Sonic Hedgehog Signaling Regulates BMP9-Induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective:To investigate the effect of Sonic hedgehog (Shh) signaling on bone morphogenetic proteins 9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs) and the possible mechanism involved.Methods:MEFs and C3H10T1/2 cells were infected with Ad-BMP9/Ad-GFP, the expression of Hh signaling related genes was detected by RT-PCR and Western Blot at 1,3 and 5 days post-infection. C3H10T1/2, MEFs and C2C12 cells were infected with Ad-BMP9/Ad-GFP, and then treated with cyclopamine (Hh signaling inhibitor) or purmorphamine (Hh signaling agonist). The early osteogenic marker ALP activity was detected by quantitative chemiluminiscence assay. The later osteogenic marker calcium deposition was detected by Alizarin Red S staining.Next, the recombinant adenovirus Ad-Shh and Ad-siShh was constructed and validated by RT-PCR and Western Blot. Then, the effect of Shh signaling on BMP9-induced osteogenic differentiation was assessed systemically in vitro. The ALP activity was measured by quantitative chemiluminiscence and histochemical staining assay. The calcium deposition was detected by Alizarin Red S staining. The expression of osteopontin (OPN) and osteocalcin (OCN) was determined by RT-PCR and Western Blot. The expression of osteogenic pivotal genes Idl,162, Id3, CTGF, Coll, Dlx5 and Runx2 was detected by RT-PCR and Western Blot. The effect of Shh signaling on BMP9-induced ectopic bone formation was analyzed by Micro-CT, H&E staining, Masson’s Trichrome staining and Alcian Blue staining. The effect of Shh signaling on BMP9-induced Smadl/5/8, MAPK was assessed by luciferae reporter assay and Western Blot. The accumulation of Smadl/5/8 and phosphor-Smadl/5/8 in nuclear was detected by Western Blot.Results:It was found that BMP9 can exert influence on Hh signaling in MSCs, leading to alteration of expression of Hh signaling-related genes. BMP9-induced early and late osteogenic markers were inhibited by cyclopamine, whereas enhanced by purmorphamine. Then we constructed recombinant adenoviruses Ad-Shh expressing exogenous Shh and Ad-siShh expressing small interfering RNA targeting Shh. We found that BMP9-induced ALP activity, matrix mineralization, the expression of OPN, OCN and osteogenic pivotal transcription factors were promoted by Ad-Shh, whereas inhibited by Ad-siShh. Ectopic bone formation induced by BMP9 was increased by Ad-Shh. Furthermore, Ad-Shh increased BMP9-induced transcriptional activity of Smadl/5/8 and accumulation of phosphor-Smad1/5/8 in nuclear.Conclusion:Shh signaling may regulate BMP9-induced osteogenic differentiation of MSCs.