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. 2013 Spring;13(1):66-75.

Kaposi sarcoma-associated herpesvirus g protein-coupled receptor enhances endothelial cell survival in part by upregulation of bcl-2

Elizabeth R Abboud  1 , Bryan D Shelby, Magdalena Angelova, Anne B Nelson, Marybeth Ferris, Harris E McFerrin, Cindy A Morris, Deborah E Sullivan
Affiliations

Kaposi sarcoma-associated herpesvirus g protein-coupled receptor enhances endothelial cell survival in part by upregulation of bcl-2

Elizabeth R Abboud et al. Ochsner J. 2013 Spring.

Abstract

Background: Kaposi sarcoma-associated herpesvirus (KSHV) encoded G protein-coupled receptor (vGPCR) is a constitutively active lytic phase protein with significant homology to the human interleukin-8 receptor. vGPCR is necessary and sufficient to induce angiogenesis as well as the spindle cell proliferation characteristic of Kaposi sarcoma (KS) lesions. We previously demonstrated that Bcl-2, an antiapoptotic protein, is upregulated in KS lesions. The aim of this study was to determine if vGPCR enhances endothelial cell survival through upregulation of Bcl-2 expression and to elucidate the signaling pathways involved.

Methods: Primary human umbilical vein endothelial cells were transduced with a recombinant retrovirus expressing vGPCR and then subjected to serum starvation. Cell viability and apoptosis were analyzed by fluorescence-activated cell sorting. Bcl-2 expression was determined by real-time quantitative reverse transcription polymerase chain reaction and immunoblotting. Specific pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) were employed to elucidate the signaling pathways involved. Bcl-2 expression was knocked down using small interfering RNA (siRNA).

Results: Endothelial cells expressing vGPCR showed increased survival after serum starvation and upregulation of Bcl-2 messenger RNA (mRNA) and protein. The vGPCR-induced increases in both Bcl-2 mRNA and protein levels were dependent on PI3K signaling but not on mTOR. Moreover, siRNA inhibition of Bcl-2 resulted in significant abrogation of the observed vGPCR-mediated cell survival advantage.

Conclusions: Taken together, the results demonstrate that Bcl-2 is a mediator of vGPCR-induced endothelial cell survival and is a downstream effector of Akt in this process.

Keywords: Bcl-2 protein; G protein–coupled receptor; Kaposi sarcoma; human herpesvirus 8; phosphatidylinositol 3-kinase.

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Conflict of interest statement

Funding: This study was supported in part by grants from the Louisiana Cancer Research Consortium (DES and HEM), HD045768/HD/NICHD NIH (CAM), P20RR016456/RR/NCRR NIH (HEM), P20GM103424/GM/NIGMS NIH (HEM), and 5G12RR026260-03/RR/NCRR NIH (HEM). ERA received support from T32-HL-O7973/HL/NHLBI NIH; ERA and BDS received matching funds from the Tulane Cancer Center.

Figures

Figure 1.
Figure 1.
vGPCR enhances cell survival in HUVECs. BABE and BABE-vGPCR transduced HUVECs were serum starved for 24 hours and then harvested and stained with Annexin-V-APC and propidium iodide for FACS analysis. (A) Representative histograms from BABE and BABE-vGPCR transduced HUVECs. (B) The percent of viable cells was determined by the number of cells negative for propidium iodide staining. (C) The percent of apoptotic cells was measured by immunostaining for Annexin-V on the cell surface. Data are presented as the mean of 3 independent experiments ± SEM (n=3) (**P<0.001, ***P<0.0001). FACS, fluorescence-activated cell sorting; HUVECs, human umbilical vein endothelial cells; SEM, standard error of the mean; vGPCR, virally encoded G protein–coupled receptor.
Figure 2.
Figure 2.
vGPCR upregulates Bcl-2 mRNA and protein levels in endothelial cells. Mock, BABE, and BABE-vGPCR transduced HUVECs were serum starved for the indicated times. (A) Bcl-2 mRNA levels were measured by real-time qRT-PCR. Each sample was analyzed in triplicate and normalized to the level of 36B4 mRNA. The data are presented as fold change (mean ± SEM) relative to the mock-infected sample at 0 hours (*P<0.05). (B) Cell lysates were prepared and analyzed by Western blot using antibodies to Bcl-2 and β-actin as a loading control. HUVECs, human umbilical vein endothelial cells; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; vGPCR, virally encoded G protein–coupled receptor.
Figure 3.
Figure 3.
vGPCR-induced upregulation of Bcl-2 mRNA and protein levels is dependent on PI3K/Akt in HUVECs. (A) Mock, BABE, and BABE-vGPCR transduced HUVECs were serum starved for 24 hours while being treated with PI3K/Akt inhibitor (+LY, 50 μM LY294002) or vehicle (-LY). Cell lysates were harvested at 0 and 24 hours following treatment and analyzed by Western blot for activation of Akt using the antibody to phospho-Akt (Ser473) (p-Akt). Blots were stripped and reprobed with antibodies to total Akt and β-actin was used as a loading control. (B) The same cell lysates were analyzed by Western blot for Bcl-2 expression and β-actin as loading control. (C) Mock, BABE, and BABE-vGPCR transduced HUVECs were serum starved for 24 hours while being treated with PI3K/Akt inhibitor (+LY, 50 μM LY294002) or vehicle (-LY). Total RNA was extracted and Bcl-2 mRNA levels were measured by real-time qRT-PCR following treatment. Each sample was analyzed in triplicate and normalized to the level of 36B4 mRNA. The data are presented as fold change (mean ± SEM) relative to the mock-infected sample without inhibitor. (*P<0.05). (D) Mock, BABE, and BABE-vGPCR transduced HUVECs were treated with 50 nM rapamycin (Rap) or vehicle under serum-starvation conditions for 24 hours. Total RNA was extracted and Bcl-2 mRNA levels were measured by real-time qRT-PCR following treatment. Each sample was analyzed in triplicate and normalized to the level of 36B4 mRNA. The data are presented as fold change (mean ± SEM) relative to the mock-infected sample without inhibitor. HUVECs, human umbilical vein endothelial cells; mRNA, messenger RNA; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; vGPCR, virally encoded G protein–coupled receptor.
Figure 4.
Figure 4.
vGPCR-enhanced cell survival in HUVECs is dependent, in part, on Bcl-2. BABE-vGPCR transduced HUVECs were transfected with siRNA targeting Bcl-2 or a scrambled siRNA as control and then serum starved for 24 hours. Cells were then harvested and stained with Annexin-V-APC and propidium iodide for FACS analysis. (A) Representative histograms from HUVECs treated with BABE, BABE-vGPCR + control siRNA, and BABE-vGPCR + Bcl-2–targeted siRNA. (B) The percent of viable cells was determined by the number of cells negative for propidium iodide staining. (C) The percent of apoptotic cells was measured by immunostaining with Anexin-V on the cell surface. (D) Knockdown of Bcl-2 mRNA in BABE-vGPCR transduced HUVECs transfected with Bcl-2–targeted siRNA was confirmed by real-time qRT-PCR. Each sample was analyzed in triplicate and normalized to the level of 36B4 mRNA. The data are presented as fold change (mean ± SEM) relative to cells transfected with control scrambled siRNA. Data are presented as the mean of 3 independent experiments ± SEM (n=3) (**P<0.001, ***P<0.0001). HUVECs, human umbilical vein endothelial cells; mRNA, messenger RNA; PI3K, phosphatidylinositol 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean; siRNA, small interfering RNA; vGPCR, virally encoded G protein–coupled receptor.
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