mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy
Rosa Bartolomeo, … , Andrea Ballabio, Carmine Settembre
Rosa Bartolomeo, … , Andrea Ballabio, Carmine Settembre
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3717-3729. https://doi.org/10.1172/JCI94130.
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Concise Communication Bone Biology Therapeutics

mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

Abstract

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associated Beclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.

Authors

Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre

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Figure 2

Hyperactivation of mTORC1 in MPS chondrocytes suppresses autophagy via a Beclin 1–Vps34–UVRAG kinase complex.

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Hyperactivation of mTORC1 in MPS chondrocytes suppresses autophagy via a...
(A) Western blot analysis of mTORC1 signaling upon amino acid stimulation in Gusb–/– and Gusb–/– Rpt+/– primary chondrocytes. (B) Quantification of S6K1 phosphorylation in primary chondrocytes isolated from mice of the indicated genotypes. n = 3. (C) Representative images of an RFP-GFP-LC3 assay in primary chondrocytes from mice of the indicated genotypes. Scale bars: 10 μm. (D and E) Quantitative analysis of RFP+GFP+ puncta (D) and RFP+GFP– puncta (E). Horizontal bars indicates the mean value. n = 3 chondrocytes donors/phenotype; n = 30 cells analyzed. (F) Western blot analysis of the indicated proteins in Gusb–/– and Gusb–/– Rpt+/– primary chondrocytes. β-Actin was used as a loading control. (G) Quantification of protein levels in cells from mice of the indicated genotypes. n = 3. (H) Representative blot of a UVRAG IP assay testing UVRAG interaction with Rubicon in control and Gusb-KO RCS chondrocytes. (I) Quantification of Rubicon co-IP with UVRAG. n = 3. (J) GFP-2xFYVE transfection in control and Gusb-KO RCS chondrocytes. Gusb-KO cells were treated with Torin1 (1 μM; 6 h) or cotransfected with Myc-UVRAG. Cells were costained for endosomes (EEA1). Boxed area in the bottom right-hand panel shows positive myc staining. Scale bars: 10 μm. (K) Quantitative analysis of PI3P puncta. n = 3; n = 25 cells analyzed. (L and M) Western blot analysis of SQSTM1/p62 in Gusb-KO cells transfected with Myc-UVRAG (L) or treated with Torin1 (1 μM, 6 h) and SAR405 (10 μM, 6 h) (M). β-Actin was used as a loading control. Bar graphs show quantification of protein levels. n = 3 (L) and n = 2 (M). Data represent the mean values derived from the indicated number of independent experiments. Error bars indicate the SEM. *P ≤ 0.05, **P ≤ 0.005, and ***P ≤ 0.0005, by ANOVA followed by Tukey’s post-hoc test (B, D, E, G, and K) and unpaired Student’s t test (I and L).