Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression - PubMed (original) (raw)
Interferon regulatory factor 4 (IRF4) interacts with NFATc2 to modulate interleukin 4 gene expression
Jyothi Rengarajan et al. J Exp Med. 2002.
Abstract
Proteins of the nuclear factor of activated T cells (NFAT) family of transcription factors are critical for lymphocyte activation in the immune system. In particular, NFATs are important regulators of inducible IL-4 gene expression. Interferon regulatory factor 4 (IRF4) is an immune system-restricted interferon regulatory factor that is required for lymphocyte activation, but its molecular functions in the T lineage remain to be elucidated. We demonstrate that IRF4 potently synergizes with NFATc2 to specifically enhance NFATc2-driven transcriptional activation of the IL-4 promoter. This function is dependent on the physical interaction of IRF4 with NFATc2. IRF4 synergizes with NFATc2 and the IL-4-inducing transcription factor, c-maf, to augment IL-4 promoter activity as well as to elicit significant levels of endogenous IL-4 production. Furthermore, naïve T helper cells from mice lacking IRF4 are compromised severely for the production of IL-4 and other Th2 cytokines. The identification of IRF4 as a partner for NFATc2 in IL-4 gene regulation provides an important molecular function for IRF4 in T helper cell differentiation.
Figures
Figure 1.
IRF4 synergizes with NFATc2 to enhance NFATc2-driven IL-4 transcriptional activity but does not regulate the IL-2 and Egr3 promoters. M12 B lymphoma cells (a) and EL4 thymoma cells (b) were transiently transfected with the IL-4 luciferase reporter and cotransfected with the indicated microgram amounts of IRF4 and/or NFATc2 expression plasmids. M12 cells were transiently transfected with (c) IL-2 luciferase reporter or (d) Egr3 luciferase reporter and cotransfected with control vectors (15 μg), NFATc2 (15 μg) or IRF4 plasmids (15 μg) individually or together (7.5 μg each). (e) M12 cells transfected with the 3×NFAT luciferase reporter and the indicated microgram amounts of IRF4 and/or NFATc2. All results represent the mean of three to five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ± SEM.
Figure 2.
Physical association between NFAT and IRF4 does not extend to IRF8. Coimmunoprecipitation of IRF4 and NFATc2. (a) 293T cells were transiently transfected with Flag-NFATc2 and HA-IRF4 or control vector and immunoprecipitated using anti-HA or control anti-myc antibodies. Proteins were run on SDS-PAGE and immunoblotted with anti-Flag antibody to detect Flag-NFATc2 in the immunoprecipitate and lysates (lys) (b) 293T cells were transiently transfected with HA-IRF4 and Flag-NFATc2 or control vector. Immunoprecipitation was performed using anti-Flag or control anti-myc antibodies and immunoblotted with anti-HA antibody to detect HA-IRF4 in the immunoprecipitate or lysate (lys). (c) Extracts from Th1 or Th2 cells were immunoprecipitated with preimmune serum (P) or anti-NFATc2 antibody and probed with anti-IRF4-HRP or reprobed with anti-NFATc2. (d) M12 cells were transiently transfected with Flag-NFATc2 and HA-IRF4 or HA-IRF8. Immunoprecipitation with anti-HA antibody and immunoblotting with anti-Flag antibody to detect immunoprecipitate. Anti-HA antibody was used to immunoblot input lysates to detect IRF4 and IRF8. (e) Comparison of IRF4 and IRF8 transcriptional synergy with NFATc2 in M12 cells. Results represent the mean of three to five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ±SEM. Control plasmid DNA was used to normalize for DNA concentration.
Figure 2.
Physical association between NFAT and IRF4 does not extend to IRF8. Coimmunoprecipitation of IRF4 and NFATc2. (a) 293T cells were transiently transfected with Flag-NFATc2 and HA-IRF4 or control vector and immunoprecipitated using anti-HA or control anti-myc antibodies. Proteins were run on SDS-PAGE and immunoblotted with anti-Flag antibody to detect Flag-NFATc2 in the immunoprecipitate and lysates (lys) (b) 293T cells were transiently transfected with HA-IRF4 and Flag-NFATc2 or control vector. Immunoprecipitation was performed using anti-Flag or control anti-myc antibodies and immunoblotted with anti-HA antibody to detect HA-IRF4 in the immunoprecipitate or lysate (lys). (c) Extracts from Th1 or Th2 cells were immunoprecipitated with preimmune serum (P) or anti-NFATc2 antibody and probed with anti-IRF4-HRP or reprobed with anti-NFATc2. (d) M12 cells were transiently transfected with Flag-NFATc2 and HA-IRF4 or HA-IRF8. Immunoprecipitation with anti-HA antibody and immunoblotting with anti-Flag antibody to detect immunoprecipitate. Anti-HA antibody was used to immunoblot input lysates to detect IRF4 and IRF8. (e) Comparison of IRF4 and IRF8 transcriptional synergy with NFATc2 in M12 cells. Results represent the mean of three to five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ±SEM. Control plasmid DNA was used to normalize for DNA concentration.
Figure 3.
Mapping the domains of IRF4 required for interaction and transcriptional synergy with NFATc2. (a) 293T cells were transiently transfected with Flag-NFATc2 and with each of the different HA-IRF4 mutants. Immunoprecipitation was performed using anti-HA antibody, and the immunoprecipitates were run on SDS-PAGE and detected by immunoblot using anti-Flag antibody to detect the association of each IRF4 mutant with NFATc2 relative to full-length IRF4 (1–450). Lysates from each transfection also were run on SDS-PAGE to detect the IRF4 proteins in the input. (b) M12 B lymphoma cells were transiently transfected with the IL-4 luciferase reporter and cotransfected with NFATc2 and each of the different IRF4 mutant expression plasmids including full-length (1–450). Results represent the mean of five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ±SEM. Control plasmid DNA was used to normalize for DNA concentration.
Figure 3.
Mapping the domains of IRF4 required for interaction and transcriptional synergy with NFATc2. (a) 293T cells were transiently transfected with Flag-NFATc2 and with each of the different HA-IRF4 mutants. Immunoprecipitation was performed using anti-HA antibody, and the immunoprecipitates were run on SDS-PAGE and detected by immunoblot using anti-Flag antibody to detect the association of each IRF4 mutant with NFATc2 relative to full-length IRF4 (1–450). Lysates from each transfection also were run on SDS-PAGE to detect the IRF4 proteins in the input. (b) M12 B lymphoma cells were transiently transfected with the IL-4 luciferase reporter and cotransfected with NFATc2 and each of the different IRF4 mutant expression plasmids including full-length (1–450). Results represent the mean of five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ±SEM. Control plasmid DNA was used to normalize for DNA concentration.
Figure 4.
IRF4 synergizes with NFATc2 and c-maf to enhance IL-4 transcriptional activity and endogenous IL-4 production. M12 cells were transiently transfected with the indicated expression plasmids along with IL-4 luciferase reporter. (a) After transfection, half of the transfected cells were cultured for 24 h and assayed for luciferase activity. (b) Cells from the other half were cultured for 72 h, and the supernatants were assayed for IL-4. Results are representatives of five independent experiments. Luciferase activity is reported as a fold increase relative to reporter alone ±SEM. IL-4 was quantified by ELISA.
Figure 5.
Compromised Th2 differentiation in IRF4−/− naïve Th cells. Naïve Thp were isolated from wild-type and IRF4−/− mice and differentiated in vitro under unskewed (U) Th1 or Th2 conditions (see Materials and Methods). (a) Intracellular cytokine staining for IL-4 and IFN-γ production on day 7 upon stimulation with PMA and ionomycin. Percentage of cells that secrete each cytokine is indicated. (b) On day 7, U, Th1, and Th2 cells from wild-type (WT) and IRF4−/− mice were stimulated with anti-CD3, and supernatants were analyzed for cytokine production 24 h later.
Figure 5.
Compromised Th2 differentiation in IRF4−/− naïve Th cells. Naïve Thp were isolated from wild-type and IRF4−/− mice and differentiated in vitro under unskewed (U) Th1 or Th2 conditions (see Materials and Methods). (a) Intracellular cytokine staining for IL-4 and IFN-γ production on day 7 upon stimulation with PMA and ionomycin. Percentage of cells that secrete each cytokine is indicated. (b) On day 7, U, Th1, and Th2 cells from wild-type (WT) and IRF4−/− mice were stimulated with anti-CD3, and supernatants were analyzed for cytokine production 24 h later.
References
- Rao, A., C. Luo, and P.G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15:707–747. - PubMed
- Crabtree, G.R. 1999. Generic signals and specific outcomes: signaling through Ca2+, calcineurin, and NF-AT. Cell. 96:611–614. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources