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Simulated Microgravity Using the Random Positioning Machine Inhibits Differentiation and Alters Gene Expression Profiles of 2T3 Pre-osteoblasts.   M.J. Patel1, S.J. Pardo1, M.O. Platt1, M.C. Sykes1, N.L. Boyd1, G.P. Sorescu1, M.Xu3, J.W.A. van Loon4, M.D. Wang1, and H. Jo1,2. 1)Department of Biomedical Engineering, Georgia Tech and Emory Univ, 2) Cardiology, Emory Univ School of Medicine, 3)Amersham Biosciences, 4)Dutch Experiment Support Center

   Exposure to microgravity causes bone loss in humans, and the underlying mechanism is believed to be at least partially due to a decrease in bone formation by osteoblasts.  Here, we examined the hypothesis that microgravity alters osteoblast gene expression profiles, resulting in bone loss.  For this study, we developed an in vitro system that simulates microgravity using the Random Positioning Machine (RPM) to study the effects of microgravity on 2T3 pre-osteoblast cells grown in gas-permeable culture disks.  Exposure of 2T3 cells to simulated microgravity using the RPM for up to 9 days significantly inhibited alkaline phosphatase activity, recapitulating an expected bone loss response, without altering cell proliferation and morphology.  Next, we carried out a DNA microarray analysis to determine the gene expression profile of 2T3 cells exposed to 3 days of simulated microgravity.  Among 10,000 genes examined with the microarray, 88 were downregulated while 52 were upregulated significantly by simulated microgravity by more than two-fold compared to static 1g.  By using real-time PCR and Western blot, we verified the microarray data for some of the expected genes.  We confirmed that microgravity induced downregulation of alkaline phosphatase, runt related transcription factor 2, osteomodulin, and parathyroid hormone 1 receptor, while confirming upregulation of cathepsin K mRNAs.  We also confirmed that bone morphogenic protein 4 and cystatin C, genes that was not altered by microgravity, did not change in protein levels.  In addition to the changes of the expected genes, the microarray data identified many more gravisensitive genes, which may provide useful insight regarding their roles not only in microgravity-induced bone loss but also in similar pathologies such as osteoporosis afflicting the general population.