Effects Of DLX1on BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Understanding the effects of homeodomain transcriptional factor DLX1on BMP9-induced osteogenic differentiation of mesenchymal stem cellsremains a significant issue to use BMP9in bone tissue engineering. BMP9was applied to induce osteogenic differentiation in mesenchymal stem cellC3H10T1/2, mouse embryonic fibroblast iMEFs and mouse myoblastC2C12. It was found by PCR and Western Blot assay that BMP9caninduce the expression of DLX1at RNA and protein level. C3H10T1/2,iMEFs and C2C12was infected with recombinant adenovirus expressingDLX1, and then stimulated with conditioned medium of BMP9.Chemoluminescence quantitative and staining assay were applied tomeasure activity of ALP. The results show that BMP9can induce activityof ALP of these three cells lines. Moreover, DLX1enhancedBMP9-induced activity of ALP of C3H10T1/2. It’s worth noting that iniMEFs, DLX1did not influence BMP9-induced activity of ALP, whereasinhibited the activity of ALP induced by BMP9in C2C12. C3H10T1/2wastreated with the same methods, and then Western Blot and PCR assay were respectively applied to measure the expression of osteogenic markersosteopontin (OPN) and osteocalcin (OCN). The results show that BMP9can induce expression of OPN and OCN, and which can be notablyenhanced by DLX1. C3H10T1/2and iMEFs were treated with DLX1andBMP9, alizarin red staining was applied to measure the later maker ofosteogenic differentiation, calcium salt deposition. The results exhibitedthat BMP9can induce calcium salt deposition of the two cell lines, andDLX1promotes this action. To further quest the effects of DLX1onBMP9-induced osteogenic differentiation, the same method was used againto treat C3H10T1/2, and PCR assay was used to analyze the expression oftarget genes of osteogenic differentiation, such as inhibitor ofdeifferentiation1(Id1), Id2, Id3, Osterix, osteoprotegerin (OPG), DLX5,connective tissue growth factor (CTGF) and Runt-related transcriptionfactor2(Runx2). It was found that DLX1can enhance the expression ofthe target genes, which are induced by BMP9. C3H10T1/2was infectedwith DLX1and conditioned medium of BMP9, luciferase reportor genesactivity assay and Western Blot were applied to reflect the relationshipbetween DLX1and Smad pathway. The results exhibited that DLX1andBMP9can both induce the luciferase activity of Smad1/5/8, and that DLX1can enhance the action of BMP9. It was found by Western Blot assay thatBMP9can induce phosphorylation of Smad1/5/8, yet DLX1did notinfluence that. Both BMP9and DLX1did not affect the protein level of total Smad1/5/8. To assay effects of DLX1on BMP9-induced osteogenicdifferentiation generally, four siRNA plasmids of DLX1were constructedand packaged to adenovirus. C3H10T1/2was infected with the adenovirusat different efficiency of infection, and stimulated with BMP9. Results ofALP activity assay show that two of the adenovirus had different effects atdifferent efficiency of infection on BMP9-induced ALP activity ofmesenchymal stem cells: they can enhance ALP activity at low efficiencyof infection, while inhibit the activity with high efficiency. However, othertwo kinds of adenovirus always inhibit ALP activity induced by BMP9.Therefore, we concluded that DLX1can promote BMP9-inducedosteogenic differentiation of mesenchymal stem cells, and this functionwould be influenced by siRNA of DLX1. Luciferase reportor genes activityassay of Smad1/5/8and Western Blot of total Smad1/5/8and pSmad1/5/8exhibited that DLX1effects BMP9-induced osteogenic differentiation ofmesenchymal stem cells maybe by enhancing transcriptional activity ofSmad1/5/8.