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Human Herpesvirus 8-Encoded vGPCR Activates Nuclear Factor of Activated T Cells and Collaborates with Human Immunodeficiency Virus Type 1 Tat

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Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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.,

1

Oxana Barabitskaya,

1

Jynho Kim,

2

B. C. Nair,

3

David Hone,

1

Jennifer Smart,

2

Ricardo A. Feldman,

2

and Marvin Reitz

1,2

*

Institute of Human Virology, University of Maryland Biotechnology Institute,1and Department of Microbiology and Immunology,

University of Maryland,2Baltimore, Maryland 21201, and Advanced BioScience Laboratory Inc., Kensington, Maryland 208953

Received 25 November 2002/Accepted 11 February 2003

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-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 cyto-kines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidyl-inositol 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). 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-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 contrib-utes 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.

In recent years, an extensive body of evidence has identified human herpesvirus 8 (HHV-8), also known as Kaposi’s sar-coma (KS)-associated herpesvirus (19), as a necessary factor in the development of KS. HHV-8 is also implicated in the eti-ology of several B-cell proliferative disorders, including diffuse B-cell lymphoma (called primary effusion lymphoma) and mul-ticentric Castleman’s disease (17, 62), both of which occur most commonly in a setting of human immunodeficiency virus type 1 (HIV-1) coinfection. KS is a neoplasm of mixed cellu-larity in which lesions are primarily composed of characteristic spindle-shaped cells of endothelial origin. The lesions are highly vascularized and contain newly formed blood vessels and infiltrates of immune cells. Inflammatory cytokines, adhe-sion molecules, and endothelial cell activation all appear to be centrally involved in the process of KS pathogenesis (67).

Until the onset of the AIDS epidemic, KS was rare and occurred in three forms, classical (in elderly males of Mediter-ranean descent), endemic (in parts of Africa), and iatrogenic (in transplant patients) (79). Individuals dually infected with HHV-8 and HIV-1 have a greatly enhanced prevalence of KS compared with those infected with HHV-8 alone (13). Host immune suppression caused by HIV infection does not entirely

explain this increased presentation of KS, since HIV-2 AIDS is not associated with an increase in KS (6). AIDS KS has a more aggressive course than other forms of KS, including those associated with iatrogenic immunosuppression in transplant patients. Studies have suggested that HIV proteins, particu-larly the HIV-1 transactivator protein Tat, contribute directly to KS pathogenesis (36, 67). Tat is secreted from HIV-1-in-fected cells, is rapidly taken up by neighboring cells (34), and activates KS cell growth, (10, 16, 30). These findings suggest that HIV-1 could influence HHV-8-infected cells by a para-crine mechanism. Inflammatory cytokines that are elevated in HIV-1-infected individuals, particularly gamma interferon (IFN-␥), also enhance lytic-phase HHV-8 replication and the proliferation of KS cells (31, 32, 56). HHV-8 and HIV-1 can reciprocally upregulate the expression of each other’s gene products (54–56). These data suggest that HIV-1 and HHV-8 collaborate in the development of KS, although there has been little evidence at the molecular level showing how this might occur.

Open reading frame 74 of HHV-8 encodes a viral G protein-coupled receptor (vGPCR), a homologue of cellular chemo-kine receptors most closely related to the interleukin-8 (IL-8) receptor CXCR2. It is an early lytic-phase gene (46) that is detected in KS lesions at low levels (39). Only about 5% of the cells in a lesion express lytic-phase gene products (14, 73). In contrast to cellular chemokine receptors, vGPCR signals in the absence of added ligand (8), although it binds both CC (␤) and * Corresponding author. Mailing address: Institute of Human

Vi-rology, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201. Phone: (410) 708-4679. Fax: (410) 706-4694. E-mail: reitz@umbi .umd.edu.

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CXC (␣) chemokines (68). A few chemokines either enhance (Gro␣) or inhibit (IFN-␥-inducible protein 10) vGPCR-medi-ated signaling (37, 38, 68). The gene for vGPCR is an attractive candidate for a gene that could contribute to KS pathogenesis by both direct and indirect mechanisms. Chemokine receptors belong to a family of receptors coupled to heterotrimeric G proteins, and a number of human diseases, including some proliferative disorders, have been ascribed to the constitutive activity of mutated cellular GPCRs (52). HHV-8 vGPCR in-duces angiogenesis and vascular endothelial growth factor ex-pression in vitro and in vivo in mice (9, 72). Transgenic mice expressing vGPCR develop angioproliferative lesions with many of the characteristics of KS (40, 78). Interestingly, when the vGPCR transgene is expressed under the regulation of a T-cell-specific promoter, expression in the tumors is largely restricted to a relatively small number of infiltrating T cells, suggesting that paracrine mechanisms cause the KS-like le-sions (78).

vGPCR spontaneously activates multiple signaling ways, including the inositol phosphate/phospholipase C path-way and the downstream mitogen-activated protein kinases p38 and JNK/SAP (8, 58, 72). We and others have recently shown that vGPCR also activates the transcription factor NF-␬B via the phosphatidylinositol 3-kinase–Akt (PI3-K/Akt) pathway (64), resulting (58, 69, 71) in induction of the expres-sion of NF-␬B-dependent proinflammatory and proangiogenic cytokines, chemokines, and cell adhesion molecules. The sol-uble factors induced by NF-␬B further activate NF-␬B in neighboring cells not expressing vGPCR and promote the che-motaxis of monocytes and lymphocytes (64).

Genes are generally regulated by multiple transcription fac-tors acting in concert. This is the case with many of the so-called NF-␬B-dependent genes, although some factors may be more crucial for their expression than others. Many such cy-tokines and adhesion molecules, including IL-2, IL-4, IL-8, tumor necrosis factor␣(TNF-␣), granulocyte-macrophage col-ony-stimulating factor (GM-CSF), and Fas ligand, are also regulated by the transcription factor nuclear factor of activated T cells (NF-AT), which is expressed in many cell types, includ-ing endothelial cells, B cells, monocytes, and T cells (44, 66). These genes, along with other similar genes (IL-3, IL-5, IL-8, IFN-␥, CD40L, and E-selectin), have NF-AT binding sites in their promoters. NF-AT, originally identified as a nuclear com-plex binding to the antigen response element of the IL-2 gene (66), is regulated by calcium and is the target of the calcium/ calmodulin-dependent protein phosphatase calcineurin. Acti-vation of the calcium-calcineurin signaling cascade is triggered by engagement of T- and B-cell antigen receptors, Fc recep-tors, and GPCRs (66). This cascade is the target of drugs like cyclosporine and tacrolimus (FK506), which are used clinically to immunosuppress organ transplant recipients (28, 53, 70). When NF-AT is dephosphorylated by calcineurin, it translo-cates to the nucleus, where it activates transcription. NF-AT forms cooperative complexes with the transcription factor AP-1 (Fos/Jun) on composite NF-AT–AP-1 DNA elements (50). There are four calcium-regulated members of the NF-AT family: AT1 (ATp, ATc2), AT2 (ATc, NF-ATc1), NF-AT3 (NF-ATc4), and NF-AT4 (NF-ATc3) (50, 66). All have discrete but overlapping functions. NF-AT has been found in a number of different cell types other than T cells,

including B cells, natural killer cells, macrophages, mast cells, and endothelial cells (66).

Expression of vGPCR mobilizes intracellular calcium, which could activate the calcium-calcineurin signaling cascade and lead to activation of NF-AT. Interestingly, calcineurin-depen-dent signaling was found to be essential for the calcium-de-pendent reactivation of HHV-8 into lytic phase (80). We present the results of our studies, which indicate that vGPCR indeed activates NF-AT in T-lymphoid cells, primary endothe-lial cells, and a KS-derived endotheendothe-lial cell line (KSIMM) (3). Activation of NF-AT by vGPCR, as with NF-␬B, was depen-dent upon the PI3-K/Akt pathway. NF-AT activation by vGPCR depended upon inactivation of glycogen synthase ki-nase 3 (GSK-3) by Akt and resulted in induction of the ex-pression of NF-AT-dependent proinflammatory and proangio-genic cytokines, chemokines, and cell adhesion molecules. We also show that the HIV-1 Tat protein collaborates with vGPCR to synergistically activate NF-AT, providing some of the first evidence that Tat and vGPCR collaborate at the molecular level.

MATERIALS AND METHODS

Cell culture and retroviral infections. Dermal microvascular endothelial cells (DMVECs) were obtained from Cell Systems Corp. (Kirkland, Wash.). DMVECs were expanded and used for experiments between passages 4 and 5.

Cells were maintained in Cell Systems Corp. complete medium in a 5% CO2

humidified incubator. A. Albini (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) kindly provided KSIMM cells, an immortalized HHV-8-negative KS cell line derived from a patient with organ transplant-related KS (3). 293 and Jurkat T cells were obtained from the Viral Biology Core Facility, Institute of Human Virology, Baltimore, Md. Jurkat T cells, 293 cells, and KSIMM cells were maintained in RPMI medium (Life Technologies Inc., Gaithersburg, Md.) with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies Inc.) in a 5%

CO2humidified incubator. Penicillin G (100 U/ml) and streptomycin (100␮g/ml)

were added to all culture media.

Supernatants containing recombinant retrovirus were prepared from the Moloney murine leukemia virus-based retroviral packaging cell line Phoenix (Ampho) by transfection with the vector pvGPCR-MIGR1 (64) or pMIGR1 (kindly provided by W. Pear, University of Pennsylvania, Philadelphia) (65). Retrovirus preparations were added to the DMVECs, which were gently rocked for 1 h. Virus titer was determined by expression of green fluorescent protein (GFP), which was translated from an independent ribosomal entry site in the retroviral vector from the same cistron containing the inserted gene. Cells were refed 24 h postinfection with fresh medium. HUT 78 cells were obtained from the Viral Biology Core Facility, Institute of Human Virology, and cultured in RPMI medium with 10% heat-inactivated FBS. For retroviral infections, HUT

78 cells or DMVECs (passage 4 to 5) were treated with Polybrene (6␮g/ml) and

seeded into a 24-well plate. Supernatants containing the retrovirus preparations were added to the wells, and the plates were spun in a Beckman GH3.8 rotor at 2,000 rpm for 1 h. Supernatants were removed and cells were refed with super-natant containing retrovirus. Plates were spun again for 45 min. This process was done a third time, after which cells were refed with RPM1 medium–10% FBS, returned to the incubator, and analyzed 3 days postinfection. Endotoxin-free recombinant HIV-1 Tat (86 kDa) protein was obtained from Advanced Bio-science Laboratories Inc. (Rockville, Md.) and confirmed as endotoxin free

(⬍0.005 endotoxin units/ml) by a kinetic chromogenic assay (Kinetic-QCL;

Bio-Whittaker, Walkersville, Md.).

Plasmids and transfections.Cells were transfected with various plasmid con-structs by using Fugene 6 (Boehringer Mannheim, Indianapolis, Ind.) or Gene-porter (GTS, San Diego, Calif.) for Jurkat cells in accordance with the manu-facturer’s instructions. Transfection efficiencies were estimated by cotransfection

with an expression vector for ␤-galactosidase (pCMV-␤-Gal; Stratagene, La

Jolla, Calif.) or GFP (Green Lantern; Life Technologies, Gaithersburg, Md.).

Lysates were assayed for␤-galactosidase activity, and cells were counted for

reporter gene expression. The NF-␬B superrepressor mutant⌱␬␤␣32A/36A was

a kind gift from J. DiDonato (University of California, San Diego, School of Medicine, La Jolla, Calif.) (26). The dominant negative (DN) mutant form of AKT [AKT(K179M)] was obtained from the laboratory of M. Greenberg

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vard Medical School, Boston, Mass.) (27). The constructs vGPCR-MIGR1 and vGPCR-pSG5 were generated as described by Pati et al. (64). The Tat101 codon-optimized expression vector for Tat and the deletion mutant form

Tat⌬30-51 were described by Agwale et al. (1). The constitutively active GSK-3

(S9A) mutant form was the kind gift of Eric Olson (University of Texas, Hous-ton) (4). HA (hemagglutinin)–NF-AT1, HA–NF-AT2, HA–NF-AT4, and GFP-VIVIT plasmids were the kind gift of Anjana Rao (Center for Blood Research, Harvard University) (5). Photographs of 293 cells with these constructs and vGPCR were taken with a digital camera (DAGE-MTI, Madison, Wis.) attached to an inverted fluorescence microscope. The PI3-K inhibitor LY294002 was

added to the cell culture medium at final concentration of 10 and 20␮M, and the

medium was incubated for 4 to 6 h on day 1 posttransfection. Gro␣(100 nM;

R&D Systems, Minneapolis, Minn.) was added to the cell culture medium at 4 h prior to harvest.

Luciferase reporter gene assays.The luciferase reporters used were

Path-Detect cisreporting vectors from Stratagene. The NF-␬B reporter construct

expresses the firefly luciferase gene under the regulation of a synthetic promoter

containing five tandem binding sites for NF-␬B. The NF-AT luciferase reporter

construct contains four tandem composite binding sites for NF-AT/AP-1 from the IL-2 promoter. The AP-1 reporter construct contains seven tandem AP-1

binding sites. KSIMM cells were transfected by using Fugene 6 with 0.5␮g of

luciferase reporter construct, vGPCR-pSG5 or pSG5 alone, various DN mutant

constructs, and pCMV-␤-Gal. The amount of total DNA transfected was

equal-ized with the appropriate amounts of control vectors. Twenty hours posttrans-fection, cells were harvested and lysed in cell lysis buffer (a proprietary formu-lation from Promega [Madison, Wis.]). Protein concentration was normalized by bicinchoninic acid assay (Pierce Biochemicals, Rockford, Ill.). Luciferase activity was determined by using Luciferase Assay reagent (Promega) and a luminometer (Turner Designs, Sunnyvale, Calif.). Transfection efficiencies were normalized to

␤-galactosidase activity from the cotransfected pCMV-␤-gal plasmid.

Western blot analysis.Cells were harvested and lysed in lysis buffer (5% Triton X-100 in PBS, pH 7.4) with protease inhibitors (complete mini-leupeptin, apro-tinin, and Pefabloc; Boehringer Mannheim). Protein was quantified by bicincho-ninic acid assay (Pierce, St. Louis, Mo.). Protein was denatured at 70°C for 10

min in 1⫻LDS loading buffer (Invitrogen, Carlsbad, Calif.). Equal amounts of

protein were loaded into each well and electrophoresed on a 4 to 12% bis-Tris gel (NuPAGE; Invitrogen) on the NOVEX X-Cell II system (Invitrogen). Trans-fer to nitrocellulose membrane (Optitran from Schleicher & Schuell, Keene, N.H.) was performed in a NOVEX transfer system X-Cell II (Invitrogen). Trans-fer bufTrans-fer contained 25 mM Bicine, 25 mM bis-Tris, 1.025 mM EDTA, 0.05 mM chlorbutanol, and 15% methanol. Blots were blocked in PBS–5% nonfat dried milk. Membranes were probed with primary antibodies to phospho-Akt (ser473),

total Akt (Cell Signaling, Beverly, Mass.), phospho-GSK-3 (␣and␤), or total

GSK-3␤(Cell Signaling, Beverly, Mass.). Secondary antibody was conjugated

with horseradish peroxidase (Cell Signaling) and detected with an ECL detection system (Amersham Pharmacia Biotech, Piscataway, N.J.) in accordance with the manufacturer’s protocol.

Cytokine quantitation by ELISA.HUT 78 cells were transduced with vGPCR-MIGR1 or with vGPCR-MIGR1 alone. Cell counts were taken to ensure equality of cell numbers in different wells. Supernatants from equal numbers of cells were

collected 3 days postinfection and centrifuged (500⫻g, 15 min) to remove cell

debris. Cytokines in supernatants were quantified by using commercial enzyme-linked immunosorbent assay (ELISA) plates precoated with the appropriate antibodies (R&D Systems). Samples were added to plates, which were incubated at room temperature (RT) for 1 h. Plates were washed four times with PBS–0.5% Tween 20, and biotinylated anticytokine antibodies were added to the plates, which were incubated at RT for 1 h. After washing, streptavidin-horseradish peroxidase was added to the plates, which were incubated for 30 min, washed again, and then incubated with TMB substrate for 20 min. Reactions were

stopped with 2N H2SO4, and absorbance at 450 to 570 nm was determined. All

readings were taken in duplicate.

Flow cytometric analysis.HUT 78 cells were transduced with vGPCR-MIGR1

or with MIGR1 alone. Cells were harvested 3 days postinfection. Aliquots (0.5⫻

106) of transduced cells were washed twice in ice-cold PBS–2% FBS.

Appropri-ate antibodies were aliquoted into each tube and incubAppropri-ated on ice in the dark for 60 min. Cells were washed twice in PBS–2% FBS and fixed in 0.5 ml of 2% paraformaldehyde. Samples were analyzed with a Becton-Dickinson FACScali-bur flow cytometer. Phycoerythrin-conjugated antibodies for CD25 (IL-2R), CD49D, CD29, Fas ligand (FasL), and CD40L were obtained from Pharmingen (San Diego, Calif.). Appropriate isotype controls were used for each antibody as a control for nonspecific antibody binding.

EMSAs.DMVECs were transduced with vGPCR-MIGR1 or with MIGR1

alone. On day 1 posttransfection or day 3 postinfection, 107cells were harvested

and nuclear extracts were prepared. Cells were washed twice with ice-cold PBS– 0.1% BSA. Nuclear extracts were prepared in accordance with the manufactur-er’s instructions for the N-PER kit from Pierce Biochemicals. For gel shifts, 200 ng of oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, Calif.) was labeled

in 70 mM Tris (pH 7.6)–10 mM MgCl2–5 mM DTT with 100␮Ci of [␥-32P]dATP

and 5 U of T4 polynucleotide kinase and incubated at 37°C for 30 min. Labeled probe was purified on Elutip-D columns (Schleicher & Schuell) and precipitated

with 2 volumes of ethanol (⫺20°C, 1 h) after addition of 10␮g of glycogen and

MgCl2to 10 mM, washed 1⫻with 70% ethanol, air dried, and resuspended in

100 ␮l of Tris-EDTA, pH 7.5. Before electrophoretic mobility shift assay

(EMSA) reactions were set up, 1␮l was counted in a scintillation counter and

diluted to 200 to 800 cpm/␮l. Gels (5% acrylamide–0.5⫻Tris-borate-EDTA)

were polymerized and prerun in 0.5⫻Tris-borate-EDTA buffer at 200 V for 2 h

at 4°C. EMSA reaction mixtures were loaded and electrophoresed at 200 V for 2 to 2.5 h at 4°C. Gels were dried at 80°C (1.5 to 2.0 h) and imaged with a Storm Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). Nuclear extract (2 to

6␮g) and 200 to 800 cpm of oligonucleotide probe were combined in 10 mM

HEPES (pH 7.9)–50 mM KCl–0.1 mM EDTA–0.25 mM DTT–0.2␮g of

poly(dI-dC)–5% normal rabbit serum–10% glycerol and incubated at RT for 20 min.

Supershifts were done with 1␮g of NF-AT2 antibody (7A6) from Santa Cruz

Biotechnology. Gel shift bands were quantitated by ImageQuant software (Mo-lecular Dynamics).

Static cell adhesion assays.KSIMM cells were plated at 2⫻103per well of a

96-well plate. Twenty-four hours after seeding, cells were refed with serum-free

medium. HUT 78 cells were labeled with 0.1␮M Calcein AM (Molecular Probes,

Eugene, Oreg.) and an equal volume of Pluronic F-127 (Molecular Probes) at 37°C for 30 min. After being loaded, the HUT 78 cells were washed twice in assay medium and resuspended in serum-free medium. Various numbers of HUT 78 cells were added to the KSIMM cells and allowed to adhere for 45 min. Control HUT 78 (see Fig. 6a) or KSIMM (see Fig. 6b) cells were treated for 4 h with 5

ng of TNF-␣per ml and then mixed with KSIMM or HUT 78 cells, respectively,

as a positive control for adhesion. Cells were then washed with minimal shear force (74). Plates were subsequently read with a Wallac Victor-2 fluorescence plate reader (Wallac, Gaithersburg, Md.). Cell numbers were correlated with fluorescence readings to generate a standard curve. This experiment was carried out both for HUT 78 cells transduced with retrovirus vGPCR-MIGR1 and independently for KSIMM transfected with vGPCR-pSG5. Controls were MIGR1 and pSG5, respectively.

RESULTS

vGPCR activates NF-AT.Past studies have shown that some chemokine receptors can signal for NF-AT activation (66). Consequently, we asked whether this was the case with vGPCR. We first tested NF-AT activation by vGPCR in T-lymphoid (Jurkat) cells because T cells express high levels of NF-AT protein, making it easy to measure NF-AT activation. T cells of patients with HHV-8-related diseases are infected, although not abundantly (41, 42, 45; M. C. Sirianni, L. Vin-cenzi, S. Topino, E. Scala, A. Angeloni, R. Gonnella, S. Uccini, and A. Faggioni, Letter, J. Infect. Dis.176:541, 1997). Infected T cells could produce soluble factors (i.e., cytokines and che-mokines) and adhesion molecules that promote KS by para-crine mechanisms or by cell-to-cell contact, a concept sup-ported by past work by our group and others (64, 78). Cotransfection of Jurkat cells with vGPCR and an NF-AT luciferase reporter plasmid showed that vGPCR activated NF-AT in a dose-dependent fashion, as judged by NF-AT luciferase reporter gene expression (Fig. 1a). The activation was calcineurin dependent because it was suppressed by cyclo-sporine A (CSA), a highly specific calcineurin inhibitor. Acti-vation was enhanced by the addition of the chemokine Gro␣, a vGPCR agonist (38, 68). NF-AT was also activated by vGPCR in KSIMM cells (data not shown). All subsequent signaling studies with T cells were also performed with KSIMM or primary endothelial cells, and the results were consistent for all cell types.

Four of the five known NF-AT proteins are activated by cell

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surface receptors coupled to Ca2⫹ mobilization. These are

NF-AT1p, NF-AT2c, NF-AT3, and NF-AT4. To determine which forms of NF-AT were activated by vGPCR, we cotrans-fected expression constructs for AT1, AT2, and NF-AT4 (5) with vGPCR-pSG5 and an NF-AT luciferase reporter plasmid into KS (KSIMM) cells. We chose an early passage of KSIMM cells with a low baseline level of NF-AT activation by vGPCR so that our luciferase reading was primarily from the exogenously transfected NF-AT constructs (1, 2, or 4). We did not include NF-AT3 because it is primarily expressed only in cardiovascular tissue. NF-AT1 and NF-AT2, but not NF-AT4, were activated by vGPCR. The constructs all express NF-AT fusion proteins that include an influenza virus HA epitope and GFP, making it possible to visualize NF-AT activation by GFP localization in the nucleus, as shown in Fig. 1c (i to ii). The nuclear localization of NF-AT1 induced by vGPCR was similar to that induced by treatment with 10 mM CaCl2and ionomycin Fig. 1c (ii).

To further confirm a functional activation of NF-AT by vGPCR, NF-AT DNA binding was assessed by EMSA with an oligonucleotide containing a consensus NF-AT/AP-1 binding site as a probe. Nuclear extracts from primary DMVECs ex-pressing vGPCR showed increased protein binding to the NF-AT probe compared with extracts from cells transduced with the control (Fig. 2a). This increase was similar to that induced by treatment of the cells with ionomycin and CaCl2, potent inducers of NF-AT activity. Supershift analysis with an antibody against AT2 (7A6) revealed the presence of NF-AT2 in the shifted complex. NF-AT DNA binding in vGPCR-expressing cells was inhibited by CSA and by cotransfection of a plasmid expressing the peptide VIVIT, which competes with NF-AT for calcineurin binding and selectively inhibits NF-AT activation without disrupting other calcineurin-dependent pathways (5). Figure 2b shows a densitometric quantification of the bands that represent NF-AT DNA binding.

Since vGPCR activates both NF-AT and NF-␬B, one ques-tion was, to what extent are the two pathways independent? To answer this, we asked whether inhibition of NF-␬B by a super-repressor mutant form of I␬B, which is a DN inhibitor of NF-␬B (26), would also block NF-AT activation. In the exper-iment whose results are shown in Fig. 3a, KSIMM cells were transfected with NF-AT luciferase or NF-␬B luciferase re-porter gene constructs and either vGPCR or a control vector. Parallel samples were cotransfected with the DN I␬B con-struct. Figure 3a shows that inhibition of NF-␬B activation by DN I␬B did not affect the activation of NF-AT. The converse was also true. KSIMM cells were transfected as in Fig. 3a but with VIVIT instead of DN I␬B. Figure 3b shows that inhibition of NF-AT activation by VIVIT did not affect activation of NF-␬B by vGPCR.

Our past studies showed that vGPCR activates the PI3-K/ Akt pathway, which is essential for NF-␬B activation by 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-␤-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 ␤-galactosidase activity 24 h after transfection. Some samples were treated with 1␮M CSA or 100 nM Gro␣4 h prior to harvest. Luciferase values are expressed as relative light units (RLU) and normalized for␤-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 detect-able 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 (vGPCR-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 CaCl2and ionomycin (1␮M) (ii) but different from that in control vector-transfected cells (iii). Cells transfected with a consti-tutively 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.

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vGPCR (58, 64, 71). To see whether the PI3-K/Akt pathway is involved in the activation of NF-AT as well, we used the PI3-K inhibitor LY294002 or a DN Akt mutant, Akt(K179M) (27). In KSIMM cells expressing vGPCR, both treatment with LY294002 and cotransfection of Akt(K179M) inhibited NF-AT activation (Fig. 3c), indicating that activation of NF-AT by vGPCR depends on signaling through PI3-K/Akt. Since there is evidence that calcineurin can also directly stimulate Akt (25), we asked whether CSA inhibited phosphorylation of Akt induced by vGPCR and found that it did not (data not shown). NF-AT activity is down-regulated by phosphorylation by gly-cogen synthetase kinase 3 (GSK-3), which is itself down-regu-lated by phosphorylation by Akt (12, 22). Dephosphorydown-regu-lated GSK-3 facilitates the export of NF-AT from the nucleus to the cytoplasm. To achieve higher efficiencies of gene expression, HUT 78 cells were transduced with a retrovirus expressing vGPCR as described in Materials and Methods. Lysates were analyzed by immunoblotting with antibodies against phosphor-ylated and total Akt and GSK-3. Consistent with the idea that vGPCR promotes NF-AT activation through the inactivation of GSK-3 by Akt, HUT 78 cells expressing vGPCR showed increased phosphorylation of GSK-3␣, GSK-3␤, and Akt (Fig. 3d). The increase in GSK-3 and Akt phosphorylation was

pre-vented by LY294002, further suggesting that PI3-K/Akt-medi-ated activation of NF-AT by vGPCR involves GSK-3.

AP-1 and NF-AT are partners in transcription and can bind cooperatively to composite DNA sites in promoter-enhancer regions. Since there is evidence that AP-1 activation can also be down-regulated by GSK-3 via phosphorylation of c-jun (63), we asked whether the PI3-K/Akt pathway is involved in the activation of AP-1 by vGPCR. KSIMM cells were transfected with an AP-1 luciferase reporter construct and either vGPCR or the control vector. Parallel samples were treated with the PI3-K inhibitor LY294002 or cotransfected with DN Akt(K179M). Figure 3e shows that inhibition of the PI3-K/Akt pathway indeed inhibits AP-1 activation by vGPCR, suggesting that phosphorylation of GSK-3 in cells expressing vGPCR leads to activation of both NF-AT and AP-1.

To further establish the role of GSK-3 in the activation of NF-AT by vGPCR, KSIMM cells were transiently transfected with NF-AT1, an NF-AT luciferase reporter gene, and either vGPCR or a control vector. In parallel, samples were treated with LiCl (a 3 inhibitor [59]) or cotransfected with GSK-3(S9A) (a constitutively active mutant form of GSK-3 that has a serine-to-alanine substitution at amino acid position 9 and cannot be inactivated by phosphorylation [4]). Consistent with a role for GSK-3 inactivation, constitutively active GSK-3 sup-pressed NF-AT activation by vGPCR and LiCl enhanced it (Fig. 3f). This finding was confirmed by visualization of the cytoplasmic location of NF-AT1 in cells transfected with vGPCR and GSK-3(S9A) (Fig. 1c [iv]). The constitutively ac-tive mutant form of GSK-3 partially inhibited NF-␬B activa-tion by vGPCR and completely inhibited AP-1 activaactiva-tion (Fig. 3g), confirming that GSK-3 is involved in multiple signaling pathways originating with vGPCR.

vGPCR expression in T cells induces the expression of NF-AT-dependent cytokines.Sequence inspection, in vitro binding assays, and ectopic expression studies have identified cytokine genes that are regulated by NF-AT, including those for IL-2, IL-4, IL-8, IFN-␥, GM-CSF, and TNF-␣ (Table 1) (66). To determine whether vGPCR affects expression levels of NF-AT-dependent cytokines, HUT 78 cells were transduced with a retroviral vGPCR vector or a control. Expression of vGPCR in HUT 78 T cells indeed stimulated production of IL-2, IL-4, GM-CSF, and TNF-␣ as determined by ELISA of superna-tants from these cells. In contrast, IFN-␥expression was un-changed (data not shown). Cytokine levels were further in-creased by addition of the vGPCR agonist Gro␣ (data not shown).

vGPCR expression in HUT 78 cells induces the expression of cell surface activation markers and adhesion molecules.

[image:5.603.44.282.67.296.2]

Cell adhesion molecules that are subject to regulation by NF-AT and NF-␬B could mediate interactions among different cell types within KS lesions. To determine whether expression of vGPCR in HUT 78 T cells induces expression of T-cell-specific activation markers and adhesion molecules, we ana-lyzed their expression by flow cytometry by using a retroviral construct expressing GFP from the same cistron as vGPCR and providing a marker with which to identify transduced cells. Approximately 30% of the cells infected with the vGPCR or control retrovirus were GFP positive (Fig. 4a). vGPCR-trans-duced cells, detected by GFP expression, had significantly in-creased levels of cell surface ICAM-1 (Fig. 4b) and CD25 (IL-2 FIG. 2. Enhancement of NF-AT1 DNA binding activity in

endo-thelial cells expressing vGPCR. (a) DMVECs were transduced with MIGR1 or vGPCR-MIGR1. Cells were harvested 24 h posttransfec-tion, 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␮M 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␮M) 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 Phosphor-Imager (see Materials and Methods).

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[image:6.603.50.530.82.494.2]

FIG. 3. NF-AT activation by vGPCR and NF-␬B activation by vGPCR are independent. (a) KS cells (105) were transfected with either an NF-␬B or an NF-AT reporter construct and combinations of vGPCR-pSG5, control (ctrl) vector (pSG5), and DN I␬B (0.1 and 0.2␮g). Inhibition of NF-␬B by DN I␬B did not affect NF-AT activation by vGPCR but did inhibit NF-␬B activation. (b). KS cells were transfected with either an NF-␬B 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-␬B 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␮g]. Parallel samples expressing vGPCR were treated with the PI3-K inhibitor LY294002 (10 and 20␮M 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-3␣and -␤. 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-3␤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␮M) 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␮g of DN Akt(K179M) or treated with the PI3-K inhibitor LY294002 (10 and 20␮M) 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-␬B, and AP-1 activation by vGPCR. KSIMM cells (105) were transfected with an NF-AT luciferase (f), AP-1 luciferase (g), or NF-␬B luciferase (g) reporter construct (100 ng) and 500 ng of vGPCR-pSG5. The indicated samples were cotransfected with 0.4␮g 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.

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receptor) (Fig. 4c) and modestly increased expression of CD29 (Fig. 4d) and Fas ligand (Fig. 4e). There was no appreciable change in CD49d (VLA-4) or CD40 ligand (data not shown). Transduction of GFP with control retrovirus did not change the expression levels of any of these cell surface markers (not shown). Interestingly, we noted morphological changes in cul-tures of HUT 78 T cells transduced with vGPCR, as evidenced by large aggregates of cells readily visible to the naked eye in the population expressing vGPCR. Figure 5a shows these ho-motypic cell aggregates of vGPCR-expressing cells but not of cells infected with the control vector (Fig. 5b).

Expression of vGPCR enhances T-cell–KS cell adherence.

To determine if the enhanced expression of cell surface mark-ers is physiologically relevant, we used a static binding assay (see Materials and Methods) to measure differences in the adhesion of HUT 78 cells expressing vGPCR to KS cells. Tumor-infiltrating lymphocytes and monocytes found in KS lesions may enhance lesion growth through the production of soluble cytokines and growth factors (35) and can also be infected with HHV-8, potentially representing a source for introduction of virus into KS lesions. Figure 6a shows that expression of vGPCR in HUT 78 T cells enhanced their ad-herence to KS cells relative to that of the control. This increase in adhesion was equivalent to that of control HUT 78 cells treated with TNF-␣ as a positive control. The enhanced ad-herence was partially NF-AT dependent, since pretreatment with CSA partially inhibited the enhanced binding. Pretreat-ment of the HUT 78 cells with the PI3-K inhibitor LY204002, which inhibits activation of both NF-␬B and NF-AT by vGPCR, completely inhibited the enhanced ability of HUT 78 cells to adhere to KS cells. This suggests that enhanced adher-ence depends on signaling through the PI3-K/Akt pathway.

HUT 78 T cells expressing vGPCR demonstrated enhanced adherence to KS cells; the converse was true as well. We transfected KSIMM cells with vGPCR or a control vector (Fig. 6b). Past studies showed that vGPCR expression in KS cells upregulates several adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, that mediate interactions with im-mune cells (35, 64). Interestingly E-selectin expression on en-dothelial cells is dependent not only on NF-␬B activation but also on NF-AT activation (21). Figure 6b shows that expression of vGPCR in KS cells indeed resulted in an increase in adhe-sion to HUT 78 T cells. This increase in adheadhe-sion was similar to that of control KSIMM cells treated with TNF-␣as a pos-itive control. Pretreatment with CSA or LY204002 inhibited the enhanced binding to HUT 78 cells, suggesting that genes regulated by NF-AT and by the PI3-K/Akt pathway are

in-volved. Thus, not only do HUT 78 T cells expressing vGPCR show enhanced binding to KS cells, as seen in Fig. 6a, but KS cells expressing vGPCR also show enhanced binding to HUT 78 T cells (Fig. 6b). Similar interactions between HHV-8-infected immune cells and KS cells are likely to occur within the KS microenvironment and may help promote lesion devel-opment.

HIV-1 Tat synergistically enhances NF-AT activation by vGPCR. As mentioned previously, the prevalence of KS is much higher in HIV-1-coinfected individuals than in those infected with HHV-8 alone, suggesting a collaborative effect between the two viruses in promoting KS. HIV-1 Tat is se-creted by HIV-infected cells and can be taken up by uninfected cells. Tat enhances KS spindle cell growth, proliferation, and migration (2, 29) and can activate signal transduction path-ways, including NF-␬B and Akt (15, 23, 24). We therefore asked whether Tat can collaborate with vGPCR to enhance activation of NF-AT or NF-␬B. This was true for both path-ways, as shown in Fig. 7a and b. NF-␬B and NF-AT activation by vGPCR, measured by luciferase activity, was synergistically enhanced by Tat. Results similar to those obtained with Tat protein were obtained when a Tat expression vector (Tat101) (1) was cotransfected with vGPCR. NF-AT DNA binding, measured by EMSA, was increased in primary endothelial cells (DMVECs) expressing vGPCR when treated with Tat protein, as shown in Fig. 8.

To begin to delineate which region of Tat is important for its ability to collaborate with vGPCR signaling, we used a deletion mutant form of Tat101 in which amino acids 30 to 51 have been deleted, designated Tat⌬30-51 (1). The deletion removes a cysteine-rich region in Tat, eliminates its ability to transacti-vate the HIV-1 long terminal repeat, and eliminates some, but not all, of its immunosuppressive properties in mice (1). Tat⌬30-51 could still moderately stimulate vGPCR-mediated NF-␬B signaling (data not shown). Surprisingly, Tat⌬30-51 had the opposite effect of wild-type Tat on NF-AT signaling by vGPCR; it was strongly inhibitory (Fig. 7b).

HIV-1 Tat enhances the vGPCR-dependent phosphorylation of Akt and GSK-3.The mechanism of stimulation of vGPCR-dependent signaling by Tat is unclear. By Western blotting, we confirmed that Tat does not increase the expression of vGPCR (not shown). One possibility is that it increases vGPCR signal-ing activity through the PI3-K/Akt pathway. Tat alone can activate Akt in serum-starved Jurkat T cells and in vincristine-treated KS cells (15, 24). Figure 7c shows that, indeed, inhibi-tion of Akt or constitutive activainhibi-tion of GSK-3 inhibited Tat’s collaborative effects on NF-AT activation by vGPCR. Since GSK-3 is a downstream target of Akt, we asked whether Tat’s augmentation of vGPCR-dependent NF-AT activity is associ-ated with increases in the phosphorylation of Akt or GSK-3. We found that exogenous Tat protein does collaborate with vGPCR to activate the phosphorylation of both Akt and its downstream substrate, GSK-3 (Fig. 9). There was no clear enhancement of GSK-3 phosphorylation in cells treated with Tat alone; the contributions to the phosphorylation of GSK-3 by Tat and vGPCR thus appear to be synergistic.

DISCUSSION

[image:7.603.44.283.91.162.2]

Past studies by our group and others have shown that vGPCR constitutively activates NF-␬B via Akt (58, 64, 71), TABLE 1. Effect of vGPCR expression in T cells on expression of

NF-AT-dependent cytokinesa

Cytokine Level (pg/ml)

Control vGPCR

IL-2 20 800

IL-4 2 11

GM-CSF 112 520

TNF-␣ 8 95

aHUT 78 cells were transduced with vGPCR-MIGR1 or a control virus.

Supernatants were harvested 3 days postinfection, and cytokine levels were determined by ELISA as described in Materials and Methods.

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[image:8.603.45.535.102.637.2]

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).

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resulting in the expression of NF-␬B-dependent inflammatory cytokines, chemokines, and adhesion molecules that could pro-mote KS pathogenesis by paracrine mechanisms (64, 69). Ex-pression of these inflammatory proteins generally depends on more than one transcription factor, although NF-␬B often plays a crucial role. Expression of many inflammatory cyto-kines and adhesion molecules also depends on the transcrip-tion factor NF-AT, and since other GPCRs mobilize calcium and activate NF-AT, we thought vGPCR likely to also activate NF-AT. We hypothesized that this would result in elevated expression levels of NF-AT-dependent factors that could con-tribute to the development of KS by both paracrine and auto-crine mechanisms.

To carry out these studies with relevant cell types, we used KSIMM (an HHV-8 negative KS endothelial cell line) (3),

[image:9.603.303.542.70.309.2]

primary human endothelial cells (DMVECs), and HUT 78 cells (a T-lymphoid cell line). Our studies indicated that vGPCR indeed activates NF-AT in all three cell types in the absence of added ligand. vGPCR specifically activated NF-AT1 and -2 but not NF-AT4. NF-NF-AT1 is present in most resting immune cells, whereas NF-AT2 is present mostly in activated cells (66). Expression of vGPCR transactivated an NF-AT-dependent promoter and induced NF-AT binding to DNA sites, as judged by luciferase activity and gel shift assays, respectively. Activation of NF-AT by vGPCR was inhibited by CSA, an inhibitor of the NF-AT-activating protein calcineurin. FIG. 5. HUT 78 cells expressing vGPCR form homotypic

aggre-gates. HUT 78 cells were transduced with the retrovirus vGPCR-MIGR1 or a control (vGPCR-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. Expression of vGPCR in HUT 78 T cells results in en-hanced 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, 1106, 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⫻106cells. For these samples, HUT 78 cells were pretreated as indicated with CSA (1␮M) or LY294002 (20␮M) 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-␣ 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 con-trol. 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, 1106, and 0.5106) 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-␣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 cor-related with cell numbers. Each value is the result of readings from eight wells. KS cell samples were pretreated as indicated with 1␮M CSA or 20␮M LY294002 for 1 h prior to seeding with the HUT 78 cells. Pretreatment was only performed with 2⫻106cells.

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The activation of NF-AT may be important to HHV-8 lytic-phase replication, since recent work by Zoetweweij et al. (80) has shown that calcineurin-dependent signaling is essential for the calcium-dependent reactivation of HHV-8 into lytic phase. Another HHV-8 lytic-phase gene (K1) also activates NF-AT through the presence of an ITAM-like sequence in its cyto-plasmic tail (47–49).

Our previous studies and those of others have shown that Akt is a critical factor in NF-␬B activation by vGPCR (58, 64, 71). With an I␬B mutant superrepressor of NF-␬B activation and the VIVIT peptide, a specific NF-AT inhibitor, we showed that the vGPCR activation pathways for the two factors branch at some point; neither depends on activation of the other. The activation of NF-␬B and NF-AT does, however, appear to be linked at the level of PI3-K and Akt, since inhibition of PI3-K or Akt inhibits both pathways. PI3-K activation by cell surface receptors has been implicated in a number of cellular

func-tions. Some GPCRs, including the IL-8 receptor (CXCR2), activate PI3-K and its downstream target kinase Akt through G protein␤-␥dimers (61, 75). Akt, in turn, can regulate cell sur-vival, glycogen metabolism, and cellular transformation (27, 33). Involvement of the PI3-K/Akt pathway in vGPCR-mediated NF-AT activation is consistent with reports that the nuclear export of NF-AT is regulated by GSK-3 (12), a downstream target of Akt (22). GSK-3␣and -␤are closely related protein-serine kinases that act as inhibitory components of Wnt (60) and promote the nuclear export of NF-AT by phosphorylation (22). Consistent with the idea that GSK-3 is an important intermediate between Akt and NF-AT in vGPCR signaling, a constitutively active mutant form of GSK-3 [GSK-3(S9A)] sup-pressed NF-AT activity induced by open reading frame 74. In contrast, LiCl, a GSK-3 inhibitor (59), enhanced NF-AT ac-tivity under the same conditions. Further support for the in-volvement of GSK-3 is provided by the increase in phosphor-FIG. 7. HIV-1 Tat synergistically enhances the activation of both NF-␬B and NF-AT by vGPCR. (a) KSIMM cells were transfected with 100 ng of a NF-␬B 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-␤-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-␬B 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 Tat⌬30-51 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 (Tat⌬30-51) 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-3␤[GSK-3(S9A)]. The values shown are averages from three independent samples, with the standard deviations represented by error bars.

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ylation of GSK-3␣and -␤in cells expressing vGPCR. This, at least in part, appears to explain the enhancement of NF-AT activity by vGPCR. There is evidence that GSK-3 can also regulate NF-␬B activity at the level of the transcriptional com-plex (43). We found partial inhibition of vGPCR-dependent NF-␬B activation by constitutively active GSK-3, but the inhi-bition was much less dramatic than that of NF-AT.

The effect of vGPCR on NF-AT activity may also be due to enhancement of AP-1 activation via PI3-K/Akt, since inhibi-tion of this pathway also inhibited AP-1 activainhibi-tion by vGPCR. This would perhaps be expected, since GSK-3 phosphorylates c-jun, one of the components of AP-1, and inhibits its activity (63). Consistent with this, we found that expression of con-stitutively active GSK-3(S9A) inhibited AP-1 activation by vGPCR, suggesting that AP-1 activation by vGPCR also occurs through the PI3-K/Akt/GSK-3 pathway. NF-AT-mediated transcription, in most cases, requires concomitant activation of NF-AT and AP-1 (50), although it is important to note that

activation of AP-1 alone is not sufficient (50). vGPCR activa-tion of NF-AT most likely depends on both a positive contrib-utory pathway that activates calcineurin and AP-1 and inacti-vation of an inhibitory factor, GSK-3. vGPCR inactiinacti-vation of GSK-3 likely increases NF-AT activity induced by vGPCR at two levels, one by blocking the nuclear export of NF-AT and the other by increasing AP-1 activity by blocking the nuclear export of c-jun. Figure 10 is a schematic diagram illustrating these potential pathways of NF-AT activation by vGPCR.

[image:11.603.47.283.67.323.2]

The dynamics and interactions between different cell types within KS lesions are obviously quite complex. KS lesions con-sist of a mixed population of immune cells, such as lympho-cytes, monolympho-cytes, endothelial cells, and spindle-shaped KS cells of endothelial cell origin. All can be infected with HHV-8 and are likely to be relevant to KS pathogenesis. The immune cells in KS lesions may stimulate KS cell growth by binding to KS cells and activating them and/or by secreting factors that promote KS cell growth by paracrine mechanisms. Direct con-tact of T cells with the endothelium can activate endothelial cells by inducing adhesion molecule expression and cytokine release (57). Others have shown that expression of vGPCR in immune cells may lead to the upregulation of NF-AT-depen-dent cytokines, such as IL-2 and IL-4 (69). We found that expression of vGPCR in HUT 78 T cells led to increases in NF-AT-dependent cytokines and cell surface markers, which are of clearly established relevance to KS pathogenesis (31, 32, 35). The enhanced activation of factors by vGPCR translated 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␮M 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 compar-ison 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 Phosphor-imager (see Materials and Methods).

FIG. 9. HIV-1 Tat and vGPCR collaborate to enhance phosphor-ylation of Akt and its downstream targets GSK-3␣and -␤. 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 phos-phorylated GSK-3␣and -␤. 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-3␤.

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into a functional increase in KS cell–T-cell adhesion. The in-crease was eliminated by inhibition of PI3-K and calcineurin, thereby indicating the importance of these pathways in medi-ating these interactions.

The increased expression of Fas ligand by vGPCR-express-ing cells is of interest. Although the data conflict somewhat, several tumor types have recently been shown to express Fas ligand; this may represent a mechanism of tumor escape from the immune system (77). Similarly, increased expression of Fas ligand by vGPCR may be an immune escape mechanism for cells infected with HHV-8.

In terms of the contribution of HIV-1 to KS, our findings suggest that there are direct collaborative effects at the molec-ular level between HIV-1 Tat and HHV-8 vGPCR. Tat is an attractive candidate for an HIV-1 protein that not only may enhance KS cell growth directly (29) but also may collaborate with HHV-8 to promote KS pathogenesis. Tat is secreted from HIV-1-infected cells and Tat-transfected cells by a leaderless secretory pathway (18, 30). A highly basic tract of 11 amino acids mediates uptake into uninfected cells. Several properties

of Tat may promote KS pathogenesis (11, 20). Tat affects signal transduction pathways, including those that lead to the activa-tion of NF-␬B and Akt (15, 23, 24). Addition of extracellular Tat protein to cells or cotransfection of a Tat expression vector (Tat101) into cells enhanced NF-AT activation by vGPCR, whereas a deletion mutant form of Tat (Tat⌬30-51) strongly suppressed activation of NF-AT by vGPCR. This suggests that the cysteine-rich amino acid 30 to amino acid 51 domain of Tat, which is important fortransactivation of viral RNA elon-gation, may also be important for activation of NF-AT in collaboration with vGPCR. Results obtained with transfected Tat and exogenously added Tat protein were substantially sim-ilar, consistent with previous reports that Tat enters and exits cells readily (18, 34). Tat alone did not affect NF-AT activity. Tat-mediated enhancement of NF-AT activation was blocked by the DN mutant form Akt(K179M), confirming that en-hancement of NF-AT activity by Tat also requires signaling through the PI3-K/Akt pathway and suggesting that the effect of Tat is mediated further upstream. Stimulation of NF-AT activation by Tat also likely involves GSK-3, since Tat en-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 G␣qG 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.

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hanced the phosphorylation of GSK-3␣and -␤by vGPCR. Our finding that constitutively active GSK-3 suppressed NF-AT activity induced by Tat and vGPCR further supports the hy-pothesis that GSK-3 is a critical component of the activation pathway stimulated by Tat.

Others have shown that Tat can enhance NF-AT transcrip-tional activity by binding to NF-AT1 (51). This is likely not the case in our studies, since we did not see an NF-AT band of altered mobility by EMSA in Tat-treated samples but, instead, saw an enhanced signal from the band already present. In addition, the enhancement of Akt phosphorylation by Tat lends further support to the idea that Tat acts upstream from Akt. In our studies, we found that Tat alone did not generally affect NF-AT activity, although in some passages of Jurkat T cells, we did see some activation by Tat alone. This could possibly be attributed to differences in the basal level of acti-vation of the cells. Vacca et al. have shown that Tat activates NF-AT in cells pretreated with phorbol ester and ionomycin (76). This suggests that preactivation of cells by other factors (e.g., vGPCR) is required for Tat-mediated augmentation of NF-AT activity and is consistent with the hypothesis that Tat augments NF-AT transcriptional activity by inhibiting GSK-3 and blocking NF-AT export from the nucleus. The mechanism of Tat-mediated enhancement of NF-AT obviously merits fur-ther study.

In summary, we have shown that the HHV-8 lytic-phase gene vGPCR activates the transcription factor NF-AT in KS cells, HUT 78 T-lymphoid cells, and primary endothelial cells. The activation of NF-AT is very likely relevant to KS patho-genesis, since NF-AT has been shown to be important not only for regulation of the inflammatory response of B and T cells but also for mediation of endothelial cell activation and angio-genesis (7). Activation of NF-AT by vGPCR is not dependent upon activation of NF-␬B, but activation of the two factors is interconnected at the level of PI3-K and Akt and appears to partly diverge at the level of GSK-3. Expression of vGPCR resulted in the expression of NF-AT-dependent cytokines and cell surface markers, consistent with a paracrine model of KS pathogenesis in which infected cells can affect neighboring through the release of soluble factors (64). We also noted a functional increase in KS cell–T-cell adhesion, an interaction likely to contribute to KS pathogenesis. This enhanced adhe-sion was also dependent on calcineurin and PI3-K, since it was partially inhibited by the specific inhibitors CSA and LY294002. Tat synergistically enhanced the activation of NF-AT and NF-␬B by vGPCR, which provides the first evi-dence that HIV-1 Tat collaborates with HHV-8 vGPCR at the molecular level. This synergy could possibly translate into en-hancement of KS pathogenesis by the two viral gene products.

ACKNOWLEDGMENTS

This work was supported by National Cancer Institute grants P01 CA78817 to M.R. and RO1 CA55293 to R.A.F. S.P. was supported by a predoctoral training grant from the National Cancer Center.

We gratefully acknowledge the editorial assistance of Paula Dean.

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Figure

Fig. 1c (ii).
FIG. 2. Enhancement of NF-AT1 DNA binding activity in endo-thelial cells expressing vGPCR
FIG. 3. NF-AT activation by vGPCR and NF-vGPCR-pSG5. The indicated samples were cotransfected with 0.4of NF-NF-NF-AT inhibitory peptide VIVIT
TABLE 1. Effect of vGPCR expression in T cells on expression ofNF-AT-dependent cytokinesa
+5

References

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