Research ArticleImmunology

PDLIM2 Inhibits T Helper 17 Cell Development and Granulomatous Inflammation Through Degradation of STAT3

See allHide authors and affiliations

Science Signaling  06 Dec 2011:
Vol. 4, Issue 202, pp. ra85
DOI: 10.1126/scisignal.2001637
  • Fig. 1

    Enhanced liver granuloma formation and TH17 cell differentiation in Pdlim2−/− mice. (A and B) Representative H&E-stained liver sections on day 7 after intraperitoneal injection of heat-killed P. acnes (0.3 mg). Scale bars, 100 μm. (C and D) ELISA analysis of IL-17 production and real-time RT-PCR analysis of the abundances of mRNAs of IL-21, IL-22, IFN-γ, and IL-4 in CD4+ T cells from mice that were untreated (−) or were treated (+) with heat-killed P. acnes (1 mg for IL-17 experiments and 0.3 mg for IL-21, IL-22, IFN-γ, and IL-4 experiments). Three to seven mice per group were analyzed. Shown are the mean values ± SEM. NS, not significant.

  • Fig. 2

    Enhanced in vitro TH17 differentiation of cells from Pdlim2−/− mice. (A) Real-time RT-PCR analysis of genes expressing TH17-type cytokines by Pdlim2+/+ and Pdlim2−/− CD4+ T cells in P. acnes–induced in vitro TH17 cell differentiation experiments. (B) Heat-killed P. acnes–induced expression of the gene encoding IL-17A by CD4+ T cells cocultured with CD11c+ dendritic cells (DC) isolated from Pdlim2+/+ and Pdlim2−/− mice in the presence of heat-killed P. acnes was analyzed by real-time RT-PCR assay. (C) ELISA analysis of IL-17A production (left) and flow cytometric analysis of intracellular IL-17A and IFN-γ (right) in naïve CD4+ T cells from Pdlim2+/+ and Pdlim2−/− mice. Numbers in the flow cytometry dot plots indicate the percentages of IL-17A+ cells. (D) Real-time RT-PCR analysis of the expression of the genes encoding IL-17A and IL-21 in naïve CD4+ T cells from Pdlim2+/+ and Pdlim2−/− mice. (E) Real-time RT-PCR analysis of IL-6–induced expression of the gene encoding RORγt in CD4+ T cells from Pdlim2+/+ and Pdlim2−/− mice. Data are representative of at least three independent experiments and are shown as means ± SD. *P < 0.05; **P < 0.01.

  • Fig. 3

    PDLIM2 physically associates with STAT3 and suppresses STAT3 signaling. (A and B) Luciferase activity in (A) HEK 293T cells cotransfected with an α2-macroglobulin luciferase reporter and either control or PDLIM2 expression plasmids that were stimulated by LIF (100 ng/ml) for 8 hours, or in (B) HEK 293T cells cotransfected with plasmid encoding STAT3-C. (C and D) Interaction between PDLIM2 and (C) wild-type (WT) STAT3 or (D) STAT3 Y705F mutant (YF) in the lysates of HEK 293T cells that had been transfected with the indicated plasmids and then were stimulated without or with LIF (100 ng/ml) for 30 min. Samples were subjected to immunoprecipitation (IP) with antibody against FLAG followed by Western blotting analysis (IB) with antibody against the c-Myc tag. TCL, total cell lysates. (E) Critical region of STAT3 for interaction with PDLIM2. Western blotting analysis of the lysates of HEK 293T cells transfected with plasmids encoding PDLIM2 and GST-fused STAT3 deletion mutants, which were pulled down with GSH-Sepharose (GSH-bound). The constructs of the STAT3 deletion mutants are shown on the top. (F) Critical region of PDLIM2 that is required for its association with STAT3. Western blotting analysis of lysates from HEK 293T cells transfected with plasmids encoding FLAG-STAT3 and the indicated PDLIM2 mutants, which were subjected to immunoprecipitation with antibody against the FLAG tag. The constructs of the PDLIM2 deletion mutants are shown on the top. (G) In vitro binding of the LIM domain of PDLIM2 to STAT3. Western blotting analysis of lysates from HEK 293T cells that were transfected with plasmid encoding FLAG-STAT3, incubated in vitro with GST-LIM, and subjected to pull-down assay with GSH-Sepharose. Data are representative of at least three independent experiments and are shown as means ± SD. **P < 0.01.

  • Fig. 4

    PDLIM2 promotes the ubiquitination and degradation of STAT3. (A to C) Ubiquitination assay for STAT3 in HEK 293T cells cotransfected with plasmid encoding histidine-tagged ubiquitin (His-Ub) and the indicated plasmids. His-tagged proteins were purified by Ni-NTA resin (Ni-pull down). Polyubiquitination of STAT3 (A and B) and autoubiquitination of PDLIM2 (C) were detected with the indicated antibodies. (D) In vitro polyubiquitination of STAT3 by the LIM domain of PDLIM2 (GST-LIM). The lysates from HEK 293T cells cotransfected with plasmids encoding FLAG-STAT3, E1 enzyme, and E2 enzyme were incubated in vitro with GST-LIM and ubiquitin and were then subjected to Western blotting analysis with antibody against the FLAG tag to detect ubiquitinated STAT3. (E) Western blotting analysis for STAT3 in HEK 293T cells transfected with plasmid encoding c-Myc–PDLIM2, which were left untreated or were treated with MG132 (20 μM) for 3 hours. (F) Western blotting analysis for STAT3 in HEK 293T cells transfected with plasmids encoding the indicated PDLIM2 mutants. Data are representative of at least three independent experiments.

  • Fig. 5

    Enhanced STAT3 activation in the context of PDLIM2 deficiency. (A) STAT3 protein amounts in Pdlim2+/+ or Pdlim2−/− MEFs treated with cycloheximide (CHX) (50 ng/ml). Bottom shows the densitometric analysis of the intensities of the bands corresponding to STAT3 protein relative to those of actin. (B) Luciferase activity in Pdlim2+/+ or Pdlim2−/− MEFs transfected with plasmid encoding the α2-macroglobulin promoter–driven luciferase reporter, which were stimulated without or with LIF (30 or 100 ng/ml) for 8 hours. (C) STAT3 protein abundance in Hep3B cells transfected with control siRNA or PDLIM2-specific siRNA. (D) Luciferase activity in Hep3B cells first transfected with control or PDLIM2-specific siRNA and then with plasmid encoding the α2-macroglobulin promoter–driven luciferase reporter, which were then stimulated without or with IL-6 (10 or 30 ng/ml) for 8 hours. Data in (C) and (D) represent the means ± SD. *P < 0.05; **P < 0.01. (E and F) Cytoplasmic and nuclear STAT3 protein amounts in WT CD4+ T cells pretreated without or with MG132 (20 μM) for 1.5 hours and stimulated with IL-6 for 1 hour (E) or, in the case of Pdlim2+/+ or Pdlim2−/− CD4+ T cells, stimulated with IL-6 for 1 hour (F). Data are representative of at least three independent experiments.

Additional Files

  • Supplementary Materials for:

    PDLIM2 Inhibits T Helper 17 Cell Development and Granulomatous Inflammation Through Degradation of STAT3

    Takashi Tanaka,* Yu Yamamoto, Ryuta Muromoto, Osamu Ikeda, Yuichi Sekine, Michael J. Grusby, Tsuneyasu Kaisho, Tadashi Matsuda*

    *To whom correspondence should be addressed. E-mail: ttanaka{at}rcai.riken.jp (T.T.); tmatsuda{at}pharm.hokudai.ac.jp (T.M.)

    This PDF file includes:

    • Fig. S1. PDLIM2 in T helper cell subsets.
    • Fig. S2. P. acnes–induced granuloma formation in Il-6– and Pdlim2-deficient mice.
    • Fig. S3. Enhanced P. acnes–induced IL-6 production by Pdlim2–/– dendritic cells.
    • Fig. S4. IL-6 induces tyrosine phosphorylation of STAT3, but not STAT1.
    • Fig. S5. Reduction in Pdlim2 mRNA abundance by PDLIM2-specific siRNA.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 340 KB


    Citation: T. Tanaka, Y. Yamamoto, R. Muromoto, O. Ikeda, Y. Sekine, M. J. Grusby, T. Kaisho, T. Matsuda, PDLIM2 Inhibits T Helper 17 Cell Development and Granulomatous Inflammation Through Degradation of STAT3. Sci. Signal. 4, ra85 (2011).

    © 2011 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article