Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes / 浙江大学学报·医学版

OBJECTIVE@#To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs).@*METHODS@#Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot.@*RESULTS@#Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (<0.01).@*CONCLUSIONS@#Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.

MicroRNA-152 and microRNA-448 inhibit proliferation of colorectal cancer cells by targeting Rictor / 南方医科大学学报

OBJECTIVE@#To screen the microRNAs (miRNAs) targeting Rictor and investigate their effects in regulating the biological behaviors of colorectal cancer (CRC).@*METHODS@#Human colorectal cancer cell line KM12SM was transfected with the miRNAs targeting Rictor identified by prediction software to test inhibitory effects of these miRNAs on Rictor expression using qRT-PCR and Western blotting. Dual luciferase reporter assay was used to further confirm the binding of these miRNAs to the 3'UTR of Rictor mRNA. Cell survival and colony formation assays were used to investigate the effects of these miRNAs on survival and colony formation in KM12SM cells.@*RESULTS@#miR-152 and miR-448 were identified as the Rictor-targeting miRNAs, which significantly inhibited the expression of Rictor in KM12SM cells ( < 0.05). The two miRNAs were confirmed to bind to the 3'UTR of Rictor mRNA and significantly inhibited luciferase activity in KM12SM cells ( < 0.01, < 0.05); they also showed activities of posttranscriptional modulation of Rictor. Overexpression of miR-152 and miR-448 both significantly inhibited the growth and colony formation of KM12SM cells.@*CONCLUSIONS@#miR-152 and miR-448 can down-regulate the protein expression of Rictor by targeting Rictor mRNA to negatively regulate the growth and colony formation of colorectal cancer cells.

Role of Rictor in Hematopoietic Stem Cells during Fetal Liver Hematopoiesis / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of Rictor on the hematopoiesis of fetal liver by specific knock-out of Rictor in hematopoietic cells of Vav-Cre mice.@*METHODS@#E12.5 0.08ee fetal liver cells from the experimental group Vav-Cre; Rictor embryos and control group Rictor or Rictor embryos were transplanted to recipients respectively to observe the effect of Rictor on reconstitution ability of hematopoietic stem cells. In the meantime, E14.5 0, 10, 20, 40, 60, 80 sorted hematopoietic stem cells from the Vav-Cre; Rictor fetal liver of experimental group and Rictor or Rictor fetal liver of control group were transplanted in to recipients to analyze the numbers of functional hematopoietic stem cells after Rictor was knocked-out. Furthermore, the self-renewal capacity was investigated by secondary transplantation of BM cells from primary recipients that had been successfully repopulated with E12.5 fetal liver-derived cells and by cell cycle analysis.@*RESULTS@#All the recipients receiving E12.5 Rictor or Rictor cells were repopulated (8/8, from 2 independent experiments) with an average chimerism of 77.2%±11.1% at 4 months post-transplantation, which resulted in 57 LT-RU per FL. In comparison, 8 out of 8 recipients receiving Vav-Cre; Rictor cells were repopulated with significantly reduced chimerism (37.0%±16.3%) (P＜0.01), which was equivalent to 8 LT-RU per FL. The limiting dilution transplantation experiment showed that there was one functional hematopoietic stem cell out of 17 sorted SLAM cells in the control group, and one functional hematopoietic stem cell out of 39 sorted SLAM cells in the experimental group. The secondary transplantation experiments showed that 2 out of 4 recipients were reconstructed in the control group after 1 month, and 0 was reconstructed in the experimental group by transplanting 4×10 donor cells respectively. What＇s more, the percentage of S/G/M cells in the experimental group increased when compared with controls.@*CONCLUSION@#In the process of fetal liver hematopoiesis, the specifically knocking-out the Rictor in hematopoietic system can lead to defect of reconstitution ability, decrease of the functional hematopoietic stem cell numbers and reduction of self-renewal ability of hematopoietic stem cells.

Expression pattern of mTOR subunits Raptor and Rictor in mouse hair follicle cycle / 中南大学学报(医学版)

To detemine the expression pattern of mTOR complex subunits Raptor and Rictor in the hair follicles of mice at different hair follicle stages, and to explore its significance. 
 Methods: Immunostaining of Ki-67, a proliferative marker, was used to determine the precise hair follicle stages of mouse dorsal skin at different postnatal time points. Real-time PCR was used to detect the mRNA expression of Raptor and Rictor in mouse dorsal skin at 43 days after birth (P43, early telogen), 56 days after birth (P56, mid-telogen), 69 days after birth (P69, late telogen) and 74 days after birth (P74, early anagen). The expression intensity and localization of Raptor and Rictor at different stages of hair cycle were tested by co-immumostaining.
 Results: Ki-67 immunostaining showed that the time points (P43, P56, P69, P74) and hair follicle stages (early telogen, mid-telogen, late telogen, early anagen) of the dorsal skin were consistent with each other. The results of real-time PCR and immunostaining were consistent, showing that the expression of Raptor and Rictor did not changed in the early-, mid-, late telogen, and early anagen. However, Raptor was specifically expressed in the bulge where hair follicle stem cells (HFSCs) are residing in, and Rictor was mainly detected in inner root sheath (IRS) cells. 
 Conclusion: The expression of Raptor and Rictor does not altered in the hair follicles at different hair follicle stages, but Raptor and Rictor are specifically expressed in the HFSCs and IRS cells, respectively, indicating that Raptor might be a molecular marker for HFSCs, and Rictor might be involved in the maintenance of IRS and formation of hair shaft.

Expression of Rictor and mTOR in colorectal cancer and their clinical significance / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the expression of Rictor and mTOR in the colorectal cancer and their clinical significance.</p><p><b>METHODS</b>The expression levels of Rictor and mTOR in HCT116, SW480, LoVo and HCoEpiC cells were detected by indirect immunofluorescence and Western blotting. Sixty-two paraffin-embedded surgical specimens of colorectal cancer tissue and adjacent tissues were examined for Rictor expression using immunohistochemistry. The association of the expression levels of Rictor protein with the clinicopathologic features and the overall survival of the patients was analyzed.</p><p><b>RESULTS</b>The expression level of Rictor was significantly higher in colorectal cancer tissues than in the adjacent tissues (P<0.05). The expression levels of Rictor and mTOR in the colon cancer cell lines were higher than those in human normal colon epithelial cell line HCoEpiC. The expression of Rictor was correlated with Dukes stage and lymphatic metastasis of the tumors but not with other clinicopathological parameter (P>0.05). Patients with Rictor expression had a lower overall survival rate than those without Rictor expression.</p><p><b>CONCLUSION</b>Rictor overexpression is associated with the carcinogenesis and progression of colorectal cancer and can be an independent indicator for evaluating the prognosis of colorectal cancer patients.</p>

Regulatory mechanisms of PI3K/AKT signaling pathway in acute leukemia / 中国实验血液学杂志

This study was aimed to analyze the expression profiles of PI3K/AKT signaling pathway genes from bone marrow samples of AML and ALL patients and normal samples. AML, ALL and normal bone marrow samples were collected from 6 AML, 6 ALL patients and 4 normal persons. The expression of PI3K/AKT signaling pathway genes including PTEN, CCND1, mTOR, RICTOR, FOXO1 were detected by real-time fluorescent quantification RT-PCR while GAPDH gene expression was used as an internal reference. The relative gene expression level was calculated by the method of the 2(-ΔΔCt). The results showed that the gene expression profiles were different between normal and leukemic groups. PTEN, mTOR and RICTOR expression levels were down-regulated, while FOXO1 and CCND1 levels were up-regulated in AML and ALL. PTEN was down-regulated in 10 out of the 12 samples; mTOR was down-regulated in 9 out of the 12 samples; RICTOR was down-regulated in 7 out of the 12 samples; FOXO1 was up-regulated in 9 out of the 12 samples and CCND1 was up-regulated in 7 out of the 12 samples. It is concluded that PI3K/AKT signal pathway is activated in both AML and ALL leukemic cells.

Phosphorylation of Rictor at Thr1135 impairs the Rictor/Cullin-1 complex to ubiquitinate SGK1

The Rictor/mTOR complex plays a pivotal role in a variety of cellular functions including cellular metabolism, cell proliferation and survival by phosphorylating Akt at Ser473 to fully activate the Akt kinase. However, its upstream regulatory pathways as well as whether it has additional function(s) remain largely unknown. We recently reported that Rictor contains a novel ubiquitin E3 ligase activity by forming a novel complex with Cullin-1, but not with other Cullin family members. Furthermore, we identified SGK1 as its downstream target. Interestingly, Rictor, but not Raptor or mTOR, promotes SGK1 ubiquitination. As a result, SGK1 expression is elevated in Rictor(-/-) MEFs. We further defined that as a feedback mechanism, Rictor can be phosphorylated by multiple AGC family kinases including Akt, S6K and SGK1. Phosphorylation of Rictor at the Thr1135 site did not affect its kinase activity towards phosphorylating its conventional substrates including Akt and SGK1. On the other hand, it disrupted the interaction between Rictor and Cullin-1. Consequently, T1135E Rictor was defective in promoting SGK1 ubiquitination and destruction. This finding further expands our knowledge of Rictor's function. Furthermore, our work also illustrates that Rictor E3 ligase activity could be governed by specific signaling kinase cascades, and that misregulation of this process might contribute to SGK overexpression which is frequently observed in various types of cancers.

Antiproliferation of cardamonin associated with mRNA expression of mTOR, Raptor and Rictor / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the antiproliferation effect of cardamonin (CAR) and its possible mechanisms on human umbilical artery smooth muscle cells (HUASMCs) cultured in the mimicking insulin resistance (IR) medium.</p><p><b>METHOD</b>Proliferation of HUASMCs was assayed by MTT method. The mRNA expression of mTOR, Raptor and Rictor was detected by a real-time PCR. The expression content was calculated by Livak method using internal control of beta-actin.</p><p><b>RESULT</b>The proliferation of HUASMCs cultured in the mimicking IR medium was significantly increased. Both in normal and mimic IR culture medium, cells proliferation was inhibited by CAR (1 x 10(-5), 1 x 10(-4) mol x L(-1)). Pretreated with PD98059 and LY294002, cell proliferation induced by phosphatidic acid (PA) was inhibited, and the mRNA expression of mTOR, Raptor and Rictor was significantly decreased by CAR in the mimic IR medium.</p><p><b>CONCLUSION</b>It is implicated that antiproliferation of CAR is involved in mRNA expression decrease of mTOR and its relative protein Raptor and Rictor.</p>