mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

Abstract

BackgroundmTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling.Methodology/Principal FindingsMice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1β, IFN-γ and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways.Conclusions/SignificanceWe conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation and mortality in response to endotoxin, and may occur via multiple pathways.

Early molecular and behavioral response to lipopolysaccharide in the WAG/Rij rat model of absence epilepsy and depressive-like behavior, involves interplay between AMPK, AKT/mTOR pathways and neuroinflammatory cytokine release

“…In the present study, in addition to measuring the early effects of LPS on brain cytokine levels, we have also demonstrated that LPS can activate the mTOR pathway in several brain areas (Cao et al, 2013;Lee et al, 2010) and for the first time, we have demonstrated that such activation is linked to a regulation of at least two important upstream regulators of mTORC-1, AMPK and AKT. Briefly, AKT phosphorylation is generally considered to increase mTOR activity through the TSC complex (Chong et al, 2012;Russo et al, 2012a); however, AKT can inhibit mTORC-1 complex activity by phosphorylating PRAS40 (proline-rich AKT substrate of 40 kDa), Fig.…”

“…Effects of LPS and RAP on downstream mTOR activity measured by p-mTOR and p-p70S6K levels p70 ribosomal protein S6 kinase (p70S6K) represents the best characterized downstream kinase target of mTOR activity (Russo et al, 2012a). To test for the possible activation of this pathway by LPS and its modulation by RAP in our experiments (Lee et al, 2010), we quantified the effects of both drugs alone and in combination, by Western blot (WB) analysis of the phosphorylated products phospho-mTOR (p-mTOR) and phospho-p70S6K (p-p70S6K) in the brain of WAG/Rij rats (n = 5 for each treatment-group and time point). We found that RAP (0.5 mg/kg) administered alone had no effects on the amount of p-mTOR expressed in the cortex, hippocampus or thalamus (relative to control untreated WAG/Rij rats), up to 90 min after drug administration ( Fig.…”

Functional kinomics establishes a critical node of volume-sensitive cation-Cl− cotransporter regulation in the mammalian brain

“…In contrast, AKT1 KO and ROCK2 KO cells exhibited no apparent change in either KCC3 P-Thr 991 /Thr 1048 or NKCC1 P-Thr 203 /Thr 207 /Thr 212 . Moreover, TSC1 KO and TSC2 KO cells (containing reduced levels of activated AKT1 and SGK131323334, showed apparently increased levels of KCC3 P-Thr 991 /Thr 1048 (>1.3-2 fold, p  <  0.05 ; Fig. 2C and Supplementary Figure 2B).…”

“…To identify kinases that might directly phosphorylate KCC3, we limited the tertiary (validation) screen to only serine-threonine kinases. We examined KCC3 P-Thr 991 using phospho-antibodies in cell lines knocked out for WNK3, PRKD1 (i.e., encoding Protein Kinase D1 [PKD1], also known as PKC-Mu), AKT1, ROCK2, and TSC1 and TSC2 (as models harboring significantly decreased AKT1 and SGK1 activity31323334. KCC3 P-Thr 1048 and NKCC1 P-Thr 203 /Thr 207 /Thr 212 were also monitored with phospho-antibodies2428 (Fig.…”

Folate-Conjugated Rapamycin Slows Progression of Polycystic Kidney Disease

“…Because any therapeutic approach for patients with ADPKD would be relevant only for adult patients, we next tested the ability of FC-rapa to inhibit mTOR pathway activity in different tissues in adult mice. Since mTOR activity is typically low in most normal adult tissues, we used short-term LPS treatment 32 as a method to stimulate mTOR activity (Supplemental Figure 5). Animals were pretreated with different doses of FC-rapa or unconjugated rapamycin, followed by short-term LPS treatment.…”