Induction of CCL20 production by Kaposi sarcoma-associated herpesvirus: role of viral FLICE inhibitory protein K13-induced NF-kappaB activation

Abstract

Kaposi sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8, is the etiologic agent of Kaposi sarcoma (KS), an angioproliferative lesion characterized by dramatic angiogenesis and inflammatory infiltration. In this study, we report that expression of chemokine CCL20, a potent chemoattractant of dendritic cells and lymphocytes, is strongly induced in cultured cells either by KSHV infection or on ectopic expression of viral FLICE inhibitory protein K13. This induction is caused by transcriptional activation of CCL20 gene, which is mediated by binding of the p65, p50, and c-Rel subunits of the transcription factor nuclear factor-kappaB (NF-kappaB) to an atypical NF-kappaB-binding site present in the CCL20 gene promoter. The CCL20 gene induction is defective in K13 mutants that lack NF-kappaB activity, and can be blocked by specific genetic and pharmacologic inhibitors of the NF-kappaB pathway. CCR6, the specific receptor for CCL20, is also induced in cultured cells either by KSHV infection or on K13 expression. Finally, expression of CCL20 and CCR6 is increased in clinical samples of KS. These results suggest that KSHV and K13-mediated induction of CCL20 and CCR6 may contribute to the recruitment of dendritic cells and lymphocytes into the KS lesions, and to tumor growth and metastases.

Figure 1

KSHV up-regulates CCL20 expression in HUVEC and PEL cell lines. (A) HUVECs were infected with KSHV for 48 hours, and expression of CCL20 was measured by quantitative RT-PCR analysis and normalized to GNB2L1 (housekeeping control). PCRs were performed in triplicate and the data presented as fold change in target gene expression (mean ± SE). The results of the quantitative RT-PCR analysis were confirmed by separating the products (5.0 μL) on a 1.5% agarose gel followed by staining with ethidium bromide (bottom panel). (B,C) Cellular supernatants from KSHV-infected HUVECs were collected 3 days (B) and 6 days (C) after infection and used to measure the secretion of CCL20 by ELISA. The values shown are averages (mean ± SE) of 1 representative experiment of 3 in which the level of CCL20 secretion was measured in duplicate. (D) Kinetics of CCL20 up-regulation in KSHV-infected iHUVEC as measured by ELISA. (E) Level of CCL20 mRNA expression, as measured by quantitative RT-PCR, in PEL cell lines naturally infected with KSHV (BC-1, BCBL-1, and JSC-1). The KSHV-negative BJAB cell line was used as negative control.

Figure 2

K13 induces CCL20 expression in HUVEC and PEL cell lines. (A) HUVECs stably expressing a control vector or K13-ER^TAM were mock-treated or treated with 4-OHT (50 nM) for 48 hours after which RNA was extracted and used for quantitative RT-PCR. The results of the quantitative RT-PCR analysis were confirmed by agarose gel electrophoresis (bottom panel). (B) Supernatants from cells treated in panel A were used for the measurement of CCL20 protein by ELISA. (C) Quantitative RT-PCR analysis showing increased expression of CCL20 mRNA in BCBL-1 and K562 cells stably transduced with a Flag-K13–expressing retroviral vector (top panel). The expression of Flag-tagged K13 in cell lysates was confirmed by immunoblotting (bottom panel). (D) Quantitative RT-PCR analyses showing down-regulation of K13 and CCL20 mRNAs in BCBL-1 cells infected with a K13 shRNA-expressing lentiviral vector compared with cells infected with a control shRNA vector.

Figure 3

K13-induced NF-κB activity is critical for the activation of CCL20 promoter. (A) The 293T cells were transfected with an empty vector or the indicated vFLIPs (250 ng/well) along with a WT CCL20 promoter luciferase construct (75 ng/well) and a pRSV/LacZ (β-galactosidase) reporter construct (75 ng/well), and the reporter assay performed as described in “Luciferase reporter assay.” The values shown are averages (mean ± SE) of 1 representative experiment of 3 in which each transfection was performed in duplicate. (B) Ectopic expression of WT K13 but not its NF-κB–defective mutants (K13 58AAA and K13 67AAA) induces CCL20 promoter activity. The experiment was performed essentially as described in panel A. (C) The NF-κB site in the CCL20 promoter is critical for activation by K13. The 293T cells were transfected with a control vector or a vector encoding K13 along with either CCL20-WT-Luc or CCL20-ΔNF-κB-Luc reporter constructs, and the luciferase reporter assay performed as described in panel A. The values shown are averages (mean ± SE) of 1 representative experiment of 3 in which each transfection was performed in duplicate.

Figure 4

K13 triggers the recruitment of p65, p50, and c-Rel to the CCL20 promoter. (A) An ELISA-based NF-κB binding assay showing increased binding of p65, p50, and c-Rel subunits in nuclear extracts derived from K13-expressing cells to the NF-κB site present in the CCL20 promoter. (B) K13 fails to activate CCL20 promoter in p65 and p50 knockout cells. WT and p65^−/− and p50^−/− MEFs were transfected with an empty vector or K13 along with a CCL20-WT-Luc (75 ng/well) and a Renilla reporter construct using Lipofectamine. The luciferase assay was performed 48 hours after transfection as described previously.

Figure 5

Role of NF-κB pathway in K13-induced CCL20 promoter activation. (A) Dominant-negative mutants of IκBα (IκBαΔN and IκBαSS32/36AA) block K13-induced CCL20 promoter activity. The 293T cells were transfected either with an empty vector or K13, along with a CCL20 luciferase reporter construct and a β-galactosidase reporter construct, as described in Figure 3A. The amount of IκBα mutant plasmids (500 ng/well) was 5 times the amount of vector or K13 (100 ng/well) plasmid, and the total amount of transfected DNA was kept constant by adding empty vector. The values shown are averages (mean ± SE) of 1 representative experiment of 3 in which each transfection was performed in duplicate. (B,C) Pharmacologic inhibitors of NF-κB block K13-induced CCL20 promoter activation. The 293T cells were transfected with an empty vector or a vector encoding K13, and 30 minutes after transfection treated with dimethyl sulfoxide (vehicle) or the indicated compounds for 16 hours before cell lysis. Reporter assay was performed as described for Figure 3A. (D) HUVECs were pretreated with Bay-11-7082 (5 μM) or As₂O₃ (2.5 μM) for 2 hours and subsequently infected with KSHV as described previously. Four hours after infection, the medium was changed with fresh medium containing Bay-11-7082 or As₂O₃. After overnight incubation, supernatant was collected and CCL20 secretion was measured as described in Figure 1B.

Figure 6

KSHV infection and K13 expression induce the expression of CCL20 receptor CCR6 in HUVECs. Quantitative RT-PCR analysis showing induction of CCR6 mRNA in HUVECs after infection with KSHV (A) and in HUVEC-K13-ER^TAM cells on treatment with 4-OHT (B). The experiments were performed essentially as described in Figures 1A and 2A, respectively.

Figure 7

Up-regulation of CCL20 and its receptor CCR6 in KS samples. (A) Box-whisker plots with 95% confidence intervals for the RNA expression of CCL20, K13, LANA, and CCR6 in KSHV-infected patients' lymph nodes (KS lymph nodes, n = 5) and skin samples (KS skin samples, n = 10) along with corresponding benign lymph nodes (n = 6) and normal skin (n = 10) samples. Box-whisker plot indicates the median expression of respective genes relative to β-actin (line in the box) and their interquartile range (box). The whiskers present an entire range of expression level of indicated genes in a group, the upper value being the largest observation ≥ 75th percentile + 1.5 × (interquartile range), with the lowest value being the smallest observation ≤ 25th percentile − 1.5 × (interquartile range). The RNA was isolated from flash frozen tissues, and the expression levels of the indicated genes were determined by quantitative RT-PCR. The expression levels of CCL20 and its receptor CCR6 were significantly higher in the KS samples compared with corresponding uninfected controls (*P < .05, Mann-Whitney U test). Similarly, the expression level of LANA and K13, 2 key genes of KSHV infectivity, were significantly higher in KS samples than the corresponding healthy skin or lymph node samples (*P < .05, Mann-Whitney U test). (B) Hematoxylin and eosin staining shows the presence of characteristic spindle cells in a KS biopsy specimen. (C) Immunohistochemistry shows the presence of LANA-positive cells with typical brown punctate nuclear staining (black arrows) in the KS lesion. (D) Double immunostaining with LANA and CCL20 antibodies shows that a majority of CCL20-positive cells (red; white arrows) are positive for LANA (brown). (B-D) original magnification ×400.