ABSTRACT
The small G-protein Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) in response to growth factor signals. mTORC1 is a master regulator of cellular growth and metabolism; aberrant mTORC1 signaling is associated with fibrotic, metabolic, and neurodegenerative diseases, cancers, and rare disorders. Point mutations in the Rheb switch II domain impair its ability to activate mTORC1. Here, we report the discovery of a small molecule (NR1) that binds Rheb in the switch II domain and selectively blocks mTORC1 signaling. NR1 potently inhibits mTORC1 driven phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) but does not inhibit phosphorylation of AKT or ERK. In contrast to rapamycin, NR1 does not cause inhibition of mTORC2 upon prolonged treatment. Furthermore, NR1 potently and selectively inhibits mTORC1 in mouse kidney and muscle in vivo. The data presented herein suggest that pharmacological inhibition of Rheb is an effective approach for selective inhibition of mTORC1 with therapeutic potential.
Subject(s)
Mechanistic Target of Rapamycin Complex 1/metabolism , Ras Homolog Enriched in Brain Protein/antagonists & inhibitors , Signal Transduction/drug effects , Small Molecule Libraries/pharmacology , Animals , Cell Line, Tumor , Crystallography, X-Ray , HEK293 Cells , Humans , Jurkat Cells , MCF-7 Cells , Male , Mice, Inbred C57BL , Molecular Structure , Phosphorylation/drug effects , Ras Homolog Enriched in Brain Protein/genetics , Ras Homolog Enriched in Brain Protein/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Small Molecule Libraries/chemistryABSTRACT
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.
Subject(s)
Autophagy , Brain/metabolism , MicroRNAs/metabolism , Ras Homolog Enriched in Brain Protein/metabolism , Spondylitis, Ankylosing/pathology , TOR Serine-Threonine Kinases/metabolism , Adult , Autophagy/drug effects , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Case-Control Studies , Cell Line, Tumor , Cytokines/blood , Down-Regulation , Female , Humans , Jurkat Cells , Male , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Middle Aged , RNA Interference , Ras Homolog Enriched in Brain Protein/antagonists & inhibitors , Ras Homolog Enriched in Brain Protein/genetics , Signal Transduction/drug effects , Sirolimus/pharmacology , Spondylitis, Ankylosing/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , Up-RegulationABSTRACT
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.