title: Melatonin Suppresses Autophagy Induced by Clinostat in Preosteoblast MC3T3-E1 Cells.
abstract: Microgravity exposure can cause cardiovascular and immune disorders, muscle atrophy, osteoporosis, and loss of blood and plasma volume. A clinostat device is an effective ground-based tool for simulating microgravity. This study investigated how melatonin suppresses autophagy caused by simulated microgravity in preosteoblast MC3T3-E1 cells. In preosteoblast MC3T3-E1 cells, clinostat rotation induced a significant time-dependent increase in the levels of the autophagosomal marker microtubule-associated protein light chain (LC3), suggesting that autophagy is induced by clinostat rotation in these cells. Melatonin treatment (100, 200 nM) significantly attenuated the clinostat-induced increases in LC3 II protein, and immunofluorescence staining revealed decreased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating a decrease in autophagosomes. The levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (Ser2448), phosphorylation of extracellular signal-regulated kinase (p-ERK), and phosphorylation of serine-threonine protein kinase (p-Akt) (Ser473) were significantly reduced by clinostat rotation. However, their expression levels were significantly recovered by melatonin treatment. Also, expression of the Bcl-2, truncated Bid, Cu/Zn- superoxide dismutase (SOD), and Mn-SOD proteins were significantly increased by melatonin treatment, whereas levels of Bax and catalase were decreased. The endoplasmic reticulum (ER) stress marker GRP78/BiP, IRE1α, and p-PERK proteins were significantly reduced by melatonin treatment. Treatment with the competitive melatonin receptor antagonist luzindole blocked melatonin-induced decreases in LC3 II levels. These results demonstrate that melatonin suppresses clinostat-induced autophagy through increasing the phosphorylation of the ERK/Akt/mTOR proteins. Consequently, melatonin appears to be a potential therapeutic agent for regulating microgravity-related bone loss or osteoporosis.
authors.affiliation: Yonsei-Fraunhofer Medical Device Laboratory, Department of Biomedical Engineering, Yonsei University, Wonju, 26493 Gangwon-do, Korea. yyeongm@hanmail.net.
authors.forename: Yeong-Min
authors.initials: YM
authors.lastname: Yoo
authors.affiliation: Fraunhofer Institute IKTS-MD, Maria-Reiche-Str.2, 01109 Dresden, Germany. tae-young.han@ikts-md.fraunhofer.de.
authors.forename: Tae-Young
authors.initials: TY
authors.lastname: Han
authors.affiliation: Yonsei-Fraunhofer Medical Device Laboratory, Department of Biomedical Engineering, Yonsei University, Wonju, 26493 Gangwon-do, Korea. hanskim@yonsei.ac.kr.
authors.forename: Han Sung
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008550:Melatonin / Q000494:pharmacology*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020011:Protective Agents / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D018474:Weightlessness Simulation / Q000009:adverse effects*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717248:Hspa5 protein, mouse; D020011:Protective Agents; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D008550:Melatonin
keywords: Akt; ERK; MC3T3-E1 cells; autophagy; clinostat; mTOR; melatonin