Hmox1 Regulates Osteogenic And Adipogenic Differentiation Of C3H10T1/2 Induced By BMP9

Objective: To investigate the effect of Heme oxygenase 1(Hmox1)on the bi-directional differentiation of C3H10T1/2 into osteoblasts and adipocytes induced by Bone Morphogenetic Protein 9(BMP9).Methods: C3H10T1/2 were infected with Ad-BMP9,then the gene expression of Hmox1 was detected by fluorescence quantitative PCR(q PCR).The expression of HMOX1 protein was detected by Western Blot.The effect of Hmox1 agonist COPP and inhibitor ZNPP onosteogenic and adipogenic differentiation of C3H10T1/2 induced by BMP9 were measured by alkaline phosphatase(ALP)staining as well as quantitative analysis,Alizarin red S staining and Oil red O staining.The recombinant adenovirus expressing Hmox1 was constructed and the effectiveness of Ad-Hmox1 was verified by q PCR and Western Blot.C3H10T1/2 were infected with Ad-Hmox1 or Ad-RFP,and treated with BMP9 conditioned medium,osteogenic markers(ALP and calcium deposition,Runx2,Dlx5,Id,OCN,OPN)and adipogenic markers(lipid droplets,C/EBP?,C/EBP?,PPAR ?2)were measured at the scheduled time.The effect of Hmox1 on Smad1/5/8,MAPKs,PI3K/AKT and Wnt/?-catenin signaling were examined by Western Blot.The nuclear translocation of ?-catenin was measured by immunofluorescence method.Results: BMP9 promoted the expression of Hmox1.COPP enhanced the ALP and calcium deposition,whereas decreased the formation of lipid droplets induced by BMP9 in C3H10T1/2.ZNPP did the opposite.Hmox1 strengthened the osteogenic markers(ALP,calcium deposition,Runx2,Dlx5,Id2,OPN,OCN)and weakened the adipogenic markers(lipid droplets,C/EBP?).While had no effect on BMP9-induced phosphorylation of Smad1/5/8,Hmox1 enhanced phospho-p38 and phospho-AKT(Ser473),increased total ?-catenin protein level,and promoted the nuclear translocation of ?-catenin.Conclusion: Our results show that Hmox1 may play an important role in regulating bi-directional differentiation induced by BMP9 in C3H10T1/2.Hmox1 is likely to potentiate the osteogenic differentiation and yet decrease the adipogenic differentiation induced by BMP9 through regulation of Wnt signaling,MAPKs and PI3K/AKT.