Exposure to microgravity is supposed to affect almost all biological systems, and we speculated that microgravity is potentially involved in autophagy regulation. A clinostat was used to simulate microgravity, and HEK293 cells that stably express GFP-LC3 were used for sensitive monitoring of autophagy induction. The clinorotation of GFP-LC3 cells resulted in autophagosome formation in the cytoplasm and a change in autophagosomal marker expression. Autophagy induction was accompanied by phosphorylation of AMPK (Thr 172) and by the dephosphorylation of mammalian target of rapamycin. To elucidate the role of AMPK in microgravity-induced autophagy, we suppressed AMPK expression by knockdown via siRNA, which inhibited the induction of autophagy upon exposure to microgravity. In addition, the clinorotation of C2C12 myotube cells resulted in the enlarged and distinctive LC3 spots in the cytoplasm and AMPK activation. These results indicate that simulated microgravity possibly contributes to autophagy induction by regulating AMPK.
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References
1.
AdamsJ., ChenZ.P., Van DenderenB.J., MortonC.J., ParkerM.W., WittersL.A., StapletonD., and KempB.E. (2004). Intrasteric control of AMPK via the gamma1 subunit AMP allosteric regulatory site. Protein Sci, 13, 155–165.
2.
AraseY., NomuraJ., SugayaS., SugitaK., KitaK., and SuzukiN. (2002). Effects of 3-D clino-rotation on gene expression in human fibroblast cells. Cell Biol Int, 26, 225–233.
3.
BodineS.C., LatresE., BaumhueterS., LaiV.K., NunezL., ClarkeB.A., PoueymirouW.T., PanaroF.J., NaE., DharmarajanK., et al. (2001). Identification of ubiquitin ligases required for skeletal muscle atrophy. Science, 294, 1704–1708.
4.
CortonJ.M., GillespieJ.G., and HardieD.G. (1994). Role of the AMP-activated protein kinase in the cellular stress response. Curr Biol, 4, 315–324.
5.
GalluzziL., AaronsonS.A., AbramsJ., AlnemriE.S., AndrewsD.W., BaehreckeE.H., BazanN.G., BlagosklonnyM.V., BlomgrenK., BornerC., et al. (2009). Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ, 16, 1093–1107.
6.
GrimmD., BauerJ., KossmehlP., ShakibaeiM., SchobergerJ., PickenhahnH., Schulze-TanzilG., VetterR., EillesC., PaulM., et al. (2002). Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells. FASEB J, 16, 604–606.
7.
HemdanD.I., HirasakaK., NakaoR., KohnoS., KagawaS., AbeT., Harada-SukenoA., OkumuraY., NakayaY., TeraoJ., et al. (2009). Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes. J Med Invest, 56, 26–32.
8.
HirasakaK., NikawaT., YugeL., IshiharaI., HigashibataA., IshiokaN., OkuboA., MiyashitaT., SuzueN., OgawaT., et al. (2005). Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-kappa B signaling. Biochim Biophys Acta, 1743, 130–140.
9.
IkemotoM., NikawaT., TakedaS., WatanabeC., KitanoT., BaldwinK.M., IzumiR., NonakaI., TowatariT., TeshimaS., et al. (2001). Space shuttle flight (STS-90) enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway. FASEB J, 15, 1279–1281.
10.
KabeyaY., MizushimaN., UenoT., YamamotoA., KirisakoT., NodaT., KominamiE., OhsumiY., and YoshimoriT. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J, 19, 5720–5728.
11.
KangC.Y., ZouL., YuanM., WangY., LiT.Z., ZhangY., WangJ.F., LiY., DengX.W., and LiuC.T. (2011). Impact of simulated microgravity on microvascular endothelial cell apoptosis. Eur J Appl Physiol, 111, 2131–2138.
12.
KimJ., KunduM., ViolletB., and GuanK.L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol, 13, 132–141.
13.
KlionskyD.J., AbeliovichH., AgostinisP., AgrawalD.K., AlievG., AskewD.S., BabaM., BaehreckeE.H., BahrB.A., BallabioA., et al. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 4, 151–175.
14.
KroemerG., and JaattelaM. (2005). Lysosomes and autophagy in cell death control. Nat Rev Cancer, 5, 886–897.
15.
KudoN., BarrA.J., BarrR.L., DesaiS., and LopaschukG.D. (1995). High rates of fatty acid oxidation during reperfusion of ischemic hearts are associated with a decrease in malonyl-CoA levels due to an increase in 5′-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J Biol Chem, 270, 17513–17520.
16.
LevineB., and KlionskyD.J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell, 6, 463–477.
17.
MihaylovaM.M., and ShawR.J. (2011). The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol, 13, 1016–1023.
18.
MirandaN., TovarA.R., PalaciosB., and TorresN. (2007). [AMPK as a cellular energy sensor and its function in the organism]. Rev Invest Clin, 59, 458–469.
NingJ., XiG., and ClemmonsD.R. (2011). Suppression of AMPK activation via S485 phosphorylation by IGF-I during hyperglycemia is mediated by AKT activation in vascular smooth muscle cells. Endocrinology, 152, 3143–3154.
21.
PankivS., ClausenT.H., LamarkT., BrechA., BruunJ.A., OutzenH., OvervatnA., BjorkoyG., and JohansenT. (2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem, 282, 24131–24145.
22.
RileyD.A., EllisS., SlocumG.R., SatyanarayanaT., BainJ.L., and SedlakF.R. (1987). Hypogravity-induced atrophy of rat soleus and extensor digitorum longus muscles. Muscle Nerve, 10, 560–568.
23.
RuestL.B., MarcotteR., and WangE. (2002). Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis. J Biol Chem, 277, 5418–5425.
24.
SanchezA.M., CandauR.B., CsibiA., PaganoA.F., RaibonA., and BernardiH. (2012). The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis. Am J Physiol Cell Physiol, 303, C475–C485.
25.
SanchezA.M., CsibiA., RaibonA., CornilleK., GayS., BernardiH., and CandauR. (2011). AMPK promotes skeletal muscle autophagy through activation of Forkhead FoxO3a and interaction with Ulk1. J Cell Biochem, 113,.695–710.
26.
SandriM., SandriC., GilbertA., SkurkC., CalabriaE., PicardA., WalshK., SchiaffinoS., LeckerS.H., and GoldbergA.L. (2004). Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell, 117, 399–412.
27.
SarkarD., NagayaT., KogaK., KambeF., NomuraY., and SeoH. (2000). Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells. J Gravit Physiol, 7, P71–P72.
28.
SolomonV., and GoldbergA.L. (1996). Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts. J Biol Chem, 271, 26690–26697.
29.
TammaR., ColaianniG., CamerinoC., Di BenedettoA., GrecoG., StrippoliM., VergariR., GranoA., ManciniL., MoriG., et al. (2009). Microgravity during spaceflight directly affects in vitro osteoclastogenesis and bone resorption. FASEB J, 23, 2549–2554.
30.
WoodsA., VertommenD., NeumannD., TurkR., BaylissJ., SchlattnerU., WallimannT., CarlingD., and RiderM.H. (2003). Identification of phosphorylation sites in AMP-activated protein kinase (AMPK) for upstream AMPK kinases and study of their roles by site-directed mutagenesis. J Biol Chem, 278, 28434–28442.
31.
YoonJ.H., HerS., KimM., JangI.S., and ParkJ. (2011). The expression of damage-regulated autophagy modulator 2 (DRAM2) contributes to autophagy induction. Mol Biol Rep, 39, 1087–1093.
32.
ZhaoJ., BraultJ.J., SchildA., CaoP., SandriM., SchiaffinoS., LeckerS.H., and GoldbergA.L. (2007). FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab, 6, 472–483.
