Microgravity control of autophagy modulates osteoclastogenesis

Evidence indicates that astronauts experience significant bone loss during space mission. Recently, we used the NASA developed rotary cell culture system (RCCS) to simulate microgravity (mu Xg) conditions and demonstrated increased osteoclastogenesis in mouse bone marrow cultures. Autophagy is a cellular recycling process of nutrients. Therefore, we hypothesize that mu Xg control of autophagy modulates osteoclastogenesis. Real-time PCR analysis of total RNA isolated from mouse bone marrow derived non-adherent cells subjected to modeled mu Xg showed a significant increase in autophagic marker Atg5, LC3 and Atg16L mRNA expression compared to ground based control (Xg) cultures. Western blot analysis of total cell lysates identified an 8.0-fold and 7.0-fold increase in the Atg5 and LC3-II expression, respectively. Confocal microscopy demonstrated an increased autophagosome formation in mu Xg subjected RAW 264.7 preosteoclast cells. RT2 profiler PCR array screening for autophagy related genes identified that mu Xg upregulates intracellular signaling molecules associated with autophagy, autophagosome components and inflammatory cytokines/growth factors which coregulate autophagy in RAW 264.7 preosteoclast cells. Autophagy inhibitor, 3-methyladenine (3-MA) treatment of mouse bone marrow derived non-adherent mononuclear cells showed a significant decrease in mu Xg induced Atg5 and LC3 mRNA expression in the presence or absence of RANK ligand (RANKL) stimulation. Furthermore, RANKL treatment significantly increased (8-fold) p-CREB transcription factor levels under mu Xg as compared to Xg cultures and 3-MA inhibited RANKL increased p-CREB expression in these cells. Also, 3-MA suppresses mu Xg elevated osteoclast differentiation in mouse bone marrow cultures. Thus, our results suggest that mu Xg induced autophagy plays an important role in enhanced osteoclast differentiation and could be a potential therapeutic target to prevent bone loss in astronauts during space flight missions. (C) 2014 Elsevier Inc. All rights reserved.