Convergence of the mammalian target of rapamycin complex 1- and glycogen synthase kinase 3-β-signaling pathways regulates the innate inflammatory response

Abstract

The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-β converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-β. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that GSK3-β associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-β. Inhibition of S6K1 abrogated the phosphorylation of GSK3-β while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-κB p65 associated with CREB-binding protein. Inhibition of NF-κB p65 attenuated rapamycin's ability to influence the levels of pro- or anti-inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3.

FIGURE 1

Rapamycin inhibits the phosphorylation of GSK3-β (S9) in LPS-stimulated cells. A, Human monocytes were stimulated with LPS (1 μg/ml) in the presence or absence of rapamycin (100 ng/ml) for 60 min, and the levels of phosphorylated GSK3-β (S9) and total GSK3-β were analyzed using a phospho-Ab array. B, The mean ratio of GSK3-β (S9) to total GSK3-β is shown ± SD of six experiments. C, Wild-type and Rictor-deficient MEFs were treated with LPS (1 μg/ml) over a 2-h time course, and the levels of phosphorylated GSK3-β (S9) were analyzed by Western blot. D, Densitometry scans were performed and the mean ratios of phosphorylated and total GSK3-β are shown ± SD of three experiments. E, Human monocytes were stimulated with LPS (1 μg/ml) in the presence or absence of rapamycin (100 ng/ml) for up to 24 h, and the levels of phosphorylated GSK3-β (S9) were analyzed by Western blot. F, Densitometry scans were performed, and the mean ratios of phosphorylated and total GSK3-β are shown ± SD of three experiments. G, The levels of phosphorylated GSK3-β (S9) in LPS-stimulated human monocytes treated with or without rapamycin (100 ng/ml) were monitored by flow cytometry. H, The effects of rapamycin (100 ng/ml) on the levels of phosphorylated GSK3-β (S9) and the GSK3-specific substrate, glycogen synthase (S641), in LPS-stimulated cells were monitored by Western blot. I, Inhibition of mTORC1 using rapamycin (100 ng/ml) and its effects on the levels of phosphorylated mTOR, p70S6K, p85S6K, and 4E-BP1 in LPS-stimulated monocytes. J, Inhibition of GSK3 using SB216763 (10 μM) and its effects on the phosphorylated levels of mTOR, p85S6K, and p70S6K in LPS-stimulated monocytes. Data are representative of three to six separate experiments. **p < 0.01, statistically significant differences between monocytes stimulated with LPS in the presence or absence of rapamycin.

FIGURE 2

Ability of rapamycin to regulate the LPS-mediated inflammatory response is dependent upon GSK3. The levels of TNF (A), IL-12 (B), and IL-10 (C) produced by LPS-stimulated monocytes, with or without the mTORC1 inhibitor rapamycin (100 ng/ml), the GSK3 inhibitor SB216763 (10 μM), or both. For A–C, the GSK3 inhibitor SB216763 attenuated the capacity of rapamycin to increase TNF and IL-12 production and suppress IL-10 levels produced by LPS-stimulated monocytes. The levels of IL-12 (D) and IL-10 (E) produced by LPS-stimulated DCs expressing a constitutively active GSK3 knockin, with or without rapamycin. Data represent the arithmetic mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, statistically significant differences.

FIGURE 3

p85S6K associates with and phosphorylates GSK3-β (S9) in LPS-stimulated monocytes. A, Interaction of GSK3-β with p70S6K or p85S6K, as determined by immunoprecipitation of GSK3-β followed by Western blot for p70S6K or p85S6K in LPS-stimulated monocytes treated with or without rapamycin (100 ng/ml). B, An in vitro kinase assay demonstrating that active p85S6K phosphorylates GSK3-β on S9, as determined by Western blot. C, Monocytes were pretreated with the S6K1 inhibitor PF-4708671 (0.5 μM) for 2 h, stimulated with LPS, and assessed for phosphorylated GSK3-β and 4E-BP1 levels by Western blot. D and E, Inhibition of S6K1 augmented the production of IL-12 (D) and decreased the production of IL-10 (E) by LPS-stimulated monocytes. For D and E, data represent the arithmetic mean ± SD of three separate experiments. **p < 0.01, ***p < 0.001, statistically significant differences.

FIGURE 4

STAT3 activation and the ability of rapamycin to affect STAT3 phosphorylation in LPS-stimulated cells are dependent upon IL-10 feedback. A, Monocytes were treated with rapamycin (100 ng/ml) and stimulated with LPS (1 μg/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated STAT3. B, Monocytes were treated with IC or neutralizing anti–IL-10 Ab, stimulated with LPS (1 μg/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated STAT3. C, Wild-type and IL-10 KO macrophages were stimulated with LPS (1 μg/ml) for up to 12 h, and cell lysates were probed by Western blot for phosphorylated STAT3. D, Monocytes were stimulated with IL-10 (10 ng/ml) in the presence or absence of rapamycin (100 ng/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated STAT3. Data are representative of three to five separate experiments. IC, isotype control; WT, wild-type.

FIGURE 5

GSK3 and mTORC1 inhibition differentially affect the nuclear DNA-binding levels of CREB (S133). A, Transcription factor binding assay measuring the levels of CREB (S133) in 10 μg of nuclear extract from LPS-stimulated monocytes treated with SB216763 (10 μM) or rapamycin (100 ng/ml). B, Rapamycin (100 ng/ml) increases and decreases the association of NF-κB p65 (S276) and CREB (S133) with the coactivator of transcription CBP, respectively, as determined by immunoprecipitation of CBP followed by Western blot for NF-κB p65 (S276) or CREB (S133). C and D, The functional effect of rapamycin (100 ng/ml) increasing the levels of NF-κB p65 (S276) activity in LPS-stimulated monocytes was assessed by pretreatment of cells with a control peptide or NF-κB p65 (S276) inhibitory peptide (100 μM), stimulated with LPS for 20 h; the levels of IL-12 (C) or IL-10 (D) were determined by ELISA. For A, C, and D, data represent the arithmetic mean ± SD of three separate experiments. *p < 0.05, ***p < 0.001, statistically significant differences.

FIGURE 6

Convergence of mTORC1 and GSK3-β in LPS-stimulated cells. LPS stimulation induces PI3K/Akt activity, which, in turn, mediates activation of the mTORC1 pathway. Activation of mTORC1 results in the phosphorylation and activation of p85S6K. Activated p85S6K binds with and phosphorylates GSK3-β, resulting in the suppression of GSK3-β activity. Suppressed GSK3-β activity enhances the nuclear binding levels of CREB that promote IL-10 and decrease IL-12 production. The phosphorylation of STAT3 in LPS-stimulated cells depends upon IL-10.