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

• Published in: Osteoarthritis and cartilage 2017-12, Vol.25 (12), p.2134-2146
• Main Authors: Ito, M., Yurube, T., Kakutani, K., Maeno, K., Takada, T., Terashima, Y., Kakiuchi, Y., Takeoka, Y., Miyazaki, S., Kuroda, R., Nishida, K.
• Format: Article
• Language: English
• Subjects: Intervertebral disc; Mammalian target of rapamycin (mTOR); Nucleus pulposus cells; Regulatory-associated protein of mTOR (RAPTOR); RNA interference (RNAi); Spine
• ISSN: 1063-4584; 1522-9653
• DOI: 10.1016/j.joca.2017.08.019

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. 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. Western blotting showed significant decreases in specific proteins by each RNAi (all P