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. 2015 Sep;29(9):3737-49.
doi: 10.1096/fj.15-270462. Epub 2015 May 20.

Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure

Huaxin Zhou  1 , Shegan Gao  1 , Xiaoxian Duan  1 , Shuang Liang  1 , David A Scott  1 , Richard J Lamont  2 , Huizhi Wang  2
Affiliations

Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure

Huaxin Zhou et al. FASEB J. 2015 Sep.

Abstract

Serum- and glucocorticoid-regulated kinase (SGK)1 is associated with several important pathologic conditions and plays a modulatory role in adaptive immune responses. However, the involvement and functional role of SGK1 in innate immune responses remain entirely unknown. In this study, we establish that SGK1 is a novel and potent negative regulator of TLR-induced inflammation. Pharmacologic inhibition of SGK1 or suppression by small interfering RNA enhances proinflammatory cytokine (TNF, IL-12, and IL-6) production in TLR-engaged monocytes, a result confirmed in Cre-loxP-mediated SGK1-deficient cells. SGK1 inhibition or gene deficiency results in increased phosphorylation of IKK, IκBα, and NF-κB p65 in LPS-stimulated cells. Enhanced NF-κB p65 DNA binding also occurs upon SGK1 inhibition. The subsequent enhancement of proinflammatory cytokines is dependent on the phosphorylation of TGF-β-activated kinase 1 (TAK1), as confirmed by TAK1 gene silencing. In vivo relevance was established in a murine endotoxin model, in which we found that SGK1 inhibition aggravates the severity of multiple organ damage and enhances the inflammatory response by heightening both proinflammatory cytokine levels and neutrophil infiltration. These findings have identified an anti-inflammatory function of SGK1, elucidated the underlying intracellular mechanisms, and establish, for the first time, that SGK1 holds potential as a novel target for intervention in the control of inflammatory diseases.

Keywords: LPS; SGK1; TAK1; inflammatory cytokines.

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Figures

Figure 1.
Figure 1.
TLR4 activation induces PI3K-dependent phosphorylation of SGK1 in human monocytes. A) Western blot of cell lysates of human monocytes pretreated with LY294002 and stimulated with LPS. Blots were probed with antibodies to phospho-SGK1 (p-SGK1) and GAPDH as a loading control. B) Densitometric quantification of the mean (sd) ratio of phospho-to-total SGK1. *P < 0.05.
Figure 2.
Figure 2.
SGK1 inhibition enhances TLR-mediated inflammatory cytokine production in human monocytes. Human monocytes were pretreated with a series of concentrations of SGK1 inhibitor EMD638683 for 2 h and then stimulated with LPS, Pam3CSK4, or flagellin. After 24 h of stimulation, cell-free supernatants were collected, and LPS-stimulated production of (A) TNF, (B) IL-12, and (C) IL-6 was determined by ELISA. D) Message levels of TNF, IL-12, and IL-6 were detected by real-time PCR at 3 h after LPS stimulation. E and F) Production of TNF, IL-12, and IL-6 in human monocytes after SGK1 inhibition with EMD638683 and upon stimulation with Pam3CSK4 (E) and flagellin (F). Data represent the arithmetic means ± sd of 3 biologic replicates. *P < 0.05; ***P < 0.001.
Figure 3.
Figure 3.
SGK1 deficiency leads to the elevated production of TNF, IL-12, and IL-6 in TLR4-stimulated innate immune cells. BMDCs with SGK1 deficiency were prepared by culturing bone marrow cells from cd11c-Cre-sgk1-fl/fl mice. Purified human monocytes were pretreated with nontarget or SGK1-specific siRNA and then stimulated with LPS. Whole-cell lysates and cell-free supernatants were collected to determine the transfection efficiency and cytokine levels, respectively. A) siRNA-mediated knockdown of SGK1 protein and total GAPDH levels were assessed by Western blot. Ctrl, control. B) The ratio of total SGK1-to-GAPDH was determined by densitometry. C) Production of TNF, IL-12, and IL-6 in LPS-stimulated cells following RNA silencing of SGK1. D) Western blot of whole-cell lysates from wild-type mice and Cre-LoxP-mediated SGK1-deficient mice probed for the total level of SGK1 and GAPDH. E) TNF, (F) IL-12, and (G) IL-6 levels in supernatants of LPS-stimulated (24 h) BMDCs harvested from wild-type (WT) and Cre-loxP-mediated SGK1 knockout (KO) mice. Data represent the arithmetic means ± sd of 3 biologic replicates. *P < 0.05; ***P < 0.001.
Figure 4.
Figure 4.
Inhibition of SGK1 enhances the phosphorylation and DNA-binding activity of NF-κB in LPS-stimulated human monocytes. A) Western blot of human monocyte lysates pretreated with EMD638683 and stimulated with LPS. Blots were probed for phospho (p)- and total IKK, IκBα, and NF-κB P65 and total GAPDH. B and C) Densitometric quantification of the mean (SD) ratio of phospho-to-total proteins for IKK and NF-κB P65 and phospho- and total IκB-to-GAPDH, *P < 0.05. D and E) Western blot of lysates of human monocytes pretreated with nontarget or SGK1-specific siRNA and then stimulated with LPS. Blots were probed with antibodies to SGK1, phospho- and total IKK, IκBα, and NF-κB p65, and total GAPDH as indicated. F and G) Densitometric quantification of the mean (sd) ratio of phospho-to-total proteins for IKK and NF-κB P65 and phospho- and total IκB-to-GAPDH. Data are representative of 3 biologic replicates, *P < 0.05. H) DNA binding of NF-κB in nuclear lysates of monocytes stimulated with LPS for 2 h in the presence or absence of EMD638683. Data represent the arithmetic means ± SD of 3 biologic replicates, ***P < 0.001.
Figure 5.
Figure 5.
SGK1 inhibition enhances TLR4-mediated proinflammatory cytokine production dependent on the activity of TAK1. A) Western blot of human monocytes pretreated with EMD638683 for 2 h and then stimulated with LPS. Blots were probed with antibodies to phospho (p)- and total TAK1 and GAPDH as a loading control. B) Densitometric quantification of the mean (sd) ratio of phospho-to-total TAK1 upon LPS stimulation in the presence and absence of EMD638683. C) Western blots of lysates of human monocytes pretreated with nontarget siRNA or specific siRNA targeting SGK1 (as in Fig. 4D), then stimulated with LPS. Blots were probed with antibodies to SGK1, phospho- and total TAK1, and GAPDH as indicated. Data are representative of 3 biologic replicates, and densitometric quantification of the mean (sd) ratio of phospho-to-total TAK1 was performed (D). E) siRNA-mediated knockdown of TAK1 protein and total GAPDH levels were assessed by Western blots. F and G) The effects of siRNA-mediated TAK1 knockdown on the phosphorylation of SGK1 and NF-κB were monitored by Western blots. H) siRNA-mediated TAK1 knockdown and its effects on the DNA binding of NF-κB in nuclear lysates of LPS-stimulated monocytes in the presence and absence of EMD638683. Data represent the arithmetic means ± sd of 3 biologic replicates. *P < 0.05; ***P < 0.001.
Figure 6.
Figure 6.
SGK1 inhibition-enhanced TAK1 phosphorylation and elevated activity of NF-κB heighten the secretion of inflammatory cytokines by LPS-stimulated human monocytes. A) TNF, (B) IL-12, and (C) IL-6 levels in the supernatants of monocytes pretreated with nontargeted or TAK1-specific siRNA and stimulated with LPS. D) IL-12, (E) TNF, and (F) IL-6 levels in LPS-stimulated monocytes pretreated with a control peptide or an NF-κB inhibitory peptide. Data represent the arithmetic means ± sd of 3 biologic replicates. *P < 0.05; ***P < 0.001.
Figure 7.
Figure 7.
Inhibition of SGK1 aggravates inflammatory responses and enhances neutrophil infiltration in the murine model of endotoxic shock. TNF, IL-12, and IL-6 levels at 3 h (A) or 24 h (B) postinjection of LPS in C57BL/6 mice with or without EMD638683 pretreatment. H&E staining of serial sections of liver (C) and lung (D) from the mice injected with PBS (control), EMD638683, LPS, or LPS plus EMD638683 showing the infiltration of inflammatory cells (6 h; area with red arrows) and the severity of tissue damage (24 h) (×20 magnification; insets amplify the area indicated by yellow arrows). E and F) Polymorphonuclear neutrophil infiltration was assessed by staining tissue sections of liver and lung with FITC-conjugated anti-Ly6G (magnification, ×20). (A), (B), and (C)–(F) are separate, and each group has a total of 7 mice for analysis. G and H) Levels of infiltrated neutrophils presented by the average number of Ly6G-positive cells in 5 different views. Data represent the arithmetic means ± sd of 3 biologic replicates. *P < 0.05; ***P < 0.001.
Figure 8.
Figure 8.
Model of SGK1 regulation of proinflammatory cytokine production in LPS-stimulated monocytes. TLR4-mediated activation of PI3K phosphorylation activates SGK1 in a similar way to phosphorylation of Akt, converting phosphatidylinositol (PIP)2 to PIP3 that will recruit PDK1 and PDK2 and then fully phosphorylate SGK1. Inhibition of SGK1 enhances phosphorylation of TAK1 upon LPS stimulation and in turn elevates phosphorylation of IKK, IκB, and NF-κB, which cumulatively augment proinflammatory cytokine production in LPS-stimulated monocytes.
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References

    1. Schulte W., Bernhagen J., Bucala R. (2013) Cytokines in sepsis: potent immunoregulators and potential therapeutic targets—an updated view. Mediators Inflamm. 2013, 165974 - PMC - PubMed
    1. Kellum J. A., Kong L., Fink M. P., Weissfeld L. A., Yealy D. M., Pinsky M. R., Fine J., Krichevsky A., Delude R. L., Angus D. C.; GenIMS Investigators (2007) Understanding the inflammatory cytokine response in pneumonia and sepsis: results of the Genetic and Inflammatory Markers of Sepsis (GenIMS) Study. Arch. Intern. Med. 167, 1655–1663 - PMC - PubMed
    1. Kawai T., Akira S. (2007) TLR signaling. Semin. Immunol. 19, 24–32 - PubMed
    1. Lee M. S., Kim Y. J. (2007) Signaling pathways downstream of pattern-recognition receptors and their cross talk. Annu. Rev. Biochem. 76, 447–480 - PubMed
    1. O’Neill L. A. (2008) ‘Fine tuning’ TLR signaling. Nat. Immunol. 9, 459–461 - PubMed

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