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

Abstract

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 3' untranslated region (UTR) of TWEAKR suggest a targeting of TWEAKR by miR-K10a. The mutation of two predicted miR-K10a recognition sites within the 3' 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.

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.

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.

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 μg/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.

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 inhibitor-transfected 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-γ 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-γ 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.

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-γ 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-γ. 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-γ, normalized to the percent FITC-positive cells in untreated samples. Averages and SD were calculated from four independent experiments. no, no miRNA.

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-α, IL-6, IL-8, and MCP-1 were measured, normalized to levels of β-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.