The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates 1 – 3 . However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy 4 , 5 , is phosphorylated by mTORC1 via a substrate-specific mechanism mediated by RagGTPases. Thus, TFEB phosphorylation is strictly dependent on amino acid-mediated activation of RagC/D GTPase but, unlike other mTORC1 substrates such as S6K and 4E-BP1, insensitive to growth factor-induced Rheb activity. This mechanism plays a crucial role in Birt-Hogg-Dubé (BHD) syndrome, a disorder caused by mutations of the RagC/D activator folliculin (FLCN) and characterized by benign skin tumors, lung and kidney cysts and renal cell carcinoma 6 , 7 . We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and paradoxical mTORC1 hyperactivity observed in BHD syndrome. Remarkably, depletion of TFEB in a kidney-specific mouse model of BHD syndrome fully rescued the disease phenotype and associated lethality and normalized mTORC1 activity. Together, these findings identify a substrate-specific control mechanism of mTORC1, whose dysregulation leads to kidney cysts and cancer.
The mechanistic Target Of Rapamycin Complex 1 (mTORC1) is recruited to the lysosome by Rag GTPases and regulates anabolic pathways in response to nutrients. Here we find that MiT/TFE transcription factors, master regulators of lysosomal and melanosomal biogenesis and autophagy, control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyper-proliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.
Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.
Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs.
Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in the SUMF1 gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in the Sumf1 gene. Sulfatase activities are completely absent in Sumf1 ؊/؊ mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients, Sumf1 ؊/؊ mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans. Sumf1 ؊/؊ mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.apoptosis ͉ macrophages ͉ sulfatase modifying factor 1
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