Pluripotent mesenchymal stem cells (MSCs) are bone marrow stromal progenitor cells that can differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Several signaling pathways have been shown to regulate the lineage commitment and terminal differentiation of MSCs. Here, we conducted a comprehensive analysis of the 14 types of bone morphogenetic protein (BMPs) for their abilities to regulate multilineage specific differentiation of MSCs. We found that most BMPs exhibited distinct abilities to regulate the expression of Runx2, Sox9, MyoD, and PPARγ2. Further analysis indicated that BMP-2, BMP-4, BMP-6, BMP-7, and BMP-9 effectively induced both adipogenic and osteogenic differentiation in vitro and in vivo. BMP-induced commitment to osteogenic or adipogenic lineage was shown to be mutually exclusive. Overexpression of Runx2 enhanced BMP-induced osteogenic differentiation, whereas knockdown of Runx2 expression diminished BMP-induced bone formation with a decrease in adipocyte accumulation in vivo. Interestingly, overexpression of PPARγ2 not only promoted adipogenic differentiation, but also enhanced osteogenic differentiation upon BMP-2, BMP-6, and BMP-9 stimulation. Conversely, MSCs with PPARγ2 knockdown or mouse embryonic fibroblasts derived from PPARγ2^−/− mice exhibited a marked decrease in adipogenic differentiation, coupled with reduced osteogenic differentiation and diminished mineralization upon BMP-9 stimulation, suggesting that PPARγ2 may play a role in BMP-induced osteogenic and adipogenic differentiation. Thus, it is important to understand the molecular mechanism behind BMP-regulated lineage divergence during MSC differentiation, as this knowledge could help us to understand the pathogenesis of skeletal diseases and may lead to the development of strategies for regenerative medicine.

Promoting osteogenic differentiation and efficacious bone regeneration have the potential to revolutionize the treatment of orthopaedic and musculoskeletal disorders. Mesenchymal Stem Cells (MSCs) are bone marrow progenitor cells that have the capacity to differentiate along osteogenic, chondrogenic, myogenic, and adipogenic lineages. Differentiation along these lineages is a tightly controlled process that is in part regulated by the Bone Morphogenetic Proteins (BMPs). BMPs 2 and 7 have been approved for clinical use because their osteoinductive properties act as an adjunctive treatment to surgeries where bone healing is compromised. BMP-9 is one of the least studied BMPs, and recent in vitro and in vivo studies have identified BMP-9 as a potent inducer of osteogenic differentiation in MSCs. BMP-9 exhibits significant molecular cross-talk with the Wnt/ β-catenin and other signaling pathways, and adenoviral expression of BMP-9 in MSCs increases the expression of osteogenic markers and induces trabecular bone and osteiod matrix formation. Furthermore, BMP-9 has been shown to act synergistically in bone formation with other signaling pathways, including Wnt/ β-catenin, IGF, and retinoid signaling pathways. These results suggest that BMP-9 should be explored as an effective bone regeneration agent, especially in combination with adjuvant therapies, for clinical applications such as large segmental bony defects, non-union fractures, and/or spinal fusions.

Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells Bone morphogenetic proteins;Mesenchymal stem cells;Mitogen activated protein kinases;Osteogenic differentiation;Smads; Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation of mesenchymal stem cells, the molecular mechanism involved remains to be fully elucidated. In this study, we showed that BMP9 simultaneously promotes the activation of Smad1/5/8, p38 and ERK1/2 in C3H10T1/2 cells. Knockdown of Smad4 with RNA interference reduced nuclear translocation of Smad1/5/8, and disrupted BMP9-induced osteogenic differentiation. BMP9-induced osteogenic differentiation was blocked by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. SB203580 decreased BMP9-activated Smads singling, and yet PD98059 stimulated Smads singling in C3H10T1/2 cells. The effects of inhibitor were reproduced with adenovirus expressing siRNA targeted p38 and ERK1/2, respectively. Taken together, our findings revealed that Smads, p38 and ERK1/2 are involved in BMP9-induced osteogenic differentiation. Also, it is noteworthy that p38 and ERK1/2 may play opposing regulatory roles in mediating BMP9-induced osteogenic differentiation of C3H10T1/2 cells.

Although previous studies have demonstrated that BMP9 is highly capable of inducing osteogenic differentiation and bone formation, the precise molecular mechanism involved remains to be fully elucidated. In this current study, we explore the possible involvement and detail effects of p38 and ERK1/2 MAPKs on BMP9-indcued osteogenic differentiation of mesenchymal progenitor cell (MPCs). We find that BMP9 simultaneously stimulates the activation of p38 and ERK1/2 in MPCs. BMP9-induced early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as matrix mineralization and osteocalcin (OC) are inhibited by p38 inhibitor SB203580, whereas enhanced by ERK1/2 inhibitor PD98059. BMP9-induced activation of Runx2 and Smads signaling are reduced by SB203580, and yet increased by PD98059 in MPCs. The in vitro effects of inhibitors are reproduced with adenoviruses expressing siRNA targeted p38 and ERK1/2, respectively. Using mouse calvarial organ culture and subcutaneous MPCs implantation, we find that inhibition of p38 activity leads to significant decrease in BMP9-induced osteogenic differentiation and bone formation, however, blockage of ERK1/2 results in effective increase in BMP9-indcued osteogenic differentiation in vivo. Together, our results reveal that p38 and ERK1/2 MAPKs are activated in BMP9-induced osteogenic differentiation of MPCs. What is most noteworthy, however, is that p38 and ERK1/2 act in opposition to regulate BMP9-induced osteogenic differentiation of MPCs.

Although there are many therapeutic strategies such as surgery and chemotherapy, the prognosis of osteosarcoma (OS) is still far from being satisfactory. It is urgent to develop more effective, tolerable and safe drugs for the treatment of OS. In the present study, we investigated the anti-OS activity of Alantolactone (ALT), a natural eucalyptone sesquiterpene lactone mainly exists in Inula helenium, and probed the possible mechanism involved. We demonstrated that ALT significantly inhibited cell proliferation of various human OS cell lines while had relative lower cytotoxicity against normal cells. Then, we validated that ALT reduced migration, decreased invasion possibly through reversing epithelial mesenchymal transition (EMT) process and suppressing Matrix metalloproteinases (MMPs). Moreover, we confirmed that ALT promoted apoptosis and arrested cell cycle at G2/M phase of human OS cells in vitro. In addition, we confirmed that ALT restrained tumor growth and metastasis of OS 143 cells in a xenograft model in vivo. Mechanistically, ALT inhibited the activity of Wnt/β-catenin and p38, ERK1/2 and JNK Mitogen Activated Protein Kinases (MAPKs) signal pathway. Notably, the combination of ALT and Wnt/β-catenin inhibitor, as well as the combination of ALT and MAPKs inhibitors resulted in a synergistically effect on inhibiting the proliferation, migration and invasion of OS cells. Collectively, our results validate the ALT may inhibit proliferation, metastasis and promotes apoptosis of human OS cells possibly through suppressing Wnt/β-Catenin and MAPKs signaling pathways.

Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-β receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-β receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-β receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-β receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.