Nrf2-SHP Cascade-Mediated STAT3 Inactivation Contributes to AMPK-Driven Protection Against Endotoxic Inflammation

Abstract

Signal transducer and activator of transcription 3 (STAT3) is implicated in inflammation processing, but the mechanism of its regulation mostly remains limited to Janus kinase (JAK)-mediated phosphorylation. Although AMP-activated protein kinase (AMPK)-mediated STAT3 inactivation has got documented, the molecular signaling cascade connecting STAT3 inactivation and the anti-inflammatory role of AMPK is far from established. In the present study, we addressed the interplay between AMPK and STAT3, and revealed the important role of STAT3 inactivation in the anti-inflammatory function of AMPK in lipopolysaccharide-stressed macrophages and mice. Firstly, we found that pharmacological inhibition of STAT3 can improve the anti-inflammatory effect of AMPK in wild-type mice, and the expression of STAT3 in macrophage of mice is a prerequisite for the anti-inflammatory effect of AMPK. As to the molecular signaling cascade linking AMPK to STAT3, we disclosed that AMPK suppressed STAT3 not only by attenuating JAK signaling but also by activating nuclear factor erythroid-2-related factor-2 (Nrf2), a redox-regulating transcription factor, which consequently increased the expression of small heterodimer protein (SHP), thus repressing the transcriptional activity of STAT3. In summary, this study provided a unique set of evidence showing the relationship between AMPK and STAT3 signaling and explored a new mechanism of AMPK-driven STAT3 inactivation that involves Nrf2-SHP signaling cascade. These findings expand our understanding of the interplay between pro- and anti-inflammatory signaling pathways and are beneficial for the therapeutic development of sepsis treatments.

Keywords: AMPK; LPS; Nrf2; SHP; STAT3; inflammation.

Figure 1

LPS-induced inflammatory response accompanied with altered activities of STAT3 and AMPK. RAW264.7 cells were treated with LPS (100 ng/ml) for indicated hours. (A) Relative mRNA levels of Il1b, Tnfa, and Nos2. (B) The level of phosphorylated STAT3 at Y705 site (pSTAT3) and phosphorylated AMPKα at T172 site (pAMPK). (C,D) The quantifications of immunoblots from 3 repeats of the experiments. (E) Subcellular localization of STAT3 (F,G) Relative mRNA levels of Socs3, Cox2, Cpt1, and Fas. Data are presented as means ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared to LPS 0h group. Bar represents 50 μm.

Figure 2

STAT3 inactivation mimics and improves the effect of AMPK in macrophages. (A–D) Cells were pre-treated with BBR (10 μM) and AG490 (35 μM) or S3I-201 (50 μM) for 2 h and then with LPS (100 ng/ml) for 6 h. (A,B) Relative mRNA levels of the Il1b, Tnfa, and Nos2. (C,D) Cells were transfected with siSTAT3 or siCTL for 36 h, followed by BBR and LPS treatments as described above. Relative mRNA levels of the Il1b, Tnfa, and Nos2. Data are presented as means ± SD from 3 independent experiments **p < 0.01, ***p < 0.001, compared to LPS alone group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared to LPS + BBR group.

Figure 3

STAT3 inhibition and AMPK activation showed similar anti-inflammatory effect in mice. Mice were intraperitoneally injected with LPS (15 mg/kg) alone or LPS with BBR (10 mg/kg) or S3I-201 (5 mg/kg) and were sacrificed 6 h after LPS injection. (A) HE-staining images of lung tissue. Black arrows indicate infiltration of inflammatory cells. (B) Relative mRNA levels of inflammatory genes in lung tissue. (C) Relative mRNA levels of the STAT3 downstream genes Socs3 and Cox2 in lung tissue. Data are presented as means ± SD from 3 independent experiments **p < 0.01, ***p < 0.001, compared to LPS alone group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, compared to LPS + BBR group. Bar represents 50 μm.

Figure 4

STAT3 deficiency eliminates the anti-inflammatory effect of AMPK in mice. Peritoneal macrophages were obtained from wild-type and STAT3-deficient mice, in which the STAT3 gene was specifically deleted in myeloid cells. Mice were intraperitoneally injected with LPS (15 mg/kg) alone or combined with BBR (10 mg/kg) for 6 h. (A) STAT3 protein levels in peritoneal macrophages. WT represents wild-type mice; STAT3^−/−#1 and STAT3^−/−#2 are two STAT3-deficient mice. (B–D) Relative mRNA levels of inflammatory genes in peritoneal macrophages. (E) HE-staining images of lung tissue. (F) Relative mRNA levels of inflammatory genes in lung tissue. Data are presented as means ± SD from 3 independent experiments. **p < 0.01, ***p < 0.001 compared to indicated group. Bar represents 50 μm.

Figure 5

AMPK inhibits STAT3 activity associated with JAK-mediated signal. (A–D) RAW264.7 were pre-treated with BBR (10 μM) and Compound C (CC, 10 mM) alone or in combination for 2 h, then treated with LPS (100 ng/ml) for 6 h. (A) The level of pAMPK (T172), pSTAT3 (Y705) were shown; (B,C) the quantifications of immunoblots from 3 repeats; (D,E) relative mRNA levels of Cpt1, Fas, Socs3, and Cox2; (F) subcellular localization of STAT3 in RAW264.7 cells. (G) RAW264.7 cells were transfected with constitutive activated AMPKα1 (CA-AMPK) or dominate negative AMPKα1 (DN-AMPK) or the empty plasmid pcDNA3.1 for 36 h, followed by LPS treatment for 6 h, the levels of pAMPK (T172) and pSTAT3 (Y705) were shown; (H,I) the quantifications of immunoblots from 3 repeats of the experiments. (J–P) RAW264.7 were pre-treated with BBR (10 μM) or AG490 (35 μM) for 2 h, then with LPS (100 ng/ml) for 6 h, (J) the protein levels of pAMPK (T172), pJAK2 (Y1007 + Y1008) and pSTAT3 (Y705) were shown; (K,L) the quantifications of immunoblots from 3 repeats of the experiments; (M) the levels of pJAK2 (Y1007 + Y1008) and pSTAT3 (Y705) were shown; (N,O) the quantifications of immunoblots from 3 repeats of the experiments; (P) relative mRNA levels of Socs3 and Cox2. Data are presented as means ± SD from 3 independent experiments *p < 0.05, **p < 0.01, ***p < 0.001, compared to indicated group. Bar represents 50 μm.

Figure 6

AMPK inhibits STAT3 activity associated with the induction of SHP. (A) RAW264.7 cells were treated with LPS (100 ng/ml) for the indicated time. Relative mRNA levels for the Shp were shown. (B) RAW264.7 cells were treated with LPS for 6 h, alone or combined with BBR (10 μM) or BBR plus CC (10 mM). The relative mRNA level of the Shp is shown. (C) RAW264.7 cells were transfected with scrambled siRNA (siCTL) and Shp siRNA (siShp) for 48 h, and the relative mRNA level of the Shp was shown. (D,E) siCTL or siShp was co-transfected with a Luc-reporter plasmid containing STAT3-binding cis-element on its promoter for 48 h, a 6 h treatment of LPS alone or combined with BBR was performed, (D) the activity of the Shp promoter, and (E) the relative mRNA levels of the Socs3 and Cox2 are shown. Data are presented as means ± SD from 3 independent experiments. **p < 0.01, ***p < 0.001, compared to indicated group. NS means no significance.

Figure 7

AMPK promotes SHP transcription and STAT3 inactivation through Nrf2. (A–C) RAW264.7 cells were treated with LPS and BBR as described in Figure 2. (A) The phosphorylation of Nrf2 protein at serine 40 site (S40). (B) Immunofluorescent images of Nrf2 in RAW264.7 cells. (C) Relative mRNA levels of the Nrf2 target gene Nqo1. (D–F) RAW264.7 cells were treated with Nrf2 inhibitor ML385 (20 μM), (D,E) the relative mRNA levels of Nqo1 and Shp, and (F) the activity of the Shp promoter. (G,H) RAW264.7 cells were transfected with pcDNA3.1 plasmid, S40 mutated Nrf2-expressing construct (DN-Nrf2) and KEAP1 for 36 h, followed by LPS treatment for 6 h. (G) The relative mRNA levels of the Shp. (H) The activity of the Shp promoter. (I,J) RAW264.7 cells were treated as described in (D–H), and the relative mRNA levels of the STAT3 downstream genes Socs3 and Cox2. Data are presented as means ± SD from 3 independent experiments. NS means no significance. *p < 0.05, ***p < 0.001, compared to indicated group. Bar represents 20 μm.

Figure 8

BBR can inhibit LPS-induced inflammation response, which mediated by STAT3 inactivation and Nrf2 activation. While the expression of STAT3 is a prerequisite for the anti-inflammatory effect of AMPK, and AMPK suppresses STAT3 not only by attenuating JAK2 signal, but also by causing the activation of Nrf2 which could increase the expression of small heterodimer protein (SHP), consequently down-regulating the transcriptional activity of STAT3, followed by inflammatory response.