Kaposi's sarcoma-associated herpesvirus inhibits interleukin-4-mediated STAT6 phosphorylation to regulate apoptosis and maintain latency

Abstract

Cytokine-mediated JAK/STAT signaling controls numerous important biologic responses like immune function, cellular growth, and differentiation. Inappropriate activation of this signaling pathway is associated with a range of malignancies. Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious viral agent associated with Kaposi's sarcoma and may also contribute to B-cell disorders, which include primary effusion lymphoma (PEL) and multicentric Castleman's disease. However, regulation of cytokine-mediated lymphocytic immune response by KSHV is not fully understood. In this report, we demonstrate that KSHV suppresses the interleukin-4 (IL-4)-stimulated immune response of B-lymphocyte activation and cell proliferation. Moreover, we show that the latency-associated nuclear antigen (LANA) encoded by KSHV is essential for viral blocking of IL-4-induced signaling. LANA reduces phosphorylation of the signal transducers and activators of transcription 6 (STAT6) on Y-641 and concomitantly its DNA binding ability. Importantly, knockdown of endogenous STAT6 dramatically increases the sensitivity of PEL cells to low-serum stress or chemical-mediated cellular apoptosis and reactivation of KSHV from latent replication. Thus, these findings suggest that the IL-4/STAT6 signaling network is precisely controlled by KSHV for survival, maintenance of latency, and suppression of the host cytokine immune response of the virus-infected cells.

FIG. 1.

LANA contributes to the suppression of IL-4-induced tyrosine phosphorylation of STAT6 in PEL cells. (A) Levels of STAT6 mRNA transcripts in KSHV-positive PEL cells are not distinct from those in KSHV-negative B-lymphoma cells. KSHV-positive PEL cells (BC3, BCBL1, and JSC) and KSHV-negative B-lymphoma cells (BJAB and DG75) were individually subjected to quantitative PCR analysis for detection of STAT6 mRNA transcription. (B) IL-4 stimulates less phosphorylation of STAT6 in KSHV-infected B-lymphoma cells. Five million KSHV-negative cells (BJAB and DG75), KSHV-positive B-lymphoma cells (BC3, BCBL1, and JSC), or KSHV-infected BJAB cells (BJAB-K) were treated with or without IL-4 (5 ng/ml) for 2 h, respectively. The cell lysates were subjected to Western blotting with antibodies as indicated. The quantitation of tyrosine-phosphorylated STAT6 (p-STAT6) is presented at the bottom. The blank box indicates constitutive phosphorylation of STAT6 (p-STAT6^c). (C) KSHV-negative and -positive B-lymphoma cells were treated with 5 ng/ml IL-4 for various times. STAT6 phosphorylation was analyzed as described in the legend to panel B. Data are quantified in a graphical format. (D) Depletion of LANA enhances the response of PEL cells to IL-4 stimulation. Assays with lentivirus-mediated LANA knockdown (shLANA) or the scramble control (shCtrl) of JSC and BC3 cells were performed similar to the assays described for panel B.

FIG. 2.

LANA is critical for KSHV to reduce IL-4-induced expression of the B-cell activation marker. (A) BJAB cells infected with GFP-KSHV (BJAB-K) and uninfected BJAB cells, (B) primary B cells (CD19⁺) gated from human PBMCs with or without KSHV-GFP (Bac36) at 7 days postinfection, and (C) lentivirus-mediated LANA knockdown (shLANA) or JSC and BC3 scramble control (shCtrl) cells were individually cultured in the presence or absence of IL-4 for 2 h. The cells were fixed with and stained with anti-CD40-APC or anti-Ki-67-PE. The average ratios of each of the stained positive cells are shown on the histograms. The efficiency of KSHV infection and LANA knockdown is presented in the right panels as the percentage of GFP fluorescence detected by flow cytometry.

FIG. 3.

KSHV infection abolishes IL-4-stimulated B-cell proliferation in vitro. (A) KSHV-negative (BJAB and DG75) and -positive (BC3, BCBL1, JSC, and BJAB-K) cell lines; (B) BC3 and JSC cells with LANA constitutively knocked down. Cells were seeded in equal amounts and treated with or without 5 ng/ml IL-4 in the presence of LPS. Proliferation was measured at 24, 48, and 72 h after treatment by triplicate cell counting. These experiments were repeated twice, and average results are shown. The P values for comparisons between the untreated and treated groups were calculated by t test.

FIG. 4.

LANA suppresses IL-4-stimulated activation of STAT6 by affecting its DNA-binding ability. (A) LANA reduces the IL-4-induced phosphorylation of exogenous STAT6. DG75 cells were transiently transfected with FLAG-STAT6 (10 μg) or with LANA-myc (0, 5, 10, 15, or 20 μg). The equal amounts of DNA were made up with empty vector. Forty-eight hours posttransfection, cells were treated with 5 ng/ml IL-4 for 2 h before harvest. Whole-cell lysates (WCL) were subjected to immunoprecipitation (IP) with anti-FLAG followed by immunoblotting (IB) against p-STAT6. The membrane was stripped and reimmunoblotted (reIB) for anti-FLAG, or immunoblotting was directly performed with anti-myc as indicated on the figure. (B) Luciferase (Luc) reporter assays of IL-4-induced transcriptional activity of phosphorylated STAT6. BJAB cells with LANA (YLF) or GFP (Vector) stable expression, or with STAT6 knockdown (shSTAT6) or scramble control (shCtrl) generated by lentivirus-mediated transduction followed by 2 μg/ml puromycin selection (Western analysis was performed to detect LANA expression and STAT6 knockdown efficiency), were individually transfected with FLAG-STAT6 (0, 10 μg) in the presence of the reporter 3×stat6-luc (10 μg). At 48 h posttransfection, cells were treated or not treated with IL-4 for 2 h before harvest. Cell lysates were performed reporter assays. The results are presented as the fold change compared with vector alone. RLU, relative luciferase units. Asterisks indicate the fold change after IL-4 induction. (C) In vitro DNA-binding assays of phosphorylated STAT6 in the presence of LANA. Whole-cell extracts with preadjusted equally IL-4-induced phosphorylated STAT6 from BJAB/vector or BJAB/YLF were individually incubated with wild-type or mutant STAT6-binding DNA oligonucleotide followed by three washings, and the precipitates were Western blotted with p-STAT6 antibody. Five percent input of DNA oligonucleotide is shown at the bottom. RD, relative density.

FIG. 5.

STAT6 knockdown increases the sensitivity of KSHV-infected cells to extracellular stress. (A) PEL cell lines with STAT6 knockdown. Two BC3 cell lines with STAT6 knockdown (shSTAT6) or the scramble control (shCtrl) were generated from parental BC3 cells by lentivirus-mediated transduction followed by selection with 2 μg/ml puromycin, and Western analysis was performed to detect STAT6 knockdown efficiency. (B and C) STAT6 knockdown increases the response of PEL cells to low-serum stress. BC3 cells (parent, shSTAT6, or shCtrl) cultured in normal (7%) or low-serum (0.1%) medium overnight were subjected to cell cycle analysis. Apoptosis was indicated by average percentage of the sub-G₁ population from three independent experiment repeats. (D) STAT6 knockdown increases the sensitivity of PEL cells to etoposide (Etop)-induced apoptosis. BC3/shSTAT6 and BC3/shCtrl cells were treated with various concentration of eptopside for 3 h. Cell lysates were subjected to immunoblotting against PARP1 and GAPDH. The relative density (RD) of the 89-kDa active form of PARP1 was quantified and is shown at the bottom.

FIG. 6.

Inhibition of STAT6 expression contributes to reactivation of KSHV lytic replicaton. (A) Stress-induced RTA expression in PEL cells is correlated with inhibition of STAT6 expression. PEL cells (BC3 and BCBL1) and KSHV-negative BJAB cells were individually subjected to treatment with hypoxia (1% O₂ or 100 μM CoCl₂) or TPA and sodium butyrate (NaBu) (20 ng/ml and 1.5 mM, respectively) for 18 h. Cell lysates were subjected to immunoblotting against STAT6, RTA, and GAPDH. The relative density (RD) of RTA was quantified and is shown at the bottom. (B and C) STAT6 knockdown enhances the expression of RTA. PEL BC3 cells (parent, shSTAT6, or shCtrl) were subjected to treatment with TPA and NaB (20 ng/ml and 1.5 mM, respectively) for various times (0, 12, or 24 h). Cell lysates were subjected to the immunoblotting assay as in panel A, or the treated cells were fixed and subjected to an immunofluorescence assay with anti-RTA and DAPI (4′,6-diamidino-2-phenylindole) staining. The numbers shown in the figure indicate the percentages of RTA-positive cells.

FIG. 7.

A proposed model of KSHV-mediated suppression of IL-4/STAT6 signaling in B cells. Generally, IL-4 binds to its receptor, IL-4Rα, and the activation of IL-4Rα recruits the kinase JAK1 or -2, which directly phosphorylates and activates STAT6 to translocation into the nucleus and enhances IL-4-responsive gene expression (like IL-4 and CD40) in terms of immune cell growth and activation. In KSHV-infected cells, the latent antigen LANA encoded by KSHV inhibits IL-4-induced STAT6 phosphorylation and DNA binding affinity, which blocks the immune response of the host cell to IL-4 stimulation (autocrine and paracrine). The dephosphorylated STAT6 contributes to KSHV-infected cell survival under extracellular stress and maintenance of viral latency.