Cancer Research Piracy of Prostaglandin E2/EP Receptor ......with noncytotoxic concentrations (Supplementary Fig. S1A– I) of well-characterized competitive EP receptor blockers SC-51322

Published OnlineFirst April 13, 2010; DOI: 10.1158/0008-5472.CAN-09-3934

Therapeutics, Targets, and Chemical Biology
Cancer
Research

Piracy of Prostaglandin E2/EP Receptor–Mediated Signalingby Kaposi's Sarcoma-Associated Herpes Virus (HHV-8)for Latency Gene Expression: Strategy of aSuccessful Pathogen

Arun George Paul1, Neelam Sharma-Walia1, Nagaraj Kerur1, Carl White2, and Bala Chandran1

Abstract

Authors' ADepartmenPhysiologyUniversity o

Note: SupResearch O

Corresponand Immunof MedicinIL 60064.chandran@

doi: 10.115

©2010 Am

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Kaposi's sarcoma-associated herpes virus (KSHV) is implicated in the pathogenesis of KS, a chronicinflammation-associated malignancy. Cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2(PGE2), two pivotal proinflammatory/oncogeneic molecules, are proposed to play roles in the expressionof major KSHV latency-associated nuclear antigen-1 (LANA-1). Microsomal PGE2 synthase, PGE2, and itsreceptors (EP1, EP2, EP3, and EP4) were detected in KS lesions with the distinct staining of EP2/EP4 inKS lesions. In latently infected endothelial TIVE-LTC cells, EP receptor antagonists downregulated LANA-1expression as well as Ca2+, p-Src, p-PI3K, p-PKCζ/λ, and p-NF-κB, which are also some of the signalmolecules proposed to be important in KS pathogenesis. Exogenous PGE2 and EP receptor agonistsinduced the LANA-1 promoter in 293 cells, and YY1, Sp1, Oct-1, Oct-6, C/EBP, and c-Jun transcriptionfactors seem to be involved in this induction. PGE2/EP receptor-induced LANA-1 promoter activitywas downregulated significantly by the inhibition of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, and p-NF-κB. Thesefindings implicate the inflammatory PGE2/EP receptors and the associated signal molecules in herpesvirus latency and uncover a novel paradigm that shows the evolution of KSHV genome plasticity touse inflammatory response for its survival advantage of maintaining latent gene expression. These dataalso suggest that potential use of anti-COX-2 and anti-EP receptor therapy may not only ameliorate thechronic inflammation associated with KS but could also lead to elimination of the KSHV latent infectionand the associated KS lesions. Cancer Res; 70(9); 3697–3708. ©2010 AACR.

Introduction

Kaposi's sarcoma-associated herpes virus (KSHV; HHV-8)is etiologically associated with KS, the most common and ag-gressive AIDS-defining malignancy (1–3). KS is characterizedby a proinflammatory microenvironment (1–3). Thus, unra-veling the biology of KSHV pathogenesis and, therefore, KS isclosely tied to understanding the chronic inflammatoryconditions that set the stage for KS.Previous reports had shown that cyclooxygenase-2 (COX-

2), a proinflammatory molecule, was highly upregulated

ffiliations: 1H.M. Bligh Cancer Research Laboratories,t of Microbiology and Immunology and 2Department ofand Biophysics, Chicago Medical School, Rosalind Franklinf Medicine and Science, North Chicago, Illinois

plementary data for this article are available at Cancernline (http://cancerres.aacrjournals.org/).

ding Author: Bala Chandran, Department of Microbiologyology, Chicago Medical School, Rosalind Franklin Universitye and Science, 3333 Green Bay Road, North Chicago,Phone: 847-578-8822; Fax: 847-578-3349; E-mail: bala.rosalindfranklin.edu.

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in vitro by KSHV infection (4, 5). The tumorigenic propertiesof COX-2 are attributed to its metabolite prostaglandin E2(PGE2) that exerts its effect through eicosonoid (EP) recep-tors (EP1–EP4; refs. 6–12). COX-2 inhibition significantlyabrogated expression of the major KSHV latent gene laten-cy-associated nuclear antigen-1 (LANA-1) during de novoKSHV infection of fibroblast (HFF) and endothelial(HMVEC-d) cells, and exogenous PGE2 reversed this down-regulation (5). These studies have indicated that COX-2/PGE2–mediated inflammation is crucial for KSHV latencyprogram. Although, the role of COX-2 and PGE2 in herpesviral lytic cycle is shown, their role in viral latency has beenobserved only in KSHV.However, the mechanistic aspects of how COX-2/PGE2

mediates KSHV latent gene expression is not known andthe role of EP receptors is unexplored in herpes virus bio-logy. Our study shows that Ca2+, Src, phosphoinositide 3-kinase (PI3K), PKCζ/λ, and NF-κB signal molecules areregulated by EP receptors in latently infected cells and block-ing EP receptors downregulated LANA-1 and COX-2 geneexpression. PGE2 stimulated the LANA-1 promoter via a net-work of Ca2+, Src, PI3K, PKCζ/λ, and NF-κB activation. Col-lectively, these studies show that KSHV uses the hostproinflammatory COX-2/PGE2/EP receptor pathway for its

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own advantage of establishing and maintaining latent geneexpression.

Materials and Methods

Cells and KSHV. TIVE-LTC (long term–infected telomer-ase immortalized umbilical vein endothelial cells) TIVE cells,a gift from Dr. Rolf Renne (University of Florida), and 293cells were cultured as described before (13). KSHV wasprepared and assessed for its infectivity, Mycoplasma, andlipopolysaccharide, as described before (5).Plasmids. LANA-1 promoter sequence (pGL3.6 or

p-LANA-1-Luc) and the LANA-1 promoter deletion sequences(pGL3.4, pGL3.3, pGL3.2, and pGL3.1) cloned in pGL3.0 vector(Promega) with the reporter gene Firefly luciferase were giftsfrom Dr. Yuan Chang, University of Pittsburgh (14).Reagents. Akt 1/2 inhibitor, TMB-8, PD98059, wortman-

nin, Ly290042, U0126, and LPA were from Sigma. GFX,GO:6976, PP2, and Bay11-7085 were from Calbiochem.PGE2, EP1-4 agonists, AH6809, and GW627368X were fromCayman Chemical. Fura-2AM was from Invitrogen. SC-51322 was from Enzo Life Sciences.Antibodies. Anti-mouse (COX-1 and COX-2) antibodies as

well as anti-rabbit [microsomal PGE2 synthase (mPGES),EP1, EP2, EP3, and EP4] antibodies were from Cayman Che-micals. Anti-mouse (PI3K, α-tubulin, and p-Src) antibodieswere from BD Biosciences, Sigma, and Calbiochem, respec-tively. Anti-mouse (p-NF-κB, p-Akt, and p-ERK 1/2) andantirabbit (Akt, Src, NF-κB p65, p-PKCζ/λ, and p-PI3K)antibodies were from Cell Signaling Technology, Inc. Anti-rabbit PGE2 was from Abcam. Anti-rabbit [PKCζ and extra-cellular signal-regulated kinase 2 (ERK2)] antibodies werefrom Santa Cruz Biotechnology, Inc. Production and charac-terization of antibodies against full-length KSHV LANA-1protein have been described before (15).Transfection and luciferase reporter assay. Transfec-

tions on 293 cells were conducted as described before (5).The luciferase assays were conducted as per the manufac-turer's guidelines (Promega). The relative LANA-1 promoteractivity or relative luciferase units (RLU) were normalized toRenilla luciferase protein levels.Fluorescence-activated cell sorting. Samples for fluores-

cence-activated cell sorting (FACS) analysis were preparedas per manufacturer's guidelines (BD Biosciences). The datawere collected using FACSCalibur flow cytometer (BectonDickinson) and analyzed with CellQuest Pro software (Bec-ton Dickinson) at the RFUMS flow cytometry core facility.Western blotting and measurement of PGE2. Total

cell lysates prepared from cells after respective treatmentswere used for Western blotting and quantified as describedbefore (5). α-Tubulin was used as the loading control for allthe blots. Secreted amounts of PGE2 were measured using aPGE2 enzyme-linked immunosorbent assay (ELISA) kit asper the manufacturer's guidelines (R&D Systems).Real-time reverse transcription–PCR. LANA-1, COX-2,

and COX-1 transcripts were detected by real-time reversetranscription–PCR (RT-PCR) as described before (5).

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Confocal microscopy and immunohistochemistry.Confluent TIVE and TIVE-LTC cells were used for confocalmicroscopy using EP1-4 antibodies as before (13). Tissue sec-tions from three healthy subjects and three KS+ patientswere obtained from AIDS and Cancer Specimen Resource,National Cancer Institute. Immunohistochemistry in Fig. 1was done by similar method as described before (13).Measurement of Ca2+. 293 or TIVE-LTC cells plated onto

coverslips and placed in six-well plates were mounted on thestage of an inverted microscope (Olympus IX71) and incubat-ed with 5 μmol/L of Ca2+ indicator fura-2AM (Invitrogen) for30 minutes at 37°C in HBSS (Sigma; pH 7.4). Cells were thencontinuously perfused with Ca2+ free HBSS (pH 7.4). Whencytoplasmic Ca2+ levels increase after respective treatments,if any, Ca2+ will bind to fura-2AM in the cytoplasm, inducinga fluorescence signal that can be used as a measure of thelevel of cytoplasmic calcium. The calcium-induced fluores-cence signal of fura-2AM was measured by alternatively ex-citing at 340 and 380 nm and collecting the emittedfluorescence at 510 nm using a CCD-based imaging systemrunning SimplePCI software (Hamamatsu Corporation).Changes in cytoplasmic Ca2+ ([Ca2+]i) are reported as the ra-tio of 340:380 emissions. Data analysis was performed usingOrigin Pro software (Origin Lab Corporation). The Ca2+ stud-ies were conducted at RFUMS Ca2+ imaging facility.Statistical analysis. In Fig. 2, the statistical significance

(t test) was conducted with respect to untreated or uninfect-ed cells. In Fig. 3, the statistical significance (t test) wasconducted with respect to supernatant treatment. In Fig. 4,the statistical significance (t test) was conducted similar toFig. 2. In Fig. 5, the statistical significance (t test) was calcu-lated with respect to PGE2 alone treatment. In Fig. 6, thestatistical significance (t test) was conducted similar toFig. 2 (*, P < 0.01; **, P < 0.001; ***, P < 0.0001).

Results

PGE2, mPGES, and EP1-4 receptors are detected in KSlesions. We used immunohistochemistry to first investigatethe presence of mPGES, PGE2, and EP1-4 in serial sectionsof biopsies from three healthy subjects and three KS+ patients.The presence of KSHV in KS lesions was confirmed by the de-tection of characteristic nuclear staining of LANA-1 (Fig. 1A,b). Strong cytoplasmic mPGES, PGE2, and EP1-4 reactivitieswere detected in KS lesions (Fig. 1B–D). However, KS lesionsamples exhibited distinct staining for EP2 and EP4 receptorscompared with normal samples (Fig. 1C, b and D, b). Collec-tively, these results for the first time show the presence ofinflammation-associated EP receptors in KS lesions.KSHV infection upregulates EP1, EP3, and EP4 receptors

and downregulates EP2 receptors. We next examined therole of EP receptors in maintaining latent gene expressionin TIVE-LTC cells, which sustains expression of latency genes(16). In a separate study, we have observed the upregulationof COX-2 and mPGES proteins and PGE2 secretion in TIVE-LTC cells compared with control TIVE cells and downregula-tion of LANA-1 expression in TIVE-LTC cells treated withCOX-2 inhibitor NS-398 (13). Western blot analysis shows

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PGE2/EP Receptor Signaling Regulates KSHV Latency Gene


that, compared with TIVE cells, EP1, EP3, and EP4 receptorswere significantly upregulated in TIVE-LTC cells, whereasEP2 receptor was downregulated in TIVE-LTC cells (Fig.2A, a–d). Confocal microscopy also confirmed the presenceand cellular localization of all four EP receptors in TIVEand TIVE-LTC cells (Fig. 2B).

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KSHV uses EP receptors to maintain LANA-1 and COX-2gene expression and PGE2 secretion. We measured LANA-1,COX-2, and COX-1 gene expression in TIVE-LTC cells treatedwith noncytotoxic concentrations (Supplementary Fig. S1A–I) of well-characterized competitive EP receptor blockers SC-51322 (EP1 antagonist; 50 μmol/L), AH6809 (EP2 antagonist;

Figure 1. Characterization ofmPGES, PGE2, and EP receptorsin KS Lesions. A–D, serial sectionsof normal and KS lesions fromthree healthy subjects and threeKS+ patients were stained byimmunohistochemistry for rabbitIgG1 (A, a), LANA-1 (A, b), mPGES(B, a), PGE2 (B, b), EP1 (C, a),EP2 (C, b), EP3 (D, a), andEP4 (D, b).

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50 μmol/L), or GW 627368X (EP4 antagonist; 5 μmol/L) at 2,8, and 24 hours posttreatment and observed no significantchange in COX-1 gene expression (Fig. 2C, b). COX-1 gene ex-pression was conducted as a control, because COX-1 promoteris constitutively active. EP1, EP2, and EP4 antagonists down-regulated LANA-1 gene expression significantly by 69%, 82%,and 73% and by 55%, 64%, and 40% at 8 and 24 hours posttreat-ments, respectively (Fig. 2C, a). COX-2 gene expression was



downregulated by EP2 and EP4 antagonists at 2 hours post-treatment and by EP4 antagonist at 8 hours posttreatment(Fig. 2C, c). At 2 hours posttreatment, EP2 and EP4 antagonistsdownregulated PGE2 secretion significantly with no signifi-cant changes at 8 and 24 hours posttreatments (Fig. 2D).KSHV uses EP1 receptor–mediated cytoplasmic Ca2+

([Ca2+]i) signaling to maintain LANA-1 gene expression.From the above results showing the downregulation of

Figure 2. Effect of EP receptor antagonists on LANA-1, COX-1, COX-2, gene expression, and PGE2 secretion in TIVE-LTC cells. A, cells were serum starvedfor 48 hours, immunoblotted for EP1 (a), EP2 (b), EP3 (c), and EP4 (d) receptor levels with respect to tubulin to find the relative difference betweenTIVE and TIVE-LTC cells. B, permeabilized TIVE and TIVE-LTC cells were stained for EP1to EP4 receptors and analyzed by confocal microscopy. C and D,TIVE-LTC cells were serum starved for 48 hours and treated with EP1, EP2, and EP4 antagonists. Total RNA and supernatant were collected at 2, 8,and 24 hours posttreatment to examine the gene expression levels of LANA-1 (C, a), COX-1 (C, b), and COX-2 (C, c) by real-time RT-PCR and PGE2 byELISA (D). The percentage change was calculated with respect to untreated cells for each time point.

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LANA-1 gene expression by EP1 blockade (Fig. 2C, a) to‐gether with the fact that EP1 receptor is a well-characterizedCa2+-inducing GPCR (10), we hypothesized that PGE2 secret-ed in the supernatant of TIVE-LTC cells might be inducing



Ca2+ signaling via the EP1 receptor. Therefore, we predictedthat, if we block the EP1 receptor, the potential of the PGE2in the supernatant to induce Ca2+ signaling via the EP1receptor, if any, would also be blocked. The purpose of our

Figure 3. Effect of EP receptor antagonists on Ca2+, Src, PI3K, PKCζ/λ, Akt, NF-κB, and ERK 1/2 in TIVE-LTC cells. A, schematic of the experimentaldesign for Ca2+ experiments. B, TIVE-LTC cells were grown on coverslips placed in six-well plates and serum starved for 48 hours. The cells were washed andtreated with fresh serum-free media for 24 hours. Coverslip 1 was removed and loaded with fura-2AM (5 μmol/L) with or without different EP receptorantagonists. The cells were then washed and incubated with serum-free media (a) or with the supernatant produced by coverslip 2 TIVE-LTC cells with orwithout EP1, EP2, and EP4 antagonists or DMSO (b). Transient elevation in [Ca2+]i was measured for 35minutes. For each graph, five representative TIVE-LTCcells were selected to portray the transient [Ca2+]i signal. d, summary results showing the peak amplitude (mean ± SEM) of transient elevation in [Ca2+]iobserved in different treatments. n refers to the number of cells that showed a change in [Ca2+]i signal within the field studied. C and D, TIVE-LTC cells wereserum starved for 48 hours and treated with EP1, EP2, and EP4 antagonists or DMSO. At 8 and 24 hours posttreatment, total cell lysates were prepared,immunoblotted for p-Src (C, a), p-PI3K (C, b), p-PKCζ/λ (C, c), p-Akt (D, a), p-NF-κB (D, b), and p-ERK 1/2 (D, c), and normalized with respect to the levels oftotal protein levels. The fold change was calculated with respect to the signal intensity for untreated (UN) cells for each time point.

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experiment was not to test the effect of downregulatingPGE2 secretion in the supernatant by EP receptor antago-nists and consequently the calcium signal because, even at2 hours posttreatment with EP2 and EP4 antagonists,



PGE2 is present within the range of 80 to 100 pg/mL(Fig. 2D). However, our goal was to investigate the effectof blocking EP receptors on the supernatant-induced cal-cium signal, if any. By doing so, we are answering the role

Figure 4. Effect of KSHV infection and exogenous PGE2 treatment on LANA-1 promoter activity and the effect of de novo KSHV infection on 293 cells.A, schematic of the LANA-1 promoter constructs (p-LANA-1-Luc or pGL3.6). b, 293 cells transfected with p-LANA-1-Luc were infected with live KSHV or heparintreated KSHV, UV-KSHV, or left uninfected for 2 hours, washed with PBS, and supplemented with serum-free DMEM. c, permeabilized 293 cells were stainedfor EP1, EP2, EP3, and EP4 receptors by FACS. B, 293 cells transfected with p-LANA-1-Luc or empty vector (pGL3.0) were treated with 10 μmol/L PGE2 (a),different concentrations of PGE2 for 4 hours (b), or PGE2 (10 and 100 μmol/L), 10 μmol/L of EP1-4 agonists, or left untreated for 4 and 24 hours (c). A, b and B, atthe indicated times, the cells were harvested, lysed, and assayed for RLU. C and D, a, 293 cells, serum starved for 24 hours, were infected with KSHV for2 hours or left uninfected (UN), washed, and supplemented with serum-free DMEM throughout the experiment. At 2, 12, and 24 hours p.i., whole-cell lysate,supernatant, and total RNA were collected to measure for COX-1, COX-2, mPGES protein levels (C, a), PGE2 by ELISA (C, b), and LANA-1 expression (D, a),respectively. D, b, 293 cells serum starved for 24 hours were pretreated with NS-398 (100 μmol/L) for 24 hours, washed, infected with KSHV for 2 hours inthe presence of NS-398 (100 μmol/L) or NS-398 (100 μmol/L) and PGE2 (10 μmol/L), washed, and supplemented with serum-free DMEM in the presence ofNS-398 (100 μmol/L) or NS-398 (100 μmol/L) and PGE2 (10 μmol/L) 24 hours p.i. Total RNA was prepared to analyze the gene expression levels of LANA-1 byreal-time RT-PCR. The fold change and corresponding statistics (t test) were calculated with respect to untreated or uninfected (UN) 293 cells for each time point.

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of PGE2 in the supernatant in inducing calcium signalthrough EP receptors.To test our hypothesis, TIVE-LTC cells were grown on cov-

erslips and [Ca2+]i was measured as outlined in Fig. 3A. Tomeasure [Ca2+]i, coverslip 1 was removed and loaded withthe Ca2+ indicator fura-2AM for 30 minutes with and without



EP receptor antagonists in Hanks solution. However, whereasbeing loaded with fura-2AM, the cells in coverslip 1 are notexposed to the physiologic supernatant of TIVE-LTC cells.Therefore, to test whether the supernatant of TIVE-LTC caninduce Ca2+ signaling in coverslip 1 cells, we used the super-natant produced by cells grown in parallel on coverslip 2. The

Figure 5. Effect of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, p-Akt 1/2, p-NF-κB, and p-ERK 1/2 inhibition on PGE2-mediated LANA-1 promoter activity. A and B, 293 cellstransfected with p-LANA-1-Luc was pretreated with indicated pharmacologic signal inhibitors for 2 hours and supplemented with 10 μmol/L PGE2 for 4 hoursor left untreated and assayed for RLU. The fold change was calculated with respect to untreated cells. C, 293 cells serum starved for 24 hours were loadedwith fura-2AM,washedbyperfusion, and treatedwith 10μmol/L PGE2orHBSS (pH 7.4), and the [Ca2+]i levels weremeasured for 25minutes. Representative cellsfrom a field of ∼50 to 60 cells. D, 293 cells serum starved for 24 hours were treated with 100 ng of LPA and 10 μmol/L PGE2, infected with 20 DNA copiesper cell of KSHV, or left untreated (UN) for 4 hours. Total cell lysate was prepared, immunoblotted for p-Src, p-PI3K, p-PKCζ/λ, Akt 1/2, NF-κB, and ERK 1/2, andnormalized with respect to the levels of total protein levels, and the fold change was calculated with respect to (UN) cells for each time point.

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supernatant of coverslip 2 TIVE-LTC cells incubated in thepresence of DMSO (Fig. 3B, b) induced a significant tran-sient elevation in [Ca2+]i. We did not observe any significantchange between the observed peak amplitude of transient



elevation in [Ca2+]i induced by the supernatant alone orwith solvent control (DMSO; Fig. 3B, d). In contrast,serum-free medium used as a negative control had no sig-nificant effect on [Ca2+]i (Fig. 3B, a), indicating that factors

Figure 6. PGE2-responsive regions of LANA-1 promoter. A, PGE2-responsive TF binding sites on p-LANA-1-Luc. The LANA-1 promoter sequence (14)was analyzed by Alibaba 2.1 software. The schematic shows the different TFs predicted by Alibaba 2.1 software, the TF binding sites predicted byref. 14, and by both. The numbers for each TF refer to the beginning and end sequences of the binding site within the 774-bp LANA-1 promoter sequencestudied, with 128,674 bp and 127,900 bp as the start and end sites of the LANA-1 promoter, respectively, in the KSHV genome. The site of origin ofeach LANA-1 promoter deletion construct is marked. B, identification of PGE2-responsive regions of LANA-1 promoter. Sequential series of LANA-1promoter deletion constructs with luciferase reporter or empty vector (pGL3.0) were transfected into 293 cells and treated with 10 μmol/L PGE2 for4 hours or left untreated. The cells were harvested, lysed, and assayed for RLU. The fold change and corresponding statistics (t test) were calculated withrespect to untreated cells for each time point. C, schematic model depicting the potential mechanism by which PGE2 and EP receptor play roles inKSHV pathogenesis. During latency, KSHV induces proinflammatory COX-2 gene expression and PGE2 secretion (5), resulting in the activation of EPreceptors in an autocrine and paracrine fashion. Activated EP receptors influence specific signal cascades that in turn influence specific TFs convergingon the LANA-1 promoter. EP2-mediated and EP4-mediated signaling might also be involved in the regulation of this process by initiating a positivefeedback loop to maintain COX-2 gene expression and therefore PGE2 synthesis/secretion resulting in the sustenance of the characteristic chronicproinflammatory environment created by KSHV infection.

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present in the physiologic supernatant of TIVE-LTC cellscan induce Ca2+ signaling.Next, we examined whether the transient [Ca2+]i induced

by TIVE-LTC cell supernatant is due to activation of EP1 re-ceptor by the supernatant PGE2. Therefore, we treated thecells on coverslip 1 with EP1, EP2, or EP4 antagonist whilebeing treated with fura-2AM and the supernatant from cov-erslip 2 cells, as outlined in Fig. 3A. Treatments with EP1 butnot EP2 or EP4 antagonists significantly abolished the super-natant-mediated Ca2+ signal in TIVE-LTC cells (Fig. 3B, c andd). These observations clearly showed that PGE2 present inthe supernatant of TIVE-LTC cells could induce a Ca2+ signalthrough the EP1 receptor.Inhibition of EP receptors downregulates p-Src, p-PI3K,

p-PKCζ/λ, p-Akt, and p-NF-κB and upregulates p-ERK inKSHV-infected TIVE-LTC cells. We next examined the signalcascades regulated by EP receptors. TIVE-LTC cells serumstarved for 48 hours were treated with EP1, EP2, and EP4 an-tagonists or DMSO. EP2 and EP4 antagonists downregulatedp-Src by 17% and 47%, and 29% and 75% at 8 and 24 hoursposttreatments, respectively (Fig. 3C, a). At 24 hours post-treatment, EP1, EP2, and EP4 antagonists downregulatedPI3K phosphorylation by 23%, 33%, and 34%, respectively,with no effect at 8 hours posttreatment (Fig. 3C, b). EP2and EP4 antagonists downregulated p-PKCζ/λ by 12% and20%, and 23% and 69% at 8 and 24 hours posttreatments, re-spectively (Fig. 3C, c). Compared with DMSO treatment, wedid not observe any changes on p-Akt by EP receptor antago-nists at 8 and 24 hours posttreatment (Fig. 3D, a). At 8 hoursposttreatment, p-NF-κB was downregulated by 39%, 46%,and 66% with EP1, EP2, and EP4 antagonists, respectively,and by 41% with EP2 antagonist 24 hours posttreatment(Fig. 3D, b). With EP4 antagonist, p-ERK 1 and p-ERK 2 wereupregulated by 1.3-fold and 2.0-fold, and 1.2-fold and 2.0-foldat 8 and 24 hours posttreatment, respectively (Fig. 3D, e).KSHV infection and exogenous PGE2 activates the LANA-1

promoter. Based on our data from TIVE-LTC cells, we hy-pothesized that PGE2-mediated signaling can upregulateLANA-1 promoter activity. To test this, 293 cells were trans-fected with a luciferase reporter gene under the control of774-bp LANA-1 promoter (p-LANA-1-Luc; Fig. 4A, a; ref.14). The efficacy of our system was first shown by the induc-tion of p-LANA-1-Luc activity by primary KSHV infection,whereas less induction of p-LANA-1-Luc activity was ob-served with entry of incompetent heparin-treated and UV-inactivated virus (Fig. 4A, b).To determine the effect of exogenous PGE2 on p-LANA-1-

Luc, we first confirmed the presence of EP1-4 receptors byFACS (Fig. 4A, c). Exogenous PGE2 (10 μmol/L) inducedp-LANA-1-Luc activity by 8.7-fold at 4 hours posttreatmentwith no significant effect on the empty vector (Fig. 4B, a).Treatments with varying concentrations of PGE2 show that10 μmol/L of PGE2 or more were necessary to activatep-LANA-1-Luc significantly with no significant differencebetween the effects of 10 and 100 μmol/L of PGE2 (Fig. 4B,b and c). Well-characterized agonists for EP1-4 receptorswere also able to induce p-LANA-1-Luc activity significantlyat 4 hours but not at 24 hours posttreatment (Fig. 4B, c).



De novo KSHV infection of 293 cells induces the COX-2/PGE2 pathway. To validate whether the 293 cells used abovewere ideal to study the paradigm, we showed that de novoKSHV infection of 293 cells induces COX-2 and mPGES pro-teins (Fig. 4C, a), PGE2 secretion (Fig. 4C, b), and LANA-1 ex-pression (Fig. 4D, a). To determine whether the COX-2/PGE2pathway is important for maintaining LANA-1 gene expres-sion in 293 cells, we showed that 10 μmol/L PGE2 can restorethe reduction in LANA-1 expression caused by COX-2–specificinhibitor NS-398 (100 μmol/L; Fig. 4D, b).Inhibition of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, p-Akt 1/2,

p-NF-κB, and p-ERK 1/2 blocks PGE2-mediated LANA-1promoter. We next examined the role of Ca2+, Src, PI3K,PKCζ/λ, Akt 1/2, NF-κB, and ERK 1/2 in PGE2-mediatedLANA-1 transcriptional regulation by measuring the LANA-1promoter activity in 293 cells pretreated with specific inhib-itors for 2 hours followed by PGE2 (10 μmol/L) treatmentfor 4 hours and then incubated with PGE2. We used pharma-cologic inhibitors of Ca2+ (BAPTA-AM and TMB-8), PI3K(wortmannin and Ly290042), Src kinase (PP2), PKC (GFXand GO:6976), Akt 1/2 (Akt 1/2 inhibitor), NF-κB (Bay 11-7085), and ERK 1/2 (PD98059 and U0126) at the indicated non-cytotoxic concentrations.Ca2+ chelation (Fig. 5A, a), Src inhibition (Fig. 5A, b), and

PKC inhibition (Fig. 5A, c) decreased PGE2-mediated p-LANA-1-Luc activity significantly (Fig. 5A, a–c). Similarly,PI3K inhibitors wortmannin (1.0 μmol/L) and Ly290042(25 and 50 μmol/L) reduced PGE2-induced p-LANA-1-Lucactivity significantly (Fig. 5A, b). Akt 1/2 and NF-κB inhi-bition downregulated PGE2-mediated p-LANA-1-Luc activi-ty significantly (Fig. 5A, d). Although, PGE2-mediatedLANA-1 promoter activity was downregulated by Akt 1/2inhibitor, we did not observe any effect on Akt phosphor-ylation by EP receptor antagonists (Fig. 3D, a). Further-more, exogenous PGE2 was able to induce Akt 1/2 in293 cells. The dichotomy between TIVE-LTC and 293 cellsindicates that, in KSHV latent TIVE-LTC cells, Akt phos-porylation might also be under the control of EP2/EP4 re-ceptor-independent mechanisms. However, in serumstarved 293 cells, PGE2 might be acting as a powerful sig-nal inducer through EP receptors to induce Akt that maysubsequently activate the LANA-1 promoter. ERK inhibi-tion by PD98059 (10 and 20 μmol/L) and U0126 (5 and10 μmol/L) reduced PGE2-induced p-LANA-1-Luc activitysignificantly by 50% to 55% with no significant inhibitionon the basal activity (Fig. 5A, e). Our promoter studiesusing ERK 1/2 inhibitors are further validated by the ob-servation that de novo KSHV infection and exogenousPGE2 activate ERK 1/2 in 293 cells (Fig. 5D, f). This isin contrast to the EP4 antagonist–induced upregulationof ERK 1/2 phosphorylation in TIVE-LTC cells. This couldbe due to the differences in the cell systems used, asthe determining factors regulating ERK phosphorylationin TIVE-LTC could be different from that of 293 cells.Under serum-starved conditions, PGE2 might be actingas a power signal inducer in 293 cells through EP receptors(Fig. 5D, f). Therefore, PGE2-induced LANA-1 promote activ-ity is inhibited by ERK inhibitors. However, in TIVE-LTC cells,

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the presence of viral proteins and a cytokine/chemokine–richsupernatant might be altering the signal transduction profileof the cell to such an effect that EP2 and EP4 receptors mightbe responsible for inhibiting ERK phosphorylation.To explore further the signal molecules studied here, next

we used different combinations of signal inhibitors (Fig. 5B)at noncytotoxic concentrations (Supplementary Fig. S1J).Blocking of PI3K and Ca2+ simultaneously showed a signifi-cant additive effect of 80% (Fig. 5B) on the decrease inLANA-1 promoter activity compared with 5 μmol/L of BAP-TA-AM (Fig. 5A, a) and 12.5 μmol/L of Ly290042 (Fig. 5A, b),when used alone.Induction of Ca2+, p-Src, p-PI3K, p-PKCζ/λ, p-Akt 1/2,

p-NF-κB, and p-ERK 1/2 by PGE2 in 293 cells. To validatethe capacity of PGE2 to induce LANA-1 promoter activitythrough the signal molecules that were blocked in Fig. 5A,we examined whether exogenous PGE2 (10 μmol/L) andKSHV infection can induce them. To test whether PGE2can induce Ca2+, we treated serum-starved 293 cells loadedwith fura-2AM with PGE2 (10 μmol/L) that evoked anoscillatory Ca2+ signal for 25 minutes (Fig. 5C, a). As a neg-ative control, we also measured the basal levels of Ca2+ in293 cells treated with Ca2+-free HBSS and did not observeany intracellular Ca2+ signals (Fig. 5C, b). Compared withuntreated cells, LPA treatment (positive control), KSHVinfection, and PGE2 (10 μmol/L) increased the phosphoryla-tion of p-Src, p-PI3K, p-PKCζ/λ, Akt 1/2, NF-κB, and ERK 1/2(Fig. 5D).Identification of candidate PGE2-responsive elements

on LANA-1 promoter. To determine the minimal LANA-1promoter region responsive to exogenous PGE2, a sequen-tial series of LANA-1 promoter deletion constructs wasassayed in a luciferase reporter experiment in 293 cells(Fig. 6A and B). We then examined the p-LANA-1-Luc se-quence using Alibaba 2.1 TF software to characterize thetranscription factor (TF) binding site profile (Fig. 6A). Exog-enous PGE2 (10 μmol/L) activated pGL3.6, pGL3.4, andpGL3.3 promoter constructs at a similar level, whereasthe pGL3.2 and pGL3.1 promoter constructs had significant-ly lower activities (Fig. 6B). Taken together, these resultssuggested that the promoter region located between −262and −159 bp with candidate TFs, such as YY1, Sp1, Oct-1,Oct-6, C/EBP, and c-Jun, is required for PGE2-mediatedLANA-1 promoter activity (Fig. 6A and B).

Discussion

The novelty of our comprehensive study is the demonstra-tion for the first time that a proinflammatory lipid metabo-lite, such as PGE2 and its receptors, plays a crucial role inherpes virus latency. Previous reports have indicated the roleof Ca2+ in KSHV lytic cycle (17–20). In contrast, our studiesshowing the downregulation of LANA-1 expression by EP1receptor antagonist, the blockage of supernatant-induced[Ca2+]i signal by EP1 antagonist, and the downregulation ofPGE2-induced LANA-1 promoter activity by calcium chela-tors are the first demonstration of a role for [Ca2+]i in KSHVlatency program. Unlike calcium, previous reports have



shown the role of Src, PI3K, PKCζ/λ, and NF-κB in KSHVlatency program (21–23). However, the novelty of our studyis that the data linking PGE2/EP receptors with KSHV LANA-1 expression and LANA-1 promoter through PGE2 via Src,PI3K, PKCζ/λ, and NF-κB signal induction provides a newframework to understand the host mechanisms used byKSHV to induce these signal cascades. Furthermore, the pro-moter region we studied accounts for the transcripts ofLANA-1, vFLIP, vCyclin, and some of the viral microRNAs(24, 25) and together with its induction by PGE2 suggeststhat KSHV might be using PGE2 via EP receptors for regulat-ing the expression of other latency genes.The downregulation of COX-2 gene expression by EP2 and

EP4 antagonists could either be due to the direct effect of sig-nal cascades on the COX-2 gene, which has an inducible pro-moter (26), or due to the downregulation of COX-2 mRNAtranscript half-life that has been shown to be mediated byp38/MK2–dependent signaling acting on the ARE sequencesin the 3′ untranslated region of the COX-2 mRNA (27) The ab-sence of any effect on COX-2 gene expression by EP receptorantagonism 24 hours posttreatment could be due to COX-2promoter induction by other factors and suggests that KSHVuses multiple pathways with specific levels of temporal hier-archy to ensure the sustained activity of COX-2/PGE2/EP axisof inflammation, including a positive feedback loop mediatedthrough EP2 and EP4 receptor signaling.The effect of exogenous PGE2 on LANA-1 promoter is the

eventuality of numerous distinct yet related signal cascadesconverging on specific TFs (Fig. 6C), which are also probablyused for the maintenance of host gene expression, such asCOX-2, which is crucial for KSHV survival. Besides COX-2/PGE2, KSHV must be also using several signature proinflam-matory and angiogenic molecules that are induced duringinfection for the sustained induction of these signal networks(2, 28–32). Nevertheless, the present study showing the PGE2/EP receptor utilization for latent gene expression is unique dueto the fact that PGE2 is a lipid signal inducer that functions byautocrine and paracrine fashion at the site of synthesis witha circulating half-life of∼30 seconds and normal plasma levelsvarying from 3 to 15 pg/mL (33). Despite the short half-life,signaling events initiated by PGE2 through EP receptors areproposed to initiate an avalanche of temporal effects on cellu-lar signaling such as Src, PI3K, PKCζ/λ, NF-κB, and Ca2+ withimmense oncogenic potential (6, 34, 35), which are the samesignal pathways that are identified here to be regulated byPGE2/EP receptors in KSHV latency program.The hallmark of KSHV interaction in human host, like in

other herpes viruses, is the establishment of lifelong latencywith periodic reactivation and reinfection. Successful reac-tivation and reinfection, however, is the consequence of thecontinuous tug of war between KSHV and the host immunesystem. Regardless of the outcome of this battle, mainte-nance and establishment of latency are crucial for herpesvirus survival. Therefore, in the course of evolution, KSHVmight have recalibrated the very purpose of inflammationfrom being a host response to eliminate the virus to thehost mechanism that enables viral survival through thecontinuous production of inflammatory cytokines and

Cancer Research





growth factors. Pirating the proinflammatory lipid metabo-lite PGE2 and EP receptors for maintaining latency gene ex-pression is a hallmark of such a phenomenon and thusgives a glimpse of the plasticity of the KSHV genomeand also a novel paradigm shift in comprehending hostmechanisms underlying KSHV latency. More excitingly, itadds a new paradigm in the understanding of the pathogen-ic mechanisms underlying chronic inflammation of KSHV-associated KS lesions. Furthermore, the study also exposesa potential “Achilles heel” of KSHV pathogenesis, whereinthe use of anti-COX-2 and anti-EP receptor therapy couldameliorate KS by simultaneously controlling latency geneexpression and chronic inflammation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.



Acknowledgments

We thank Keith Philibert for critically reading the manuscript, Rita Levinefor helping in FACS analyses, Terri Li of University of Chicago forimmunohistochemistry staining, Dr. Daniel Peterson and Scott Surridge ofthe RFUMS Core Confocal facility for helping in immunohistochemistryimaging, NIH (NCI) AIDS Research and Reference Reagent Program forproviding the normal and KS specimens, Dr. Rolf Renne (University ofFlorida) for providing the TIVE-LTC and TIVE cells, and Dr. Yuan Chang(University of Pittsburgh) for providing LANA-1 promoter constructs.

Grant Support

Public Health Service grant CA 099925 and RFUMS H.M. Bligh CancerResearch Fund (B. Chandran) and Public Health Service grant CA128560(N. Sharma-Walia).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/24/2009; revised 01/26/2010; accepted 02/23/2010; publishedOnlineFirst 04/13/2010.

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Cancer Research



2010;70:3697-3708. Published OnlineFirst April 13, 2010.Cancer Res Arun George Paul, Neelam Sharma-Walia, Nagaraj Kerur, et al. Latency Gene Expression: Strategy of a Successful PathogenKaposi's Sarcoma-Associated Herpes Virus (HHV-8) for

Mediated Signaling by−Piracy of Prostaglandin E2/EP Receptor

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