Distinct roles of transforming growth factor-beta-activated kinase 1 (TAK1)-c-Rel and interferon regulatory factor 4 (IRF4) pathways in human T cell lymphotropic virus 1-transformed T helper 17 cells producing interleukin-9

Abstract

Investigation of helper T cell markers in HTLV-1-transformed cell lines demonstrated that HuT-102 has an IL-9-producing Th17 phenotype. We confirmed the vital role of retinoic acid-related orphan receptor C, a Th17 transcription factor, in the expression of IL-17. Interferon regulatory factor 4 (IRF4), a transcription factor overexpressed in all HTLV-1-infected cells, regulated IL-17 and IL-9 concomitantly. We further demonstrated a novel pathway for the regulation of Tax-induced cytokines, IL-9 and IL-6, through TAK1-mediated nuclear accumulation of c-Rel. A microarray analysis for IRF4 knocked down HuT-102 cells showed a significant up-regulation in the set of genes related to Th1, mainly IFN-γ and several transcription factors. T-bet and IRF1, but not STAT1 and IRF9, participated in counteracting the inhibitory effect of IRF4 on the production of IFN-γ. Finally, suppression of both IRF4 and c-Rel resulted in the reduced proliferation. Collectively, these findings indicate that TAK1-c-Rel and IRF4 pathways play distinct roles in the maintenance of IL-9-producing Th17 phenotype of HTLV-1-transformed cells.

FIGURE 1.

HuT-102 cells show a Th17-like phenotype and respond to RORC knockdown. A, absolute concentration (ng/ml) of IL-17 in the supernatant collected from HTLV-1-transformed cell culture was determined by ELISA. B, protein expression of RORC, STAT3, and actin in HuT-102 cells was determined by Western blot. C, HuT-102 cells were transfected with siRNAs against RORC and Luc. At 60 h post-transfection, protein expression of RORC, IRF4, STAT3, and PCNA was determined by Western blotting. D, effects of RORC knockdown on the expression of IL-17A, IL-9, and IL-6 were examined by real-time RT-PCR.

FIGURE 2.

Distinct roles of IRF4 and c-Rel in the expression of IL-17, IL-9, and IL-6. A and C, effects of siRNAs against IRF4 (A) or c-Rel (C) on the protein expression of IRF4 and c-Rel by Western blot. B and D, effects of IRF4 (B) and c-Rel (D) siRNAs on the expression of IL-17A, IL-9, and IL-6 were examined by real-time PCR.

FIGURE 3.

TAK1 is the upstream regulator of c-Rel and controls IL-9 independently from IRF4. A, the expression of TAK1, IL-17A, IL-9, and IL-6 in HuT-shTAK1 and HuT-shLuc cells were examined by real-time RT-PCR. B and C, whole cell lysates (B) or cytoplasmic/nuclear extracts (C) prepared from HuT-shLuc, and HuT-shTAK1 cells were immunoblotted with antibodies for NF-κB/Rel subunits. D, HuT-shLuc and HuT-shTAK1 cells were transfected with siRNAs for IRF4 or Luc. At 60 h post-transfection, IL-9 expression was examined by RT-PCR. E, effects of IRF4 and c-Rel siRNAs on cell proliferation was examined in a colorimetric WST-1 assay. Data are the mean ± S.D. of triplicate determinations. The statistical significance of differences between groups was calculated by applying Tukey-Kramer method of analysis. *, p < 0.01 was statistically significant.

FIGURE 4.

IFN-γ is specifically controlled by IRF4. A, HuT-102, ED40515(−), and MT-2 cells were transfected with siRNAs against IRF4, c-Rel, RORC, and Luc. At 60 h post-transfection, IFN-γ mRNA expression was examined by RT-PCR. B, HuT-102 cells were transfected with siRNAs against IRF4 and T-bet. IRF4, T-bet, IFN-γ, and IL-17A mRNAs were quantified by real-time RT-PCR.

FIGURE 5.

IRF4 control IFNγ by an independent pathway through IRF1. A, HuT-102 cells were transfected with IRF4 siRNA. The up-regulation of Th1-related proteins was confirmed by Western blot. B, formation of the STAT1-STAT2 heterodimer was confirmed by immunoprecipitation. Whole cell lysates (WCE) were immunoprecipitated with anti-STAT1 antibody, and then the immunoprecipitates (IP) were immunoblotted with anti-STAT1 and STAT2 antibodies. C, neither STAT1 nor IRF9 knockdown reversed the up-regulation of IFN-γ in response to IRF4 knockdown in HuT-102 cells. D and E, HuT-102 cells were transfected with IRF4 and IRF1 siRNAs. Protein expression of IRF1 and IRF4 was determined by Western blot (D). IFN-γ, CXCL10, and IL-17A mRNAs were quantified by real-time RT-PCR (E). F, HuT-102 cells were transfected with an empty vector (mock) or pcDNA-IRF1. At 60 h post-transfection, the expressions of IRF1 and IFN-γ mRNAs were examined by real-time RT-PCR.