HIF1a Enhances BMP9-induced Osteogenic Differentiation And Bone Formation In Vivo And In Vitro

Background: Mesenchymal stem cells (MSCs) can self-renew anddifferentiate into osteogenic, adipogenic, myogenic and chondrogeniclineages. Efficient osteogenic differentiation and bone formation frommesenchymal stem cells (MSCs) should have clinical applications intreating nonunion fracture healing. We have identified bone morphogeneticprotein9(BMP-9) as one of the most osteogenic BMPs.Study design: Here we study the effect of HIF1a on BMP9-inducedformation of bone tissue. First step, we reconstruct and identifyHIF1a-expressing adenovirus Ad-HIF1a. Then, we do several levels ofexperiment to confirm that HIF1a can potentiate BMP9-induced osteogenicdifferentiation of MSCs in vivo and in vitro. We also probe the relationshipof BMP9and HIF1a in cross-talking mechanisms.Results: Endogenous expression of HIF1a is low in MSCs. ExogenousHIF1a expression can be mediated by adenovirus-expressing human HIF1a.Exogenous expression of HIF1a can enhance BMP9-induced osteogenicmarkers’ expression, including early marker alkaline phosphatase (ALP)activity and later markers. RNA interference of HIF1a (simHIF1a) leads to inhibition of BMP9-induced osteogenic markers expression including earlymarker alkaline phosphatase (ALP) activity and later matrix mineralization.CAY10585, an antagonist of HIF1a, antagonizes the effect of HIF1a onBMP-9-induced osteogenic differentiation in vitro. RNA interference ofHIF1a (simHIF1a) can suppress BMP-9-induced early osteogenic markeralkaline phosphatase (ALP) activity and matrix mineralization inCoCl2-mimic hypoxia environment. HIF1a has been shown to reinforceBMP9-induced ectopic bone formation in implantation of MSCs.Mechanistically, ChIP confirms that BMP-9stimulation induces therecruitment of Smad1/5/8complex to HIF1a promoter. BMP9can stimulatemRNA expression and protein product of HIF1a. HIF1a is shown toeffectively intensify activity of BMP9-induced BMPR-Smad reporter. Theresults demonstrate that BMP-9crosstalks with HIF1a through HIF1apromoter and Receptor-Smad activity during osteogenic differentiation ofMSCs.Conclusion: Taken together, our findings suggest that a combination ofBMP-9and HIF-1a may be explored as an effective bone-regenerationagent to treat large segmental bony defects, nonunion fracture, and/orosteoporotic fracture.