Mammalian target of rapamycin complex 2 regulates inflammatory response to stress

Objective and design

To explore the role of mammalian target of rapamycin 2 (mTORC2) in the activation of inflammatory and oxidative responses in rodent models of acute injury and metabolic stress.

Material

The impact of nephrilin, an inhibitor of mTORC2 complex, was assessed in three CD-1 mouse models of acute xenobiotic stress and in a hypertensive Dahl rat model of metabolic stress.

Methods

Animals received daily subcutaneous bolus injections of saline or 4?mg/kg nephrilin. Tissues were assayed by ELISA, gene arrays and immunohistochemical staining.

Results

Nephrilin significantly inhibited elevations in plasma tumor necrosis factor-alpha, kidney substance P, and CX3CR1, and urinary lipocalin-2 [urinary neutrophil gelatinase-associated lipocalin (uNGAL)] in models of acute xenobiotic stress. UCHL1 gene expression levels dropped and plasma HMGB1 levels rose in the rhabdomyolysis model. Both effects were reversed by nephrilin. The inhibitor also blocked diet-induced elevations of uNGAL and albumin-creatinine ratio (UACR) as well as kidney tissue phosphorylation of PKC-beta-2-T641 and p66shc-S36, and reduced dark ring-like staining of nuclei by anti-phos-p66shc-S36 antibody in frozen sections of diseased kidneys from hypertensive Dahl rats fed an 8?% NaCl diet for 4?weeks.

Conclusions

Taken together, our results suggest a role for mTORC2 in the inflammatory-oxidative responses to stress.     

Mammalian target of rapamycin is required for thrombopoietin-induced proliferation of megakaryocyte progenitors

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study, we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway, which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells, human CD34+, and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells, but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42), nuclear polyploidization levels, cell size, or cell survival. The downstream effectors of mTOR, p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1), are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion, these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors, without affecting differentiation or cell survival.     

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction

The mammalian target of rapamycin (mTOR) is a serine‐threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B‐cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short‐lived plasma cells, long‐lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock‐out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre‐ and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long‐lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.     

Decorin-mediated regulation of fibrillin-1 in the kidney involves the insulin-like growth factor-I receptor and Mammalian target of rapamycin

Decorin, a small leucine-rich proteoglycan, affects the synthesis of the elastic fiber component fibrillin-1 in the kidney via hitherto unknown mechanisms. Here, we show that decorin binds to and induces phosphorylation of insulin-like growth factor-I (IGF-I) receptor in renal fibroblasts. Inhibition of the IGF-I receptor tyrosine kinase and its downstream target phosphoinositide-3 kinase prevented decorin-mediated synthesis of fibrillin-1. Furthermore, decorin induced phosphorylation of phosphoinositide-dependent kinase 1, protein kinase B/Akt, mammalian target of rapamycin (mTOR), and p70 S6 kinase. Accordingly, the enhanced synthesis of fibrillin-1 was blocked by rapamycin, an inhibitor of mTOR. Notably, IGF-I, which signals through the same pathway, also stimulated fibrillin-1 synthesis. Systemic administration of rapamycin to mice subjected to unilateral ureteral obstruction, a model of renal fibrosis and increased fibrillin-1 synthesis, markedly reduced the number of interstitial fibroblasts and fibrillin-1 deposition. In streptozotocin-induced diabetes, IGF-I receptor was up-regulated in the kidneys from decorin-null mice. However, this could not compensate for the decorin deficiency, resulting ultimately in decreased fibrillin-1 content. This study provides evidence for the involvement of decorin and the IGF-I receptor/mTOR/p70 S6 kinase signaling pathway in the translational regulation of fibrillin-1.     

Mammalian target of rapamycin (mTOR) complex 2 regulates filamin A‐dependent focal adhesion dynamics and cell migration

The serine/threonine kinase mTOR forms two distinct complexes, mTORC1 and mTORC2, and controls a number of biological processes, including proliferation, survival and autophagy. Although the function of mTORC1 has been extensively studied, the mTORC2 signaling pathway largely remains to be elucidated. Here, we have shown that mTORC2 phosphorylates filamin A, an actin cross‐linking protein, at serine 2152 (S2152) both in vivo and in living cells. Treatment of HeLa cells with Torin1 (an mTORC1/mTORC2 inhibitor), but not rapamycin (an mTORC1 inhibitor), suppressed the phosphorylation of filamin A, which decreased the binding of filamin A with β7‐integrin cytoplasmic tail. Torin1 also inhibited focal adhesion formation and cell migration in A7 filamin A‐replete melanoma cells but not in M2 filamin A‐deficient cells, suggesting a pivotal role for mTORC2 in filamin A function. Finally, reduced focal adhesion formation in M2 cells was significantly rescued by expressing wild type but not S2152A nonphosphorylatable mutant of filamin A. Taken together, our results indicate that mTORC2 regulates filamin A‐dependent focal adhesions and cell migration.     

L-arginine transport at the fetal side of human placenta: effect of aspirin in pregnancy

L-Arginine transport by the fetal side of human placenta was investigated through the characterization of L-[3H]arginine uptake in isolated perfused cotyledon. Competitive inhibition experiments suggest the presence of at least two transport systems: a Na+-independent, pH-insensitive system inhibitable by cationic amino acids, similar to system y+, and a Na+-dependent system which recognizes both cationic and neutral amino acids only in the presence of Na+, i.e. a Bo,+-like system. The kinetic analysis of L-arginine uptake in the presence of Na+ revealed that the process is mediated by saturable components: a high-affinity system (Km = 167 +/- 18.0 microM; Vmax = 0.174 +/- 0.012 micromol min-1) and a low-affinity carrier (Km = 980 +/- 112 microM; Vmax = 1.60 +/- 0.12 micromol min-1). In the absence of Na+, L-arginine uptake was fitted by one model with a Michaelis-Menten constant of 200 +/- 24.8 microM. These results suggest that the high-affinity component corresponds to the Na+-independent system y+, whilst the low-affinity system may represent the activity of the Na+-dependent Bo,+ transporter. Kinetic studies in placentae taken from aspirin-treated pregnancies showed that L-arginine is transported with a significantly higher affinity (Km = 42.5 +/- 5.7 microM), but with a lower capacity (Vmax = 0.064 +/- 0.003 micromol min-1) than in the non-treated group. The latter finding suggests that aspirin would facilitate the uptake of the NO precursor only at very low arginine concentrations.     

Mammalian target of rapamycin in spinal cord neurons mediates hypersensitivity induced by peripheral inflammation

mTOR, the mammalian target of rapamycin, is a serine–threonine kinase known to regulate cell proliferation and growth. mTOR has also been implicated in neuronal synaptic plasticity as well as in pain transmission in models of chemically induced and neuropathic pain. To date, the role of mTOR as a modulator of inflammatory pain has not been examined. In this study, we investigated the role of mTOR in Sprague–Dawley rats using the carrageenan model of inflammatory pain. mRNA of Ras homolog enriched in brain (Rheb), a GTPase that positively regulates mTOR activation, was significantly increased 2 h following carrageenan injection. Four hours after induction of inflammation phosphorylation (p) of p70S6 kinase (S6K), ribosomal protein S6 (S6) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) was increased, indicating mTOR activation. Inhibition of spinal mTOR with intrathecal (i.t.) injection of rapamycin (0.1–3 μg) led to a dose-dependent decrease in carrageenan-induced thermal hyperalgesia and a reduction of mechanical allodynia. In vitro studies confirmed rapamycin inhibition of the mTOR pathway. Carrageenan-induced activation of the mTOR pathway in rats was localized predominantly to dorsal horn neurons in the superficial lamina. Taken together, these data show that the mTOR pathway is activated in dorsal horn neurons during inflammatory pain, and that inhibition of spinal mTOR attenuates inflammation-induced thermal and tactile hypersensitivity. Hence, our study indicates that spinal mTOR is an important regulator of spinal sensitization and suggests that targeting mTOR may provide a new avenue for pain therapy.     

Mammalian target of rapamycin complex 1 (mTORC1) signaling in energy balance and obesity

Energy balance is guaranteed by a complex circuitry that in the brain, and in the hypothalamus in particular, integrates and coordinates several types of signals, including hormones and nutrients, so to match energy expenditure to energy needs. Similar to individual cells, the hypothalamus also profits from intracellular pathways known to work as fuel sensors to maintain energy balance. The mammalian target of rapamycin complex 1 (mTORC1) pathway has been recently implicated in such function, due to its ability to integrate nutrient and hormonal signals to control food intake and body weight. This review therefore describes recent advances made in understanding the role of the hypothalamic mTORC1 pathway in energy balance regulation and its possible contribution to the metabolic dysregulation associated with diet-induced obesity.     

Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence

Marioni G, Staffieri A, Giacomelli L, Lionello M, Guzzardo V, Busnardo A & Blandamura S
(2011) Histopathology  58, 1148–1156
Mammalian target of rapamycin expression and laryngeal squamous cell carcinoma prognosis: novel preliminary evidence Aims: The mammalian target of rapamycin (mTOR) has a key role in regulating cancer cell proliferation, apoptosis, cell migration, and angiogenesis. The aim of this study was to assess the relationships between mTOR and clinicopathological and prognostic parameters in laryngeal squamous cell carcinoma (SCC). Methods and results: Mammalian target of rapamycin expression was determined in 103 consecutive operable laryngeal SCCs. Among the mTOR‐positive cases, the locoregional recurrence rate was higher (P = 0.048) and the disease‐free survival (DFS) rate was shorter (P = 0.031) in patients with mTOR expression >50.7%. In the N₀ subgroup, the disease recurrence rate was higher (P = 0.034) and the DFS was shorter (P = 0.009) in patients with mTOR expression >50.7%. In mTOR‐positive patients, multivariate analysis showed that N stage (P = 0.0001) and mTOR status (P = 0.042) were independent indicators of a poor prognosis. Conclusions: mTOR appeared to be a significant predictor of DFS in univariate and multivariate models. mTOR expression in laryngeal SCC may be useful for the detection of patients at higher risk for recurrence, and N₀ patients at higher risk for early locoregional recurrence who might benefit from more aggressive therapy. The role of mTOR inhibitors in multimodality or multitarget strategies against laryngeal SCC warrants investigation.     

Regulation of cellular growth by the Drosophila target of rapamycin dTOR

The TOR protein kinases (TOR1 and TOR2 in yeast; mTOR/FRAP/RAFT1 in mammals) promote cellular proliferation in response to nutrients and growth factors, but their role in development is poorly understood. Here, we show that the Drosophila TOR homolog dTOR is required cell autonomously for normal growth and proliferation during larval development, and for increases in cellular growth caused by activation of the phosphoinositide 3-kinase (PI3K) signaling pathway. As in mammalian cells, the kinase activity of dTOR is required for growth factor-dependent phosphorylation of p70 S6 kinase (p70(S6K)) in vitro, and we demonstrate that overexpression of p70(S6K) in vivo can rescue dTOR mutant animals to viability. Loss of dTOR also results in cellular phenotypes characteristic of amino acid deprivation, including reduced nucleolar size, lipid vesicle aggregation in the larval fat body, and a cell type-specific pattern of cell cycle arrest that can be bypassed by overexpression of the S-phase regulator cyclin E. Our results suggest that dTOR regulates growth during animal development by coupling growth factor signaling to nutrient availability.     

P29, an oestrogen receptor-associated protein, is down-regulated by mifepristone in first trimester human placenta and decidua.

P29 is an oestrogen receptor-associated protein which acts as a marker of oestrogen action in several systems. The concentration of P29 was measured in placenta and decidua from women following medical termination of pregnancy with the antiprogesterone steroid mifepristone (RU 38,486) and a prostaglandin E1 analogue, and compared with the concentration of P29 found in matched controls undergoing surgical aspiration of pregnancy. Oestrogen receptors were also measured in the same samples. Placental and decidual P29 concentrations (IU/mg protein) in patients treated with mifepristone were 9.6 (4.6-54) and 4.8 (1.3-13.3) (median and range), respectively. These values were significantly lower than the corresponding values, 39.5 (27-69) and 22.0 (2-107) in the surgical group. In contrast, the levels of oestrogen receptors did not change significantly in either decidua or placenta. These data show that mifepristone causes down-regulation of P29 in placenta and decidua, and therefore its action may disrupt oestrogen function in uterine tissues.     

Isolation and characterization of human fetal macrophages from placenta

Human fetal macrophages expressing class II major histocompatibility complex (MHC) antigens have been isolated from the stroma of the chorionic plate of term placentas, using enzymatic digestion procedures, and enriched by Percoll density centrifugation. These cells are adherent, phagocytic and express Fc receptors for IgG. By rosetting with bovine erythrocytes coated with IgG, they can be enriched to 77-95% purity. Placental macrophages isolated in this way stimulate the proliferation of lymphocytes from unrelated donors in mixed-cell cultures, and act as accessory cells in oxidative mitogenesis. In a family study, placental macrophages stimulated proliferation of maternal and paternal lymphocytes but there was no evidence for either priming to, or suppression by, the fetal cells when the responses of lymphocytes from the mother and her HLA identical twin were compared. The possibility that these cells can protect the fetus from infection and/or stimulate the production of maternal anti-fetal HLA-antibodies is discussed.     

Hypoxia-inducible factor-2alpha is involved in enhanced apoptosis in the placenta from pregnancies with fetal growth restriction

The aim of the present study is to investigate whether hypoxia-inducible factors (HIF-1alpha, HIF-2alpha, HIF-1beta) are involved in enhanced apoptosis in the human placenta from pregnancies with fetal growth restriction (FGR). Placental samples were obtained from women with normal term pregnancy (n = 18) or from pregnancy with FGR (n = 12). Placenta apoptosis was assessed by the terminal deoxynucleotidyl transferase deoxy-UTP-nick end labeling (TUNEL) staining. The expressions of HIF-1alpha, HIF-2alpha, and HIF-1beta were examined by immunohistochemical analysis. Enhanced apoptosis was observed in the placenta from pregnancies with FGR compared with normal term placenta. The apoptosis index in FGR group (1.45 +/- 1.26%) was significantly higher than that in the normal control group (0.18 +/- 0.16%; P < 0.01). There were no significant differences in the intensity of the staining for HIF-1alpha and HIF-1beta expressions between two groups, while HIF-2alpha was overexpressed in the placenta from pregnancies with FGR group (P < 0.05). The upregulation of HIF-2alpha protein expression in the placenta from pregnancies with FGR may, at least in part, be involved in the increased placental apoptosis.