1. National Aeronautics and Space Administration
www.nasa.gov
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
ABSTRACT
Spaceflight 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 56
Fe 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/nr
d3669_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
ACKNOWLEDGEMENT
S
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 56
Fe Radiation on percent of
apoptotic MLO-Y4 cell populations at
48H post-IR
Conclusion: Radiation causes an
increase in percent of apoptotic cells
0
10
20
30
40
50
60
70
80
90
100
110
0.0 0.5 1.0 1.5 2.0
PercentCells
Radia on Dose, Gy
*p<0.05
*
*
*
Effects of 56
Fe Radiation on cell cycle
progression in MLO-Y4 cells 48H post-IR
Conclusion: Radiation causes a shift in
cell cycle progression
0
10
20
30
40
50
60
70
%Cells in G1 %Cells in S %Cells in G2
PercentCells
Phase of Cell Cycle
Sham
0.5 Gy
1 Gy
2 Gy
*
*
(1)Exposed osteocyte-like cell line, MLO-Y4, to Sham, 0.5, 1, and 2 Gy
of 56
Fe 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 50 100
Red Fluorescence Units
NumberofEvents
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 56
Fe radiation, 24
hour post-IR
2.0 Gy produced 50% cell death
CONCLUSION
* * *
*p<0.05
*
* Used Western Blotting to examine effect of radiation on
autophagic signaling in MLO-Y4 cells
LC3B cleaved from LC3-I to LC3-II
during 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