The Regulatory Mechanism Of BMP9Induced Mesenchymal Stem Cells Osteogenic Differentiation And BMP9Promoted Osteosarcoma Growth

Objective: The most important part of improving the quality of bonetissue engineering is to keep the dynamic balance of cell proliferation anddifferentiation during osteogenic process. Our studies are①to investigatethe effects and mechanism of TGF-β1undergoing BMP9-inducedmesenchymal stem cells (MSCs) osteogenic differentiation, to analyze thesynergistic effect of TGF-β1combined with BMP9in dose-timing regularpattern in order to support the experimental basis of improving bone quality.②to explore that how Notch signaling pathway mediates BMP9-indcuedosteosarcoma (OS) proliferation and tumor growth, so that we canunderstand the regulatory mechanism of BMP9promoted OStumorigenesis and development.Method: The first part: Using mesenchymal stem cell (MSCs) lineC3H10T1/2, we constructed the MSCs osteogenic differentiated modelinfected with recombinant adenovirus BMP9and performed with differentconcentrations of TGF-β1protein. During the process, we set up concentration gradient groups of TGF-β1and series time-points forexperiments. Fresh TGF-β1protein supernatant was obtained fromconditioned medium. We tested the activity of Alkaline Phosphatase (ALP)by a modified Great Escape SEAP Chemiluminescence assay and ahistochemical staining assay, investigated the matrix mineralization byAlizarin Red staining and detected the mRNA and protein expression levelof CollagenⅠ (COLⅠ), Osteopotin (OPN) and Osteocalcin (OCN). Cellcycles were tested by flow cytometry and cell proliferation was calculatedby cell counting with trypan blue staining. At last, we explore the Smadexpression level by luciferase reporter assay.The second part:①we identified the exogenous expression of Notchreceptors and ligands of11human osteosarcoma (OS) cell lines by RT-PCRand immunohistochemistry (IHC) assay, constructed and identified arecombinant adenovirus expressing the dominant-negative mutant ofNotch1(AdR-dnNotch1).②In vitro, we selected OS cell line143B andMG63, infected them with AdBMP9and/or AdR-dnNotch1, or performedAdBMP9transduction with Compound E, a γ-secretase inhibitor for Notchsignaling, to observe the effects on cell proliferation, migration and cellcycles. Crystal violet staining and quantitive analysis were used for cellproliferating ability; Gap closure assay and flow cytometry were used forcell migration and cell cycle changing; RT-PCR was used forBMP9-induced expression of Notch receptors and ligands and the immunofluorescence staining was to observe the nuclear translocation ofNotch Intra-Cellular Domain (NICD), the hallmark of Notch pathwayactivation.③The effects of Notch signaling blockade on BMP9-stimulatedOS tumor growth and invasion were analyzed by the subperiostealxenograft tumor model injected with143B cell line. The tumors sizes weremeasured by caliper one time in every4days and by Xenogen IVIS systemone time a week; the mice were sacrificed, dissected and scanned bymicro-CT for bone3D reconstruction. Tumor tissue was cut and stained byHE staining for checking bone destruction and IHC staining for the markerof proliferating ability: Proliferating Cell Nuclear Antigen (PCNA), whichconfirmed the cell proliferating ability.Results: The first part: the concentration of5ng/ml TGF-β1synergistically induces higher ALP activity in BMP9-transducedC3H10T1/2cells and produces more pronounced matrix mineralization atearlier status. However, the increased ALP activity is diminished when theconcentrations of TGF-β1increased; Real-time PCR and Western blottingindicate that BMP9in combination with5ng/ml TGF-β1potentiates theexpression of later osteogenic markers osteopontin, osteocalcin andcollagen typeⅠ (COLⅠa2), while higher concentrations of TGF-β1decrease the expression of osteopontin and osteocalcin but not COLⅠa2.Cell cycle analysis reveals that TGF-β1inhibits C3H10T1/2proliferation inBMP9-induced osteogenesis and restricts the cells in G0/G1phase. Smad reporter assay indicates that TGF-β1combined with BMP9decreases thetranscriptional expression level of smads, especially BMPR-Smad.The second part: Majority of Notch ligands and receptors is readilydetected in the tested11OS cell lines. BMP9up-regulates the expressionof Notch receptors and ligands in OS cells and significantly increases thenuclear translocated accumulation of NICD, which can be blocked by theγ-secretase inhibitor, ComE. BMP9-promoted OS cell proliferation,migration, cell cycle S/G2progression are effectively inhibited bydnNotch1or ComE. Furthermore, BMP9-promoted tumor growth andosteolytic lesions in vivo are significantly inhibited by dnNotch1.Conclusion:①a ppropriateamount of TGF-β1promote BMP9inducedosteogenic differentiation in C3H10T1/2cell line. TGF-β1actssynergistically with BMP9in dose-dependent fashion.②BMP9plays animportant role on OS growth and development; Notch signaling pathwaymediates BMP9-promoted OS proliferation and migration ability and theOS growth with invasive behavior. BMP9may function as upstreamregulator of Notch signaling in OS tumorigenesis and interfering Notchsignaling pathway should be the new strategy for preventing human OS.