Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 May;77(10):5759-73.
doi: 10.1128/jvi.77.10.5759-5773.2003.

Human herpesvirus 8-encoded vGPCR activates nuclear factor of activated T cells and collaborates with human immunodeficiency virus type 1 Tat

Shibani Pati  1 , James S Foulke Jr, Oxana Barabitskaya, Jynho Kim, B C Nair, David Hone, Jennifer Smart, Ricardo A Feldman, Marvin Reitz
Affiliations

Human herpesvirus 8-encoded vGPCR activates nuclear factor of activated T cells and collaborates with human immunodeficiency virus type 1 Tat

Shibani Pati et al. J Virol. 2003 May.

Abstract

Human herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-kappa B, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNF alpha). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-kappa B activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Expression of vGPCR activates NF-AT. (a) Jurkat T cells (105) were transfected with an NF-AT luciferase reporter construct (100 ng). Increasing amounts (100 to 300 ng) of vGPCR-pSG5 were transfected along with the reporter construct. Total input DNA was balanced with the empty pSG5 vector. A pCMV-β-gal vector (200 ng) was cotransfected as an internal control for transfection efficiencies, which were approximately 10%. Cells were harvested and assayed for luciferase and β-galactosidase activity 24 h after transfection. Some samples were treated with 1 μM CSA or 100 nM Groα 4 h prior to harvest. Luciferase values are expressed as relative light units (RLU) and normalized for β-galactosidase. The values shown are averages of three independent samples, with the standard deviations represented by error bars. (b) KSIMM cells (105) were transfected with 400 ng of vGPCR-pSG5 or a control vector (pSG5) and 100 ng of an NF-AT luciferase reporter gene construct. Each sample was also cotransfected with 200 ng of either HA-NF-AT1, HA-NF-AT2, or HA-NF-AT4. Cells were harvested 24 h posttransfection and assayed for luciferase activity. Baseline levels of NF-AT induced by vGPCR were not detectable in this passage of KSIMM cells, thereby allowing us to attribute the luciferase reading to the transiently transfected NF-AT expression vectors. (c) 293 cells (2 × 105) were transfected with 500 ng of vGPCR-pSG5 or a control vector (pSG5) and the expression construct for a GFP-NF-AT1 (HA-NF-AT1) fusion protein. Visualization of the GFP-tagged NF-AT1 protein within the cells was performed with an inverted fluorescence microscope. (i) Cells expressing vGPCR demon-strated a nuclear localization of NF-AT1 similar to that in cells treated with 10 mM CaCl2 and ionomycin (1 μM) (ii) but different from that in control vector-transfected cells (iii). Cells transfected with a constitutively active mutant form of GSK-3 [GSK-3(S9A)] plus vGPCR (iv) demonstrated a cytoplasmic localization of NF-AT1 similar to that of the control.
FIG. 2.
FIG. 2.
Enhancement of NF-AT1 DNA binding activity in endothelial cells expressing vGPCR. (a) DMVECs were transduced with MIGR1 or vGPCR-MIGR1. Cells were harvested 24 h posttransfection, and nuclear extracts were prepared and used in an EMSA as described in Materials and Methods. The total protein in all samples was equalized. Lane 3 shows enhanced DNA binding of NF-AT in cells expressing vGPCR in comparison to that of the control (MIGR1 alone; lane 4). The enhanced binding in vGPCR-expressing cells was inhibited by addition of 0.5 μM CSA (lane 2) or by cotransfection with an expression vector for the NF-AT inhibitory peptide VIVIT (lane 1). Lane 5 shows an increase in NF-AT DNA binding in cells treated with ionomycin (2 μM) and CaCl2 (10 mM). Lane 6 shows a supershift of the NF-AT2-DNA complex with NF-AT2 antibody (Ab) 7A6 (Santa Cruz Biotechnology). ss, supershift. (b) Densitometric readings for NF-AT bands, measured by ImageQuant software for the PhosphorImager (see Materials and Methods).
FIG. 3.
FIG. 3.
NF-AT activation by vGPCR and NF-κB activation by vGPCR are independent. (a) KS cells (105) were transfected with either an NF-κB or an NF-AT reporter construct and combinations of vGPCR-pSG5, control (ctrl) vector (pSG5), and DN IκB (0.1 and 0.2 μg). Inhibition of NF-κB by DN IκB did not affect NF-AT activation by vGPCR but did inhibit NF-κB activation. (b). KS cells were transfected with either an NF-κB or an NF-AT luciferase reporter plasmid and combinations of vGPCR-pSG5, control vector (pSG5), and an expression vector for the NF-AT inhibitory peptide VIVIT. Expression of VIVIT inhibited NF-AT but not NF-κB activation by vGPCR. (c) Inhibition of the PI3-K/Akt pathway inhibited activation of NF-AT by vGPCR. KSIMM cells (105) were transfected with an NF-AT reporter construct and combinations of vGPCR and a control vector (pSG5) with or without a DN Akt mutant form [Akt(K179M); 0.1 and 0.2 μg]. Parallel samples expressing vGPCR were treated with the PI3-K inhibitor LY294002 (10 and 20 μM as indicated). Both Akt(K179M) and LY294002 inhibited NF-AT activation by vGPCR. (d) Phosphorylation of Akt and GSK-3 in HUT 78 T cells expressing vGPCR. Cell lysates (3 days postinfection) from HUT 78 T cells transduced with retroviral vectors expressing vGPCR or a control (MIGR1) were analyzed by Western blot assay (as described in Materials and Methods) with antibodies specific for phosphorylated Akt (serine 473) or phosphorylated GSK-3α and -β. Cells were analyzed by flow cytometry for GFP, which is coexpressed from the same vectors. GFP was expressed in approximately 30% of both vGPCR-transduced and control cells. Blots were stripped and analyzed for total Akt and total GSK-3β to ensure that differences in levels of phosphorylated protein did not simply reflect the presence of more protein. Cells expressing vGPCR were treated with the PI3-K inhibitor LY294002 (20 μM) to demonstrate the involvement of this pathway. (e) Inhibition of the PI3-K pathway inhibits AP-1 activation by vGPCR. KSIMM cells (105) were transfected with an AP-1 luciferase reporter construct (100 ng) and 500 ng of vGPCR-pSG5 or a control vector. Transfection efficiencies were approximately 20%. vGPCR-transfected cells were cotransfected with 0.1 to 0.2 μg of DN Akt(K179M) or treated with the PI3-K inhibitor LY294002 (10 and 20 μM) as indicated for 6 h. Readings are expressed as relative light units (RLU). The values shown are averages of three independent samples, with the standard deviations represented by error bars. (f and g) Constitutive activation of GSK-3 inhibits NF-AT, NF-κB, and AP-1 activation by vGPCR. KSIMM cells (105) were transfected with an NF-AT luciferase (f), AP-1 luciferase (g), or NF-κB luciferase (g) reporter construct (100 ng) and 500 ng of vGPCR-pSG5. The indicated samples were cotransfected with 0.4 μg of the constitutively active GSK-3 mutant form GSK-3(S9A) or treated with the GSK-3 inhibitor LiCl (10 mM). Transfection efficiencies were approximately 20%. Readings are expressed as RLU (f) or fold of the control value (g). The values shown are averages of three independent samples, with the standard deviations represented by error bars.
FIG. 4.
FIG. 4.
vGPCR-induced expression of ICAM-1, CD25, CD29, and Fas ligand in HUT 78 cells. HUT 78 cells were transduced with the retroviral construct vGPCR-MIGR1 and the control MIGR1 (see Materials and Methods). Samples were analyzed with a Becton-Dickinson FACScalibur flow cytometer for GFP+ cells. (a) At 3 days postinfection, approximately 30% of the cells infected with vGPCR were GFP positive. This was typical of all samples. Phycoerythrin-conjugated antibodies for ICAM-1, CD25, CD29, and Fas ligand (FasL) were from Pharmingen. An appropriate isotype-matched control for each antibody was used as a control for nonspecific antibody binding. Panels: b, cell surface ICAM-1; c, cell surface CD25; d, cell surface CD29; e, cell surface Fas ligand. Arrows indicate the isotype control- and MIGR1 (control)- or vGPCR-transduced cells. Expression of these markers in uninfected HUT 78 cells was no different from that in cells infected with control virus MIGR1 (not shown).
FIG. 5.
FIG. 5.
HUT 78 cells expressing vGPCR form homotypic aggregates. HUT 78 cells were transduced with the retrovirus vGPCR-MIGR1 or a control (MIGR1) (see Materials and Methods). Panel a shows the aggregates present in cultures infected with vGPCR in comparison with the control (b). Original magnification, ×20.
FIG. 6.
FIG. 6.
Expression of vGPCR in HUT 78 T cells results in enhanced adherence to KS cells. (a) HUT 78 cells were transduced with the retroviral construct vGPCR-MIGR1 or a control (ctrl), MIGR1. KSIMM cells (2 × 103) were seeded into each well of a 96-well plate. Twenty-four hours later (48 h posttransduction), different numbers of calcein-labeled, infected HUT 78 cells (2 × 106, 1 × 106, and 0.5 × 106) were seeded onto these wells and allowed to adhere for 45 min. The plates were washed (see Materials and Methods) and read on a fluorimeter, and calcein readings were correlated with cell numbers. Each value shown is the result of readings of eight wells. Samples treated as indicated with CSA or LY294002 (LY) also were seeded with 2 × 106 cells. For these samples, HUT 78 cells were pretreated as indicated with CSA (1 μM) or LY294002 (20 μM) for 1 h prior to being seeded onto the KS cells. Control HUT 78 cells (2 × 106) were treated for 4 h with 5 ng of TNF-α per ml and then seeded onto KSIMM cells as a positive control for adhesion. (b) Expression of vGPCR in KS cells enhanced adherence of HUT 78 T cells. KSIMM cells were transfected with the retroviral construct vGPCR or a control. KSIMM cells (2 × 103) were seeded into each well of a 96-well plate. Twenty-four hours later, different amounts of calcein-labeled HUT 78 cells (2 × 106, 1 × 106, and 0.5 × 106) were seeded onto these wells and allowed to adhere for 45 min. Control KSIMM cells were treated for 4 h with 5 ng of TNF-α per ml and mixed with HUT 78 cells as a positive control for adhesion. Plates were washed (see Materials and Methods) and read on a fluorimeter. Calcein readings were correlated with cell numbers. Each value is the result of readings from eight wells. KS cell samples were pretreated as indicated with 1 μM CSA or 20 μM LY294002 for 1 h prior to seeding with the HUT 78 cells. Pretreatment was only performed with 2 × 106 cells.
FIG. 7.
FIG. 7.
HIV-1 Tat synergistically enhances the activation of both NF-κB and NF-AT by vGPCR. (a) KSIMM cells were transfected with 100 ng of a NF-κB luciferase reporter construct and 500 ng of vGPCR-pSG5 or a control vector (pSG5). Total input DNA was balanced with the empty pSG5 vector. The pCMV-β-gal vector (200 ng) was cotransfected as an internal control for transfection efficiency, which was generally 20%. Some samples were treated as indicated with lipopolysaccharide-free recombinant Tat protein (86 amino acids) 24 h posttransfection at the indicated concentrations. Six hours later, samples were assayed for luciferase activity. The values shown are averages of three independent samples, with the standard deviations represented by error bars. RLU, relative light units. Tat synergistically enhanced NF-κB activation by vGPCR. (b) KSIMM cells were transfected with an NF-AT luciferase reporter construct (100 ng) and 500 ng of vGPCR or a control (ctrl) vector (pSG5). Transfection efficiencies were approximately 20%. Some samples were also cotransfected with wild-type Tat101 or TatΔ30-51 as indicated. Cells were harvested and assayed for luciferase activity. The values shown are averages of three independent samples, with the standard deviations represented by error bars. Full-length Tat (101 amino acids) enhanced NF-AT activation by vGPCR, but mutant Tat (TatΔ30-51) suppressed it. Treatment of cells with Tat protein yielded results similar to those obtained with Tat101 transfection (not shown). (c) Inhibition of Akt or constitutive activation of GSK-3 inhibited Tat's collaborative effects on NF-AT activation by vGPCR. KSIMM cells were transfected with an NF-AT luciferase reporter construct (100 ng) and 500 ng of vGPCR-pSG5 or a control vector. Transfection efficiencies were approximately 20%. Some samples were also cotransfected with wild-type Tat101. In parallel, Tat and vGPCR-transfected samples were cotransfected with 200 ng of DN Akt (AktK179M) or constitutively active GSK-3β [GSK-3(S9A)]. The values shown are averages from three independent samples, with the standard deviations represented by error bars.
FIG. 8.
FIG. 8.
Induction of NF-AT DNA binding activity in DMVECs expressing vGPCR. (a) DMVECs were transduced with retrovirus vGPCR-MIGR1 or a control (MIGR1) alone. Cells were harvested 3 days postinfection, and nuclear extracts were prepared and used in an EMSA as described in Materials and Methods. Lane 3 shows enhanced DNA binding of NF-AT in cells expressing vGPCR compared to lane 1, which shows the control vector. This enhanced binding in vGPCR-expressing cells was inhibited by addition of 1 μM CSA (lane 2). A mutant oligonucleotide (Oligo) failed to bind NF-AT (lane 7). An excess of the unlabeled wild-type (Wt.) oligonucleotide competed for NF-AT DNA binding with the labeled wild-type probe (lanes 8 and 9). As expected, the labeled wild-type probe with no nuclear extract did not bind (lane 6). Addition of extracellular Tat protein (100 ng/ml) to vGPCR-expressing cells for 1 h before harvest resulted in enhanced NF-AT DNA binding in cells expressing vGPCR (lane 5) in comparison with control cells treated with the same concentration of Tat (lane 4). Panel b shows the densitometric quantification of NF-AT1 binding in the five lanes as measured by ImageQuant software and a Phosphorimager (see Materials and Methods).
FIG. 9.
FIG. 9.
HIV-1 Tat and vGPCR collaborate to enhance phosphorylation of Akt and its downstream targets GSK-3α and -β. Lysates of HUT 78 cells transduced with vGPCR-MIGR1 or a control virus were analyzed by Western blotting (as described in Materials and Methods) with an antibody specific for phosphorylated Akt (serine 473) or phosphorylated GSK-3α and -β. Samples were treated with Tat protein at 50 ng/ml (designated vGPCR+ Tat and control+ Tat). Blots were stripped and reprobed with antibodies to total Akt and GSK-3β.
FIG. 10.
FIG. 10.
Schematic diagram of pathways activated by vGPCR that contribute to the activation of NF-AT. A model depicting the pathways activated by vGPCR that promote NF-AT activation is shown. GPCRs such as vGPCR activate calcium signaling through Gαq G proteins, which subsequently activate phospholipase C (PLC) and the downstream effectors diacylglycerol (DAG) and inositol triphosphate (IP-3). Inositol triphosphate mobilizes intracellular calcium, which activates the calcium/calmodulin-dependent phosphatase calcineurin. Calcineurin activates NF-AT, which leads to its nuclear translocation. PI3-K is also activated by vGPCR, which results in the activation of Akt and the inactivation of GSK-3 by phosphorylation. Since activated GSK-3 normally promotes the nuclear export of NF-AT, inactivation of GSK-3 by vGPCR blocks the nuclear export NF-AT. GSK-3 also affects NF-AT activity at the level of activation of AP-1 (dimers of fos and jun), a transcription partner of NF-AT. c-jun activity is normally inactivated by GSK-3 via phosphorylation, which is blocked by activation of the PI3-K pathway. Thus, GSK-3 represents a point of divergence of disparate signals that lead to activation of NF-AT by vGPCR. MAPK, mitogen-activated protein kinase.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Agwale, S. M., M. T. Shata, M. S. Reitz, Jr., V. S. Kalyanaraman, R. C. Gallo, M. Popovic, and D. M. Hone. 2002. A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type 1 Tat protein in mice. Proc. Natl. Acad. Sci. USA 99:10037-10041. - PMC - PubMed
    1. Albini, A., G. Barillari, R. Benelli, R. C. Gallo, and B. Ensoli. 1995. Angiogenic properties of human immunodeficiency virus type 1 Tat protein. Proc. Natl. Acad. Sci. USA 92:4838-4842. - PMC - PubMed
    1. Albini, A., I. Paglieri, G. Orengo, S. Carlone, M. G. Aluigi, R. DeMarchi, C. Matteucci, A. Mantovani, F. Carozzi, S. Donini, and R. Benelli. 1997. The β-core fragment of human chorionic gonadotropin inhibits growth of Kaposi's sarcoma-derived cells and a new immortalized Kaposi's sarcoma cell line. AIDS 11:713-721. - PubMed
    1. Antos, C. L., T. A. McKinsey, N. Frey, W. Kutschke, J. McAnally, J. M. Shelton, J. A. Richardson, J. A. Hill, and E. N. Olson. 2002. Activated glycogen synthase-3β suppresses cardiac hypertrophy in vivo. Proc. Natl. Acad. Sci. USA 99:907-912. - PMC - PubMed
    1. Aramburu, J., M. B. Yaffe, C. Lopez-Rodriguez, L. C. Cantley, P. G. Hogan, and A. Rao. 1999. Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285:2129-2133. - PubMed

Publication types

MeSH terms