Robo2 promotes osteoblast differentiation and mineralization through autophagy and is activated by parathyroid hormone induction

Background: As a systemic skeletal disorder, osteoporosis can increase fracture risk. This study wants to discuss the mechanism of osteoporosis and find possible molecular therapy. Bone morphogenetic protein 2 (BMP2) was utilized to stimulate MC3T3-E1 to establish a cellular osteoporosis model in vitro.

Methods: Initially, the viability of BMP2-induced MC3T3-E1 was assessed with a Cell counting kit-8 (CCK-8) assay. By real-time quantitative PCR (RT-qPCR) and western blot, Robo2 expression after roundabout (Robo) silencing or overexpression was estimated. Besides, alkaline phosphatase (ALP) expression, mineralization level and LC3II green fluorescent protein (GFP) expression were evaluated using ALP assay, Alizarin red staining and immunofluorescence staining, separately. Additionally, the expression of proteins related to osteoblast differentiation and autophagy was analyzed by RT-qPCR and western blot. Then, following autophagy inhibitor 3-methyladenine (3-MA) treatment, osteoblast differentiation and mineralization were measured again.

Results: MC3T3-E1 cells were differentiated into osteoblasts under BMP2 induction and Robo2 expression was greatly ascended. After Robo2 silencing, Robo2 expression was markedly diminished. ALP activity and mineralization level in BMP2-induced MC3T3-E1 cells were declined after depleting Robo2. Robo2 expression was conspicuously enhanced after overexpressing Robo2. Robo2 overexpression promoted the differentiation and mineralization of BMP2-induced MC3T3-E1 cells. Rescue experiments revealed that Robo2 silence and its overexpression could regulate the autophagy of BMP2-stimulated MC3T3-E1 cells. After 3-MA treatment, the increased ALP activity and mineralization level of BMP2-induced MC3T3-E1 cells with Robo2 upregulation were reduced. Furthermore, parathyroid hormone 1-34 (PTH1-34) treatment enhanced the expression of ALP, Robo2, LC3II and Beclin-1 and reduced the levels of LC3I and p62 of MC3T3-E1 cells concentration-dependently.

Conclusion: Collectively, Robo2, which was activated by PTH1-34, promoted osteoblast differentiation and mineralization through autophagy.