Method: Cultured MC3T3-E1 cells were treated with ascorbic acid (AA) and globular adiponectin. The adiponectin signaling pathway in osteoblast differentiation was investigated by qPCR, western blots and co-immunoprecipitation. To study adiponectin mediated BMP/Smad signaling pathway, stromal cell-derived factor-1 (SDF1) mRNA expression and Smad1/5/8 phosphorylation were analyzed. The specificity of adiponectin signaling pathway was also studied by using APPL1 siRNA and BMP signaling inhibitor LDN-193189.
Result: Following AA induction, Smad1/5/8 phosphorylation was up-regulated and peak at 30min after adiponectin treatment. SDF1 mRNA expression was significantly increased, indicating that SDF1 might be the target gene of adiponectin. Pretreatment with LDN-193189, but not suppression of APPL1 with its siRNA, abolished the adiponectin-mediated pSmad1/5/8 increases. Interestingly, we found that adiponectin receptor 1 (AdipoR1) directly bound Smad1/5/8 and casein kinase CK2. The bindings of AdipoR1 with either Smad1/5/8 or CK2 were decreased while Smad1/5/8 was phosphorylated and translocated after adiponectin stimulation.
Conclusion: We concluded that globular adiponectin increases Smad1/5/8 phosphorylation during osteoblast differentiation. Adiponectin also upregulates SDF1 expression, suggesting an additional effect of adiponectin in promoting BMSC migration. We found for the first time that Smad1/5/8 is a novel intracellular mediator of AdipoR1. Smad/CK2 are involved in the crosstalk between adiponectin and BMP signaling pathways whereas, surprisingly, Appl1, the conventional adiponectin pathway adapter, is not involved. These results provide new insight into molecular mechanism of adiponectin in promoting osteogenesis. Adiponectin can serve as a therapeutic target for bone formation and regeneration.