Mammalian Target of Rapamycin Complex 2 (mTORC2) Negatively Regulates Toll-like Receptor 4-mediated Inflammatory Response via FoxO1

Abstract

Activation of the PI3K pathway plays a pivotal role in regulating the inflammatory response. The loss of mTORC2 has been shown to abrogate the activation of Akt, a critical downstream component of PI3K signaling. However, the biological importance of mTORC2 in innate immunity is currently unknown. Here we demonstrate that rictor, a key component of mTORC2, plays a critical role in controlling the innate inflammatory response via its ability to regulate FoxO1. Upon LPS stimulation, both rictor-deficient mouse embryonic fibroblasts (MEFs) and rictor knockdown dendritic cells exhibited a hyperinflammatory phenotype. The hyperinflammatory phenotype was due to a defective Akt signaling axis, because both rictor-deficient MEFs and rictor knockdown dendritic cells exhibited attenuated Akt phosphorylation and kinase activity. Analysis of downstream Akt targets revealed that phosphorylation of FoxO1 was impaired in rictor-deficient cells, resulting in elevated nuclear FoxO1 levels and diminished nuclear export of FoxO1 upon LPS stimulation. Knockdown of FoxO1 attenuated the hyperinflammatory phenotype exhibited by rictor-deficient MEFs. Moreover, FoxO1 deletion in dendritic cells attenuated the capacity of LPS to induce inflammatory cytokine expression. These findings identify a novel signaling pathway by which mTORC2 regulates the TLR-mediated inflammatory response through its ability to regulate FoxO1.PI3K is a heterodimeric enzyme that consists of a regulatory (p85) and a catalytic (p110) subunit (1). PI3K has the ability to phosphorylate both lipids and proteins. The activation of PI3K drives the generation of phosphatidylinositol 3,4,5-trisphosphate. A major downstream target of PI3K is the AGC family member and kinase Akt (Protein Kinase B). Upon the generation of phosphatidylinositol 3,4,5-trisphosphate, Akt is recruited to the plasma membrane through its pleckstrin homology domain. Sequential phosphorylation of Akt on both serine 473 and threonine 308 are required for full activation. Phosphorylation of Akt on threonine 308 is mediated by PDK1 (2, 3). Several kinases including DNA-activated Protein Kinase, integrin-linked kinase, and PKC-␤ have been proposed to be involved in the phosphorylation of Akt on serine 473 (4 -6). Studies utilizing cells deficient in rictor, a component of the mammalian target of rapamycin complex 2 (mTORC2), 4 have shown that mTORC2 is the primary kinase involved in phosphorylation of serine 473 on Akt. Upon insulin or serum stimulation, rictor-deficient MEFs displayed defective Akt Ser-473 phosphorylation compared with wild type cells (7-9).mTOR can be present in two distinct complexes, mTORC1 and mTORC2 (8, 10). The mTORC1 complex consists of mTOR, raptor, mLST8, and Deptor, which is a negative regulator mTOR (11-13). mTORC1 is the target of the immunosuppressant macrolide drug rapamycin and functions in a wide variety of cellular processes including cell growth via its phosphorylation of two downstream targets, S6 kinase 1, and eIF-4E-binding protein (14). mTORC1 has been shown to p...

Gene expression analysis of mTOR pathway: association with human longevity

“…Rictor, an important component of mTORC2, contributes to glucose homeostasis in murine muscle tissue (Kumar et al, 2008), TORC2 C. elegans mutants show modulation of lifespan by affecting feeding and metabolism of different diets (Soukas et al, 2009), TORC2 is a mediator of proliferative and survival signals in cancer cells (Fang et al, 2012) and a study in mouse embryonic fibroblasts showed that mTORC2 regulates the TLR-mediated inflammatory response via FoxO1 (Brown et al, 2011). In addition, PRR5L, which is part of mTORC2, has been suggested to play a role in apoptosis in HeLa cells, but whether it is pro-apoptotic or antiapoptotic remains unclear (Thedieck et al, 2007).…”

Dynamic Modulation of Innate Immune Response by Varying Dosages of Lipopolysaccharide (LPS) in Human Monocytic Cells

“…While FoxO1 contributes to the expression of pro-inflammatory mediators (23,29), CREB is largely involved in the expression of anti-inflammatory mediators (25,41). Pharmacological activation of Akt corresponds with suppression of FoxO1 (42), which, in light of GSK3 suppression by Akt (26), points to a connection between GSK3 and FoxO1 activity.…”

“…The anti-inflammatory effects of Akt on TLR4 signaling appear to be mediated through FoxO1 (23), suggesting that GSK3 may be important for the activation of FoxO1. GSK3 can potently suppress CREB (24).…”

“…Blocking of inhibitory phosphorylation of FoxO1 results in increased proinflammatory cytokine production by macrophages upon challenge with a TLR2 ligand (31). Defective Akt activation results in increased FoxO1 activity and pro-inflammatory cytokine production (23). The broadly anti-inflammatory effects of PI3K signaling in TLR4 stimulation (32) can thus be ascribed to the tandem activation of CREB and suppression of FoxO1 by Akt, but the competition between GSK3 and Akt has not been well characterized in the context of LPS challenge.…”

“…Activation of Akt by rapamycin blunts the sensitivity of pro-inflammatory genes to LPS and increases CREB activity (28), and mTOR/Akt signaling additionally suppresses inflammation by inactivating the transcription factor forkhead box O1 (FoxO1) (23). The role of FoxO1 in TLR4 signaling is predominantly pro-inflammatory.…”