title: Activation of autophagy via Ca(2+)-dependent AMPK/mTOR pathway in rat notochordal cells is a cellular adaptation under hyperosmotic stress.
abstract: Nucleus pulposus (NP) cells experience hyperosmotic stress in spinal discs; however, how these cells can survive in the hostile microenvironment remains unclear. Autophagy has been suggested to maintain cellular homeostasis under different stresses by degrading the cytoplasmic proteins and organelles. Here, we explored whether autophagy is a cellular adaptation in rat notochordal cells under hyperosmotic stress. Hyperosmotic stress was found to activate autophagy in a dose- and time-dependent manner. SQSTM1/P62 expression was decreased as the autophagy level increased. Transient Ca(2+) influx from intracellular stores and extracellular space was stimulated by hyperosmotic stress. Activation of AMPK and inhibition of p70S6K were observed under hyperosmotic conditions. However, intercellular Ca(2+) chelation inhibited the increase of LC3-II and partly reversed the decrease of p70S6K. Hyperosmotic stress decreased cell viability and promoted apoptosis. Inhibition of autophagy led to SQSTM1/P62 accumulation, reduced cell viability, and accelerated apoptosis in notochordal cells under this condition. These evidences suggest that autophagy induction via the Ca(2+)-dependent AMPK/mTOR pathway might occur as an adaptation mechanism for notochordal cells under hyperosmotic stress. Thus, activating autophagy might be a promising approach to improve viability of notochordal cells in intervertebral discs.
authors.affiliation: a Department of Orthopedic Surgery; Zhongshan Hospital ; Fudan University ; Shanghai , China.
authors.forename: Li-Bo
authors.initials: LB
authors.lastname: Jiang
authors.forename: Lu
authors.initials: L
authors.lastname: Cao
authors.forename: Xiao-Fan
authors.initials: XF
authors.lastname: Yin
authors.forename: Miersalijiang
authors.initials: M
authors.lastname: Yasen
authors.forename: Mumingjiang
authors.initials: M
authors.lastname: Yishake
authors.forename: Jian
authors.initials: J
authors.lastname: Dong
authors.forename: Xi-Lei
authors.initials: XL
authors.lastname: Li
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000222:Adaptation, Physiological; D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D020868:Gene Silencing; D006360:Heat-Shock Proteins / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D008954:Models, Biological; D009672:Notochord / Q000166:cytology*; D009997:Osmotic Pressure; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D000071456:Sequestosome-1 Protein; D015398:Signal Transduction; D013194:Staining and Labeling; D013312:Stress, Physiological; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: C545865:Atg5 protein, rat; D000071187:Autophagy-Related Protein 5; D006360:Heat-Shock Proteins; D011506:Proteins; D034741:RNA, Small Interfering; D000071456:Sequestosome-1 Protein; C491079:Sqstm1 protein, rat; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D002118:Calcium
keywords: AMPK, AMP activated Protein Kinase; Adaptation; Apoptosis; Autophagy; BAPTA-AM, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetra(acetoxymethyl) ester; DAPI, 4′, 6-diamidino-2-phenylindole; ECM, extracellular matrix; EGTA, ethyleneglycol-bis (2-aminoethylether)-N,N,N′, N-tetraacetic acid; FITC, fluorescein isothiocyanate; Hyperosmotic stress; IDD, Intervertebral disc degeneration; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide; MAPK, mitogen-activated protein kinase; MDC, Monodansylcadaverine; MMP, mitochondrial membrane potential; MPT, mitochondrial permeability transition; NP, nucleus pulposus; Notochordal cells; OD, optical density; PI, propidium iodide; PLC, phospholipase C; SQSTM1, Sequestosome-1; TRPV4, Transient receptor potential vanilloid 4; fluo3-AM, fluo3 acetoximethylester; mTOR, mammalian target of rapamycin