The Mechanism Of DLL1 In BMP9-Induced Osteogenic Differentiation Of MSCS

Bone marrow mesenchymal stem cells(MSCs), belonging to multipotent stem cells, possess the ability to differentiate into diverse tissues and potent capacity of self-renewing. It has been extentisvely explored over years largely in that it’s convenient to obtain samples, which are easily separated and cultured with proper conditions, therefore, based on these palpable advantages, nowadays MSCs have become preferable seed cells for treatment of bone-related diseases as well as applied in tissue engineering. Bone morphogenetic protein(BMP) family can induce MSCs into osteoblasts, among which BMP9 has been found to be the stronggest mediator that can induce osteogenic differentiation so far.Notch signaling, as a conservative pathway, has great implications in vertebrates and non-vertebrates, and it plays a pivot role in regulating embryogenesis and postnatal development. In the meanwhile,Notch pathway has great influences on enormous biological and physiological processes such as cell proliferation, differentiation, apoptosis, adhesion and epidermal cells to mesenchymal transformation.There are studies reporting that Notch signaling is involved in regulating osteogenesis of MSCs. And in our preivous study, we demonstrated that Notch pathway could synergy BMP-induced osteogenic differentiation in MSCs, however, the underlying mechanism is not yet elucidated.DLL 1 is one of ligands in Notch pathway, it has been recently identified that DLL1 can promote tumor cell proliferation as well as depress cell differentiation in gliomas; In melanoma, DLL1 can regulate embryonic arteriovenous vascular differentiation and induce angiogenesis of adult injured vessels. But there are no studies illustrating the role of DLL1 in osteogenic differentiation process and its related mechanisms.The main objectives in part one of this study lie in exploring the role of DLL 1 in BMP9-induced osteogenesis of MSCs; it was observed that DLL1 significantly promoted BMP-9 induced osteogenic differentiation, BMP9 receptor ALK2 was highly increased by DLL1 while other receptors remained unchanged; In addition, ALK2 was adversely regulated when Notch pathway inhibitor DAPT was used or Notch 1 receptor was a dominant negative mutant. Therefore, in part two we focus on investigating if Notch signaling could coordinate BMP9-mediated osteogenic differentiation via upregulating ALK2.PART1 DLL PLAYS A ROLE IN BMP9-INDUCED OSTEOGENIC DIFFERENTIATION OF MSCSObjectives:To demonstrate the role of Delta-like 1 (DLL 1) in BMP9-mediated osteogenesis in MSCs and its possible mechanisms.Methods:In Vitro, recombinant adeno virus were used to overexpress BMP9 and DLL1 respectively, early-stage index alkaline phosphatase(ALP)and late-stage indicator calcium deposits were evaluated by cytochemical staining; In Vivo, ectopic bone formation assay was conducted in nude mice, then microCT and HE staining were employed to analyze osteogenic status so as to determine DLL1’s role in the process. On the other hand, qRT-PCR was conducted to detect BMP receptors expression levels, Western Blotting was used to detect p-Smadl/5/9 expression level, while luciferase reporter assay was adpoted to test Smad-binding element(SBE) activity, thus addressing the underlying mechanisms that DLL might have implications in BMP9-induced osteogenesis via three cellular levels.Results:Compared with control group, osteogenic indicators ALP and calcium deposits were increased by DLL1; In Vivo, DLL promoted ectopic bone formation in nude mice, in which BMP receptors ALK2 was elevated significantly; It was also observed that total protein level of Smadl/5/8 didn’t change, but phosphorylated Smadl/5/9 expression level was increased, SBE activity was also increased to some degree as well(P<0.05).Conclusion:Both in vitro and in vivo, DLL1 could enhance BMP-induced osteogenesis in MSCs, and DLL1 might have impact on upregulating canonical BMP-Smad pathway through three cellular levels.PART2NOTCH SIGNALING FACILITATES BMP9 INDUCING MSCS DIFFERENTIATION INTO OSTEOBLASTS VIA UNREGULATING ALK2objectives:To explore if Notch signaling collaborates BMP9-mediated osteogenic differentiation in a ALK2-dependent manner. Methods: recombinant adenovirus were used to upregulate BMP9 and DLL1 respectively, DAPT and recombinant adenovirus dnNotchl were applied to downregulate Notch, recombinant adenovirus siALK1 and si ALK2 were adopted to downregulate ALK1 and ALK2 respectively.RT-PCR/qRT-PCR was conducted to measure receptors expression levels; Osteogenic indicators were identified by cytochemical staining; Cell cycle and apoptosis were assessed by Flow Cytometry Assay; Cell proliferation was measured by Edu Assay.Results:In the process of BMP inducing MSCs differentiation into osteoblasts, upregulating or downregulating Notch could cause alteration of ALK2 expression level rather than that of ALK1 expression level; DLL1 could reverse attenuation of osteogenic markers caused by ALK2 deletion, but it had no impact on siALK 1-treated group; Flow cytometric analysis and indicated that siALK2-induced decreased proliferation could be rescued by DLL (P< 0.05), while no such change was found in siALKl-treated group:However, cell apoptosis showed no significant influence by DLL in both siALKl-treated and siALK2-treated groups.Conclusion:During BMP9-mediated osteogenesis, Notch signaling may regulate cell proliferation and promote osteogenic differentiation via upregulating ALK2 expression level.