Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

Abstract

OBJECTIVE:

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells.

DESIGN:

The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1β) stimulation.

RESULTS:

Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1β-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1β-induced senescence-associated beta-galactosidase (SA-β-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1β-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages.

CONCLUSION:

Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

• Authors: M Ito, T Yurube, K Kakutani, K Maeno, T Takada, Y Terashima, Y Kakiuchi, Y Takeoka, S Miyazaki, R Kuroda, K Nishida
• Journal: Osteoarthritis and cartilage (Osteoarthritis Cartilage) Vol. 25 Issue 12 Pg. 2134-2146 (12 2017) ISSN: 1522-9653 [Electronic] England
• PMID: 28888905 (Publication Type: Journal Article)
• Copyright: Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chemical References

• Interleukin-1beta
• MAP1LC3A protein, human
• Microtubule-Associated Proteins
• RPTOR protein, human
• Regulatory-Associated Protein of mTOR
• SQSTM1 protein, human
• Sequestosome-1 Protein
• Mechanistic Target of Rapamycin Complex 1
• Mechanistic Target of Rapamycin Complex 2
• Proto-Oncogene Proteins c-akt
• Ribosomal Protein S6 Kinases, 70-kDa
• Sirolimus

Topics

• Apoptosis (drug effects)
• Autophagy (drug effects)
• Blotting, Western
• Cellular Senescence (drug effects)
• Extracellular Matrix (drug effects, metabolism)
• Gene Knockdown Techniques
• Humans
• Interleukin-1beta (pharmacology)
• Intervertebral Disc (cytology, drug effects, metabolism)
• Mechanistic Target of Rapamycin Complex 1 (antagonists & inhibitors, genetics)
• Mechanistic Target of Rapamycin Complex 2
• Microtubule-Associated Proteins (drug effects, metabolism)
• Nucleus Pulposus (cytology, drug effects, metabolism)
• Phosphorylation
• Proto-Oncogene Proteins c-akt (drug effects, metabolism)
• RNA Interference
• Regulatory-Associated Protein of mTOR (antagonists & inhibitors, genetics)
• Ribosomal Protein S6 Kinases, 70-kDa
• Sequestosome-1 Protein (drug effects, metabolism)
• Sirolimus (pharmacology)