Fig 1.
Endogenous PTHrP protects trabecular osteoblasts from Space microgravity-induced apoptosis. (6-day experiments).
(A) Automated flight-ready tray for conducting cell culture experiments aboard Foton M3 satellite. Osteoblasts were attached onto CC2 culture slides inside three bioreactors (lower right) in a sealed pathway. (B) Proportion of apoptotic cells in trabecular osteoblasts in bioreactors after 6 days in Space microgravity (0g) or in normal Earth gravity (1g). ** p < 0.01. White bars: untreated; black bars: 2-h daily treatment with PTHrP1-36 10-8M. (C) Proportion of apoptotic cells in calvarial osteoblasts in bioreactors after 6 days in Space microgravity (0g) or in normal Earth gravity (1g). No PTHrP1-36 treatment was conducted on calvarial cells. Scale bar A: 15 cm.
Fig 2.
Allelic effect of endogenous PTHrP levels in simulated microgravity, and compensation by exogenous PTHrP 1–36 (6-day experiments).
(A) Microgravity simulation apparatus (RCCS) with one rotating culture unit (HARV). (B) MicroHex carriers with attached trabecular osteoblasts in culture. (C) Cell-induced MicroHex aggregate in culture (occurs for both trabecular and calvarial osteoblasts). (D) Trabecular osteoblast viability after 6 days on MicroHex carriers in normal gravity (1g) or in simulated microgravity (0g) (triplicate 6-day experiments). White bars: untreated; black bars: 2-h daily treatment with PTHrP1-36 10-8M. ** p < 0.01, *** p < 0.001. Scale bars: B, C: 200 μm.
Fig 3.
Long-term exposure to simulated microgravity is detrimental to wild-type trabecular osteoblasts but compensated for by exogenous intermittent PTHrP1-36 treatment.
Pthrp+/+ trabecular osteoblasts at 1g (■) or 0g (▼). Pthrp+/+ trabecular osteoblasts treated with PTHrP1-36 10-8M.at 1g (▲) or 0g (♦). (triplicate 6-week experiments). Viability estimated by Trypan blue. ** p < 0.01, *** p < 0.001.
Fig 4.
Long-term exposure to simulated microgravity has little negative effect on calvarial osteoblasts.
Phtrp+/+ calvarial osteoblasts at 1g (▼) or 0g (■). Pthrp-/- calvarial osteoblasts at 1g (●) or 0g (▲). (triplicate 6-week experiments). No exogenous PTHrP1-36 treatment was applied in this experiment. Viability estimated by Trypan blue. * p < 0.05, ** p < 0.01.
Fig 5.
Venn diagrams illustrating the number of trabecular osteoblast genes significantly up- or downregulated by microgravity exposure (6 days) or by Pthrp ablation, and reversed by PTHrP1-36 treatment.
(A) 52 genes are upregulated by both 0g (yellow) and Pthrp ablation (blue). Fold change > 2.0, p < 0.05. (B) 115 genes are downregulated by both 0g (yellow) and Pthrp ablation (blue). Fold change < 0.5, p < 0.05. (C) 24 genes are upregulated by both 0g (yellow) and Pthrp ablation (blue), and are also downregulated (central intersect) by PTHrP1-36 treatment (red). Fold change < 0.65, p < 0.05. (D) 50 genes are downregulated by both 0g (yellow) and Pthrp ablation (blue), and upregulated (central intersect) by PTHrP1-36 treatment (red). Fold change >1.5, p < 0.05.
Table 1.
Genes (52) upregulated by both microgravity and Pthrp ablation.
Table 2.
Genes (115) downregulated by both microgravity and Pthrp ablation.
Table 3.
Genes (24) upregulated by both Pthrp ablation and microgravity and downregulated by PTHrP1-36 treatment.
Table 4.
Genes (50) downregulated by both Pthrp ablation and microgravity and upregulated by PTHrP1-36 treatment.