The Kaposi’s Sarcoma-associated Herpesvirus G Protein-coupled Receptor Promotes Endothelial Cell Survival through the Activation of Akt/Protein Kinase B

INTRODUCTION

Tumor viruses can induce cell transformation by overcoming cellular defense mechanisms and promoting the ungoverned proliferation of the infected cells (1 , 2) . Indeed, functionally related viral oncogenes have evolved in different viruses to override key intracellular regulatory pathways, thus assuring efficient viral replication and contributing to tumor formation (3 , 4) . This includes the disruption of a number of molecular mechanisms involved in triggering cell cycle arrest upon viral infection or DNA damage (3) . In addition, the protection from early apoptosis is a fundamental strategy that most oncogenic viruses have developed to ensure the survival of the target cells and to promote tumor progression (1, 2, 3) . In this regard, KSHV/HHV8 3 is an exemplary model of an oncogenic virus that includes, within its genome, homologues of key cellular genes implicated in the regulation of cell cycle progression and apoptosis, thus promoting viral surveillance and pathogenicity (3 , 5 , 6) .

KSHV/HHV8 is the infectious etiological agent of KS as well as other lymphoproliferative disorders, including body cavity-based B-cell lymphoma and multicentric Castleman’s syndrome (7, 8, 9, 10) . In particular, KS is a multifocal neovascular neoplasm, frequently associated with AIDS, in which KSHV infection causes the appearance of spindle-shaped cells believed to be derived from an endothelial lineage. These cells subsequently secrete different chemotactic and angiogenic factors, including VEGF, which stimulate the inflammatory and neovascular responses typical of KS and are critical for spindle cell proliferation and KS lesion development (11, 12, 13, 14) .

One of the KSHV genes believed to play a significant role in KSHV-mediated tumorigenesis is KSHV-GPCR, encoded by open-reading frame 74 (15, 16, 17) . KSHV-GPCR is a member of the family of CXC chemokine G protein-linked receptors, with significant homology to the CXCR2 receptor for IL-8, which exhibits ligand-independent activities as a result of the presence of a Val138Asp mutation in a highly conserved DRY sequence among GPCRs (16 , 18) . This virally encoded GPCR acts as a potent angiogenic activator by inducing the expression and secretion of VEGF in an autocrine manner (11 , 19) , and it is also sufficient for cell transformation when ectopically expressed in murine fibroblasts (11) . Furthermore, the expression of KSHV-GPCR in transgenic mice has been shown to induce the appearance of KS-like lesions, strongly suggesting that KSHV-GPCR is critically involved in KS pathogenesis through the promotion of VEGF-driven angiogenesis and spindle cell formation and growth (20 , 21) . It has been also demonstrated that the expression of this viral receptor can activate a number of signal-transducing pathways (11 , 19 , 22) . However, the molecular mechanisms by which this receptor is able to trigger cell transformation are still poorly understood.

Recently, a number of studies have indicated that Akt/PKB represents a critical molecule involved in the control of cell survival and in tumor development. Indeed, different oncogenes and tumor suppressor genes promote tumorigenicity by targeting Akt, and its function has been found to be deregulated in a variety of neoplasias (23 , 24) . This serine-threonine kinase is activated in response to different extracellular stimuli in various cellular systems by a mechanism that involves the activity of members of the PI3K family. In this regard, the direct binding of the PI3K-generated phospholipids to the PH domain of Akt stimulates its translocation to the membrane, where it is activated by phosphorylation by 3-phosphoinositide-dependent protein kinase and PDK2 (23 , 25) . Ultimately, the enhancement of the Akt kinase activity provokes the inactivation of different pro-apoptotic proteins and the concomitant activation of transcription factors that increase the expression of survival genes (24) . Of interest, KSHV-GPCR stimulates the expression and secretion of VEGF, and VEGF has been shown to be able to rescue cells from apoptosis induced by serum starvation through activation of the Akt/PKB pathway (26 , 27) . Thus, we decided to investigate whether KSHV-GPCR could promote cell survival pathway(s) in endothelial cells in an attempt to further elucidate the nature of the transforming intracellular mechanisms activated by this virally encoded receptor.

MATERIALS AND METHODS

Expression Plasmids and Reagents.

The expression plasmids for KSHV-GPCR, m1 and m2 human muscarinic receptors as well as for an epitope-tagged mouse wild-type Akt (pCEFL-HA-Akt) have been described previously (11 , 28) . The cDNA for the dominant-negative mutant of Akt, Akt K179M, was generously provided by Dr. P. N. Tsichlis (Thomas Jefferson University, Philadelphia, PA) and subcloned into the pCEFL expression vector. The cDNA for the human VEGF₁₆₅ was kindly provided by Dr. G. Neufeld (Israel Institute of Technology, Haifa, Israel) and subcloned into the pCEFL expression vector to obtain the sense and antisense constructs pCEFL-VEGF and pCEFL-AS-VEGF, respectively. To generate the pCEFL-EGFP-AH-Akt construct, the AH domain of the mouse wild-type Akt spanning the PH motif was obtained by PCR as a BamHI-NotI insert, including a four glycine residue cross-linker after the BamHI restriction site. The previous construct was then subcloned into the pCEFL-EGFP vector. Other expression plasmids have been described (28) . Recombinant human IL-8, IP-10, and SDF-1α were purchased from PrepoTech, Inc. _rhVEGF₁₆₅ and the polyclonal antibody against _rhVEGF₁₆₅ were obtained from R&D Systems. Wortmannin was purchased from Sigma and Ptx from List Biological Laboratories, Inc. Secreted human VEGF₁₆₅ was measured in cell culture supernatants using an immunoassay kit from R&D Systems.

Cell Lines and Transfections.

COS-7 cells were maintained in DMEM supplemented with 10% FBS. Cells were transfected by the DEAE-dextran technique in 60-mm cell culture dishes. The total amount of DNA was adjusted to 1–4 μg/plate with pCEFL-AU5-GFP in each experiment when necessary (29) .

HUVECs were obtained from Clonetics and cultured in Endothelial Cell Basal Medium-2 supplemented with 2% FBS and other supplements provided by the manufacturer. Cell transfections were performed in 100-mm cell culture dishes using the Fugene reagent (Roche Molecular Biochemicals), according to the manufacturer’s protocol. The total amount of DNA was adjusted to 1–5 μg/plate with pCEFL-AU5-GFP when necessary.

Akt Assay and Western Blots.

Akt activity was determined after transfection of cells with an expression vector for an epitope-tagged wild-type Akt (pCEFL-HA-Akt). Thirty-six h after transfection, cells were serum starved overnight in the corresponding medium with 10 mm HEPES (pH 7.5), and treated with the different drugs or stimuli when necessary. Cells were washed twice in cold PBS and lysed on ice with 900 μl of lysis buffer containing 1% Triton X-100, 10% glycerol, 137 mm NaCl, 20 mm Tris-HCl (pH 7.5), 1 μg/ml aprotinin and leupeptin, 1 mm phenylmethylsulfonyl fluoride, 20 mm NaF, 1 mm Na₂PP_i and 1 mm Na₃VO₄. Lysates were immunoprecipitated with 1 μl of anti-HA-specific monoclonal antibody HA.11 (Babco), after the samples were precleared by centrifugation. Gamma-binding beads (Amersham Pharmacia Biotech) were added for 60 min, and samples were washed three times with cold lysis buffer, once with cold water, and once with kinase buffer [20 mm HEPES (pH 7.4), 10 mm MgCl₂, 10 mm MnCl₂]. Reactions were performed for 30 min at 25°C under continuous agitation in kinase buffer containing 0.05 mg/ml histone 2B (Roche Molecular Biochemicals), 5 μm ATP, 1 mm DTT, and 10 μCi of [γ-³²P]ATP. Samples were analyzed in a 15% SDS-polyacrylamide gel, transferred to nylon membrane (Immobilon), and exposed. Resulting autoradiograms were quantified. Data for the kinase activity are expressed as fold induction with respect to the activity exhibited by control transfected cells. To assess the level of expression of HA-Akt, the same membranes were subsequently examined by Western blot, using mouse anti-HA antibody HA.11 (1:500; Babco). Bands were developed by an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech) using a secondary antibody coupled to horseradish peroxidase (Cappel).

Fluorescence Microscopy.

HUVECs or COS-7 cells were grown in 24-well plates on coverslips and transfected with the pCEFL-EGFP-AH-Akt plasmid along with additional expression vectors. Thirty-six h later, cells were serum starved for 10 h, washed with 1× PBS, and fixed with 2% paraformaldehyde. Cells were then mounted and visualized using an Axioplan2 microscope (Zeiss).

Apoptosis Assay.

HUVECs were seeded on 100-mm cell culture dishes and transfected at 70% confluence, using the Fugene reagent (Roche Molecular Biochemicals). The efficiency of transfection was examined after 36 h, based on the expression of GFP. Plates were serum starved in the presence or absence of 100 ng/ml _rhVEGF₁₆₅ for an additional 48 h. Wortmannin (50 nm) was added 2 h before serum deprivation. When necessary, samples from cell culture supernatants were collected to measure VEGF₁₆₅ secretion using an immunoassay kit. Attached and nonadherent cells were then collected and fixed in cold 70% ethanol. Samples were washed in PBS and stained with 25 μg/ml propidium iodide. Samples were analyzed on a FACScan (Becton Dickinson), and the percentage of apoptotic cells was evaluated from the ratio of the population in the sub-G₀-G₁ peak and the total cell population.

Reporter Gene Assays.

COS-7 cells were seeded on 60-mm cell culture dishes and transfected with the different expression vectors together with 0.5 μg of pcDNAIII-βgal and 0.5 μg of 5 x κB-LUC reporter plasmid, using the DEAE-dextran method. The next day, cells were serum starved overnight, and then washed twice with PBS and lysed in reporter lysis buffer (Promega). The luciferase and β-galactosidase activities present in cellular lysates were assayed as described previously (30) . The data for luciferase activity, normalized by the β-galactosidase activity, are expressed as fold induction with respect to control cells.

[³H]Thymidine Incorporation.

HUVECs were grown in 24-well plates and transfected with the pCEFL-AU5-GFP or pCEFL-KSHV-GPCR expression vectors. Thirty-six h later, cells were serum starved overnight, stimulated with 20% FBS for 16 h where appropriate, and labeled with 1 μCi/ml [methyl-³H]-thymidine for 4 h. Cells were washed twice with ice-cold PBS and twice with ice-cold 6% (w/v) trichloroacetic acid. After resuspension in 0.25 m NaOH, the radioactivity present in the trichloroacetic acid-insoluble material was determined by liquid scintillation counting.

RESULTS

KSHV-GPCR Promotes Cell Survival Independently of its Ability to Regulate VEGF Secretion.

KS is an angioproliferative tumor whose development is preceded by the infection of endothelial cells by KSHV/HHV8. We recently focused on the molecular mechanisms by which the KSHV-encoded GPCR could mediate pathogenic effects. Indeed, we have shown that KSHV-GPCR harbors transforming potential and promotes the expression and secretion of VEGF, a critical mitogen and angiogenic stimulator in KS lesions that acts in an autocrine fashion (11 , 19) . Furthermore, VEGF activates the Akt pathway and has been shown to protect endothelial cells from apoptosis induced by serum deprivation (26 , 27) . Thus, we decided to investigate whether KSHV-GPCR could be involved in the promotion of cell survival in endothelial cells. To that end, we transiently transfected HUVECs with an expression plasmid for KSHV-GPCR, using the Fugene compound. Cells were placed in basal medium with or without serum for 48 h, stained with propidium iodide, and analyzed by FACScan. As shown in Fig. 1A ⇓ , we observed that the expression of KSHV-GPCR effectively protected cells from the apoptosis induced by serum deprivation to nearly the same extent as that in response to treatment with VEGF. As a control, we excluded the possibility that this result could be attributable to an effect of KSHV-GPCR on cell proliferation because we did not detect any significant changes in the [³H]thymidine incorporation in cells transfected with the receptor or a DNA control (Fig. 1B) ⇓ . Thus, KSHV-GPCR was able to promote cell survival when overexpressed in HUVECs without inducing cell proliferation. However, when the same experiment was performed in the presence of an anti-human VEGF antibody, the ability of KSHV-GPCR to rescue HUVECs from apoptosis was not significantly affected, whereas VEGF was no longer able to increase cell survival under identical experimental conditions (Fig. 1C) ⇓ .

To further address the possibility that the induction of cell survival by KSHV-GPCR is independent of its ability to enhance VEGF secretion, we decided to examine whether expression of an antisense cDNA for human VEGF₁₆₅ affects the antiapoptotic activity of this viral GPCR. As shown in Fig. 2A ⇓ , the KSHV-GPCR-induced secretion of VEGF into the medium was effectively inhibited by the transfection of increasing amounts of pCEFL-AS-VEGF but not by control DNAs (not shown). Under these conditions, we observed that KSHV-GPCR was still able to induce cell survival in endothelial cells, even when the secretion of VEGF was abolished by the transfection of the ant-sense construct (Fig. 2B) ⇓ .

In addition, we studied the effect of the anti-VEGF antibody in the induction of cell survival promoted by the expression of KSHV-GPCR as well as a cDNA for VEGF₁₆₅ (Fig. 2, C and D) ⇓ . We observed that KSHV-GPCR induces a remarkable increase in the release of VEGF from expressing endothelial cells and that similar levels of VEGF secretion could be achieved by expression of a VEGF₁₆₅ cDNA (Fig. 2C) ⇓ . Furthermore, expression of VEGF in primary human endothelial cells was sufficient to rescue a fraction of the cells from programmed cell death upon serum removal (Fig. 2D) ⇓ . As expected, the anti-VEGF antibody prevented the antiapoptotic effect of VEGF expression. In contrast, as shown above, in parallel experiments, KSHV-GPCR was still able to rescue cells from apoptosis induced by serum deprivation even the presence of the anti-VEGF antibody. All together these results suggested that KSHV-GPCR is able to activate cell survival pathways in primary human endothelial cells but that the antiapoptotic activity of KSHV-GPCR may be independent of its ability to enhance the expression of VEGF.

KSHV-GPCR Activates Akt and Induces Its Translocation to the Membrane.

The finding that KSHV-GPCR was able to induce cell survival prompted us to investigate the nature of the intracellular routes implicated in this effect. In this regard, GPCRs can activate Akt in certain cell types, and this signaling pathway can promote cell survival under physiological as well as pathological conditions (28 , 31, 32, 33) . Thus, we decided to explore whether KSHV-GPCR was able to stimulate the activity of this serine-threonine kinase in endothelial cells. We transiently transfected HUVECs with an expression vector encoding for the wild-type form of Akt tagged with the HA epitope (pCEFL-HA-Akt), along with increasing doses of the plasmid pCEFL-KSHV-GPCR. As shown in Fig. 3A ⇓ , KSHV-GPCR was able to induce the kinase activity of Akt in a dose-dependent manner, as judged by immune-complex kinase reactions using histone 2B as a substrate. Stimulation of the Akt activity by the overexpression of an activated form of the H-Ras protein, RasV12, was included as a positive control. In parallel, we ran identical experiments using COS-7 cells, which frequently are used as a highly efficient in vivo reconstitution system (28 , 29) . In this cell line, the expression of the viral receptor also increased the kinase activity of Akt in a dose-dependent manner. Moreover, this induction was similar to that induced by the stimulation of the m2 muscarinic GPCR, a typical G_i-coupled receptor, by the agonist carbachol (Ref. 28 ; Fig. 3B ⇓ ). These results indicated that the expression of KSHV-GPCR is sufficient to activate the Akt signaling pathway.

Upon activation, Akt changes its subcellular distribution and translocates rapidly to the plasma membrane, where it binds to PI3K-generated phospholipids and undergoes phosphorylation by PDK1 and PDK2 (24) . Thus, we next set out to determine whether the expression of KSHV-GPCR could induce a change in the intracellular localization of the Akt kinase. As an approach, we used the pCEFL-EGFP-AH-Akt plasmid, in which GFP is fused to the AH domain of the wild-type form of Akt. This domain includes the Akt NH₂-terminal regulatory region spanning the PH domain, which mediates the interaction of Akt with the 3′ phosphorylated phosphoinositide lipids localized in the membrane (34) . The pCEFL-EGFP-AH-Akt plasmid was cotransfected, along with expression vectors for KSHV-GPCR, into both HUVECs and COS-7 cells (Fig. 3C) ⇓ . When cells were visualized under the UV microscope, we observed that the GFP-AH-Akt fusion protein was localized in the nucleus in control cells, in agreement with previous data (34) . However, the expression of the constitutively active KSHV-GPCR promoted the translocation of the PH domain of Akt to the plasma membrane, in a fashion similar to that induced by RasV12.

The Activity of PI3K Is Required for the Stimulation of Akt by KSHV-GPCR.

The signaling pathway leading to the activation of Akt generally involves the activity of members of the PI3K family, which are critically involved in cell survival as well as in many other biological effects. Nevertheless, the activation of Akt may also be triggered through other intracellular biochemical routes (31 , 35, 36, 37) . Thus, we decided to examine whether the activation of Akt by KSHV-GPCR could be blocked by wortmannin, a potent inhibitor of PI3K (38) . As shown in Fig. 4 ⇓ , activation of Akt by the expression of KSHV-GPCR was dramatically diminished by the treatment of cells with wortmannin. As a control, this compound also showed an inhibitory effect on the activation of Akt following stimulation of the m2 receptor by carbachol, as described previously (28) . These observations suggest that the induction of Akt by KSHV-GPCR requires the activity of PI3K.

The Activation of Akt by KSHV-GPCR Is Dependent on βγ Subunits Released from Ptx-sensitive and -insensitive Heterotrimeric G Proteins.

GPCRs can be linked to a variety of heterotrimeric GTP-binding proteins. Both α subunits and βγ complexes that are released upon the activation of these receptors are critically involved in the regulation of a complex array of intracellular signaling pathways (39) . Regarding the family of CXCR receptors, the vast majority have been shown to be coupled to the G_i family of α subunits (40) . Conversely, signaling through KSHV-GPCR appears to be mediated by G_q because the accumulation of inositol phosphates derived from the activation of phospholipase C by this receptor is not blocked by the treatment with Ptx, a specific inhibitor of G_i-dependent signaling pathways (16 , 18) . To assess whether the activation of Akt by KSHV-GPCR was a G_i- or G_q-mediated event, we transfected COS-7 cells with the expression vector for the tagged Akt along with the cDNA for KSHV-GPCR. The m1 and m2 muscarinic receptors, typical examples of G_q- and G_i-coupled receptors, respectively, were used as controls. Cells were then serum starved and treated with 50 ng/ml Ptx overnight. As shown in Fig. 5A ⇓ , the Ptx was able to block the activation of Akt induced by m2 but had no effect on the activation caused by m1. In the same experiments, the enhancement of the Akt activity induced by the constitutively active KSHV-GPCR was partially inhibited by the Ptx treatment. This implies that, although this receptor may signal primarily through G_q proteins, they may also engage G_i proteins to activate certain downstream signaling pathways, such as Akt.

To study whether βγ subunits were required for the activation of Akt in response to the expression of KSHV-GPCR, we used a chimeric molecule, CD8-βARK, which is endowed with the ability to sequester free βγ complexes, thus blocking βγ-dependent signaling pathways (41) . As shown in Fig. 5B ⇓ , we found that the expression of the CD8-βARK chimera inhibited the activation of Akt by KSHV-GPCR and by the m2 receptor, although this βγ sequestering molecule did not affect the Akt activation induced by RasV12. These data indicate that signaling from KSHV-GPCR to Akt requires the βγ subunits of heterotrimeric G proteins, likely upon release from heterotrimeric G proteins of the G_q and G_i families.

Inverse Agonists Inhibit the Activation of Akt Induced by KSHV-GPCR.

Although KSHV-GPCR is a constitutively active G protein-linked receptor characterized by increased ligand-independent activity, it also binds a variety of chemokines that can modulate its function in a positive or negative manner (42, 43, 44) . In particular, several inverse agonists have been shown to decrease the constitutive activity of this viral receptor, thereby blocking different downstream intracellular signaling pathways. IP-10 and SDF-1α are two examples of this type of chemokine (22 , 45 , 46) . Therefore, we investigated whether the stimulation of Akt by KSHV-GPCR could be modulated by different cytokines. We expressed KSHV-GPCR along with the tagged Akt protein in COS-7 cells, and then treated the cells with IL-8, IP-10, or SDF-1α for 15 min. As shown in Fig. 6 ⇓ , the activation of Akt by the viral receptor was diminished by the addition of IP-10 and SDF-1α. However, the Akt signaling pathway was not significantly affected by IL-8, in agreement with previous data (44) . These results show that the activation of Akt induced by KSHV-GPCR can still be modulated by the treatment with inverse agonists, such as IP-10 and SDF-1α.

KSHV-GPCR Stimulates the Transcriptional Activity of NF-κB through the Induction of Akt.

Once we had found that the expression of KSHV-GPCR could activate Akt, we decided to explore which downstream targets of this kinase could be involved in this cell survival response. A variety of proteins have been shown to act as effector molecules downstream of Akt, eliciting different potential mechanisms by which this serine-threonine kinase can regulate cell survival (24) . Of interest, the transcription factor NF-κB, an important component of biochemical routes rescuing cells from apoptosis in diverse cellular systems, has been shown to be a target for Akt (47 , 48) . Thus, to investigate whether the transcriptional activity of NF-κB could be regulated by KSHV-GPCR, we transfected COS-7 cells with the expression vectors pCEFL-AU5-GFP, pCEFL-AU5-cdc42QL, pCEFL-HA-rasV12, or pCEFL-AU5-KSHV, along with the plasmid pCDNAIII-βgal and the reporter 5 x κB-LUC, in which the luciferase gene is under the control of the κB site. As shown in Fig. 7A ⇓ , the overexpression of the constitutively active KSHV-GPCR was able to induce the transcriptional activity of NF-κB. Activated GTPase-deficient forms of Cdc42 or Ras proteins were included as positive controls (49) . Coexpression of the viral receptor along with a kinase-deficient mutant of Akt, Akt K179M, and the κB-dependent reporter, resulted in the inhibition of NF-κB, suggesting that the ability to activate this nuclear response was mediated by Akt (Fig. 7B) ⇓ . The activation of NF-κB triggered by TPA was not blocked under the same conditions (48) and served as a control for the specificity of this approach. These results indicate that the NF-κB transcription factor is a possible downstream effector molecule involved in the PI3K-Akt signaling route activated by KSHV-GPCR.

The Activation of Akt Is Necessary for the Rescue from Apoptosis Induced by KSHV-GPCR in Endothelial Cells.

To investigate whether the PI3K-Akt pathway mediates the survival pathway elicited by KSHV-GPCR, we transfected the cDNA encoding for the viral receptor into HUVECs and maintained cells in basal medium with or without serum, in the absence or presence of wortmannin, for 48 h. As shown in Fig. 8A ⇓ , KSHV-GPCR was no longer able to stimulate cell survival when the PI3K inhibitor was added during the starvation period. Similarly, expression of the dominant-negative mutant of Akt, AktK179M, blocked the ability of KSHV-GPCR to prevent cell death (Fig. 8B) ⇓ . The inhibition of the Akt pathway also impaired the rescue from apoptosis induced by VEGF, as described previously (26 , 27) . Taken together, these results show that the activation of the PI3K-Akt signaling pathway is required for the promotion of cell survival induced by the expression of KSHV-GPCR in endothelial cells.

DISCUSSION

KS is an angioproliferative disorder that has been under intense investigation during the last few years because of its increased prevalence associated with the AIDS epidemic. HHV8, also known as KSHV, has been discovered as a necessary etiological agent for all forms of KS because the infection of endothelial cells by KSHV precedes the development of the lesions and coincides with KS risk (1 , 7 , 20 , 50) . Of note, compelling data point to the constitutively active CXC chemokine GPCR (KSHV-GPCR) as a key molecule in the pathogenesis of KS (20 , 21) . KS is a highly neovascularized neoplasm, and KSHV-GPCR may be responsible for the elevated expression and secretion of the angiogenic factor VEGF through the activation of different signal-transduction pathways (11 , 19) . Interestingly, KSHV-GPCR is also a viral oncogene that induces cell transformation when ectopically expressed and is able to induce the appearance of KS-like lesions in transgenic mice (11 , 21) . Although the potential mechanisms of KSHV-GPCR-mediated angiogenesis and spindle cell proliferation need further investigation (20) , these data suggest that KSHV-GPCR must be acting on different transforming, growth-regulating signaling pathways within the cell.

In general, abnormal cell proliferation is sustained by the deregulation of intracellular routes that control cell death. The induction of apoptosis is a highly regulated process that occurs during development, but it also occurs as a consequence of the exposure of cells to a diverse array of external stresses. In particular, programmed cell death is one of the most immediate responses of the host cell to viral infection (51 , 52) , and thus is one of the targets that oncogenic viruses have to counteract to induce tumor progression. In this study, we found that the expression of the G protein-linked receptor from KSHV is able to promote endothelial cell survival. Indeed, we found that the expression of KSHV-GPCR in human primary endothelial cells is able to rescue cells from apoptosis induced by serum deprivation. Interestingly, experiments using VEGF antisense constructs and specific antibodies against VEGF showed that this effect is mostly independent of the ability of KSHV-GPCR to increase the level of expression and secretion of VEGF in these primary cells. However, we cannot discard the possibility that under physiological conditions both pathways could cooperate to provide cell survival. We also found that the PI3K-Akt pathway is required for the prevention of cell death induced by KSHV-GPCR because both treatment with wortmannin and expression of a dominant-negative interfering mutant of Akt are sufficient to abolish the inhibition of apoptosis in response to the expression of KSHV-GPCR. These results provide further knowledge in the elucidation of the intracellular signaling pathways activated by KSHV-GPCR and support its likely contribution to KSHV-induced neoplasias by promoting the survival and uncontrolled cell proliferation of infected cells.

The mechanism whereby the constitutively active KSHV-GPCR enhances the kinase activity of Akt in endothelial cells may involve the translocation of this protein to the plasma membrane by means of its PH domain. This intracellular relocalization of Akt has been described as an important step in the activation of this serine-threonine kinase (23 , 24) . Additionally, we observed that the βγ complexes that are released as a consequence of the receptor activity are required for the activation of Akt because sequestration of βγ subunits by a chimeric molecule containing the βγ-binding domain of βARK, CD8-βARK, prevents the induction of the Akt activity by KSHV-GPCR. On the other hand, although this receptor has been shown to be linked to G_q-dependent biochemical events, our results using Ptx treatment suggests that in addition to G_q, members of the G_i family of heterotrimeric G proteins can also contribute to signaling by KSHV-GPCR.

Confirming the importance of the antiapoptotic strategy developed by KSHV, other survival genes, including those encoding v-bcl2, v-FLIP, v-IL6 and LANA, are also expressed from the genome of this infectious agent (1 , 53, 54, 55, 56) . Of interest, oncogenic forms of the catalytic subunits of PI3K and Akt have been isolated from avian sarcoma virus 16 and the AKT8 retrovirus, respectively (57 , 58) . Moreover, different pathogenic viruses also contain viral oncogenes that promote cell transformation and activate Akt (59, 60, 61, 62) . Thus, activation of this serine/threonine kinase by KSHV-GPCR may represent a critical intracellular pathway in the blockade of cell death because this kinase acts on a large number of target molecules involved in the execution of apoptotic signals or in the promotion of cell survival. In this regard, we provide evidence that the transcription factor NF-κB, recently described as a target of Akt (47 , 48) , is one of the downstream molecules activated by the PI3K-Akt signaling pathway upon KSHV-GPCR expression. In line with these observations, NF-κB activity recently has been reported to be necessary for survival of KSHV-infected lymphoma cells (63) . Whether other putative Akt substrates are also affected by this viral receptor is currently under investigation.

Certain inverse agonists that bind the extracellular domains of the receptor can modulate the constitutive activation of the Akt signaling pathway by KSHV-GPCR. These findings suggest that the ability of KSHV to induce cell survival can be amenable to regulation by pharmacological intervention. Furthermore, by investigating the signaling pathways used by KSHV-GPCR and by other survival genes encoded by KSHV, we may begin to unravel the complexity of the mechanism by which this elusive virus initiates endothelial cell transformation, promotes spindle cell proliferation, and thus ultimately causes KS lesion development. This, in turn, may help identify novel therapeutic targets for the treatment of this devastating disease.