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Expression of pro-osteoclastogenic genes and oxidative stress genes increased in irradiated MLO-Y4 cells

Role of Oxidative Stress and Autophagy Signaling in Osteocyte Response to Spaceflight-like Radiation

Victoria E. Rael1,2, Ruth K. Globus4, and Candice Tahimic3,4

1 Space Life Sciences Training Program (SLSTP), NASA Ames Research Center, 2 Biological Sciences Collegiate Division, University of Chicago, Chicago, IL, 3 Wyle Laboratories, 4 Space Biosciences Division, NASA Ames Research Center

ABSTRACTSpaceflight leads to bone loss, which is attributed to increased activity of bone-resorbing osteoclasts and changes in osteoblasts, the cells that give rise to mature osteocytes. Our current understanding of the mechanisms controlling bone loss is incomplete. However, it is known that oxidative stress is elevated during radiation exposure, a risk associated with spaceflight. Here, we examine oxidative stress responses of osteocytes following exposure to spaceflight-like radiation. We hypothesize that (1) oxidative stress, as induced by radiation, decreases osteocyte survival and increases pro-osteoclastogenic signals and that (2) autophagy is one of the key cellular defenses against oxidative stress. Autophagy is the process in which cellular components including organelles and proteins are broken down and recycled. To test our hypothesis, we exposed the osteocyte-like cell line, MLO-Y4, to 0.5, 1, and 2 Gy of 56Fe radiation (600 MeV/n) and observed reduced cell numbers in the cells that received 1 and 2 Gy doses. Analysis of cell cycle by flow cytometry showed that all radiation doses caused a shift in the cell cycle distribution consistent with growth arrest. We are currently examining expression of other markers of oxidative stress and autophagy as well as pro-osteoclastogenic and apoptotic genes.

BACKGROUND

• Spaceflight bone loss• Bone loss is due to the increased

activity of bone-resorbing osteoclast cells and changes in bone-forming osteoblast cells

• Osteocytes - mature bone cells

• Autophagy: process in which organelles are broken down and recycled [2]

• Could be key defense mechanism against damages from spaceflight, including oxidative stress [1]

• Autophagic degradation increases under stressful conditions [2]

Image: http://www.nature.com/nrd/journal/v11/n3/fig_tab/nrd3669_F1.html

Image: http://www.nexcelom.com/Nexcelom-Blog/autophagy/

EXPERIMENTAL AIMS(1)Determine the role of autophagy in osteocyte function

(2)Determine the relationship between autophagy and oxidative stress in osteocytes

Understanding mechanisms underlying spaceflight-induced bone loss may facilitate development of countermeasures

RESULTS: Cell Growth

RESULTS: Gene Expression

ACKNOWLEDGEMENTS

REFERENCES

This project was supported by the Space Life Sciences Training Program (SLSTP) at NASA Ames Research Center and by Wyle Laboratories. Thank

you to my mentors for their constant support and guidance!

1. Yumoto et al., 2010, “Short-Term Effects of Whole-Body Exposure to 56Fe Ions in Combination with Musculoskeletal Disuse on Bone Cells.

2. Hocking et al., 2012, “Autophagy: A New Player in Skeletal Maintenance?“.

RESULTS: Markers of Autophagy

METHODS

Effects of 56Fe Radiation on percent of apoptotic MLO-Y4 cell populations at 48H post-IR

Conclusion: Radiation causes an increase in percent of apoptotic cells

*p<0.05

***

Effects of 56Fe Radiation on cell cycle progression in MLO-Y4 cells 48H post-IR

Conclusion: Radiation causes a shift in cell cycle progression

*

*

(1)Exposed osteocyte-like cell line, MLO-Y4, to Sham, 0.5, 1, and 2 Gy of 56Fe radiation and sampled at 4, 24, and 48 hour post-IR

(2)Performed cell count, FACS, gene expression, and western blot analyses

0 50 100 0 50 100 0 50 100 0 50 100Red Fluorescence Units

Num

ber o

f Eve

nts

Flow Cytometry to observe effects of radiation on cell cycle progression

Survival of MLO-Y4 cells exposed to 0, 0.5, 1.0, and 2.0 Gy 56Fe radiation, 24 hour post-IR2.0 Gy produced 50% cell death

CONCLUSION

* * *

*p<0.05

** Used Western Blotting to examine effect of radiation on

autophagic signaling in MLO-Y4 cellsLC3B cleaved from LC3-I to LC3-IIduring autophagy

Found decrease in LC3B-II, suggesting decrease in autophagy

Radiation appeared to decrease survival and cause cell cycle arrest in osteocyte-like cells. Pro-osteoclast signals and markers of oxidative stress were increased in irradiated cells, alongside decreased levels of autophagy. This suggests a correlation between oxidative stress and autophagy, with decreased autophagic degradation as a potential mechanism contributing to bone loss.

Sham 0.5 Gy 1 Gy 2 Gy