Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Role of Raptor Gene Variants in Hypertension: Influence on Blood Pressure Independent of Salt Intake in White Population.

BACKGROUND: The mTOR (mechanistic target of rapamycin) is an essential regulator of fundamental biological processes. mTOR forms 2 distinct complexes, mTORC1 (mTOR complex 1) when it binds with RAPTOR (Regulatory-associated Protein of mTOR) and mTORC2 (mTOR complex 2) when it associates with RICTOR (Rapamycin-insesitive companion of mTOR). Due to the previous link between the mTOR pathway, aldosterone, and blood pressure (BP), we anticipated that variants in the mTOR complex might be associated with salt-sensitive BP. METHODS: BP and other parameters were assessed after a one-week liberal Na+ (200 mmol/d) and a one-week restricted Na+ (10 mmol/d) diet in 608 White subjects from the Hypertensive Pathotype cohort, single-nucleotide variants in MTOR, RPTOR, and RICTOR genes were obtained for candidate genes analyses. RESULTS: The analysis revealed a significant association between a single nucleotide variants within the RPTOR gene and BP. Individuals carrying the RPTOR rs9901846 homozygous risk allele (AA) and heterozygous risk allele (GA) exhibited a 5 mm Hg increase in systolic BP on a liberal diet compared with nonrisk allele individuals (GG), but only in women. This single nucleotide variants effect was more pronounced on the restricted diet and present in both sexes, with AA carriers having a 9 mm Hg increase and GA carriers having a 5 mm Hg increase in systolic BP compared with GG. Interestingly, there were no significant associations between MTOR or RICTOR gene variants and BP. CONCLUSIONS: The RPTOR gene variation is associated with elevated BP in White participants, regardless of salt intake, specifically in females.

Selective disruption of mTORC1 and mTORC2 in VTA astrocytes induces depression and anxiety-like behaviors in mice.

Dysfunction of the mechanistic target of rapamycin (mTOR) signaling pathway is implicated in neuropsychiatric disorders including depression and anxiety. Most studies have been focusing on neurons, and the function of mTOR signaling pathway in astrocytes is less investigated. mTOR forms two distinct complexes, mTORC1 and mTORC2, with key scaffolding protein Raptor and Rictor, respectively. The ventral tegmental area (VTA), a vital component of the brain reward system, is enrolled in regulating both depression and anxiety. In the present study, we aimed to examine the regulation effect of VTA astrocytic mTOR signaling pathway on depression and anxiety. We specifically deleted Raptor or Rictor in VTA astrocytes in mice and performed a series of behavioral tests for depression and anxiety. Deletion of Raptor and Rictor both decreased the immobility time in the tail suspension test and the latency to eat in the novelty suppressed feeding test, and increased the horizontal activity and the movement time in locomotor activity. Deletion of Rictor decreased the number of total arm entries in the elevated plus-maze test and the vertical activity in locomotor activity. These data suggest that VTA astrocytic mTORC1 plays a role in regulating depression-related behaviors and mTORC2 is involved in both depression and anxiety-related behaviors. Our results indicate that VTA astrocytic mTOR signaling pathway might be new targets for the treatment of psychiatric disorders.

p300 nucleocytoplasmic shuttling underlies mTORC1 hyperactivation in Hutchinson-Gilford progeria syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.

Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide.

The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following ß-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

Iron drives anabolic metabolism through active histone demethylation and mTORC1.

All eukaryotic cells require a minimal iron threshold to sustain anabolic metabolism. However, the mechanisms by which cells sense iron to regulate anabolic processes are unclear. Here we report a previously undescribed eukaryotic pathway for iron sensing in which molecular iron is required to sustain active histone demethylation and maintain the expression of critical components of the pro-anabolic mTORC1 pathway. Specifically, we identify the iron-binding histone-demethylase KDM3B as an intrinsic iron sensor that regulates mTORC1 activity by demethylating H3K9me2 at enhancers of a high-affinity leucine transporter, LAT3, and RPTOR. By directly suppressing leucine availability and RAPTOR levels, iron deficiency supersedes other nutrient inputs into mTORC1. This process occurs in vivo and is not an indirect effect by canonical iron-utilizing pathways. Because ancestral eukaryotes share homologues of KDMs and mTORC1 core components, this pathway probably pre-dated the emergence of the other kingdom-specific nutrient sensors for mTORC1.

O-GlcNAcylation of Raptor transduces glucose signals to mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates metabolism and cell growth in response to nutrient levels. Dysregulation of mTORC1 results in a broad spectrum of diseases. Glucose is the primary energy supply of cells, and therefore, glucose levels must be accurately conveyed to mTORC1 through highly responsive signaling mechanisms to control mTORC1 activity. Here, we report that glucose-induced mTORC1 activation is regulated by O-GlcNAcylation of Raptor, a core component of mTORC1, in HEK293T cells. Mechanistically, O-GlcNAcylation of Raptor at threonine 700 facilitates the interactions between Raptor and Rag GTPases and promotes the translocation of mTOR to the lysosomal surface, consequently activating mTORC1. In addition, we show that AMPK-mediated phosphorylation of Raptor suppresses Raptor O-GlcNAcylation and inhibits Raptor-Rags interactions. Our findings reveal an exquisitely controlled mechanism, which suggests how glucose coordinately regulates cellular anabolism and catabolism.

Discovery of the First Raptor (Regulatory-Associated Protein of mTOR) Inhibitor as a New Type of Antiadipogenic Agent.

Raptor, a regulatory-associated protein of mTOR, has been genetically proved to be an important regulator in lipogenesis. However, its druggable potential is rarely investigated, largely due to the lack of an inhibitor. In this study, the antiadipogenic screening of a daphnane diterpenoid library followed by target fishing led to the identification of a Raptor inhibitor, 1c (5/7/6 carbon ring with orthoester and chlorine functionalities). Pharmacodynamic studies verified that 1c is a potent and tolerable antiadipogenic agent in vitro and in vivo. Mechanistic studies revealed that the targeting of Raptor by 1c could block the formation of mTORC1 and then downregulate the downstream S6K1- and 4E-BP1-mediated C/EBPs/PPARγ signaling, eventually retarding adipocyte cell differentiation at the early stage. These findings suggest that Raptor can be explored as a novel therapeutic target for obesity and its related complications, and 1c as the first Raptor inhibitor may provide a new therapeutic option for these conditions.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

Phosphorylation of OTUB1 at Tyr 26 stabilizes the mTORC1 component, Raptor.

Raptor plays a critical role in mTORC1 signaling. High expression of Raptor is associated with resistance of cancer cells to PI3K/mTOR inhibitors. Here, we found that OTUB1-stabilized Raptor in a non-canonical manner. Using biochemical assays, we found that the tyrosine 26 residue (Y26) of OTUB1 played a critical role in the interaction between OTUB1 and Raptor. Furthermore, non-receptor tyrosine kinases (Src and SRMS kinases) induced phosphorylation of OTUB1 at Y26, which stabilized Raptor. Interestingly, phosphorylation of OTUB1 at Y26 did not affect the stability of other OTUB1-targeted substrates. However, dephosphorylation of OTUB1 destabilized Raptor and sensitized cancer cells to anti-cancer drugs via mitochondrial reactive oxygen species-mediated mitochondrial dysfunction. Furthermore, we detected high levels of phospho-OTUB1 and Raptor in samples of patients with renal clear carcinoma. Our results suggested that regulation of OTUB1 phosphorylation may be an effective and selective therapeutic target for treating cancers via down-regulation of Raptor.

RBM3 interacts with Raptor to regulate autophagy and protect cardiomyocytes from ischemia-reperfusion-induced injury.

Acute myocardial infarction (AMI) is a common disease with high morbidity and mortality worldwide. However, postinfarction pathogenesis remains unclear, and it is particularly important to identify new therapeutic targets. The RNA-binding motif protein RBM3 (also known as cold-inducible protein) is known to promote translation and is associated with tumor proliferation and neuroprotection. However, little is known about the biological effects of RBM3 on myocardial infarction. In the present study, we found that RBM3 expression was significantly upregulated in ischemia-reperfusion (I/R) condition and downregulation of RBM3 inhibited autophagy and promoted apoptosis in cardiomyocytes. We confirmed that RBM3 interacts with Raptor to regulate the autophagy pathway. Taken together, these findings illustrate the protective effects of RBM3 against I/R-induced myocardial apoptosis through the autophagy pathway.

mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.

The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.

Inducible deletion of raptor and mTOR from adult skeletal muscle impairs muscle contractility and relaxation.

Skeletal muscle weakness has been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and is accompanied by altered mammalian target of rapamycin (mTOR) signalling. We wanted to elucidate the functional role of mTOR in muscle contractility. Most loss-of-function studies for mTOR signalling have used the drug rapamycin to inhibit some of the signalling downstream of mTOR. However, given that rapamycin does not inhibit all mTOR signalling completely, we generated a double knockout for mTOR and for the scaffold protein of mTORC1, raptor, in skeletal muscle. We found that double knockout in mice results in a more severe phenotype compared with deletion of raptor or mTOR alone. Indeed, these animals display muscle weakness, increased fibre denervation and a slower muscle relaxation following tetanic stimulation. This is accompanied by a shift towards slow-twitch fibres and changes in the expression levels of calcium-related genes, such as Serca1 and Casq1. Double knockout mice show a decrease in calcium decay kinetics after tetanus in vivo, suggestive of a reduced calcium reuptake. In addition, RNA sequencing analysis revealed that many downregulated genes, such as Tcap and Fhod3, are linked to sarcomere organization. These results suggest a key role for mTOR signalling in maintaining proper fibre relaxation in skeletal muscle. KEY POINTS: Skeletal muscle wasting and weakness have been associated with different pathological conditions, including sarcopenia and muscular dystrophy, and are accompanied by altered mammalian target of rapamycin (mTOR) signalling. Mammalian target of rapamycin plays a crucial role in the maintenance of muscle mass and functionality. We found that the loss of both mTOR and raptor results in contractile abnormalities, with severe muscle weakness and delayed relaxation following tetanic stimulation. These results are associated with alterations in the expression of genes involved in sarcomere organization and calcium handling and with an impairment in calcium reuptake after contraction. Taken together, these results provide a mechanistic insight into the role of mTOR in muscle contractility.

Raptor knockdown concurrently increases the electrical resistance and paracellular permeability of Caco-2 cell monolayers.

AIMS: As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. Intestinal barrier integrity ensures tissue homeostasis by closely regulating the permeability of the epithelium to lumenal contents. The role of mTORC1 in the regulation of intestinal barrier function and permeability remains to be fully elucidated. MATERIALS AND METHODS: In this study, we employed lentivirus-mediated knockdown of mTORC1 signaling-associated proteins Raptor (regulatory-associated protein of mTOR) and TSC2 (tuberin) to ascertain the effects of constitutive activation or repression of mTORC1 activity on barrier function in Caco-2 cell monolayers. KEY FINDINGS: Results showed that the loss of Raptor concomitantly raised the transepithelial electrical resistance (TEER) and para/transcellular permeability leading to a cell monolayer that is leaky for dextran yet electrically resistant to the movement of ions. Paracellular permeability was linked to the downregulation of tight junction protein expression and enhanced autophagy. Raptor-depleted cells had the highest abundance of myosin binding subunit MYPT1 concomitantly with the lowest abundance of p-MYPT1 (Thr696) and phosphorylated myosin light chain (p-MLC, Ser19) implying that MLC phosphatase activity was increased resulting in MLC relaxation. Although rapamycin suppressed mTORC1 activity and decreased the abundance of tight junction proteins in control cells, rapamycin caused a modest increase of TEER compared to Raptor knockdown. SIGNIFICANCE: The study showed that epithelium paracellular permeability of small molecular weight dextran is dissociated from TEER.

AAA + ATPase Thorase inhibits mTOR signaling through the disassembly of the mTOR complex 1.

The mechanistic target of rapamycin (mTOR) signals through the mTOR complex 1 (mTORC1) and the mTOR complex 2 to maintain cellular and organismal homeostasis. Failure to finely tune mTOR activity results in metabolic dysregulation and disease. While there is substantial understanding of the molecular events leading mTORC1 activation at the lysosome, remarkably little is known about what terminates mTORC1 signaling. Here, we show that the AAA + ATPase Thorase directly binds mTOR, thereby orchestrating the disassembly and inactivation of mTORC1. Thorase disrupts the association of mTOR to Raptor at the mitochondria-lysosome interface and this action is sensitive to amino acids. Lack of Thorase causes accumulation of mTOR-Raptor complexes and altered mTORC1 disassembly/re-assembly dynamics upon changes in amino acid availability. The resulting excessive mTORC1 can be counteracted with rapamycin in vitro and in vivo. Collectively, we reveal Thorase as a key component of the mTOR pathway that disassembles and thus inhibits mTORC1.

ZNF143 regulates autophagic flux to alleviate myocardial ischemia/reperfusion injury through Raptor.

The exact role of autophagy in myocardial ischemia/reperfusion (I/R) injury is still controversial. Excessive or insufficient autophagy may lead to cell death. Therefore, how to regulate autophagic balance during myocardial ischemia/reperfusion is critical to the treatment of myocardial I/R injury. Raptor is an mTOR regulatory related protein and closely related to the induction of autophagy. ZNF143 is widely expressed in various cells and acts as a transcription factor, which is involved in the regulation of autophagy, cell growth and development. In this study, we aimed to explore the mechanism by which ZNF143 regulated autophagy in myocardial I/R injury and the relationship between ZNF143 and Raptor. In our results, we found that ZNF143 expression was down-regulated in myocardial I/R. Inhibition of ZNF143 expression further enhanced autophagy and restored the deficiency of autophagic flux caused by myocardial I/R, subsequently alleviating myocardial I/R injury. On the other hand, overexpression of ZNF143 up-regulated Raptor expression and reduced autophagic activity, consequently exacerbating myocardial I/R injury. Taken together, our study revealed that ZNF143 might be a key target of the regulation of autophagy and a novel therapeutic target of myocardial I/R injury.

Raptor downregulation rescues neuronal phenotypes in mouse models of Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in the TSC1 or TSC2 genes, which encode proteins that negatively regulate mTOR complex 1 (mTORC1) signaling. Current treatment strategies focus on mTOR inhibition with rapamycin and its derivatives. While effective at improving some aspects of TSC, chronic rapamycin inhibits both mTORC1 and mTORC2 and is associated with systemic side-effects. It is currently unknown which mTOR complex is most relevant for TSC-related brain phenotypes. Here we used genetic strategies to selectively reduce neuronal mTORC1 or mTORC2 activity in mouse models of TSC. We find that reduction of the mTORC1 component Raptor, but not the mTORC2 component Rictor, rebalanced mTOR signaling in Tsc1 knock-out neurons. Raptor reduction was sufficient to improve several TSC-related phenotypes including neuronal hypertrophy, macrocephaly, impaired myelination, network hyperactivity, and premature mortality. Raptor downregulation represents a promising potential therapeutic intervention for the neurological manifestations of TSC.

Chylomicron production is repressed by RPTOR knockdown, R-α-lipoic acid and 4-phenylbutyric acid in human enterocyte-like Caco-2 cells.

Although the role of mechanistic target of rapamycin complex 1 (mTORC1) in lipid metabolism has been the subject of previous research, its function in chylomicron production is not known. In this study, we created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low, or high mTORC1 kinase activity, and used these cells to investigate the consequences of manipulating mTORC1 activity on enterocyte differentiation and chylomicron-like particle production. Constitutively active mTORC1 induced Caco-2 cell proliferation and differentiation (as judged by alkaline phosphatase activity) but weakened transepithelial electrical resistance (TEER). Repressed mTORC1 activity due to the knockdown of RPTOR significantly decreased the expression of lipogenic genes FASN, DGAT1, and DGAT2, lipoprotein assembly genes APOB and MTTP, reduced protein expression of APOB, MTTP, and FASN, downregulated the gene expression of very long-chain fatty acyl-CoA ligase (FATP2), acyl-CoA binding protein (DBI), and prechylomicron transport vesicle-associated proteins VAMP7 (vesicle-associated membrane protein 7) and SAR1B (secretion associated Ras related GTPase 1B) resulting in the repression of apoB-containing triacylglycerol-rich lipoprotein secretion. Exposure of Caco-2 cells harboring a constitutively active mTORC1 to short-chain fatty acid derivatives, R-α-lipoic acid and 4-phenylbutyric acid, downregulated chylomicron-like particle secretion by interfering with the lipidation and assembly of the particles, and concomitantly repressed mTORC1 activity with no change to Raptor abundance or PRAS40 (Thr246) phosphorylation. R-α-lipoic acid and 4-phenylbutyric acid may be useful to mitigate intestinal lipoprotein overproduction and associated postprandial inflammation.

Retinoic acid increases the cellular cholesterol predominantly in a mTOR-independent manner.

Retinoic acid (RA) plays a role in the mounting immune response and controls several functions of the human body, including cholesterol homeostasis. The synthesis, uptake, and efflux of cellular cholesterol are significantly linked to the mammalian target of rapamycin complex-1 (mTORC1). Activation of mTORC1 promotes the synthesis and uptake of the cholesterol and suppresses its efflux, thus causing accumulation of cellular cholesterol. It is intriguing to know the effect of a high dose of RA on cholesterol accumulation in macrophages (mφ) and whether it is via mTOR activation. It is important to note that the long-term treatment of RA in humans is safe. Therefore, we chose a high dose of RA to observe its effect, which may be implicated in diseases like visceral leishmaniasis, where cholesterol deficiency is established. In the present study, we found the increased expression of RAPTOR, a regulatory component of the mTORC1 complex, in mφ upon treatment with RA. We observed the increased expression of SREBP2, LDLR, and PCSK9 in RA-treated mφ under sufficient cholesterol conditions, which further increased cellular cholesterol levels. Notably, their expressions were decreased when the mTOR pathway was inhibited by rapamycin. However, treatment with rapamycin did not result in the loss of cellular cholesterol in RA-treated mφ. Comparison with rapamycin-treated mφ suggests that RA induces cellular cholesterol levels in a mTORC1-independent manner.

RPTOR methylation in the peripheral blood and breast cancer in the Chinese population.

BACKGROUND: Altered regulatory-associated protein of mTOR, complex 1 (RPTOR) methylation levels in peripheral blood was originally discovered as breast cancer (BC)-associated risk factor in Caucasians. OBJECTIVE: To explore the relationship between RPTOR methylation and BC in the Chinese population, we conducted two independent case-control studies. METHODS: Peripheral blood samples were collected from a total of 333 sporadic BC cases and 378 healthy female controls for the DNA extraction and bisulfite-specific PCR amplification. Mass spectrometry was applied to quantitatively measure the levels of methylation. The logistic regression, Spearman's rank correlation, and Non-parametric tests were used for the statistical analyses. RESULTS: In our study, we found an association between BC and RPTOR_CpG_4 hypomethylation in the general population (per-10% of methylation, OR 1.29, P = 0.012), and a weak association between BC and RPTOR_CpG_8 hypomethylation in the women with older age (per-10% of methylation, OR 2.34, P = 0.006). We also identified age as a confounder for the change of RPTOR methylation patterns, especially at RPTOR_CpG_4, which represented differential methylation comparing age groups especially in the BC cases (age < 50 years vs age ≥ 50 years by Mann-Whitney U test, P < 0.0001 for BC cases and P = 0.079 for controls). CONCLUSION: Our study validated the association between hypomethylation of RPTOR and BC risk in the Chinese population also with weak effect and mostly for postmenopausal women. In addition, our findings provided novel insight for the regulation of DNA methylation upon aging or the change of hormone levels.