rapamycin (10 ng/ml) or vehicle for 12 h. Subsequently EC were stimulated with VEGF (20 ng/ml) for 30 min. Cell lysates were analyzed by Western blot for pFoxo1 and total Foxo1 (panel A) and pFoxo3a and total Foxo3a (panel B). The illustrated blots are representative of four with similar findings. The relative expression of pFoxo1/total Foxo1 and pFoxo3a/total Foxo3a was assessed by densitometry. The bar graphs above each blot illustrate the mean -fold change in pFoxo1 (panel A) and pFoxo3a (panel B) from the four experiments (*, p < 0.005 versus untreated cells).

To examine whether mTOR- and Akt-induced inhibition of the FoxO pro-apoptotic pathway is of physiological significance, we treated EC with rapamycin and assessed apoptosis using propidium iodide staining in the absence or presence of serum withdrawal. As illustrated in Fig. 4A, in untreated EC, serum withdrawal resulted in 21% of cells undergoing apoptosis after 24 h, 35% of cells undergoing apoptosis after 48 h, and 49% after 72 h. In contrast, in the presence of rapamycin, apoptosis of EC was augmented after 48 and 72 h (Fig. 4A, p < 0.001 in n = 3 experiments). Consistent with this finding, we also observed by Western blot that the inhibition of Akt, Foxo1, and Foxo3a phosphorylation persisted for 48 and 72 h after prolonged treatment with rapamycin (Fig. 4B). To further confirm an effect of Akt on cell survival, we transiently transfected EC with an expression vector in which the Ser-473 and Thr-308 sites of Akt are mutated to encode a constitutively active form of the kinase (called 2DAkt, Fig. 4C) (37). As controls, we also transfected EC with a wild type Akt construct or an empty vector. Transfection efficiency was determined by Western blot (Fig. 4D). After transfection, EC were serum-starved and treated with rapamycin, and after 48 h cell death was determined by propidium iodide staining (Fig. 4C). Consistent with its known pro-survival effects, we found that the transfection of EC with wild type Akt reduced apoptosis after serum starvation but that rapamycin markedly enhanced apoptosis in these Akt-transfected cells (Fig. 4C). However, overexpression of the 2DAkt mutant not only suppressed apoptosis induced by serum starvation but also prevented the pro-apoptotic function of rapamycin. Transfection of EC with the rapamycin-resistant mTOR plasmid (RRmTOR) also protected cells from the pro-apoptotic effects of rapamycin (Fig. 4C). We interpret these observations to indicate that the ability of rapamycin to inhibit the assembly of the mTORC2 complex and the subsequent inhibition of Akt phosphorylation and activity results in apoptosis of EC.

FIGURE 4. Rapamycin inhibits the anti-apoptotic effect of Akt.

FIGURE 4

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A, EC were serum-starved and treated with rapamycin (Rapa, 10 ng/ml) for 24, 48, or 72 h. Floating and attached EC were collected and analyzed for apoptosis by fluorescence-activated cell sorter using propidium iodide staining. The sub-G1 population contains cells undergoing apoptosis and is calculated and shown in each plot as a percent of total cells. One of three similar experiments is shown. B, EC were serum-starved and treated with rapamycin (10 ng/ml) for 48 or 72 h. Cell lysates were examined for pAkt(Ser-473), total Akt, pFoxo1(Ser-256), total Foxo1, pFoxo3a(Ser-253), and total Foxo3a by Western blot. The illustrated blots are representative of three experiments with similar findings. C, EC were transfected with vector alone (pcDNA3), the WT Akt construct, the dominant active Akt construct (2DAkt), or the rapamycin-resistant mTOR construct (RRmTOR). After transfection, EC were serum-starved and treated with rapamycin (10 ng/ml) for an additional 48 h. EC were harvested and stained with propidium iodide for the evaluation of apoptosis. The bar graph illustrates the mean percent apoptosis ±1 S.D. as assessed by fluorescence-activated cell sorter in three separate experiments. D, the transfection of the plasmids used in the apoptosis studies (illustrated in panel C) was assessed by Western blot (WB). 48 h after transfection lysates of EC were analyzed for the expression of mTOR, Akt, and β-tubulin by Western blot. The illustrated blot is representative of three with similar findings.

To next determine whether VEGF- or TNF-mediated survival of EC involves mTOR-dependent regulation of pAkt, serum-starved EC were stimulated with VEGF or TNF alone in the absence or presence of rapamycin. Apoptosis of EC was determined by propidium iodide staining and fluorescence-activated cell sorter analysis (as above). As illustrated in Fig. 5A, we observed that both VEGF and TNF protected EC from undergoing apoptosis and that this effect was totally blocked by rapamycin.

FIGURE 5. Effect of rapamycin on VEGF- and TNF-mediated EC survival.

FIGURE 5

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A, EC were serum-starved and treated with rapamycin (10 ng/ml) for 12 h. Subsequently, the cells were stimulated with VEGF (20 ng/ml) or TNF (100 units/ml) for an additional 48 h. Attached and floating EC were collected and analyzed for apoptosis by propidium iodide staining. B, EC were transfected with the vector alone (pcDNA3) or the dominant active 2DAkt construct, serum-starved, and treated in the absence or presence of TNF (100 units/ml) with or without rapamycin (10 ng/ml) for an additional 48 h as illustrated. The cells were harvested and stained with propidium iodide to determine the number of EC undergoing apoptosis. Results illustrate the mean percent apoptosis ±1 S.D. of three individual experiments (*, p < 0.001 versus untreated cells in panel A or control in panel B).

Finally, we transiently transfected EC with the constitutively active mutant of Akt (2DAkt). As illustrated in Fig. 5B, we observed that this mutant prevented rapamycin from inducing apoptosis in the presence of TNF. Collectively, these results demonstrate that mTOR-Akt-induced signals are critical for the survival of EC and for protection from apoptosis.

Inhibition of EC Migration and Proliferation by Rapamycin

Our findings indicate that mTOR mediates upstream Akt-dependent activation responses in EC. Other signals resulting from mTOR-Raptor interactions (mTORC1) have been reported to be functional in EC proliferation and migration (14, 15). However, the effect of mTORC2 on this response is not known. We next performed proliferation and migration assays using EC transfected with the dominant active mutant of Akt (2DAkt). We found that overexpression of 2DAkt resulted in an increase in EC proliferation and that rapamycin significantly inhibited this proliferative response (Fig. 6A). Similarly, the augmented effects of 2DAkt on EC migration were inhibited by rapamycin (Fig. 6B). Taken together, these findings indicate that proliferative and migratory responses in EC are regulated by mTORC1 downstream of Akt.

FIGURE 6. Inhibition of EC proliferation and migration by rapamycin.

FIGURE 6

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A, EC were serum-starved, transfected with an empty vector (pcDNA3), or with the dominant active mutant of Akt (2DAkt) and treated for 36 h with rapamycin (10 ng/ml) or vehicle as a control. Proliferation of EC was measured by [3H]thymidine uptake. Results were expressed as the mean cpm of triplicate wells ±1 S.D. (representative of n = 3 experiments). B, EC were transfected with an empty vector (pcDNA3) or with the dominant active mutant of Akt (2DAkt). Twelve hours after transfection, rapamycin (10 ng/ml) or vehicle was added to cultures for 24 h, and the migration assay was performed as described under “Materials and Methods.” Migrated EC were quantified by cell counting. Results are illustrated as the mean cell count ±1 S.D. per three fields at high power magnification (HPF; ×400). The experiment illustrated is representative of three with similar findings.

DISCUSSION

In this study we observed that mTOR regulates Akt activity in EC. We found that rapamycin potentiated serum withdrawal-mediated apoptosis and also attenuated the protective effects of cytokine and growth factor-inducible responses in EC. These effects were mediated via upstream inhibition of Akt phosphorylation, presumably by inhibition of mTORC2 complex formation. Rapamycin also inhibited the proliferation and migration of EC, which was a result of inhibition of mTORC1-mediated signaling.

The interrelationship between Akt and mTOR involves positive and negative regulatory feedback loops (39). As discussed earlier, mTORC2 is composed of a protein complex among Rictor, Sin-1, and mTOR that regulates the phosphorylation of Akt via the Ser-473 residue in the carboxyl domain. Although mTORC2 is insensitive to rapamycin, our study is in agreement with others (26, 32) that long-term depletion of mTOR within a cell by rapamycin can inhibit mTORC2 complex formation. In contrast, mTORC1 mediates biological effects downstream of Akt but also facilitates a negative feedback loop to inhibit phosphatidylinositol 3-kinase-mediated Akt activation. Our data suggest that this negative feedback loop exists in EC in as much as we found that short exposure of EC to rapamycin inhibits mTORC1-mediated signals while increasing Akt phosphorylation. In contrast, long-term exposure of EC to rapamycin inhibits the association between mTOR and rictor and inhibits Akt phosphorylation (8). However, the precise molecular mechanisms involved in this negative feedback loop have not been fully characterized (34), but it is postulated that p70 S6 kinase can in part phosphorylate and inactivate downstream effectors of the pathway (40).

Several reports have documented that the anti-angiogenic properties of rapamycin result from its effects to inhibit mTORC1-mediated EC proliferation. Moreover, rapamycin has been shown to inhibit VEGF expression and VEGF-mediated EC proliferation (41, 42). mTOR inhibitors may reduce VEGF expression in different cell types in part by inhibiting the expression of HIF-1α, which is well recognized to function in VEGF gene transactivation (41, 43–45). We find that rapamycin also inhibits upstream Akt-induced signaling in EC, suggesting that its effect as an anti-angiogenic agent may relate to its ability to inhibit both upstream and downstream Akt-induced responses. Furthermore, we find that rapamycin inhibits the prosurvival functions of Akt and sensitizes EC to serum withdrawal-mediated apoptosis. Therefore, the effects of rapamycin on EC are most likely related to its ability to inhibit several Akt-inducible responses.

Akt is typically recognized as a major pro-survival signal, and its ability to protect cells from undergoing apoptosis is well established (3, 11, 12, 46). The FoxO subfamily of forkhead transcription factors as well as BAD are known substrates of Akt that in part facilitate its anti-apoptotic effects. Foxo1 and Foxo3a are known to be expressed by mature EC (47). The stimulation of EC with VEGF results in the phosphorylation and inactivation of Foxo factors in EC, indicating a potential major role for these anti-apoptotic genes in VEGF-inducible EC survival. To this end, the overexpression of an active mutant of Foxo3a has been found to inhibit VEGF-inducible EC survival (48). We observed that treatment of EC with rapamycin induced hypophosphorylation of base line as well as VEGF-inducible phosphorylation of Foxo1 and Foxo3. Although we could not detect BAD in EC by Western blot (data not shown), we also found that rapamycin inhibited the phosphorylation and inactivation of another apoptotic gene, the YES-associated protein (YAP protein, data not shown). Together, these data are suggestive that mTOR may regulate EC survival via Akt-inducible inactivation of the FoxO transcription factors. Indeed, consistent with this interpretation, Foxo1-deficient mice are embryonic lethals and show impaired vascular development (47, 49).

Finally, although Akt plays an important role in VEGF-mediated EC migration (14), we observed that the inhibition of EC migration by rapamycin may be independent of its effect on mTORC2. We found that overexpression of a dominant active mutant of Akt resulted in EC proliferation and migration, which was inhibitable by rapamycin. Therefore, the effect of rapamycin on cell motility likely involves inhibition of down-stream p70 S6 kinase and 4E-BP1 (50). Similarly, the inhibition of EC proliferation by rapamycin was independent of its ability to inhibit the phosphorylation of Akt and is likely associated with the ability of mTOR to control cell cycle progression through p70 S6 kinase and 4E-BP1 (51). Nevertheless, because rapamycin also inhibits upstream activation of Akt, we cannot rule out the possibility that it inhibits migration and proliferation via inhibition of mTORC1 and mTORC2 in vivo under physiological conditions. To this end it is noteworthy that it was recently found that high doses of rapamycin in vivo can inhibit the phosphorylation of pAkt (32).

In summary, these studies define mTOR as a novel regulatory kinase in EC that is of critical importance in EC survival, migration, and cell proliferation. We show that rapamycin inhibits the upstream phosphorylation and activation of Akt in EC as well as downstream Akt-inducible responses. These data suggest that mTOR is a key kinase involved in Akt-inducible EC activation and survival responses. Our results are among the first to demonstrate that the inhibition of Akt-inducible signals in EC by rapamycin results in enhanced apoptosis. The ability of rapamycin to inhibit these signals identifies this agent as a potent drug to target EC in vivo.

Acknowledgments

We acknowledge Dr. Soumitro Pal for critical review of the manuscript, and we thank members of our laboratory for constructive criticisms. We also thank the maternity staff at South Shore Hospital, Weymouth for help in obtaining umbilical cords for the generation of endothelial cells, and we thank Dr. Robert Abraham, William Sellers, and Debabrata Mukhopadhyay for sharing plasmids for these studies. Finally, we thank Christopher Geehan and Katiana Calzadilla for outstanding technical assistance.

Footnotes

*

This work was supported by National Institutes of Health Grant R01AI046756 (to D. M. B.).

3

The abbreviations used are: EC, endothelial cell(s); VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; RRmTOR, rapamycin-resistant mTOR; WT, wild type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; p, phosphorylated.

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