Constitutive Activation of Interleukin-13/STAT6 Contributes to Kaposi's Sarcoma-Associated Herpesvirus-Related Primary Effusion Lymphoma Cell Proliferation and Survival

Abstract

Activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway has been associated with numerous human malignancies, including primary effusion lymphomas (PELs). PEL, a cancerous proliferation of B cells, is caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Previously we identified constitutive phosphorylation of STAT6 on tyrosine 641 (p-STAT6(C)) in PEL cell lines BC3 and BCBL1; however, the molecular mechanism leading to this activation remains unclear. Here we demonstrate that STAT6 activation tightly correlates with interleukin-13 (IL-13) secretion, JAK1/2 tyrosine phosphorylation, and reduced expression of SHP1 due to KSHV infection. Moreover, p-STAT6(C) and reduction of SHP1 were also observed in KS patient tissue. Notably, blockade of IL-13 by antibody neutralization dramatically inhibits PEL cell proliferation and survival. Taken together, these results suggest that IL-13/STAT6 signaling is modulated by KSHV to promote host cell proliferation and viral pathogenesis.

Importance: STAT6 is a member of signal transducer and activator of transcription (STAT) family, whose activation is linked to KSHV-associated cancers. The mechanism through which STAT6 is modulated by KSHV remains unclear. In this study, we demonstrated that constitutive activation of STAT6 in KSHV-associated PEL cells results from interleukin-13 (IL-13) secretion and reduced expression of SHP1. Importantly, we also found that depletion of IL-13 reduces PEL cell growth and survival. This discovery provides new insight that IL-13/STAT6 plays an essential role in KSHV pathogenesis.

FIG 1

KSHV induces constitutive phosphorylation of STAT6 (p-STAT6^C). (A) Relative levels of p-STAT6^C/STAT6 in KSHV-positive (BC1, BC3, BCP1, BCBL1, JSC1, K-BJAB^Low, and K-iSLK) and KSHV/EBV-negative (DG75, BJAB, LCL1, and iSLK) cell lines. (B) p-STAT6^C is blocked by JAK inhibitor in PEL cells. Representative data for immunoblotting of treated BC3 cells are shown at the bottom. (C) The levels of IL-4Rα expression and phosphorylated JAK (p-JAK1 and p-JAK2) are elevated in KSHV-infected cells. The relative protein level is quantitated in the histogram (bottom). RD, relative density.

FIG 2

KSHV downregulates SHP1 expression. (A) Low expression of SHP1 in PEL cells. Cell lysates from equal amounts of KSHV-positive and -negative cells were subjected to immunoblotting analysis with antibodies as indicated. GAPDH was used as an internal control. (B) Analysis of SHP1 promoter sequence from B lymphoma and PEL cells. (C) Transcriptional level of SHP1 and SHP2 in KSHV-positive and -negative cells. Total RNA was extracted from cultured cells for quantitative PCR analysis for SHP1 and SHP2. GAPDH was used as an internal control. RD, relative density.

FIG 3

p-STAT6^C is associated with SHP1 downregulation by KSHV infection. (A) Loss of the KSHV episome due to inhibition of KAP1 reduced the level of p-STAT6^C. Cell lysates from BC3 cells (clone 1 and 2) with constitutive knockdown of KAP1 (shKAP1), a luciferase control (shKAP1), or supplementation with exogenous KAP1 with a FLAG tag were subjected to immunoblotting as indicated. The relative density (RD) of p-STAT6^C was quantitated and is shown in the graph in the middle. (B) Immunoblotting analysis. Whole-cell lysates of human PBMCs subjected to GFP-KSHV infection for 3 or 5 days or left uninfected were immunoblotted with antibodies against p-STAT6^C, STAT6, and GAPDH. The relative density of p-STAT6^C/STAT6 and LANA is individually quantitated, and relative trend line is predicted and shown at the bottom.

FIG 4

Expression levels of p-STAT6^C and SHP1 in KS patient tissues. (A) Representative images of immunohistochemistry assays of KS patient tissue and normal skin tissue against p-STAT6^C, SHP1, and LANA. A larger magnification (×400) of the image is also shown. (B) The relative intensities of p-STAT6^C and SHP1 in KS (n = 6) and normal skin tissue (n = 3) were individually quantitated by nuclear and cytoplasmic staining (double positive percentage: +, 10 to 20%; +/−, 1 to 10%; −, <1%) of 100 cells.

FIG 5

EBV infection reduces p-STAT6^C expression in PEL cells. (A) Lysates from BC3 cells infected with GFP-EBV for 2, 7, or 21 days or left uninfected (mock) were subjected to immunoblotting as indicated. (B) Relative ratio of p-STAT6^C to STAT6 during EBV infection. The results are the average relative fold compared with that for uninfected cells from 3 independent experiments. A representative image of BC3 cells with GFP-EBV infection at each time point is shown at the top. (C) Latent (EBNA1, EBNA2, and LMP1) and lytic (BZLF1, BALF5, and BcLF1) gene expression during GFP-EBV infection. The relative mRNA levels of the genes, including GAPDH as a control, were examined by quantitative PCR. The fold change was calculated by the threshold cycle (ΔΔC_T) method.

FIG 6

IL-13 expression is correlated with p-STAT6^C in PEL cells with KSHV infection alone. (A) Equal amounts of KSHV-positive and-negative B lymphoma cells were subjected to immunoblotting analysis with antibodies as indicated. (B) The transcription level of IL-13 but not IL-4 is upregulated by KSHV. (C) IL-13 but not IL-4 depletion reduces the level of p-STAT6^C. PEL cells were individually incubated with or without antibodies against IL-4, IL-13, or the same isotype IgG control for 12 h, followed by immunoblotting analysis as indicated. (D) The putative STAT6-binding sites within IL-13 and IL-4 promoters are indicated in the top portion. DNA sequencing reveals four hot mutation spots within the IL-13 promoter from PEL cells. nd, not determined. (E) p-STAT6^C has a higher affinity for the IL-13 promoter than for the IL-4 promoter. Chromatin immunoprecipitation (ChIP) with p-STAT6^C from BC3 cells was performed, and the relative density (RD) of p-STAT6^C bound to IL-13 or IL-4 promoter was detected by quantitative PCR. The specific amplicon was verified by agarose gel electrophoresis (top).

FIG 7

IL-13 is crucial for triggering PEL cell proliferation and survival. (A) Equal amounts (2 million) of BC3, BCBL1, BJAB, and K-BJAB^Low cells were individually treated with either 20 μg/ml of anti-IL-4, anti-IL-13 antibody or an IgG isotype control. Proliferation was measured at 24, 48, and 72 h by cell vitality counter. The proliferation rate of the treated cells is presented as a percentage of the corresponding untreated control and was calculated as the mean of triplicate samples. The statistical significance was evaluated, and P values of <0.05 are indicated by double asterisks. (B) IL-13 enhances PEL cell survival. BC3 cells were individually subjected to treatment with anti-IL-13 or control antibody (as for panel A) in combination or not with sera starved (0.1%) overnight, followed by analysis of the cell cycle profile. The average percentages of different phases (sub-G1, G₁, S, G₂/M) from three repeats are presented in a histogram.

FIG 8

Schematic representation of constitutive activation of IL-13/STAT6 signaling in PEL cells. In KSHV-associated B lymphoma cells, KSHV not only significantly blocks IL-4-induced activation of STAT6 (high) for suppressing immune cell growth and activation (24) but also downregulates SHP1 and constitutively activates IL-13-mediated phosphorylation of STAT6 (low) to a certain extent via selective induction of IL-13 but not IL-4 expression, for enhancing host cell proliferation and survival. However, EBV coinfection dramatically blocks KSHV-induced activation of IL-13/STAT6 signaling.