JOURNAL OF VIROLOGY, Dec. 2010, p. 12139–12151 0022-538X/10/$12.00 doi:10.1128/JVI.00884-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 23
Regulation of Tumor Necrosis Factor-Like Weak Inducer of Apoptosis Receptor Protein (TWEAKR) Expression by Kaposi’s Sarcoma-Associated Herpesvirus MicroRNA Prevents TWEAK-Induced Apoptosis and Inflammatory Cytokine Expression䌤† Johanna R. Abend, Thomas Uldrick, and Joseph M. Ziegelbauer* HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Received 23 April 2010/Accepted 2 September 2010
Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, the second most common AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs) during latency. To identify cellular targets of KSHV miRNAs, we have analyzed a previously reported series of microarrays examining changes in cellular gene expression in the presence of KSHV miRNAs. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR) was among the most consistently and robustly downregulated genes in the presence of KSHV miR-K12-10a (miR-K10a). Results from luciferase assays with reporter plasmids containing the 3ⴕ untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3ⴕ UTR of TWEAKR completely disrupts inhibition by miR-K10a. The expression of TWEAKR was downregulated in cells transfected with miR-K10a as well as during de novo KSHV infection. In a KS tumor-derived endothelial cell line, the downregulation of TWEAKR by miR-K10a resulted in reduced levels of TWEAK-induced caspase activation. In addition, cells transfected with miR-K10a showed less induction of apoptosis by annexin V staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. Finally, the downregulation of TWEAKR by miR-K10a in primary human endothelial cells resulted in a decrease in levels of expression of the proinflammatory cytokines interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to TWEAK. These results identify and validate an important cellular target of KSHV miRNAs. Furthermore, we demonstrate that a viral miRNA protects cells from apoptosis and suppresses a proinflammatory response, which may have significant implications in the complex context of KS lesions.
Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) (human herpesvirus 8 [HHV-8]), a gammaherpesvirus, is the causative agent of KS. The different forms of KS range from classical KS, an indolent, highly vascularized tumor of endothelial cells confined primarily to the legs and arms, to AIDS-associated KS, a far more aggressive presentation which can result in substantial morbidity and death. Despite a dramatically decreased incidence of KS since the introduction of effective therapy for HIV, it remains the second most common tumor in people with HIV/AIDS in the United States and the most common tumor in sub-Saharan Africa, where the prevalences of both HIV and KSHV are high and access to HIV therapy remains limited (8, 31, 33). In addition to KS, KSHV is also the suspected causative agent of primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD), two relatively rare tumors that arise in patients with AIDS. In KS tumors, the majority of cells are latently infected with KSHV. During latency, however, only a few viral protein-en* Corresponding author. Mailing address: Building 10, Room 6N106 MSC 1868, 10 Center Drive, Bethesda, MD 20892-1868. Phone: (301) 594-6634. Fax: (301) 480-5955. E-mail:
[email protected]. † Supplemental material for this article may be found at http://jvi .asm.org/. 䌤 Published ahead of print on 15 September 2010.
coding genes are expressed, which function in the maintenance of the viral genome, driving cellular proliferation, and the activation of NF-B and p38 mitogen-activated protein kinase (MAPK) signaling cascades. Also expressed during latency are the 12 viral pre-microRNAs (miRNAs). miRNAs are small (21- to 23-nucleotide [nt]), noncoding RNAs that regulate gene expression posttranscriptionally. Viral miRNAs may represent a unique mechanism for viruses to modulate host gene expression without generating additional antigenic viral proteins. In general, viral miRNA biogenesis is believed to be identical to that of other species. During biogenesis, the strands of the miRNA duplex are separated, and one or both are loaded into the RNA-induced silencing complex (RISC), which mediates either transcriptional silencing or, less frequently, the degradation of the target transcript based on the imperfect complementarity of the miRNA for its target sequence, typically within the 3⬘ untranslated region (UTR) (3, 12, 14). The 12 KSHV pre-miRNAs give rise to at least 18 mature miRNAs such that five pre-miRNAs donate both strands of the duplex to the RISC (miR-K12-3, -4, -6, -9, and -12) and one (miRK12-10a) undergoes RNA editing, resulting in a single-base change in the seed sequence (miR-K12-10b). Studies using microarrays and deep-sequencing techniques have revealed
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that the individual KSHV miRNAs are present at different levels during latency (20, 35, 45). Since miRNAs bind to their target sequences with imperfect complementarity, it is difficult to predict what cellular genes will be regulated by KSHV miRNAs. Thus far, few cellular targets have been identified and validated for the KSHV miRNAs. Samols et al. previously identified thrombospondin, a glycoprotein involved in cell-to-cell and cell-to-matrix interactions, as a target of KSHV miRNAs (37). Two independent reports identified BACH1, a transcriptional repressor, as a target of miR-K12-11 (16, 41). Very recently, several groups identified new cellular targets for KSHV miRNAs: miRK12-11 and -6 were found to target an endothelial cell-specific transcription factor, musculoaponeurotic fibrosarcoma oncogene homolog (MAF; 20); miR-K12-1 was reported to target IB␣ to promote NF-B signaling (28); miR-K12-4-5p was found to inhibit a repressor of DNA methyl transferases, Rbl2 (29); and miR-K12-1 was found to inhibit the cellular cyclindependent kinase inhibitor p21 (15). In addition, Ziegelbauer et al. previously identified bcl-2-associated factor 1 (BCLAF1) as a target of KSHV miR-K12-5, -9, -10a, and -10b by using a series of microarrays to examine changes in cellular gene expression in the presence of KSHV miRNAs under the following conditions: transient transfection of B cells with KSHV pre-miRNAs, stable transduction of B cells with clusters of pre-miRNAs, de novo infection of endothelial cells, and transient transfection of latently infected B cells with inhibitors of KSHV miRNAs (54). This expression profiling data set was utilized to identify additional cellular targets of KSHV miRNAs. In this report, we present validation and functional data for a new cellular target of a KSHV miRNA, the cytokine receptor tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) receptor (TWEAKR). TWEAK (also known as TNFSF12, APO3L, or CD255) has only one known functional receptor, TWEAKR (also known as Fn14, TNFRSF12A, or CD266). TWEAK and TWEAKR belong to the TNF and TNF receptor (TNFR) superfamilies and are widely expressed in a variety of cell types, including human vascular endothelial cells (49, 50). TWEAKR expression has been shown to be upregulated on human tumor specimens and various tumor cell lines (1, 4, 11, 19, 32, 43, 48). Similarly, levels of TWEAK mRNA and protein were reported previously to be elevated in human breast, colon, liver, and kidney tumors (24–26, 32, 53) but not in glioblastoma samples (43). These reports support the relevance of TWEAK and TWEAKR signaling in the context of cancer. TWEAKR is a type I transmembrane protein of approximately 14 kDa with a single TNFR-associated factor binding domain in the cytoplasmic tail. TWEAK-mediated signaling can have vastly different effects depending on the type of cell stimulated (4, 50). TWEAK signaling promotes cell proliferation, migration, and survival in many primary cells, including endothelial cells, indicating its roles in angiogenesis, tumorigenesis, and wound repair. A number of cell types are induced to express proinflammatory molecules upon TWEAK stimulation. TWEAK signaling can also lead to the induction of apoptosis, particularly in tumor cell lines. These proapoptotic effects often require long incubations and presensitization with other cytokines, like gamma interferon (IFN-␥), or inhibitors, such as cycloheximide. Interestingly, TWEAKR does not have
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a canonical death domain within its cytoplasmic tail, which has led to speculation that the proapoptotic effects of TWEAK stimulation are indirect, resulting from the induction of TNF-␣ (34, 38, 47). Using the above-mentioned array data detailing changes in cellular gene expression in the presence of KSHV miRNAs (54), we have taken an unbiased approach using rank sum analysis to prioritize potential targets to pursue. Unlike other approaches to identify miRNA targets, our method is dependent only on expression changes in response to miRNA expression and does not make the assumption that a target gene must have an miRNA seed-matching sequence in the 3⬘ UTR. TWEAKR was one of the top predicted targets for KSHV miR-K12-10a (miR-K10a) based on expression changes. In this report, we validate TWEAKR as a target of miR-K10a. To understand the biological purpose for the regulation of TWEAKR expression in the context of viral infection, we postulated that the most plausible benefit would be from preventing apoptosis in KS tumor-derived endothelial cells and from preventing proinflammatory signaling in response to TWEAK stimulation. Indeed, the miR-K10a-mediated knockdown of TWEAKR coincided with a reduced level of apoptosis upon TWEAK stimulation. Furthermore, the miR-K10a-mediated knockdown of TWEAKR in primary endothelial cells reduced interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) production in response to treatment with TWEAK. These results have important implications not only for an understanding KSHV pathogenesis and the complex nature of KS lesions but also for discovering new functions of viral miRNAs. MATERIALS AND METHODS Cells and reagents. Primary human umbilical vein endothelial cells (HUVECs) (Lonza) were maintained for up to five passages in EGM-2 (Lonza). 293 cells, the KS-derived human endothelial cell line SLK (23), and KSHV latently infected SLK cells (SLK⫹K) (46, 54) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1⫻ Pen Strep glutamine solution (Gibco). SLK⫹K cells were maintained under selection with 10 g/ml puromycin. Synthetic KSHV miRNA mimics were obtained from Ambion, and miRCURY locked nucleic acid (LNA) miRNA inhibitors were obtained from Exiqon. Mutant miR-K10a miScript miRNA mimics (MUTa and MUTb) were obtained from Qiagen. ON-TARGETplus SMARTpool small interfering RNAs (siRNAs) targeting TWEAKR and the ON-TARGETplus nontargeting pool were obtained from Dharmacon. HUVECs were transfected in six-well plates using 1.5 l of DharmaFECT 1 reagent (Dharmacon) and 10 nM KSHV miRNA. SLK and SLK⫹K cells were transfected in six-well plates using 6 l DharmaFECT 1 and 100 nM KSHV miRNA or 50 nM LNA inhibitor, respectively. Luciferase assays. 293 cells were reverse transfected in 96-well plates using 0.5 l Lipofectamine 2000 (Invitrogen), 13 nM each KSHV miRNAs (or a negativecontrol miRNA, neg2), and 0.5 ng/l of the luciferase reporter plasmid, which expresses firefly luciferase driven by the simian virus 40 (SV40) promoter as an internal control and Renilla luciferase fused to the 3⬘ UTR of TWEAKR driven by the herpes simplex virus (HSV) TK promoter as the reporter. The TWEAKR 3⬘ UTR luciferase reporter was cloned by the Protein Expression Laboratory (SAIC, Frederick, MD). A luciferase reporter plasmid without the TWEAKR 3⬘ UTR served as a control for any nonspecific response of luciferase expression to the KSHV miRNAs. Site-directed mutagenesis was performed on the luciferase reporter plasmid containing the TWEAKR 3⬘ UTR using the QuikChange II kit (Stratagene) according to the manufacturer’s instructions. The following primers and their reverse complements were used to introduce mutations into the TWEAKR 3⬘ UTR: 5⬘-GGGGTTAGGGACCTATTTTTAAGCAAAGGGGG CTGGCCCAC-3⬘ for mut1 and 5⬘-GACTTGGGGGGCAGACTTGAGCAAA GGCCCCACTCACTC-3⬘ for mut2. Assays were performed by using the DualLuciferase reporter system (Promega) at 24 and 48 h posttransfection (hpt). Each transfection was performed at least three times and was assayed in triplicate.
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De novo infections. KSHV was harvested from BCBL-1 cultures 6 days after induction with valproic acid and purified by centrifugation. HUVECs at 60 to 70% confluence were infected with KSHV diluted in EGM-2 containing 8 g/ml polybrene for 6 h at 37°C. Cells were washed twice, and medium was changed every other day throughout the course of infection. Cell lysates were harvested at 7 days postinfection. Western blot analysis. Total cell protein was harvested by using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma) supplemented with 1⫻ Halt protease and a phosphatase inhibitor cocktail (Thermo Scientific). Cells were lysed on ice for 10 min, and cell debris was removed by centrifugation at 13,000 rpm for 10 min. The Li-Cor Odyssey system was used for the detection and quantitation of protein bands. The following primary antibodies were used: rabbit anti-TWEAKR (catalog number 4403; Cell Signaling), goat anti-BACH1 (catalog number sc-14700; Santa Cruz), and mouse anti-actin (catalog number A5316; Sigma). The following secondary antibodies conjugated to infrared (IR) fluorescing dyes were obtained from Li-Cor: goat anti-rabbit IR800CW, goat anti-mouse IR680, and donkey anti-goat IR800CW. Results are presented as TWEAKR or BACH1 expression normalized to actin, relative to mock-infected or negative-control miRNA-transfected cells. Caspase activation assay. At 24 hpt, SLK cells transfected with miR-K10a or siRNAs targeting TWEAKR or SLK⫹K cells transfected with LNA inhibitors were trypsinized, resuspended in growth medium diluted 1:10 with Opti-Mem I (Gibco), and replated at a low density (2,500 to 5,000 cells/well) in 96-well plates. Cells were either mock treated, treated with 20 ng/ml IFN-␥ (Peprotech) or 500 ng/ml TWEAK (Peprotech), or treated simultaneously with IFN-␥ and TWEAK. At 48 h posttreatment, cells were incubated with caspase-Glo 3/7 Glo reagent (Promega) for 1 h, and luminescence was detected by using a Modulus luminometer (Turner), with relative light units (RLU) being indicative of caspase activity. Results are presented as the fold change in caspase activity relative to the activity in untreated cells. Annexin V and TUNEL assays. At 24 hpt, SLK cells transfected with miRK10a or siRNAs targeting TWEAKR were trypsinized, resuspended in growth medium diluted 1:10 with Opti-Mem I, and replated at a low density (75,000 cells/well) in six-well plates. Cells were either mock treated or treated with 20 ng/ml IFN-␥ and 500 ng/ml TWEAK. At 72 h posttreatment, cells were collected and stained with phycoerythrin (PE)-conjugated annexin V and 7-amino-actinomycin (7-AAD) as a viability stain using PE-annexin V apoptosis detection kit I (BD Pharmingen). In parallel, cells were collected, fixed, and assayed for DNA fragmentation by a terminal deoxynucleotidyltransferase (TdT)-mediated dUTPbiotin nick end labeling (TUNEL) reaction by using an in situ cell death detection kit with fluorescein (fluorescein isothiocyanate [FITC]) (Roche). Samples were analyzed with a FACSCalibur instrument (BD) using CellQuest Pro for collection and FlowJo for analysis. Results of the annexin V assay are presented as a percentage of the gated cell population that was PE-annexin V positive and 7-AAD negative (early-stage apoptotic), or PE annexin V positive and 7-AAD positive (late-stage apoptotic). Results of the TUNEL assay are presented as the fold change in the percentage of FITC-positive cells with TWEAK and IFN-␥ treatment relative to untreated cells. Bio-Plex assays for IL-8 and MCP-1. At 24 hpt, HUVECs transfected with miR-K10a or negative-control miRNA were trypsinized, resuspended in growth medium diluted 1:10 with Opti-Mem I, and replated at a low density (50,000 cells/well) in 12-well plates. Cells were either mock treated or treated with 500 ng/ml TWEAK. Supernatants were collected at 24 h posttreatment and analyzed by using Bio-Plex Pro assays for IL-8 and MCP-1 (Bio-Rad) according to manufacturer’s instructions. Results are presented as the concentration of IL-8 or MCP-1 present in 25 l of supernatant. Quantitative RT-PCR assays for cytokine expression. At 24 hpt, HUVECs transfected with miR-K10a, a mutant of miR-K10a (MUTb), or negative-control miRNA (neg2) were trypsinized, resuspended in growth medium diluted 1:10 with Opti-Mem I, and replated at a low density (120,000 to 150,000 cells/well) in six-well plates. Cells were either mock treated or treated with 500 ng/ml TWEAK. Cell pellets were collected at 24 h posttreatment, and total RNA was isolated by using the RNeasy microkit (Qiagen). cDNA was generated from 500 ng RNA in reverse transcription (RT) reactions by using the high-capacity RNA-to-cDNA kit (Applied Biosystems). PCRs were performed with a total volume of 25 l using SYBR green PCR 2⫻ master mix (Applied Biosystems), 1 M each primer, and 5 ng of a cDNA template. The ABI Prism 7000 sequence detection system was used for amplification with the following PCR program: 2 min at 50°C, 10 min at 95°C, 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Results are presented as the fold change in IFN-␣, IL-6, IL-8, or MCP-1 transcript levels in cells treated with TWEAK relative to untreated cells, normalized to -actin transcript levels using the comparative threshold cycle (CT) (⌬⌬CT) method. The following primer sets
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were used: 5⬘-CTTCAACCTCTTTACCACAAAAGATTC-3⬘ and 5⬘-TGCTGG TAGAGTTCGGTGCA-3⬘ for IFN-␣1, 5⬘-AAATGCCAGCCTGCTGACGAA G-3⬘ and 5⬘-AACAACAATCTGAGGTGCCCATGCTAC-3⬘ for IL-6, 5⬘-TTG GCAGCCTTCCTGATTTC-3⬘ and 5⬘-AACTTCTCCACAACCCTCTG-3⬘ for IL-8, 5⬘-CCCCAGTCACCTGCTGTTAT-3⬘ and 5⬘-TGGAATCCTGAACCCA CTTC-3⬘ for MCP-1, and 5⬘-GTGACATTAAGGAGAAGCTGTGCTA-3⬘ and 5⬘-CTTCATGATGGGAGTTGAAGGTAGTT-3⬘ for -actin. Statistical analysis. A Student’s t test (unpaired and two tailed) was used to determine the statistical significance of data wherever indicated, and P values of less than 0.05 were considered significant. Microarray data accession numbers. MIAME-compliant HUVEC array data are available at http://puma.princeton.edu/ under accession number 5824. BJAB and BCBL-1 array data are available under accession number GSE24069.
RESULTS Previously, a series of microarray analyses examined changes in cellular gene expression in response to the presence of KSHV miRNAs in B cells and endothelial cells as well as the inhibition of KSHV miRNAs during latent infection of B cells (54). Those studies provided a large data set that can be used to make unbiased predictions of miRNA targets based on miRNA-dependent changes in gene expression. We analyzed the microarray data using rank sum analysis, ignoring potential 3⬘ UTR sequence matches to the seed region of the miRNA, and identified TWEAKR as a high-probability target for KSHV miR-K10a. Figure 1 shows the change in the expression of TWEAKR across the arrays involving each of the KSHV miRNAs, with TWEAKR downregulated in the presence of miR-K10a and upregulated with the miRNA inhibitor of miRK10a during latency (arrows). The scatter plot shows the fold change of TWEAKR (Fig. 1, arrows and encircled dot) during de novo KSHV infection of HUVECs relative to other downregulated genes in the array (black dots). TWEAKR was among the top 2% of genes downregulated during infection, demonstrating its strength as a potential target. miR-K10a targets the 3ⴕ UTR of TWEAKR. To begin target validation studies, we first wanted to determine if miR-K10a regulates TWEAKR using luciferase assays with reporter plasmids containing the 3⬘ UTR of TWEAKR. In the presence of miR-K10a, luciferase activity was consistently downregulated by approximately 2-fold (0.58 and 0.48 at 24 and 48 h posttransfection [hpt], respectively, relative to a negative-control miRNA), suggesting that miR-K10a specifically targets the 3⬘ UTR of TWEAKR (Fig. 2A). miR-K10b, which is identical to miR-K10a except for a single-base change at nucleotide position 2, had no effect on luciferase activity, indicating the specificity of miR-K10a targeting of TWEAKR. We searched the 3⬘ UTR of TWEAKR for binding sites of miR-K10a and identified two potential target sites predicted by the miRanda (9) program; one of the sites (mut1) was also predicted by the TargetScan (17) program. Using site-directed mutagenesis, we mutated these sites to disrupt miR-K10a binding in the context of the luciferase reporter plasmid (mut1 and mut2) (Fig. 2B). Compared to the wild-type TWEAKR 3⬘ UTR luciferase reporter, both reporters with a single mutated site (mut1 or mut2) showed a partial restoration of luciferase activity (Fig. 2C). A reporter containing mutations at both predicted binding sites (mut1 plus mut2), however, showed a full restoration of luciferase activity in the presence of miR-K10a. In addition, a mutant of miR-K10a (MUTa), in which complementarity to the binding sites in mut1 and mut2 was restored (Fig. 2B),
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FIG. 1. TWEAKR is a predicted target for miR-K10a. (Top) Results for the average fold change in TWEAKR expression in each of the cellular gene expression arrays, denoted as follows: a-X, latently infected B cells transfected with inhibitors of KSHV miR-X; p-X, B cells transiently transfected with KSHV miR-X; stable-X, B cells stably transduced with KSHV miR-X. Arrows highlight the arrays using miR-K10a. (Bottom) Results for the cellular gene expression array performed on RNA isolated during de novo KSHV infection of HUVECs, with each point representing one probe. The arrows and encircled dot indicate the average fold changes in TWEAKR expression. Each array was performed in duplicate.
showed no inhibition of the wild-type reporter and a strong inhibition of luciferase activity from the double mutant (mut1 plus mut2) reporter. These data show that miR-K10a binds directly to the 3⬘ UTR of TWEAKR at two distinct sites. miR-K10a downregulates expression of TWEAKR. We next wanted to investigate the regulation of TWEAKR at the level of protein expression in response to viral miRNA expression. HUVECs were transfected with each of the KSHV miRNAs individually, and whole-cell lysates were analyzed by quantitative Western blotting. We observed that the expression of TWEAKR in the presence of miR-K10a was downregulated by 3.5-fold (Fig. 3A). We also saw consistent downregulation in the presence of miR-K2, suggesting the involvement of miR-K2 in modulating TWEAKR expression. Since there was no regulation by miR-K2 in the luciferase assays (Fig. 2A), we speculate that either miR-K2 must bind to a region outside the
3⬘ UTR or miR-K2 affects TWEAKR expression indirectly by repressing a protein that induces the expression of TWEAKR. It is unclear at this time why TWEAKR expression was upregulated in the presence of miR-K3*. However, others have reported a similar activation by miR-K3* in luciferase assays (2). HUVECs transfected with siRNAs targeting TWEAKR showed a decrease in the level of TWEAKR expression similar to that seen with miR-K10a (Fig. 3B). These results were confirmed by cell surface staining assays for TWEAKR expression with analysis by flow cytometry (see Fig. S1A in the supplemental material) and in-cell Western blotting (Fig. S1B). Furthermore, TWEAKR was consistently downregulated in HUVECs undergoing de novo infection with KSHV (Fig. 3C). These lysates were also probed for BACH1, a previously identified target for miR-K12-11 (16, 41), as a positive control for KSHV infection and miRNA function. This result correlates
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FIG. 2. miR-K10a targets the 3⬘ UTR of TWEAKR. (A) 293 cells were transfected with each of the KSHV miRNAs and the reporter plasmid expressing Renilla luciferase fused to the 3⬘ UTR of TWEAKR. Lysates were analyzed by a luciferase assay at 24 and 48 hpt, and results are presented as the change in normalized RLU relative to negative-control miRNA (neg2). Averages and standard deviations (SD) were calculated from three independent experiments. (B) The sequence of miR-K10a and its putative binding sites within the 3⬘ UTR of TWEAKR are shown, based on predictions from miRanda and TargetScan. The same 4-bp mutation (underlined lowercase letters) was made at two different sites in the reporter plasmid containing the 3⬘ UTR of TWEAKR (mut1 and mut2) to disrupt the binding of miR-K10a. A mutant of miR-K10a (MUTa) was designed with the complementary mutation to restore binding to the mut1 and mut2 sites. (C) Luciferase assays were performed at 24 hpt as described above for A, using only neg2, miR-K10a, and MUTa and the reporter plasmid containing the wild-type 3⬘ UTR (TWEAKR), the 3⬘ UTR mutated at either the mut1 or mut2 sites (mut1 or mut2), or the 3⬘UTR containing mutations at both sites (mut1 plus mut2). Averages and SD were calculated from three independent experiments.
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with the observed downregulation of TWEAKR at the mRNA level in the arrays (Fig. 1) and is important for showing the inhibition of TWEAKR expression under physiologically relevant conditions. In addition, a lymph node biopsy sample from a patient with KS had lower levels of TWEAKR than a normal lymph node tissue lysate (Fig. S1C). TWEAKR was undetectable in two other clinical samples, both skin biopsy specimens from patients with KS. Taken together, the results validate TWEAKR as a target for KSHV miR-K10a. miR-K10a-mediated downregulation of TWEAKR prevents TWEAK-induced caspase activation. The reported functions of TWEAKR signaling vary widely from proliferation and angiogenesis to the induction of apoptosis in tumor cell lines. We hypothesized that one of the most beneficial results of inhibiting TWEAKR expression, from the viral perspective, would be to protect infected cells from apoptosis. For these studies, we used SLK cells, a KS tumor-derived endothelial cell line that has lost the KSHV genome upon passaging in culture (23). The transfection of SLK cells with miR-K10a or siRNAs targeting TWEAKR resulted in over a 3-fold downregulation of TWEAKR expression (Fig. 4A). To examine the ability of miR-K10a to protect cells from TWEAK-induced apoptosis, SLK cells were transfected with miR-K10a or siRNAs targeting TWEAKR for 24 h and then replated at a low cell density in 1/10 growth medium. These conditions allowed a better observation of the relatively weak induction of apoptosis by TWEAK (6) and reduced the stimulatory effects that certain components of medium, such as FBS and growth factors, have on the expression of TWEAKR (50). The cells were then treated with either TWEAK alone, IFN-␥ alone, or TWEAK and IFN-␥ simultaneously, since it was shown previously that TWEAK-induced apoptosis often requires costimulation with IFN-␥ (4). While IFN-␥ treatment alone had little effect on caspase activity, we saw that treatment with IFN-␥ and TWEAK resulted in an approximately 3.5-fold induction of caspase activity in negative-control miRNA-transfected cells. Cells transfected with miR-K10a were less responsive to IFN-␥ and TWEAK stimulation, having only about a 1.7-fold induction of caspase activity (Fig. 4B). Cells transfected with siRNAs targeting TWEAKR showed a similar but less robust reduction in caspase activity. These results suggest that the miR-K10a-mediated knockdown of TWEAKR may protect cells from TWEAK-induced apoptosis. We also performed a complementary loss-of-miRNA-function experiment using SLK cells latently infected with recombinant KSHV r.219 (SLK⫹K cells) (46). SLK⫹K cells transfected with locked nucleic acid (LNA) inhibitors of miR-K10a
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showed an upregulation of TWEAKR expression by over 2-fold at 24 h, suggesting that miR-K10a is a key inhibitor of TWEAKR (Fig. 4A). Transfection with a mix of inhibitors of miR-K10a and miR-K2 showed no further upregulation in TWEAKR compared to inhibitors of miR-K10a alone. The level of caspase activity upon TWEAK and IFN-␥ stimulation in infected cells transfected with negative-control miRNA inhibitors was relatively high, 3-fold above that of untreated cells. Transfection with the LNA inhibitors of miR-K10a, however, resulted in a subtle but statistically significant increase in caspase activity with IFN-␥ and TWEAK treatment (Fig. 4C). These results suggest that the expression of miR-K10a in infected cells suppresses caspase activity induced by TWEAK and IFN-␥ stimulation. miR-K10a-mediated downregulation of TWEAKR protects cells from TWEAK-induced apoptosis. The results of the caspase activation assays suggest that the miR-K10a-mediated knockdown of TWEAKR protects cells from TWEAK-induced apoptosis. To confirm this effect, we performed annexin V staining and TUNEL assays to examine later stages in the apoptosis signaling cascade. SLK cells were transfected with miR-K10a or siRNAs targeting TWEAKR for 24 h, replated as described above, and mock treated or treated with IFN-␥ and TWEAK simultaneously. Figure 5A shows a representative dot plot from the annexin V assay for SLK cells transfected with negative-control miRNA or miR-K10a. Upon cytokine stimulation, there was a clear shift in the negative-control miRNAtransfected cell population to the lower-right quadrant (PEannexin V positive and 7-AAD negative, indicating the early stages of apoptosis) and the upper-right quadrant (PE-annexin V positive and 7-AAD positive, indicating the late stages of apoptosis) of the plot. This shift was reduced in cells transfected with miR-K10a, and the reproducible effect is presented in Fig. 5B and C. Cells transfected with miR-K10a showed no increase in the percent population in the early stages of apoptosis (Fig. 5B) and a less-than-2-fold increase in the percent population in the late stages of apoptosis (Fig. 5C). In comparison, negative-control miRNA-transfected cells were induced to early-stage apoptosis by approximately 3-fold and driven to late-stage apoptosis by over 5.5-fold. Similar results were obtained by using a mutant of miR-K10a (MUTb) (Fig. 6B) as an additional negative control (see Fig. S2 in the supplemental material). The protective effect of the miR-K10amediated downregulation of TWEAKR was replicated in the TUNEL assay. Cells transfected with miR-K10a showed no increase in TUNEL staining upon treatment with IFN-␥ and TWEAK, while negative-control miRNA-transfected cells
FIG. 3. miR-K10a downregulates expression of TWEAKR. (A) HUVECs were transfected with each of the KSHV miRNAs. Total cell lysates were harvested at 48 hpt and analyzed by Western blotting. (Top) Representative images of TWEAKR and actin expression. (Bottom) Average change in normalized TWEAKR expression levels relative to levels of negative-control miRNA-transfected cells (neg2). Averages and SD were calculated from five independent experiments. The dashed line indicates TWEAKR expression in the neg2 control. no, no miRNA; neg1, additional negative-control miRNA. (B) HUVECs were transfected with either miR-K10a or siRNAs targeting TWEAKR. Total cell lysates were harvested at 48 hpt and analyzed by Western blotting. (Top) Representative images for TWEAKR and actin expression. (Bottom) Average change in normalized TWEAKR expression levels relative to levels in neg2-transfected cells. Averages and SD were calculated from data from four independent experiments. no, no miRNA. (C) HUVECs were infected with KSHV diluted 1:50 or 1:40 in EGM-2 containing 8 g/ml polybrene, and total cell lysates were harvested at 7 days postinfection for analysis by Western blotting. (Left) Average change in normalized TWEAKR (black bars) or BACH1 (white bars) expression levels relative to levels in mock-infected cells. (Right) Representative images of BACH1, TWEAKR, and actin expression. Averages and SD were calculated from three independent experiments.
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FIG. 5. miR-K10a-mediated downregulation of TWEAKR protects cells from TWEAK-induced apoptosis. SLK cells were transfected with either miR-K10a or siRNAs targeting TWEAKR. Cells were treated with IFN-␥ and TWEAK at 24 hpt and analyzed for annexin V staining or DNA fragmentation by a TUNEL assay at 72 h posttreatment. (A) Representative dot plot of cells stained with PE-annexin V (x axis) and 7-AAD (y axis). The numbers within the plot reflect the percentages of the gated cell population present in each of the quadrants. Cell populations in the lower-right quadrant are defined as early-stage apoptotic; cell populations in the upper-right quadrant are defined as late-stage apoptotic. (B and C) Results from annexin V staining showing the percentage of the gated cell population defined as early-stage apoptotic (B) or late-stage apoptotic (C). The numbers above the brackets indicate the fold changes in percentages of the gated cell population undergoing apoptosis upon treatment with TWEAK and IFN-␥. Averages and SD were calculated from three independent experiments. (D) Results from the TUNEL assay showing the fold change in the percentage of the gated cell population staining FITC positive upon treatment with TWEAK and IFN-␥, normalized to the percent FITC-positive cells in untreated samples. Averages and SD were calculated from four independent experiments. no, no miRNA.
had a 3-fold increase in the number of TUNEL-positive cells (Fig. 5D). Interestingly (see Discussion), siRNAs targeting TWEAKR displayed a diminished ability to protect cells from apoptosis by annexin V staining (Fig. 5B and C) and a TUNEL
assay (data not shown). This observation correlates somewhat with the results from the caspase activation assay, for which only a modest decrease in caspase activity was detected in cells transfected with siRNAs targeting TWEAKR (Fig. 4B). Taken
FIG. 4. miR-K10a-mediated downregulation of TWEAKR prevents TWEAK-induced caspase activation. (A) SLK cells were transfected with either miR-K10a or siRNAs targeting TWEAKR (lanes 1 to 5); SLK cells latently infected with recombinant KSHV (SLK⫹K cells) were transfected with LNA miRNA inhibitors of miR-K10a or a mix of inhibitors of miR-K10a and miR-K2 (10a ⫹ 2) (lanes 6 to 9). Total cell lysates were harvested at 24 hpt and analyzed by Western blotting. (Top) Representative images for TWEAKR and actin expression. (Bottom) Average change in normalized TWEAKR expression levels relative to levels in neg2-transfected SLK cells (left) or negative-control LNA inhibitortransfected SLK⫹K cells (neg) (right). Averages and SD were calculated from four independent experiments. no, no miRNA or no LNA inhibitor. (B) SLK cells were transfected as described above (A), treated with IFN-␥ and TWEAK at 24 hpt, and assayed for luciferase activity as a direct measurement of caspase activity at 48 h posttreatment. Results are presented as the fold change in RLU in treated samples relative to untreated samples. Averages and SD were calculated from four independent experiments. SLK no, no miRNA. (C) SLK⫹K cells were transfected as described above for A, treated with IFN-␥ and TWEAK at 24 hpt, and assayed for luciferase activity as described above for B. Averages and SD were calculated from five independent experiments. SLK⫹K no, no LNA inhibitor.
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FIG. 6. miR-K10a-mediated downregulation of TWEAKR inhibits production of IL-8 and MCP-1. (A) HUVECs were transfected with miR-K10a and treated with TWEAK at 24 hpt. Culture supernatants were harvested at 24 h posttreatment and analyzed for IL-8 (top) and MCP-1 (bottom) expression. Data are expressed as the concentration of IL-8 or MCP-1 in the supernatant, and the numbers above the brackets indicate the fold change in expression upon treatment with TWEAK. Supernatants were collected from three independent experiments and analyzed in duplicate in the same assay. (B and C) HUVECs were transfected with either miR-K10a or a mutant of miR-K10a (MUTb). (B) Total cell lysates were harvested at 24 hpt and analyzed by Western blotting. Data show the average changes in normalized TWEAKR expression relative to levels in neg2-transfected cells. Averages and SD were calculated from data from three independent experiments. (C) Transfected HUVECs were treated with TWEAK at 24 hpt, and total cell RNA was harvested at 24 h posttreatment for quantitative RT-PCR analysis. Transcript levels of IFN-␣, IL-6, IL-8, and MCP-1 were measured, normalized to levels of -actin transcripts, and reported as the fold change in transcript levels upon stimulation with TWEAK. Averages and SD were calculated from data from three independent experiments. no, no miRNA.
together, the results of the caspase activation assay, annexin V staining, and TUNEL assay demonstrate that the miR-K10amediated knockdown of TWEAKR protects cells from TWEAK-induced apoptosis. miR-K10a-mediated downregulation of TWEAKR inhibits the proinflammatory response. TWEAK-stimulated apoptosis has been reported primarily for transformed cell lines, and primary endothelial cells stimulated with TWEAK showed no change in caspase activity (see Fig. S3 in the supplemental material). Therefore, we wondered how the inhibition of TWEAKR expression would benefit the virus in primary cells. TWEAK signaling was reported previously to induce proinflammatory cytokines in certain cell types (4). More specifically, TWEAK stimulation has been shown to increase the levels of expression of IL-8, MCP-1, ICAM-1, and E-selectin in primary endothelial cells (21). We hypothesized that the miRK10a-mediated knockdown of TWEAKR would prevent proinflammatory cytokine expression by primary endothelial cells (HUVECs), which may benefit the virus by dampening
and controlling the immune response. To investigate this possibility, HUVECs were transfected with miR-K10a and treated with TWEAK. Aliquots of culture medium were removed at 24 h posttreatment and assayed for the presence of IL-8 and MCP-1. Supernatants from negative-control miRNA-transfected cells showed a robust response to TWEAK stimulation, with a 10-fold upregulation of IL-8 expression and a 9-fold upregulation of MCP-1 expression. Cells transfected with miRK10a, however, produced much less of these cytokines in response to TWEAK, with less than a 5-fold upregulation of IL-8 production and 3-fold upregulation of MCP-1 production (Fig. 6A). To demonstrate the specificity of this effect for TWEAKmediated signaling, we performed quantitative RT-PCR to analyze transcript levels of IFN-␣ and IL-6, which have not been reported to be induced by TWEAK-mediated signaling in HUVECs (4, 21, 30), in addition to IL-8 and MCP-1. The transfection of HUVECs with miR-K10a resulted in a 5.8-fold downregulation of TWEAKR expression, while a mutant of miR-K10a (MUTb) had no effect on TWEAKR (Fig. 6B).
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There was no significant difference in the fold change in transcript levels of IFN-␣ and IL-6 upon treatment with TWEAK in cells transfected with neg2, miR-K10a, or MUTb. However, the fold change in both IL-8 and MCP-1 transcript levels was decreased in cells transfected with miR-K10a compared to multiple negative controls. These findings support the data for protein expression shown in Fig. 6A and demonstrate that the reduction in IL-8 and MCP-1 expression levels is specific to TWEAK/TWEAKR-mediated signaling. These results provide evidence for a second function of miR-K10a targeting of TWEAKR expression, namely, the control of proinflammatory cytokine production in response to TWEAK. Furthermore, we have observed the functional effects of the miR-K10a-mediated knockdown of TWEAKR in two different cell types relevant for KSHV infection and disease. DISCUSSION Little is known about the cellular targets of KSHV miRNAs and how this regulation affects viral pathogenesis. KSHV is an important human pathogen that causes multiple malignancies, primarily in HIV/AIDS patients. We are interested in determining how the expression of the virus-encoded miRNAs plays a role during infection in various cell types. In this report, we have shown data from luciferase reporter assays and protein expression assays that demonstrate the direct regulation of a cellular target, TWEAKR, by KSHV miR-K10a. The functional consequence of this regulation appears to depend on the cell type examined. In a KS tumor-derived endothelial cell line, we show that the downregulation of TWEAKR by miR-K10a results in the protection of cells from TWEAK-induced apoptosis. For primary human endothelial cells, we show that the downregulation of TWEAKR by miR-K10a results in a decrease in levels of IL-8 and MCP-1 production in response to TWEAK. In addition to providing information about a particular target of miR-K10a, these results suggest that the effect of miRNA-mediated regulation can vary depending on the cell type and perhaps also the stage of infection. To investigate the functional effect of the miR-K10a-mediated knockdown of TWEAKR, we performed experiments with two different cell types since the outcome of TWEAK signaling is cell type specific. Both systems, however, are relevant to KSHV infection and pathogenesis. SLK cells are a KS tumor-derived endothelial cell line, and although the KSHV genome was lost during passage in culture, the cellular environment may still be an adequate model for transformed cells within a KS lesion. In agreement with previous reports of other tumor cell lines, SLK cells express more TWEAKR than do primary endothelial cells (see Fig. S1A in the supplemental material). The treatment of SLK cells with TWEAK and IFN-␥ is consistent with the presence of abundant inflammatory infiltrates in KS lesions (13, 18, 40). In addition, TWEAK expression levels were reported previously to be elevated in certain human tumors, including kidney, breast, hepatocellular carcinoma, and colonic adenocarcinoma (24–26, 32, 53). Thus, it is valid to investigate the effect of TWEAKR knockdown on SLK cells with TWEAK and IFN-␥ stimulation. Primary endothelial cells (HUVECs) were used to examine the induction of proinflammatory cytokines to mimic the effect of miR-K10a prior to transformation. This system is relatable to both lytic
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and latent infections since KS lesions are heterogeneous populations of cells containing infected, transformed spindle cells intertwined with uninfected cells and inflammatory infiltrates (7, 22, 36). While TWEAK signaling was reported previously to induce IL-8, MCP-1, ICAM-1, and E-selectin in primary endothelial cells (21), it can also induce the expression of these and other proinflammatory molecules in other primary cells and tumor cell lines (4, 6, 27). Therefore, the proinflammatory effects of TWEAK/TWEAKR signaling may be relevant to KS lesions. It is interesting that while transfection with miR-K10a is efficient at protecting cells from TWEAK-induced apoptosis in all of the assays described, we did not see an identical replication of this phenotype using siRNAs targeting TWEAKR. This is not a result of a less efficient knockdown of TWEAKR expression with the siRNAs, as steady-state protein levels in cells transfected with miR-K10a or siRNAs targeting TWEAKR are comparable at 24 hpt (Fig. 4A) and 96 hpt (data not shown). Instead, we speculate that the dramatic protective effect of miR-K10a results from the combination of knocking down, but not completely ablating, the expression of TWEAKR and the targeting of additional cellular genes involved in apoptosis. The knockdown of TWEAKR by miRK10a decreases the ability of TWEAK to stimulate cells, and the knockdown of additional components of the apoptosis pathway could prevent signaling and progression to apoptosis. Previous reports of miR-K10a targeting BCLAF1 showed a decrease in caspase activity in the presence of miR-K10a, thus supporting this hypothesis (54). Since siRNAs typically have only one target mRNA, siRNAs targeting TWEAKR would likely be able to inhibit apoptosis only by preventing TWEAK signaling. Even in the presence of the siRNAs, TWEAKR is still detectable by Western blotting, and therefore, it is reasonable that we observed only a modest effect on caspase activation (Fig. 4B) and a minimal effect at later stages of apoptosis by annexin V and TUNEL staining (Fig. 5B and C and data not shown). Although we are not currently aware of other miRK10a targets involved in cytokine signaling, it is possible that the reduction in levels of IL-8 and MCP-1 production upon TWEAK stimulation is also compounded by additional targets. In summary, the phenotypes of a given miRNA will not always be the result of an inhibition of a single target gene. Future studies will address other targets of miR-K10a and how these relate to the phenotypes reported here. The reduction of proinflammatory cytokine production with the knockdown of TWEAKR expression was expected based on previously reported effects of TWEAK/TWEAKR signaling (4, 21) but requires a nuanced interpretation in the context of KS lesions. Inflammation is an important characteristic of KS: a variety of cytokines are produced by both endothelial cells and infiltrating lymphocytes, including IFN-␥, TNF, IL-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-8, and MCP-1 (10). Overexpression studies indicated that the viral proteins vFLIP (ORF71) and vGPCR (ORF74) activate NF-B, leading to increased MCP-1 and IL-8 expression levels (39, 42). However, several reports have shown that during KSHV infection of HUVECs, MCP-1 production is increased while IL-8 levels remain unchanged or decrease slightly (5, 52). Nonetheless, the expression of inflammatory cytokines appears to be necessary for the development of KS
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lesions (10). So why would KSHV express miRNAs that prevent the production of IL-8 and MCP-1 in response to TWEAK in the same cells that express viral proteins that drive the expression of these cytokines? We hypothesize that it is a matter of control: the virus expresses proteins that induce IL-8 and MCP-1 production of its own accord, indicating that the cytokines are regulated to a level compatible with viral infection. The repression of TWEAKR signaling by viral miRNAs prevents the expression of IL-8 and MCP-1 in response to host cytokine stimulation, which may elicit an inflammatory response beyond what is beneficial to viral infection. In this way, KSHV may be suppressing host-mediated inflammatory responses of KSHV-infected cells. As described above, TWEAKR is upregulated on human tumors and cell lines. Given that the predominant phenotype of TWEAK signaling in tumor cells is proapoptotic, it is possible that TWEAKR overexpression is a remnant of signaling and upregulation in the primary precursor cells, in which TWEAK signaling is likely to be prosurvival, proangiogenesis, and overall protumorigenic. In this case, TWEAKR is simply a biomarker for tumors. The downregulation of TWEAKR by KSHV and the subsequent protection of cells from TWEAKinduced apoptosis support this hypothesis. However, several studies have indicated a correlation between elevated levels of TWEAKR and severity of disease, namely, in glioblastomas (43, 44) and esophageal adenocarcinomas (48). Cell lines derived from these tumors have enhanced migration and survival responses to TWEAK stimulation, suggesting that for these particular cell types, TWEAK signaling may be beneficial for the tumor (44, 51). Thus, it remains unclear whether TWEAK signaling within a tumor microenvironment is pro- or antitumorigenic (50). Our studies demonstrating that the KSHV miRNA-mediated downregulation of TWEAKR protects cells from TWEAK-induced apoptosis suggest that TWEAK signaling in KS lesions has an overall antitumor effect. In this report, we predicted cellular targets of KSHV miRNAs based on expression changes induced by miRNA expression and not by sequences or known functions of potential targets genes. This approach led to the validation of TWEAKR as a target of miR-K10a. In functional studies, the miR-K10a-mediated downregulation of TWEAKR appears to be important for preventing apoptosis in response to cytokine stimulation in KS tumor-derived cells. In primary endothelial cells, the inhibition of TWEAKR results in a decrease in levels of TWEAK-stimulated IL-8 and MCP-1 production, which may be a way to control cytokine expression within infected cells and avoid a proliferation of the immune response. Other reports have investigated the role of viral miRNA regulation in pathways recognized as being important during KSHV infections. In contrast, we first looked for the cellular targets of miRNAs and allowed these data to lead our investigations, revealing new information about KSHV. These studies are significant not only for identifying and characterizing cellular targets of KSHV miRNAs but also for understanding the complex nature of KSHV pathogenesis. ACKNOWLEDGMENTS We thank Patricia Valdez for assistance with the Bio-Plex assays and Barbara Taylor and the NCI FACS core for assistance with annexin V and TUNEL assays. We thank Don Ganem for sharing unpublished
J. VIROL. microarray data. We thank members of the Ziegelbauer laboratory for help and support and Bob Yarchoan and Thomas Zheng for critical reviews of the manuscript. This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. REFERENCES 1. Almstrup, K., C. E. Hoei-Hansen, U. Wirkner, J. Blake, C. Schwager, W. Ansorge, J. E. Nielsen, N. E. Skakkebaek, E. Rajpert-De Meyts, and H. Leffers. 2004. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling. Cancer Res. 64: 4736–4743. 2. Bellare, P., and D. Ganem. 2009. Regulation of KSHV lytic switch protein expression by a virus-encoded microRNA: an evolutionary adaptation that fine-tunes lytic reactivation. Cell Host Microbe 6:570–575. 3. Boss, I. W., K. B. Plaisance, and R. Renne. 2009. Role of virus-encoded microRNAs in herpesvirus biology. Trends Microbiol. 17:544–553. 4. Burkly, L. 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