Methods: The human osteoblasts (NHOst)(Cambrex) and murine osteoblasts (MC3T3-E1) were cultured in the osteoblast growth medium supplemented with 10% fetal bovine serum in 5% CO2 at 37 °C. The growth factors were removed from the medium. During experiment, then the cells were cultured in the presence of H2S gas(0-100ng/ml). Cell proliferation was measured by [3H]thymidine incorporation in the 5% TCA insoluble fraction. The phosphorylation of Erk1/2 and p38 were determined with Western blot analysis.
Results: After treatment of the NHOst cells with H2S for 24 hours, [3H]thymidine incorporation into the DNA was decreased significantly and H2S dose-dependently. At a concentration of 100 ng/ml H2S, [3H]thymidine incorporation decreased by 79% compared to the controls. Similar reduction was observed in MC3T3-E1 cells. The effects of H2S on signal transduction pathways for controlling cell proliferation were also determined in NHOst cells. Phoshorylation of p38 was induced by H2S. The phosphorylation of Erk1/2 was dose-dependently increased by H2S. The phosphorylation was started within 10 min after starting H2S incubation. Then, the phosphorylation slightly decreased. Activation of ERK1/2 induced by H2S was inhibited with the ERK1/2 specific inhibitor U0126.
Conclusion: These results demonstrated that H2S inhibits cell proliferation of human osteoblasts through the MAPK cascade.