RhoA inhibits the hypoxia-induced apoptosis in osteoblasts

Bone regeneration and fracture healing is dependent on local and systemic factors, including oxygen tension. Disrupted blood supply by fracture always results in reduced oxygen tension (pO2) and hypoxia in bone tissues and inhibits fracture healing. RhoA small GTPase triggers signaling cascades in a variety of cellular responses, including apoptosis suppression via regulating anti-apoptotic Bcl-2 expression. In the present study, we examined the apoptosis promotion by hypoxia in mouse osteoblastic MC3T3-E1 cells by flow-cytometry, caspase 3 activity assay and western blot analysis of apoptosis-associated markers. And then we investigated the regulatory role of RhoA in the hypoxia-promoted apoptosis in MC3T3-E1 cells with gain-of-function and loss-of-function strategies to manipulate the RhoA expression in MC3T3-E1 cells. Results demonstrated that apoptosis was significantly promoted by hypoxia in the osteoblast MC3T3-E1 cells. There were higher levels of apoptotic cells, caspase 3 activity and apoptosis-associated markers such as released cytochrome c, cleaved caspase 3 and lyzed PARP in the hypoxiatreated MC3T3-E1 cells. And such apoptosis was markedly inhibited by the RhoA overexpression with lentivirus vector, whereas was aggravated by the RhoA knockout via the transfection with RhoA-specific siRNA. In conclusion, the present study confirmed the protective role of RhoA in the hypoxia-induced apoptosis in osteoblast cells, implying the RhoA promotion might be a valuable strategy for the therapeutic intervention to the hypoxic-ischemic damage to osteoblasts.