STAT3 protein interacts with Class O Forkhead transcription factors in the cytoplasm and regulates nuclear/cytoplasmic localization of FoxO1 and FoxO3a proteins in CD4(+) T cells

Abstract

An important feature of the adaptive immune response is its remarkable capacity to regulate the duration of inflammatory responses, and effector T cells have been shown to limit excessive immune responses by producing anti-inflammatory cytokines such as IL-10 and IL-27. However, how anti-inflammatory cytokines mediate their suppressive activities is not well understood. In this study, we show that STAT3 contributes to mechanisms that control the duration of T cell proliferation by regulating the subcellular location of FoxO1 and FoxO3a, two Class O Forkhead transcription factors that mediate lymphocyte quiescence and inhibit T cell activation. We show that active FoxO1 and FoxO3a reside exclusively in the nucleus of naïve T cells whereas inactive pFoxO1 and pFoxO3a were most abundant in activated T cells and sequestered in their cytoplasm in association with unphosphorylated STAT3 (U-STAT3) and 14-3-3. We further show that FoxO1/FoxO3a rapidly relocalized into the nucleus in response to pSTAT3 activation by IL-6 or IL-10, and the accumulation of FoxO1/FoxO3a in their nuclei coincided with increased expression of p27(Kip1) and p21(WAF1). STAT3 inhibitors completely abrogated cytokine-induced translocation of FoxO1/FoxO3a into the nucleus. In naïve or resting STAT3-deficient T cells, expression of pFoxO1/pFoxO3a was predominantly in the cytoplasm and correlated with defects in p27(Kip1) and p21(WAF1) expression, suggesting requirement of STAT3 for importation or retention of FoxO in the nucleus and attenuation of lymphocyte proliferation. Taken together, these results suggest that U-STAT3 collaborates with 14-3-3 to sequester pFoxO1/pFoxO3a in cytoplasm and thus prolong T cell activation, whereas pSTAT3 activation by anti-inflammatory cytokines would curtail the duration of TCR activation and re-establish lymphocyte quiescence by inducing nuclear localization of FoxO1/FoxO3a and FoxO-mediated expression of growth-inhibitory proteins.

FIGURE 1.

STAT3 co-localized and interacted with pFoxO1 and pFoxO3a in the cytoplasm of activated T cells. A and B, WT naïve CD4⁺ T cells (A) were stimulated with anti-CD3/CD28 for 96 h (B). Cells were fixed and stained with DAPI prior to visualization of subcellular localization of FoxO (green), STAT3 (red), or nuclei (blue) by immunofluorescence on laser confocal microscope. C, whole cell extracts from naïve (lane 1) or 96 h (lane 2) TCR-stimulated primary CD4⁺ T cells were precipitated (IP) with anti-pFoxO1/pFoxO3a Abs and analyzed by Western blotting (Blot) with anti-STAT3, anti-pSTAT3, or anti-pFoxO1 Ab. D, interactions of FoxO3a with STAT3 in whole cell extracts of resting (lane 1), TCR-activated (lane 2) human Jurkat T cells, or activated mouse T cells (lane 3) were analyzed by immunoprecipitation and Western blotting. E and F, co-immunoprecipitation was performed in anti-CD3/CD28-activated CD3⁺ T cells (lane 1) and AE7 CD4⁺ T cell lines (lane 2).

FIGURE 2.

STAT3 interacts with the N-terminal 149 amino acids of FoxO1. A, T cells were transiently transfected with plasmids expressing full-length or truncated FoxO1 in-frame with GFP, and subcellular localization of STAT3 or FoxO fusion proteins was visualized by immunofluorescence and confocal microscopy. B, schematic description of the FoxO1 constructs in context of their functional domains is shown at the top of figure. DNA-binding domain (DBD), nuclear export signal (NES), and 14-3-3 binding sequences are shown. C, whole cell extracts from T cells expressing FoxO1-GFP fusion proteins were precipitated (IP) with anti-GFP Abs, and precipitates were analyzed by Western blotting (Blot; WB) with anti-STAT3 or anti-GFP Abs. Results are representative of >3 independent experiments.

FIGURE 3.

FoxO1 and STAT3 relocalized into the nucleus in response to IL-10 stimulation. Mouse naïve CD4⁺ T cells were activated with anti-CD3/CD28 for 48 h, and IL-10 was added to some cultures after 36 or 42 h. Cells were fixed and stained with DAPI, and spatial localization of FoxO (green), STAT3 (red), or nuclei (blue) was visualized by confocal microscopy (A). B and C, subcellular localization of FoxO1, FoxO3a, or p27^Kip1 protein was also detected by Western blotting. Results are representative of >3 independent experiments.

FIGURE 4.

FoxO3a and STAT3 relocalized into the nucleus in response to IL-6 by STAT3-dependent mechanism. A, mouse naïve CD4⁺ T cells were activated with anti-CD3/CD28 for 48 h, and IL-6 was added to some cultures after 42 h. B, mouse naïve CD4⁺ T cells were activated with anti-CD3/CD28 for 48 h. IL-6 and/or STAT3 inhibitors (STAT3VIII and ORLL-NIH001) were added to some cultures after 42 h as indicated. Cells were fixed and stained with DAPI prior to visualization of subcellular localization of FoxO (green), STAT3 (red), or nuclei (blue) by confocal microscopy. Results are representative of >3 independent experiments.

FIGURE 5.

Loss of STAT3 correlates with nuclear exclusion of FoxO1 and FoxO3a. A, WT or STAT3KO naïve CD4⁺ T cells were fixed and stained with DAPI, and subcellular location of FoxO (green) or nuclei (blue) was visualized by confocal microscopy. B and C, cell surface expression of activation markers on naïve or TCR-activated T cells was analyzed by FACS (B), and expression of p27^Kip1 and p21^WAF1 was analyzed by Western blotting (C). D and E, CD4⁺ T cells were propagated under Th1, Th2, or Th17 polarization condition for 4 days and then analyzed for IL-2 secretion by ELISA (D) or intracellular cytokine expression (E). Numbers in quadrants (B and E) indicate percent of T cells expressing IL-17, IFN-γ, and/or IL-2. Results are representative of at least three independent experiments.

FIGURE 6.

A model for the regulation of the duration of T cell activation. Top panel, constitutive activation of FoxO1 and FoxO3a in the nucleus contributes to maintenance of naïve or resting T cells in a quiescent state by (i) up-regulating expression of IκB and the cell cycle inhibitory protein p27^Kip1, promoting IκB-mediated sequestration of NF-κB in the cytoplasm, and inhibiting IL-2 production. Middle panel, upon TCR activation, PI3K/AKT phosphorylates FoxO, mediates their expulsion to the cytoplasm where they are sequestered in a transcription factor complex comprising of pFoxO1/pFoxO3a, 14-3-3, and U-STAT3. Bottom panel, anti-inflammatory cytokines such as IL-6 and IL-10 induce STAT3 activation. Conformational change from anti-parallel U-STAT3 into parallel pSTAT3 conformation results in: (i) disruption of pFoxO·14-3-3·U-STAT3 complex/displacement of 14-3-3; (ii) unmasking of STAT3 NLS; (iii) translocation of FoxO and STAT3 proteins into the nucleus aided by piggybacking of FoxO/pFoxO on pSTAT3 NLS and importin-α3; (iv) FoxO-mediated transcription of growth-inhibitory genes (e.g. IκB, p27^Kip1); (v) termination of T cell proliferation and reestablishment of lymphocyte quiescence.