Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 production through the activation of Bruton's tyrosine kinase and STAT3

Abstract

Previous data demonstrate that Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) enhances IL-10 to promote the survival of LMP2A-expressing B cell lymphomas. Since STAT3 is an important regulator of IL-10 production, we hypothesized that LMP2A activates a signal transduction cascade that increases STAT3 phosphorylation to enhance IL-10. Using LMP2A-negative and -positive B cell lines, the data indicate that LMP2A requires the early signaling molecules of the Syk/RAS/PI3K pathway to increase IL-10. Additional studies indicate that the PI3K-regulated kinase, BTK, is responsible for phosphorylating STAT3, which ultimately mediates the LMP2A-dependent increase in IL-10. These data are the first to show that LMP2A signaling results in STAT3 phosphorylation in B cells through a PI3K/BTK-dependent pathway. With the use of BTK and STAT3 inhibitors to treat B cell lymphomas in clinical trials, these findings highlight the possibility of using new pharmaceutical approaches to treat EBV-associated lymphomas that express LMP2A.

Keywords: B cell; Bruton's tyrosine kinase (BTK); Epstein-Barr virus; Interleukin-10; Latent Membrane Protein 2A; Signal Transducer and Activator of Transcription 3 (STAT3).

Fig 1

LMP2A uses Syk, Ras, and PI3K to increase IL-10 production. LMP2A-negative (Vector.1 and Vector.2) and LMP2A-positive (LMP2A1.1 and LMP2A1.2) B cell lines were incubated in the absence or presence of the (A) Syk inhibitor, R788 (Fostamatinib), (B) Ras inhibitor, Manumycin A, or (C) PI3K inhibitor, Wortmannin for 24 hours and supernatants were isolated for analysis using an IL-10 ELISA. Data are representative of 3 independent experiments with similar results. * indicates p<0.05 when compared to LMP2A-negative B cell lines, ** indicates p<0.05 when compared to the same cell line that was incubated in the absence of inhibitor

Fig 2

LMP2A uses PI3K to activate STAT3 to increase IL-10 production. (A) Protein from LMP2A-negative or –positive B cell lines (10 x 10⁶ cells) was isolated and analyzed for phosphorylated (Y705)-STAT3, total STAT3, or GAPDH by Western blot analysis. (B) Protein from LMP2A-negative or –positive B cell lines (10 x 10⁶ cells) was isolated after a 24 hour incubation in the absence or presence of R788 (5 uM), Manumycin A (0.5 uM), Wortmannin (10 uM), or Stattic (1.75 uM). Phosphorylated (Y705)-STAT3, total STAT3, or GAPDH were analyzed by Western Blot analysis. (C) LMP2A-negative and –positive B cell lines were incubated in the absence or presence of increasing concentrations of the STAT3 inhibitor, Stattic, for 24 hours and supernatants were isolated for analysis using an IL-10 ELISA. Data are representative of 3 independent experiments with similar results. For Western blot analysis, the ratio of phosphorylated (Y705)-STAT3/total STAT3 is given underneath each respective band. * indicates p<0.05 when compared to LMP2A-negative B cell lines, ** indicates p<0.05 when compared to the same cell line that was incubated in the absence of inhibitor

Fig 3

LMP2A activates BTK to phosphorylate STAT3 and increase IL-10 production. (A) Protein from LMP2A-negative or –positive B cell lines (10 x 10⁶ cells) was isolated after a 24 hour incubation in the absence or presence of the BTK inhibitor, Ibrutinib (10 uM). Total and phosphorylated-STAT3 were analyzed by Western Blot analysis. (B) LMP2A-negative and –positive B cell lines were incubated in the absence or presence of increasing concentrations of Ibrutinib for 24 hours and supernatants were isolated for analysis using an IL-10 ELISA. Data in (A–B) are representative of 3 independent experiments with similar results. For Western blot analysis, the ratio of phosphorylated (Y705)-STAT3/total STAT3 is given underneath each respective band. * indicates p<0.05 when compared to LMP2A-negative B cell lines, ** indicates p<0.05 when compared to the same cell line that was incubated in the absence of inhibitor

Fig 4

LMP2A increases STAT3 phosphorylation in the context of latent EBV infection. (A) Protein from LMP2A-negative (ES1) or –positive (LCL3) lymphoblastoid cell lines (10 x 10⁶ cells) was isolated after a 24 hour incubation in the absence or presence of Ibrutinib (1.75 uM) or Stattic (1.75 uM). Phosphorylated (Y705)-STAT3, total STAT3, or GAPDH were analyzed by Western Blot analysis. Data are representative of three independent experiments with similar results. For Western blot analysis, the ration of phosphorylated (Y705)-STAT3/total STAT3 is given underneath each respective band. (B) Model of the mechanism by which LMP2A increases IL-10 production. LMP2A activates Syk to increase Ras and subsequent PI3K activation. PI3K activation results in the stimulation of BTK to phosphorylate STAT3 to increase the amounts of IL-10 RNA in LMP2A-expressing cells.