1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway.

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated differentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 induces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD inhibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.

Small nucleolar RNA host gene 1 promotes development and progression of colorectal cancer through negative regulation of miR-137.

Small nucleolar RNA host gene 1 (SNHG1) is critical in the progression of cancers. However, the mechanism by which SNHG1 regulates the progression of colorectal cancer (CRC) remains unclear. Expressions of SNHG1 and miR-137 in CRC tissues and cell lines were evaluated by quantitative real-time polymerase chain reaction. A luciferase reporter gene assay was conducted to investigate miR-137 target. Additionally, RNA pull-down assay was performed to explore the physical association between miR-137, SNHG1, and RNA induced silencing complex (RISC). Cell cycling and invasion were examined by flow cytometry (FCM) and transwell assays. The in vivo carcinogenic activity of SNHG1 was examined using murine xenograft models. Expression of RICTOR, serine/threonine kinase 1 (AKT), serum and glucocorticoid-inducible kinase 1 (SGK1), p70S6K1, and LC3II/LC3I ratio was examined by Western blot analysis. SNHG1 upregulation was observed in CRC tissues and cell lines, which was associated with the lymph node metastasis, advanced TNM stage and poorer prognosis. SNHG1 increased RICTOR level in CRC via sponging miR-137. In addition, SNHG1 silencing inhibited CRC cell proliferation and migration in vitro and in vivo. SNHG1 regulated RICTOR expression by sponging miR-137 and promoted tumorgenesis in CRC.

Inhibition of 14-3-3 binding to Rictor of mTORC2 for Akt phosphorylation at Ser473 is regulated by selenoprotein W.

14-3-3 reduces cell proliferation by inhibiting the activity of proteins involved in the signaling pathway that includes Akt kinase. Activation of Akt is enhanced by activating the mammalian target of rapamycin complex 2 (mTORC2). 14-3-3 is also a negative regulator of the mTORC2/Akt pathway, by interacting with a component of mTORC2. Recently, we reported that selenoprotein W (SelW) regulated the interaction between 14-3-3 and its target protein, CDC25B. Here, we show that the binding of Rictor, a component of mTORC2, to 14-3-3, is regulated by the interaction of 14-3-3 with SelW. When SelW was down-regulated, mTORC2-dependent phosphorylation of Akt at Ser473 was decreased. However, the phosphorylation of Thr308 was not affected. The interaction of Rictor with 14-3-3 was increased in SelW-knockdown cells, as compared to control cells. SelW-knockdown cells were also more sensitive to DNA damage induced by etoposide, than control cells. This phenomenon was due to the decreased phosphorylation of Akt at Ser473. We also found that ectopic expression of SelW(U13C) reduced the interaction between Rictor and 14-3-3, leading to Akt phosphorylation at Ser473. Taken together, these findings demonstrate that SelW activates the mTORC2/Akt pathway for Akt phosphorylation at Ser473, by interrupting the binding of Rictor to 14-3-3.

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.

Effect of miR-144-5p on the proliferation, migration, invasion and apoptosis of human umbilical vein endothelial cells by targeting RICTOR and its related mechanisms.

The purpose of the present study was to investigate the effect of microRNA (miR)-144-5p on human umbilical vein endothelial cells (HUVECs) to explore the role of miR-144-5p in atherosclerosis. miR-144-5p expression was upregulated in HUVECs using miR-144-5p mimics. The relative expression level of miR-144-5p in HUVECs was detected using reverse transcription-quantitative PCR (RT-qPCR). Cell proliferation was detected by performing an MTT assay. Apoptosis was determined via flow cytometry. Cell migration ability was detected by a wound-healing assay. Cell invasion was determined by a transwell assay. The protein levels of phosphorylated (p)-PI3K, p-Akt and endothelial nitric oxide synthase (eNOS) were detected using western blot analysis. The binding sites between miR-144-5p and 3'-untranslated region of rapamycin-insensitive companion of mTOR (RICTOR) mRNA were predicted by TargetScan and confirmed by a dual luciferase reporter assay. The present study showed that miR-144-5p mimics significantly inhibited cell proliferation and induced apoptosis in HUVECs. In addition, miR-144-5p mimics could suppress migration and invasion of HUVECs. Further analysis identified that RICTOR was a direct target gene of miR-144-5p. Moreover, miR-144-5p upregulation decreased the protein level of p-PI3K, p-Akt and eNOS. In conclusion, miR-144-5p regulated HUVEC proliferation, migration, invasion, and apoptosis through affecting the PI3K-Akt-eNOS signaling pathway by altering the expression of RICTOR. These results indicated that miR-144-5p may be a potential target for the prevention and treatment of atherosclerosis.

RICTOR/mTORC2 affects tumorigenesis and therapeutic efficacy of mTOR inhibitors in esophageal squamous cell carcinoma.

Dysregulation of mTORC1/mTORC2 pathway is observed in many cancers and mTORC1 inhibitors have been used clinically in many tumor types; however, the mechanism of mTORC2 in tumorigenesis is still obscure. Here, we mainly explored the potential role of mTORC2 in esophageal squamous cell carcinoma (ESCC) and its effects on the sensitivity of cells to mTOR inhibitors. We demonstrated that RICTOR, the key factor of mTORC2, and p-AKT (Ser473) were excessively activated in ESCC and their overexpression is related to lymph node metastasis and the tumor-node-metastasis (TNM) phase of ESCC patients. Furthermore, we found that mTORC1/ mTORC2 inhibitor PP242 exhibited more efficacious anti-proliferative effect on ESCC cells than mTORC1 inhibitor RAD001 due to RAD001-triggered feedback activation of AKT signal. Another, we demonstrated that down-regulating expression of RICTOR in ECa109 and EC9706 cells inhibited proliferation and migration as well as induced cell cycle arrest and apoptosis. Noteworthy, knocking-down stably RICTOR significantly suppresses RAD001-induced feedback activation of AKT/PRAS40 signaling, and enhances inhibition efficacy of PP242 on the phosphorylation of AKT and PRAS40, thus potentiates the antitumor effect of RAD001 and PP242 both in vitro and in vivo. Our findings highlight that selective targeting mTORC2 could be a promising therapeutic strategy for future treatment of ESCC.

RICTOR expression in esophageal squamous cell carcinoma and its clinical significance.

Esophageal cancer is one of the most common malignant tumors in the world, and its incidence is the eighth highest; meanwhile, its fatality rate is the sixth highest. The PI3K/Akt/mTOR signaling pathway plays a required role in human cancer, including cell survival, metabolism and migration. As a kind of important scaffold protein in mTORC2, RICTOR has showed over-expression in several malignancies like melanoma and endometrial cancer. In this research, we selected 201 cases of paraffin specimens from patients diagnosed as esophageal squamous cell carcinoma after surgical treatment and then estimated the RICTOR expression in each esophageal squamous cell carcinoma tissue by using the immunohistochemical streptavidin-peroxidase technique. Then, we analyzed the association among the clinicopathological parameters, the prognosis and the expression of RICTOR. Eventually, we found that the percentage of RICTOR-positive expression in 201 ESCC samples is 70.6% (142/201) and the figure for RICTOR-negative or RICTOR-doubtful-positive expression is 29.4% (59/201). RICTOR expression positively correlated with ESCC patients' AJCC stage (P = 0.011) and showed an opposite trend with survival (P = 0.007). Based on univariate and multivariate Cox proportional hazards regression analysis, RICTOR-positive expression, AJCC staging III or IV and nodal metastasis are prognostic factors and the former two are independent risk factors for ESCC. In conclusion, our study showed potential that targeting RICTOR may represent new effective inhibitors for treating ESCC.

10-Gingerol as an inducer of apoptosis through HTR1A in cumulus cells: In-vitro and in-silico studies.

OBJECTIVES: Cumulus cells play a crucial role as essential mediators in the maturation of ova. Ginger contains 10-gingerol, which induces apoptosis in colon cancer cells. Based on this hypothesis, this study aimed to determine whether 10-gingerol is able to induce apoptosis in normal cells, namely, cumulus cells. METHODS: This study used an in vitro analysis by culturing Cumulus cells in M199 containing 10-gingerol in various concentrations (12, 16, and 20 µM) and later detected early apoptotic activity using an Annexin V-FITC detection kit. RESULT: The in vitro data revealed that the number of apoptosis cells increased along with the period of incubation as follows: 12 µM (63.71% ± 2.192%); 16 µM (74.51% ± 4.596%); and 20 µM (78.795% ± 1.435%). The substance 10-gingerol induces apoptosis in cumulus cells by inhibiting HTR1A functions and inactivating GSK3B and AKT-1. CONCLUSIONS: These findings indicate that further examination is warranted for 10-gingerol as a contraception agent.

Identification of rictor as a novel substrate of Polo-like kinase 1.

Plk1 has been essentially described as a critical regulator of many mitotic events. However, increasing evidence supports the notion that its molecular functions are not restricted to the cell cycle. In particular, recent reports suggest the existence of a molecular and functional link between Plk1 and the mammalian target of rapamycin (mTOR) pathway, which controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Herein, we have identified rapamycin-insensitive companion of mTOR (Rictor), a core component of mTORC2, as a new Plk1 substrate and have shown that Plk1 phosphorylates Rictor at Ser1162 in vitro and in vivo. Surprisingly, cells expressing the unphosphorylatable mutant (S1162A) of Rictor did not show any effect on well characterized canonical PI3K-mTOR pathway. However, we found that cells expressing the unphosphorylatable form of Rictor have an elevated level of mSin1 isoform (mSin1.5). Considering that mSin1.5-containing mTORC2 was reported to associate with stress signaling, we propose that phosphorylation of Rictor at Ser1162 by Plk1 might be involved in a novel signaling pathway by regulating the mSin1.5-defined mTORC2.

New insights into Notch1 regulation of the PI3K-AKT-mTOR1 signaling axis: targeted therapy of γ-secretase inhibitor resistant T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is characterized as a high-risk stratified disease associated with frequent relapse, chemotherapy resistance, and a poorer prognostic outlook than B-precursor ALL. Many of the challenges in treating T-ALL reflect the lack of prognostic cytogenetic or molecular abnormalities on which to base therapy, including targeted therapy. Notch1 activating mutations were identified in more than 50% of T-ALL cases and can be therapeutically targeted with γ-secretase inhibitors (GSIs). Mutant Notch1 can activate cMyc and PI3K-AKT-mTOR1 signaling in T-ALL. In T-ALLs with wild-type phosphatase and tensin homolog deleted on chromosome ten (PTEN), Notch1 transcriptionally represses PTEN, an effect reversible by GSIs. Notch1 also promotes growth factor receptor (IGF1R and IL7Rα) signaling to PI3K-AKT. Loss of PTEN is common in primary T-ALLs due to mutation or posttranslational inactivation and results in chronic activation of PI3K-AKT-mTOR1 signaling, GSI-resistance, and repression of p53-mediated apoptosis. Notch1 itself might regulate posttranslational inactivation of PTEN. PP2A is activated by Notch1 in PTEN-null T-ALL cells, and GSIs reduce PP2A activity and increase phosphorylation of AKT, AMPK, and p70S6K. This review focuses on the central role of the PI3K-AKT-mTOR1 signaling in T-ALL, including its regulation by Notch1 and potential therapeutic interventions, with emphasis on GSI-resistant T-ALL.

Huang-Lian-Jie-Du-Decotion induced protective autophagy against the injury of cerebral ischemia/reperfusion via MAPK-mTOR signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huang-Lian-Jie-Du-Decotion (HLJDD, Hwangryun-Hae-Dok-Decotion in Japan), an ancient antipyretic and detoxifying traditional Chinese medicine formula, was reported to have protective effect on ischemic stroke. AIM OF THE RESEARCH: To investigate the therapeutic effect of HLJDD on ischemic stroke and explore its mode of action. MATERIAL AND METHODS: A model of ischemic stroke in the rat was established after transient middle cerebral artery occlusion (MCAO) followed by reperfusion. Rats were assigned randomly to groups of control, sham, transient ischemia/reperfusion (I/R), and three treatment groups by HLJDD at 2.5, 5.0, 10.0mg/kg. The neurological deficit, the cerebral infarct size, morphology abnormality, biochemical parameters were examined, and the levels of relevant proteins were determined by immunoblotting analysis to evaluate the protective effects of HLJDD on ischemic stroke and explore the underlying mechanism. RESULTS: Compared with I/R group, HLJDD significantly ameliorated neurological deficit and histopathology changes, decreased infarct area, and restored the levels of biochemical indicators including nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), glutathione disulfide (GSSG), total superoxide dismutase (T-SOD), Cu/Zn-SOD, Mn-SOD and glutathione peroxidase (GSH-PX). HLJDD also notably elevated the levels of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and other autophagy related genes (Atgs), promoted the activation of extracellular signal-regulated kinases (ERK), protein kinase B (Akt), 3-phosphoinositide-dependent kinase (PDK1), and inhibited the activation of mammalian target of rapamycin (mTOR), c-Jun N-terminal protein kinases (JNK), p38, phosphatase and tensin homolog (PTEN). CONCLUSION: HLJDD showed neuroprotective effects on ischemic stroke, at least in part to the induced protective autophagy via the regulation of mitogen-activated protein kinase (MAPK) signals. This Akt-independent protective autophagy is favorable in the treatment of stroke, avoiding unfavorable side-effects associated with the inactivation of Akt. The efficacy of HLJDD on ischemic stroke and its safety warranted by its long-term clinical use in traditional Chinese medicine favored further study to develop HLJDD as an effective therapeutic agent to treat ischemic stroke.