Rictor Expression Research Articles

Screening high-risk individuals for primary gastric adenocarcinoma: evaluating progression-free survival probability score in the presence and absence of Rictor expression after gastrectomy

ObjectiveDeveloping nomogram-based risk stratification model to determine 3-year and 5-year progression-free survival (PFS) and to identify high-risk patients with gastric adenocarcinoma based on different Rictor statuses.Methods1366 individuals who underwent radical gastric surgery to treat gastric adenocarcinoma at Shanxi Cancer Hospital from May 2002 to December 2020 were analyzed. Cox regression analysis was employed to create the nomograms. The nomograms’ performance was assessed using C-index, time receiver operating characteristic (t-ROC) curves, calibration curves, and decision curve analysis (DCA) curves in training and validation cohorts. Subsequently, patients were categorized into high-risk and low-risk groups based on the nomogram’s risk scores.ResultsThe Rictor (-) nomogram for predicting PFS included variables such as age, number of positive lymph nodes, vascular invasion, maximum diameter of the tumor, omentum metastasis, and expression of MSH2. In the internal validation, the C-index of the Rictor (-) nomogram was 0.760 (95%CI: 0.720-0.799), which was superior to the C-index of the American Joint Committee on Cancer (AJCC) 8th edition TNM staging (0.683, 95%CI: 0.646-0.721). Similarly, the Rictor (+) nomogram for predicting PFS included variables such as gender, age, pT stage, number of positive lymph nodes, neural invasion, maximum diameter of the tumor, omentum metastasis, Clavien-Dindo classification for complications, and CGA expression. The C-index of the Rictor (+) nomogram was 0.795 (95%CI: 0.764-0.825), which outperformed the C-index of the AJCC 8th edition TNM staging (0.693, 95%CI: 0.662-0.723). The calibration curves, t-ROC curves, and decision curve analysis for both nomogram models demonstrated their excellent prediction ability.ConclusionThis study presents the first risk stratification for Rictor status in gastric adenocarcinoma. Our model identifies low-risk patients who may not require additional postoperative treatment, while high-risk patients should consider targeted therapies that specifically target Rictor-positive indicators.

FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia.

BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21).

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

Protection by selective mTORC2 inhibition of Zymosan-induced hypotension and systemic inflammation mediated via IKKα/IκB-α/NF-κB activation

Non-septic shock is a serious condition leading to multiple organ dysfunction. Although targeting the mammalian target of the rapamycin complex 1 (mTORC1) signaling pathway exerts potent anti-inflammatory activity, little is known about mTORC2’s contribution to non-septic shock. Thus, our research aims to investigate mTORC2's contribution and associated changes of IκB kinase (IKKα)/inhibitor κB (IκB-α)/nuclear factor-ĸB (NF-κB) pathway on Zymosan (ZYM)-induced non-septic rat model using the novel mTORC2 selective inhibitor JR-AB2-011. Rats were given saline (4ml/kg), dimethylsulfoxide (DMSO) (4ml/kg), ZYM (500mg/kg), and (or) JR-AB2-011 (1mg/kg). Mean arterial pressure (MAP) and heart rate (HR) of rats were recorded. JR-AB2-011 reversed both ZYM-induced reduction in MAP and increase in HR. Protein expression and/or phosphorylation of rictor, protein kinase B (Akt), IκB-α, IKKα, NF-κB p65, inducible nitric oxide synthase (iNOS), nitrotyrosine, cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, besides prostaglandin (PG) E2 levels were measured. The enhanced expression of the proteins mentioned above has been inhibited by JR-AB2-011. These data suggest mTORC2’s promising role in ZYM-induced hypotension and systemic inflammation mediated via IKKα/IκB-α/NF-κB pathway.

MTORC2 knockdown mediates lipid metabolism to alleviate hyperlipidemic pancreatitis through PPARα.

Hyperlipidemic pancreatitis (HP) is an inflammatory injury of the pancreas triggered by elevated serum triglyceride (TG) levels. The mechanistic target of rapamycin (mTOR) signaling pathway plays a crucial role in regulating lipid homeostasis and inflammation. This study aimed to investigate whether the activity of mTOR complex 2 (mTORC2) affects the progression of HP and its underlying mechanisms. In vivo, a high-fat diet and retrograde administration of sodium taurocholate were employed to establish theHP models in rats, with pancreatic tissue pathology evaluated. The expression of Rictor and peroxisome proliferator-activator receptor (PPAR) was examined. The serum levels of TG, fatty acid metabolites, inflammatory and lipid metabolism-related factors were determined. In vitro, pancreatic acinar cells (PACs) were exposed to palmitic acid and cholecystokinin-8. PAC apoptosis, pyroptosis, and ferroptosis were assessed. In the HP models, rats and PACs exhibited upregulated Rictor and downregulated PPARα, and Rictor knockdown promoted PPARα expression. In vivo, Rictor knockdown decreased the serum levels of TG, α-amylase, total cholesterol, low-density lipoprotein cholesterol, lactate dehydrogenase, and inflammatory factors, while increasing high-density lipoprotein cholesterol levels. Rictor knockdown increased ACOX1 and CPT1α and decreased SREBP-1, CD36, SCD1, ACLY, and ACACA. Rictor knockdown reduced damage to pancreatic tissue structure. In vitro, Rictor knockdown inhibited PAC apoptosis, pyroptosis, and ferroptosis. Treatment with the PPARα antagonist GW6471 abolished the beneficial effects of Rictor knockdown. Rictor/mTORC2 deficiency reduces serum TG levels, maintains lipid homeostasis, and suppresses inflammation by inhibiting PPARα expression. Weakening mTORC2 activity holds promise as a novel therapeutic strategy for HP.

KLF4 Suppresses the Progression of Hepatocellular Carcinoma by Reducing Tumor ATP Synthesis through Targeting the Mir-206/RICTOR Axis.

To address the increased energy demand, tumor cells undergo metabolic reprogramming, including oxidative phosphorylation (OXPHOS) and aerobic glycolysis. This study investigates the role of Kruppel-like factor 4 (KLF4), a transcription factor, as a tumor suppressor in hepatocellular carcinoma (HCC) by regulating ATP synthesis. Immunohistochemistry was performed to assess KLF4 expression in HCC tissues. Functional assays, such as CCK-8, EdU, and colony formation, as well as in vivo assays, including subcutaneous tumor formation and liver orthotopic xenograft mouse models, were conducted to determine the impact of KLF4 on HCC proliferation. Luciferase reporter assay and chromatin immunoprecipitation assay were utilized to evaluate the interaction between KLF4, miR-206, and RICTOR. The findings reveal low KLF4 expression in HCC, which is associated with poor prognosis. Both in vitro and in vivo functional assays demonstrate that KLF4 inhibits HCC cell proliferation. Mechanistically, it was demonstrated that KLF4 reduces ATP synthesis in HCC by suppressing the expression of RICTOR, a core component of mTORC2. This suppression promotes glutaminolysis to replenish the TCA cycle and increase ATP levels, facilitated by the promotion of miR-206 transcription. In conclusion, this study enhances the understanding of KLF4's role in HCC ATP synthesis and suggests that targeting the KLF4/miR-206/RICTOR axis could be a promising therapeutic approach for anti-HCC therapeutics.

RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition.

The main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF-mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM. After analyzing The Cancer Genome Atlas MM patients' database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAFV600E MM cell lines on their response to BRAF/MEKi. We performed proteomic screening to identify proteins modulated by changes in RICTOR expression, and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAFV600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi. Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase of BRAFV600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low RICTOR tumors. Importantly, our findings indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Lack of mTORC2 signaling in CD11c+ myeloid cells inhibits their migration and ameliorates experimental colitis.

The mammalian target of rapamycin (mTOR) pathway plays a key role in determining immune cells function through modulation of their metabolic status. By specific deletion of Rictor in CD11c+ myeloid cells (referred to here as CD11cRicΔ/Δ), we investigated the role of mTOR complex 2 (mTORC2) signaling in dendritic cells (DCs) function in mice. We showed that upon dextran sulfate sodium-induced colitis, the lack of mTORC2 signaling CD11c+ cells diminishes the colitis score and abrogates DC migration to the mesenteric lymph nodes, thereby diminishing the infiltration of T helper 17 cells in the lamina propria and subsequent inflammation. These findings corroborate with the abrogation of cytoskeleton organization and the decreased activation of Rac1 and Cdc42 GTPases observed in CD11c+-mTORC2-deficient cells. Meta-analysis on colonic samples from ulcerative colitis patients revealed increased gene expression of proinflammatory cytokines, which coincided with augmented expression of the mTOR pathway, a positive correlation between the DC marker ITGAX and interleukin-6, the expression of RICTOR, and CDC42. Together, this work proposes that targeting mTORC2 on DCs offers a key to hamper inflammatory responses, and this way, ameliorates the progression and severity of intestinal inflammatory diseases.

RICTOR amplification is associated with Rictor membrane staining and does not correlate with PD-L1 expression in lung squamous cell carcinoma.

RICTOR gene, which encodes the scaffold protein of mTORC2, can be amplified in various tumor types, including squamous cell carcinoma (SCC) of the lung. RICTOR amplification can lead to hyperactivation of mTORC2 and may serve as a targetable genetic alteration, including in lung SCC patients with no PD-L1 expression who are not expected to benefit from immune checkpoint inhibitor therapy. This study aimed to compare RICTOR amplification detected by fluorescence in situ hybridization (FISH) with Rictor and PD-L1 protein expression detected by immunohistochemistry (IHC) in SCC of the lung. The study was complemented by analysis of the publicly available Lung Squamous Cell Carcinoma (TCGA, Firehose legacy) dataset. RICTOR amplification was observed in 20% of our cases and 16% of the lung SCC cases of the TCGA dataset. Rictor and PD-L1 expression was seen in 74% and 44% of the cases, respectively. Rictor IHC showed two staining patterns: membrane staining (16% of the cases) and cytoplasmic staining (58% of the cases). Rictor membrane staining predicted RICTOR amplification as detected by FISH with high specificity (95%) and sensitivity (70%). We did not find any correlation between RICTOR amplification and PD-L1 expression; RICTOR amplification was detected in 18% and 26% of PD-L1 positive and negative cases, respectively. The TCGA dataset analysis showed similar results; RICTOR copy number correlated with Rictor mRNA and protein expression but showed no association with PD-L1 mRNA and protein expression. In conclusion, the correlation between RICTOR amplification and Rictor membrane staining suggests that the latter can potentially be used as a surrogate marker to identify lung SCC cases with RICTOR amplification. Since a significant proportion of PD-L1 negative SCC cases harbor RICTOR amplification, analyzing PD-L1 negative tumors by RICTOR FISH or Rictor IHC can help select patients who may benefit from mTORC2 inhibitor therapy.

MiR-192 inhibits the activation of hepatic stellate cells by targeting Rictor.

The activation of hepatic stellate cells (HSCs) is regarded as the primary driving factor of liver fibrosis. miR-192, a miRNA associated with hepatocellular carcinoma and enriched in HSCs, has an undisclosed role in HSC activation and liver fibrosis. In this study, a CCl4-induced rat liver fibrosis model and transforming growth factor-beta 1 (TGF-β1)-treated HSC lines (LX-2 and HSC-T6) were used to detect miR-192 and Rictor levels in vivo and in vitro. Bioinformatic analysis and a dual luciferase assay were used to predict and confirm the interaction of Rictor with miR-192. Gain- and/or loss-of-function methods evaluated molecular changes and HSC activation phenotypes, detected by quantitative real-time PCR, western blotting, and immunofluorescence. We observed a gradual downregulation of miR-192 and upregulation of Rictor during CCl4-induced liver fibrosis/cirrhosis in rats. Enriched miR-192 was downregulated, while Rictor was upregulated in TGF-β1-activated HSCs. miR-192 inhibited the activation of HSCs by directly targeting Rictor. High miR-192/low Rictor expression attenuated the fibrotic-related gene expression by AKT/mTORC2 signaling. In conclusion, miR-192 could inhibit the activation of HSCs by directly targeting Rictor in the AKT/mTORC2 signaling pathway. This study provides insights into potential therapeutic targets for liver fibrosis and cirrhosis.

Rictor mediates p53 deactivation to facilitate the malignant transformation of hepatocytes and promote hepatocarcinogenesis

BackgroundMutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear.MethodsMicroarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo.ResultsOur study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P < 0.05). In mice, xenograft tumors overexpressing miR-192 exhibited lower Rictor expression levels, leading to higher p53 activity, and these tumors displayed slower growth compared to untreated HCC cells.ConclusionsRictor dynamically shuttles between the nucleus and cytoplasm during HCC development. Its pivotal oncogenic role involves binding and inhibiting p53wt activity within the nucleus in early hepatocarcinogenesis. Targeting Rictor presents a promising strategy for HCC based on p53 status.

EZY-1 inhibits idiopathic pulmonary fibrosis by regulating M2-type macrophage differentiation

Macrophage differentiation is closely associated with idiopathic pulmonary fibrosis (IPF) initiation. Herein, we show that EZY-1, a novel peptide derived from Eucheuma, regulates macrophage differentiation to inhibit IPF. Macrophage differentiation was detected by analyzing the surface antigens of the macrophages using flow cytometry. The effect of EZY-1 on cell proliferation was assessed. Signaling molecules involved in macrophage differentiation and fibrosis were detected by Western blot and enzyme-linked immunosorbent assays. The results showed that EZY-1 suppressed BLM-induced pulmonary fibrosis, with a decrease in type M2 macrophages. Simultaneously, EZY-1 reduced the amount of TGF-β1 secreted by M2 macrophages. In addition, EZY-1 downregulated COL1A1, TGF-β, and p-Smad3 and upregulated p-β-catenin in fibroblasts induced by M2 macrophages. Our results confirmed that EZY-1 inhibits IPF by inhibiting type M2 macrophage differentiation, which may be associated with the expression of Rictor and SHP2. Thus, EZY-1 can potentially be used as a drug for treating IPF.

Selective RNA‐targeting of mTORC1 and 2 to ameliorate Alzheimer’s Disease pathogenesis

AbstractBackgroundIt has been well described that inhibition of the kinase, mechanistic target of rapamycin (mTOR), improves age‐related pathologies including Alzheimer’s Disease (AD). However, mTOR exists as a complex and exerts differential functions depending on its binding partners. Suppression of mTORC1 has yielded positive outcomes for AD‐related measures (decreased amyloid beta/tau pathologies and improved cognition)1. In contrast, increased mTORC2 activity also resulted in positive outcomes in AD models (improved brain insulin sensitivity and neuronal survival)2, suggesting that a therapeutic which inhibits mTORC1 while upregulating mTORC2 may be desirable for AD. However, no such therapeutic exists.MethodTo inhibit mTORC1, we used a raptor siRNA which has been chemically modified to be stable for in vivo purposes. To upregulate mTORC2, we bioinformatically identified natural antisense transcripts (NATs) of rictor and designed custom siRNAs to target the various transcripts. Following an in vitro screen of the custom siRNAs, we determined which NATs yield upregulation of rictor when targeted and designed more siRNAs targeting the same transcript to optimize our mTORC2‐upregulating siRNA. Cellular RNA and protein were quantified using RT‐qPCR and western blot, respectively.ResultWe demonstrate efficient knockdown of raptor in all transfected cell lines, as well as in mouse brain. Raptor knockdown has been confirmed at both the RNA and protein level. Furthermore, we successfully upregulated expression of rictor using rictor NAT‐targeting siRNAs (AntagoNATs) in multiple cell lines. Downstream analyses suggest a therapeutic effect for both raptor knockdown and rictor upregulation in the context of AD.ConclusionNucleic acid‐based therapeutics which target RNA have been outcompeting and replacing small molecule protein‐targeting drugs in recent years. However, RNA‐targeted therapeutics have been largely unexplored for AD. Chemical linkage of raptor siRNA to rictor AntagoNAT siRNA yields the first therapeutic to selectively inhibit mTORC1 and upregulate mTORC2, providing a modern RNA‐targeting therapeutic for AD.

Assessment of the anti-inflammatory mechanism of quercetin 3,7-dirhamnoside using an integrated pharmacology strategy.

Pouzolzia zeylanica (L.) Benn. is a Chinese herbal medicine widely used for its anti-inflammatory and pus-removal properties. To explore its potential anti-inflammatory mechanism, quercetin 3,7-dirhamnoside (QDR), the main flavonoid component of P. zeylanica (L.) Benn., was extracted and purified. The potential anti-inflammatory targets of QDR were predicted using network analysis. These potential targets were verified using molecular docking, molecular dynamics simulations, and invitro experiments. Consequently, 342 potential anti-inflammatory QDR targets were identified. By analyzing the intersection between the protein-protein interaction and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we identified several potential protein targets of QDR, including RAC-alpha serine/threonine-protein kinase (AKT1), Ras-related C3 botulinum toxin substrate 1 (RAC1), nitric oxide synthase 3 (NOS3), serine/threonine-protein kinase mTOR (mTOR), epidermal growth factor receptor (EGFR), growth factor receptor-bound protein 2 (GRB2), and endothelin-1 receptor (EDNRA). QDR has anti-inflammatory activity and regulates immune responses and apoptosis through chemokines, Phosphatidylinositol 3-kinase 3(PI3K)/AKT, cAMP, T-cell receptor, and Ras signaling pathways. Molecular docking analysis showed that QDR has good binding abilities with AKT1, mTOR, and NOS3. In addition, molecular dynamics simulations demonstrated that the protein-ligand complex systems formed between QDR and AKT1, mTOR, and NOS3 have high dynamic stability, and their protein-ligand complex systems possess strong binding ability. In RAW264.7 macrophages, QDR significantly inhibited lipopolysaccharides (LPS)-induced inducible nitric oxide synthase expression, nitric oxide (NO) release and the generation of proinflammatory cytokines IL-6, IL-1β, and TNF-α. QDR downregulated the expression of p-AKT1(Ser473)/AKT1 and p-mTOR (Ser2448)/mTOR, and upregulated the expression of NOS3, Rictor, and Raptor. This indicates that the anti-inflammatory mechanisms of QDR involve regulation of AKT1 and mTOR to prevent apoptosis and of NOS3 which leads to the release of endothelial NO. Thus, our study elucidated the potential anti-inflammatory mechanism of QDR, the main flavonoid found in P. zeylanica (L.) Benn.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Effects and mechanism of Rictor interference in podocyte injury induced by high glucose.

Rapamycin-insensitive companion of mTOR (Rictor) is a critical effector of mTOR protein complex 2 (mTORC2). The aim of the present study was to investigate the effect of Rictor in the mTORC2 signaling pathway in high glucose (HG)-induced diabetic podocyte injury by silencing the expression of Rictor. In the present study, mouse podocytes were treated with glucose (150 mM) and mannitol (200 mM), the Rictor gene was silenced using small interfering RNA (siRNA). Apoptosis was detected by flow cytometry, whereas podocyte cytoskeletal protein expression was detected by western blotting (WB) and immunofluorescence staining. The results demonstrated that, compared with that in the control group, the podocyte apoptotic rate was significantly increased in the mannitol group (negative group) and the groups that were treated with glucose (model groups). The podocyte apoptotic rate in the model + Rictor siRNA group was significantly decreased compared with that in the negative, model and the model glucose + siRNA negative control (NC) groups. WB indicated that the protein expression levels of podocalyxin and synaptopodin were reduced in the model and model + siRNA NC groups compared with those in the normal control and negative groups. Additionally, the protein expression levels of α-smooth muscle actin (α-SMA) and P-AKT/AKT were increased in the model and model + siRNA NC groups compared with the those in control and negative groups. Compared with those the model and model + siRNA NC groups, the protein expression levels of podocalyxin and synaptopodin were increased, whilst those of the α-SMA and P-AKT/AKT proteins were decreased, in the model + Rictor siRNA group. Results from immunofluorescence analysis were basically consistent with those of WB. Therefore, results of the present study suggest that silencing of the Rictor gene may reduce the damage to podocytes induced by HG, such that the Rictor/mTORC2 signaling pathway may be involved in the remodeling of podocyte actin cytoskeletal in diabetes.

Growth and dormancy control of myeloma cells by mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) may have contrasting impacts on the progression of multiple myeloma (MM). Priming normal MSCs, by culturing them with MM cells, mimics the MSC-induced MM growth. We studied the contrasting effects of conditioned medium (CM) from unprimed or primed MSCs on growth of MM cells from newly diagnosed cases. We elucidated potential molecular pathways using global gene expression profiling and focused on the role of the mTOR2 component, RICTOR, as a novel mediator of dormancy in MM. Primed MSCs CM consistently increased proportions of proliferating cells and supported MM growth in 3-day (n = 20) and 10-day (n = 12) cultures, effects that were partially mediated through the IGF1 axis. In contrast, unprimed MSCs CM inhibited growth of MM cells in cases mainly from stages I/II MM. The genes most overexpressed in MM cells treated with primed MSCs CM were associated with cell cycle, DNA-damage repair, and proliferation; genes most overexpressed in MM cells treated with unprimed MSCs CM were associated with dormancy pathways including RICTOR (mTOR2 pathway), CXCR4, and BCL2. RICTOR protein level was induced by unprimed MSCs CM and was lower in KI67+ proliferating MM cells treated with primed MSCs CM. RICTOR was underexpressed in clinical relapse samples compared with baseline samples of the same patients. Inhibiting RICTOR expression in primary MM cells promoted their growth, and enforced expression of RICTOR in MM cell lines inhibited their growth. Our findings suggest that, after prolonged interactions with MM cells, bone marrow MSCs shift from MM-repressive to MM-permissive. Availability of Data and MaterialsOur institutional GEP data of MM cells from newly diagnosed patients used to show RICTOR expression have been deposited at Gene Expression Omnibus (GEO: GSE2658, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2658)

Wuzi Yanzong administration alleviates Sertoli cell injury by recovering AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in aging-induced testicular dysfunction

Ethnopharmacological relevanceWuzi Yanzong Prescription (WZ), a classic traditional Chinese medicine formula, has the properties of kidney nourishing and essence strengthening, and it is widely used to treat male infertility with a long history. Sertoli cells are injured with aging, resulting in testicular dysfunction, and WZ effectively rejuvenates the age-related decline of testicular function. However, whether the therapeutic effects of WZ on aging-related testicular dysfunction are dependent on the restoration of Sertoli cell function remains unclear. Aim of the studyIn a mouse model of natural aging, we explored the protective effects of WZ and its potential mechanisms. Materials and methodsFifteen-month-old C57BL/6 mice were randomized to receive either standard diet or WZ (2 and 8 g/kg) for 3 months. Meanwhile, 10 1-month-old mice were considered the adult control group and received standard diet for 3 months. The testis and epididymis were rapidly collected, and the sperm quality, testicular histology, Sertoli cell numbers, tight junction (TJ) ultrastructure, and blood–testis barrier-associated protein expression and localization were assessed. ResultsWZ significantly increased sperm concentration and sperm viability, improved the degenerative histomorphology and elevated the seminiferous epithelium height. Furthermore, WZ increased the number of Sertoli cells, restored the ultrastructure of the Sertoli cell TJ, and upregulated the expression of TJ-associated proteins (zonula occludens-1 and Claudin11), ectoplasm specialized-associated proteins (N-Cadherin, E-Cadherin and β-Catenin), and gap junction-associated protein (connexin 43), but did not affect the expression of Occludin and cytoskeletal protein (Vimentin). In addition, WZ did not change the localization of zonula occludens-1 and β-Catenin in aged testis. Moreover, WZ increased the expression of autophagy-associated proteins (light chain 3 beta and autophagy related 5) and decreased the expression of p62, phosphorylated mammalian target of rapamycin, and phosphorylated AKT in Sertoli cells. Finally, we found that WZ attenuated mTOR complex 1 (mTORC1) activity and upregulated mTORC2 activity, as evidenced by inhibition of the expression of the regulatory-associated protein of mTOR, phosphorylated p70 S6K, and phosphorylated ribosomal protein s6 and enhancement of the expression of Rictor in the Sertoli cells of aging mice. ConclusionsWZ improves the injury of Sertoli cells by restoring AKT/mTOR-mediated autophagy and the mTORC1–mTROC2 balance in Sertoli cells during aging. Our findings provide a new mechanism of WZ in the treatment of aging-induced testicular dysfunction.

Involvement of Rictor/mTORC2/Akt/GLUT4 pathway in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats.

Diabetes mellitus can be accompanied by a variety of complications. The purpose of the present study was to characterize the Rictor/mTOR complex 2 (mTORC2)/Akt/glucose transporter 4 (GLUT4) pathway and its effects on energy metabolism in the gastric smooth muscle of diabetic rats. Diabetes was induced in rats using streptozotocin and their phenotype was compared with untreated rats. The relationship between gastric motility and energy metabolism was analyzed by comparing the contraction and ATP metabolism of muscle strips. Western blotting was used to detect the expression of key proteins in the pathway. The diabetic rats demonstrated less frequent and less powerful gastric smooth muscle contractions. The concentrations of ADP, AMP, and ATP, and the energy charge in gastric smooth muscle changed in different periods of diabetes, and these changes were consistent with changes in mechanistic target of rapamycin (mTOR) protein content. The expression of the key intermediates in signal transduction in the Rictor/mTORC2/Akt/GLUT4 pathway also underwent significant changes. Rictor protein expression increased during the development of diabetes, but the activation of mTORC2 did not increase with the increase in Rictor expression. GLUT4 translocation is regulated by Akt and its expression change during the development of diabetes. These findings suggest that altered energy metabolism is present in gastric smooth muscle that is associated with changes in the Rictor/mTORC2/Akt/GLUT4 pathway. Rictor/mTORC2/Akt/GLUT4 pathway may be involved in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats and the development of diabetic gastroparesis.

The spatial expression of mTORC2-AKT-IP3R signal pathway in mitochondrial combination of endoplasmic reticulum of maternal fetal interface trophoblast in intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is complicated by adverse fetal outcomes and even fetal death, the mechanism remains unclear. This study aims at evaluating the differential expression of mTORC2-AKT-IP3R signaling pathway, which accurately regulate Ca2+ transfer across mitochondria-associated membranes (MAMs) and determine the stress intensity experienced by endoplasmic reticulum (ER) and mitochondria, in patients diagnosed withICP. We combined western blot analysis and placental immunofluorescence co-localization detection to assess the expression and co-localization of the mTORC2-AKT-IP3R signaling pathway in severe (maternal total bile acid (TBA) levels≥40 μmol/L) and mild (maternal TBA 10-40 μmol/L) ICP. Compared with the control and mild ICP groups, phosphorylated protein kinase B (p-AKT) levels were significantly upregulated in the severe ICP group. Placental Rictor levels were lower in the mild ICP group than in the control group and were further downregulated in the severe ICP group. IP3R3 and p-IP3R3 levels were lower in placentas in the severe ICP group than in those in the mild ICP and control groups. Moreover, the co-localization of IP3R3 and p-AKT in patients in the mild and severe ICP groups was significantly elevated compared with that in patients in the control group. In patients with severe ICP, limited expression of Rictor and elevated p-AKT levels would suppress IP3R3/p-IP3R3 levels in MAMs. This inhibition might influence the transportation of Ca2+ from the ER to the mitochondria, thus weaken the stress adaptation associated with MAMs. Our results reveal the possible pathophysiological mechanism of adverse fetal outcomes in ICP.