Microgravity control of autophagy modulates osteoclastogenesis

Abstract

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 (μXg) conditions and demonstrated increased osteoclastogenesis in mouse bone marrow cultures. Autophagy is a cellular recycling process of nutrients. Therefore, we hypothesize that μXg control of autophagy modulates osteoclastogenesis. Real-time PCR analysis of total RNA isolated from mouse bone marrow derived non-adherent cells subjected to modeled μXg 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 μXg subjected RAW 264.7 preosteoclast cells. RT2 profiler PCR array screening for autophagy related genes identified that μXg 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 μXg 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 μXg as compared to Xg cultures and 3-MA inhibited RANKL increased p-CREB expression in these cells. Also, 3-MA suppresses μXg elevated osteoclast differentiation in mouse bone marrow cultures. Thus, our results suggest that μXg 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.

Microgravity modulation of syncytin‐A expression enhance osteoclast formation

“…The vessel was filled with Dulbecco's Modified Eagle Medium (DMEM) free of bubbles and rotated at a constant speed of 16 rpm for 24 h to simulate microgravity (0.008 Xg), termed µXg. Ground based cultures were considered as control gravity (Xg) …”

Microgravity, Stem Cells, and Embryonic Development: Challenges and Opportunities for 3D Tissue Generation

“…When cultured in 1 g, cells previously exposed to microgravity display an increased mesenchymal and hematopoietic differentiation, suggesting that microgravity causes an accumulation of undifferentiated precursors (Blaber et al, 2014). A role for the autophagic control of osteoclastogenesis in response to microgravity has been proposed for mouse bone (Sambandam et al, 2014). A recent review by Ulbrich et al (2014) details the impact of simulated and real microgravity on bone and mesenchymal cells and their osteogenic potential.…”

Glucocorticoids: Dose-related effects on osteoclast formation and function via reactive oxygen species and autophagy

“…Autophagy is a dynamic catabolic process that delivers cellular components to the lysosome for degradation to retrieve molecules and regain energy to maintain cellular homeostasis [24,25]. Recently, reports have shown that the upregulation of autophagy contributes to osteoclastogenesis in response to hypoxic conditions, oxidative stress and microgravity in vitro [5,26,27]. However, the role of autophagy in osteoclast formation and function with glucocorticoid exposure remains unclear.…”