mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals (original) (raw)
Related papers
2011
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.
Cardiovascular Research, 2008
Aim Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. Methods and results Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor a (TNFa). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFa-dependent Eselectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. Conclusion A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.
Molecular Immunology, 2009
Bacterial lipopolysaccharide (LPS) induces monocytes/macrophages to express proinflammatory cytokines and tissue factor (TF), the primary activator of the coagulation cascade. Anti-inflammatory signaling pathways including the phosphatidylinositol-3-kinase (PI3K)-Akt pathway inhibit proinflammatory and TF gene expression in macrophages. We determined the role of Akt, the mammalian target of rapamycin (mTOR) and interleukin-10 in the inhibition of LPS-induced proinflammatory cytokine and TF gene expression in peritoneal macrophages (PMs). We used wild type (WT) peritoneal macrophages (PMs), and PMs from PTEN flox/flox /LysMCre mice (PTEN −/− PMs), which have increased Akt activity. Pharmacologic inhibition of mTOR with rapamycin inhibited LPS induction of IL-10 mRNA and protein, and enhanced the expression of TF and the proinflammatory cytokine TNFα in WT PMs. Furthermore, neutralizing IL-10 with anti-IL-10 antibody enhanced LPS induction of TNFα and TF expression in WT PMs. The addition of recombinant IL-10 abolished rapamycin enhancement of LPS-induced TNFα and TF expression in WT PMs. Consistent with enhanced Akt activation, LPS-induced IL-10 expression was increased in PTEN −/− PMs compared to WT PMs. In contrast, LPS-induced TNFα and TF expression was significantly reduced in PTEN −/− PMs compared to WT PMs. However, the neutralizing IL-10 antibody did not completely prevent inhibition of LPS-induced TNFα and TF expression in PTEN −/− PMs. The results indicate that mTOR-dependent IL-10 expression leads to inhibition of LPS induction of TF and the proinflammatory cytokine TNFα in WT macrophages. In contrast, the decrease in LPS-induced TNFα and TF expression in PTEN −/− PMs also requires an IL-10independent pathway.
The Journal of Immunology, 2021
Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ–induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pa...
Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells
European Journal of Immunology, 2008
The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune-defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3-kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor-5 and interferon regulatory factor-7, known as master transcription factors for pro-inflammatory cytokine-and type I IFN-genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxinmediated shock, which correlated with increased levels of IL-1b. Mechanistically, mTOR suppresses caspase-1 activation, thus inhibits release of bioactive IL-1b. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.
Journal of Biological Chemistry, 2013
Background: MAP kinase phosphatase-1 (MKP-1) plays a critical role in regulating inflammation in innate and adaptive immunity. Results: mTOR inhibition leads to induction of MKP-1 through the activation of AKT1 and MEK1/MEK2 pathways. Rapamycin pretreatment of macrophages inhibits LPS-mediated p38 activation and IL-6 and nitric oxide production. Conclusion: Both AKT1 and MEK1/2 regulate rapamycin-mediated MKP-1 induction. Significance: mTORC1 inhibition regulates immunity through MKP-1 induction. Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition. Mammalian target of rapamycin (mTOR) 2 is a multifunctional kinase complex that plays a central role in cell growth and metabolism (1, 2). mTOR can interact with the regulatory-associated proteins of mTOR (raptor) to constitute the mTORC1 complex or, alternatively, interacts with rapamycin-insensitive companion (rictor) to form the mTORC2 complex (1, 2). mTORC1 and mTORC2 exert their actions by regulating several important kinases, such as S6K and AKT. Although AKT activates mTORC1, the TORC2 complex directly phosphorylates AKT at Ser-473 (3, 4). Although the role of mTORCs in cancer is well studied, the signaling input and output of mTORCs in the immune system are not well understood. Rapamycin, a natural product of the bacterium Streptomyces hygroscopicus and a macrolide antibiotic, has emerged as a potent anti-proliferative medication with immunosuppressive properties (5). Activation of both AKT and mTOR influences signaling of various receptors such as toll-like receptors (TLRs) or cytokine receptors (6, 7). The immunosuppressive effect of rapamycin has been largely attributed to inhibition of the clonal expansion of lymphocytes, an altered cytokine production, or differential regulation of effector and regulatory T cell linage commitment (6, 8). However, mTOR plays an important role in terminally differentiated macrophages and in regulation of innate immune responses (6-8). Three principal MAPK subfamilies, including the extracellular signal-regulated kinases 1 and 2 (ERK1 and-2), c-Jun aminoterminal kinases (JNKs), and p38, are essential for initiation of * This work was supported, in whole or in part, by National Institutes of Health Grants R01 HL113508 (to L. S.) and R01 DK56886 (to M. J. B.
The Journal of Immunology, 2011
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 e...
Electronic Journal of Biotechnology, 2011
The Liver X Receptor (LXR) and Pregnane X Receptor (PXR) are members of the nuclear receptor superfamily. Previously, they have been classified as important regulators of lipid homeostasis. However, recent studies have shown that they may be implicated in anti-inflammatory responses as well. This study shows that Tumour Necrosis Factor-α (TNF-α) treatment reduces both LXR-α and PXR mRNA expression. However, pre-treatment with rapamycin, an mTOR inhibitor, followed by TNF-α stimulation, significantly induces LXR-α and PXR mRNA expression to ~17-and ~2fold, respectively. This suggests that mTORC1, a multi-molecular complex of which mTOR is a member, may act as a negative regulator that inhibits the induction of LXR-α and PXRas antiinflammatory genes. It is also shown here that inhibition of JNK1 via the mTOR/Akt pathway coincides with the up-regulation of LXR-α and PXR mRNA, after TNF-α treatment. Together, these observations suggest that JNK1 possibly act downstream of mTORC1 as an LXR-α and PXR inhibitor. From the results gleaned in this study, rapamycin (and its analogues) may be used to reduce acute inflammation by promoting the induction of LXR-α and PXR as anti-inflammatory genes.
Biochemical Pharmacology, 2011
The mTOR-inhibitor rapamycin is a potent drug used in many immunosuppressive and antiinflammatory therapeutic regimes. In renal transplantation despite its beneficial roles rapamycin in some cases can promote renal fibrosis in the kidney but the underlying mechanisms are unknown. In this study, we tested for possible modulatory effects of rapamycin on the cytokine-triggered matrix metalloproteinase 9 (MMP-9)/tissue inhibitor of metalloproteinase (TIMP)-1 protease–antiprotease system which is critically involved in renal inflammation and fibrosis. Treatment of rat mesangial cells (MC) with rapamycin dose-dependently reduced the interleukin 1β (IL-1β)-triggered increase in gelatinolytic levels as demonstrated by zymography. The reduction in the extracellular MMP-9 content by rapamycin coincided with an attenuation in cytokine-induced steady-state MMP-9 mRNA levels. Conversely, rapamycin caused a dose-dependent increase in cytokine-evoked TIMP-1 expression in a Smad binding element (SBE)-dependent manner. Surprisingly, the attenuation of MMP-9 mRNA levels by rapamycin is accompanied by a potentiation of IL-1β-induced MMP-9 promoter activity in which the stimulatory effects by rapamycin are mainly attributed to a proximal AP-1 binding site. Furthermore, the rapamycin-dependent potentiation of MMP-9 expression is accompanied by an amplification of cytokine-triggered activities of nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors. Importantly, rapamycin-triggered increase in MMP-9 promoter activity is fully impaired when we used a MMP-9 reporter construct which is under the additional control of the 3′ untranslated region (3′-UTR) of MMP-9. Collectively, these data imply that rapamycin inhibits the cytokine-induced MMP-9 mainly through posttranscriptional events and thereby exerts profibrotic activities.