mTOR Senses Intracellular pH through Lysosome Dispersion from RHEB.

Acidity, generated in hypoxia or hypermetabolic states, perturbs homeostasis and is a feature of solid tumors. That acid peripherally disperses lysosomes is a three-decade-old observation, yet one little understood or appreciated. However, recent work has recognized the inhibitory impact this spatial redistribution has on mechanistic target of rapamycin complex 1 (mTORC1), a key regulator of metabolism. This finding argues for a paradigm shift in localization of mTORC1 activator Ras homolog enriched in brain (RHEB), a conclusion several others have now independently reached. Thus, mTORC1, known to sense amino acids, mitogens, and energy to restrict biosynthesis to times of adequate resources, also senses pH and, via dampened mTOR-governed synthesis of clock proteins, regulates the circadian clock to achieve concerted responses to metabolic stress. While this may allow cancer to endure metabolic deprivation, immune cell mTOR signaling likewise exhibits pH sensitivity, suggesting that suppression of antitumor immune function by solid tumor acidity may additionally fuel cancers, an obstacle potentially reversible through therapeutic pH manipulation.

MicroRNA-199a-5p Induced Autophagy and Inhibits the Pathogenesis of Ankylosing Spondylitis by Modulating the mTOR Signaling via Directly Targeting Ras Homolog Enriched in Brain (Rheb).

BACKGROUND/AIMS: Ankylosing spondylitis (AS) is an inflammatory and immune disease leading to disability. Autophagy has been identified as a potential player in understanding the pathogenesis of AS. METHODS: MiRNA-199a-5p and autophagy-related gene expression were determined by qRT-PCR or Western blot. Cytokine production was determined using ELISA assays. Proliferation was determined by MTT assay. MiRNA-199a-5p and Ras homolog enriched in brain (Rheb) were upregulated or downregulated by overexpression of plasmid or siRNA transfection. RESULTS: Expression of miRNA-199a-5p, and autophagy-related genes LC3, beclin1, and ATG5 was significantly decreased in T cells of AS patients. Serum concentrations of TNF-α, IL-17, and IL-23 were promoted in AS patients, compared to healthy controls. MiRNA-199a-5p expression levels also showed significant negative correlations with the Ankylosing Spondylitis Disease Activity Score (ASDAS) and modified Stoke Ankylosing Spon dylitis Spinal Score (mSASSS) of AS patients. In Jurkat T cells and T cells isolated from AS patients, miRNA-199a-5p overexpression promoted autophagy-related genes expression and decreased TNF-α, IL-17, and IL-23 levels, whereas inhibition of miRNA-199a-5p attenuated these effects. As a direct target of miRNA-199a-5p, Rheb inhibition led to a striking decrease in the phosphorylation of the mechanistic target of rapamycin (mTOR) and induced autophagy. Moreover, pcDNA3.1-Rheb effectively reduced the inhibiting effects of mTOR signaling caused by miRNA-199a-5p overexpression. All effects were offset by pretreating with rapamycin (an mTOR antagonist). CONCLUSIONS: AS patients with advanced spinal damage had decreased autophagy levels and that miRNA-199a-5p may induce autophagy and inhibit the pathogenesis of AS by modulating the mTOR signaling via direct targeting Rheb.

Role of RHEB in Regulating Differentiation Fate of Mesenchymal Stem Cells for Cartilage and Bone Regeneration.

Advances in mesenchymal stem cells (MSCs) and cell replacement therapies are promising approaches to treat cartilage and bone defects since substantial differentiation capacities of MSCs match the demands of tissue regeneration. Our understanding of the dynamic process requiring indispensable differentiation of MSCs remains limited. Herein, we describe the role of RHEB (Ras homolog enriched in brain) regulating gene signature for differentiation of human adipose derived mesenchymal stem cells (ASCs) into chondrogenic, osteogenic, and adipogenic lineages. RHEB-overexpression increases the proliferation of the ASCs. RHEB enhances the chondrogenic differentiation of ASCs in 3D culture via upregulation of SOX9 with concomitant increase in glycosaminoglycans (GAGs), and type II collagen (COL2). RHEB increases the osteogenesis via upregulation of runt related transcription factor 2 (RUNX2) with an increase in the calcium and phosphate contents. RHEB also increases the expression of osteogenic markers, osteonectin and osteopontin. RHEB knockdown ASCs were incapable of expressing sufficient SRY (Sex determining region Y)-box 9 (SOX9) and RUNX2, and therefore had decreased chondrogenic and osteogenic differentiation. RHEB-overexpression impaired ASCs differentiation into adipogenic lineage, through downregulation of CCAAT/enhancer binding protein beta (C/EBPß). Conversely, RHEB knockdown abolished the negative regulation of adipogenesis. We demonstrate that RHEB is a novel regulator, with a critical role in ASCs lineage determination, and RHEB-modulated ASCs may be useful as a cell therapy for cartilage and bone defect treatments.

Direct Interaction between Ras Homolog Enriched in Brain and FK506 Binding Protein 38 in Cashmere Goat Fetal Fibroblast Cells.

Ras homolog enriched in brain (Rheb) and FK506 binding protein 38 (FKBP38) are two important regulatory proteins in the mammalian target of rapamycin (mTOR) pathway. There are contradictory data on the interaction between Rheb and FKBP38 in human cells, but this association has not been examined in cashmere goat cells. To investigate the interaction between Rheb and FKBP38, we overexpressed goat Rheb and FKBP38 in goat fetal fibroblasts, extracted whole proteins, and performed coimmunoprecipitation to detect them by western blot. We found Rheb binds directly to FKBP38. Then, we constructed bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38), and examined their interaction by yeast two-hybrid assay. Their direct interaction was observed, regardless of which plasmid served as the prey or bait vector. These results indicate that the 2 proteins interact directly in vivo. Novel evidence is presented on the mTOR signal pathway in Cashmere goat cells.