Background: Bone morphogenetic protein (BMP) signaling is crucial for osteogenic (OS) differentiation, bone growth, and repair. However, the clinical use of BMPs is limited due to their cost and challenges in delivery. SB4, a potent compound of benzoxazole, is a newly introduced small molecule shown to activate the BMP signaling pathway. In this study, we investigated the effects of SB4 on OS differentiation of adipose-derived mesenchymal stem cells (ADMSCs). Methods: We first examined whether BMP signaling is active in osteoinduced ADMSCs. ADMSCs were treated with SB4 (5 and 10 μM) for 24 hr, and mRNA expression of BMP direct target genes (ID1 and ID3) was analyzed. ADMSCs were then cultured with or without SB4 (10 μM) for 7, 14, and 21 days, followed by Alizarin red staining and real-time polymerase chain reaction analysis of mRNA expression of OS markers RUNX2, BMP2, alkaline phosphatase (ALP), and Osteopontin. To investigate the most effective time period of SB4 in osteogenesis, SB4 was applied every 3.5 days during the 21-day differentiation period. Results: After 24 hr of treatment with SB4 (5 and 10 μM), increased mRNA expression of BMP direct target genes (ID1 and ID3) confirmed that SB4 acts as an active BMP agonist. SB4-treated ADMSCs showed enhanced bone matrix production and increased mRNA expression of RUNX2, BMP2, ALP, and Osteopontin. Increased mRNA expression of OS markers, bone matrix formation, and ALP activity during the first 7 days of SB4 treatment suggested the importance of SB4 at the early stage of differentiation. Conclusions These results indicate that SB4, with potential BMP-like activity, induces OS differentiation of ADMSCs, leading it to serve as a cost-effective BMP agonist for osteogenesis-related research as well as a potential therapeutic agent for bone repair and regeneration.

Background: Due to the contradictory and temporally variable effects of transforming growth factor-β (TGF-β) and the Wnt/β-catenin pathways on osteogenic differentiation in different stem cell types, we sought to examine the activity of these pathways as well as their interaction during the osteogenic differentiation of the osteo-induced adiposederived mesenchymal stem cells (AD-MSCs). Methods: The osteo-induced AD-MSCs were treated with TGF-β1 (1 ng/mL) either alone or together with its antagonist SB- 431542 (10 μM) or that of the Wnt antagonist, inhibitor of Wnt production 2 (IWP2) (3 μM), every 3 days for 21 days. Cells were then analyzed for calcium deposit, bone matrix production, and the osteogenic markers gene expression. Results: Our results showed firstly that, either of the pathways is active since the mRNA expressions of their respective target genes, PAI-1 and Cyclin D1 were detectable although the latter was at a very low level. Secondly that, treatment with TGF-β1 decreased levels of calcium deposit, bone matrix production and the osteogenic markers gene expression. Accordingly, osteogenesis was induced in those treated with SB either alone or together with the TGF-β1, pointing to inhibitory effect of TGF-β pathway on osteogenic differentiation. Thirdly that following treatment with IWP2 and TGF-β1, the inhibitory effect of TGF-β1 on bone matrix production was reversed. Fourthly, there was constantly low expression of Wnt3amRNA but progressively increasing that of its endogenous antagonist Dkk-1mRNA throughout. Conclusions Together these results suggest that TGF-β1 requires the active Wnt/β-catenin signaling pathway to exert its inhibitory effects on osteogenic differentiation of AD-MSCs.

Background: Due to the contradictory and temporally variable effects of transforming growth factor-β (TGF-β) and the Wnt/β-catenin pathways on osteogenic differentiation in different stem cell types, we sought to examine the activity of these pathways as well as their interaction during the osteogenic differentiation of the osteo-induced adiposederived mesenchymal stem cells (AD-MSCs). Methods: The osteo-induced AD-MSCs were treated with TGF-β1 (1 ng/mL) either alone or together with its antagonist SB- 431542 (10 μM) or that of the Wnt antagonist, inhibitor of Wnt production 2 (IWP2) (3 μM), every 3 days for 21 days. Cells were then analyzed for calcium deposit, bone matrix production, and the osteogenic markers gene expression. Results: Our results showed firstly that, either of the pathways is active since the mRNA expressions of their respective target genes, PAI-1 and Cyclin D1 were detectable although the latter was at a very low level. Secondly that, treatment with TGF-β1 decreased levels of calcium deposit, bone matrix production and the osteogenic markers gene expression. Accordingly, osteogenesis was induced in those treated with SB either alone or together with the TGF-β1, pointing to inhibitory effect of TGF-β pathway on osteogenic differentiation. Thirdly that following treatment with IWP2 and TGF-β1, the inhibitory effect of TGF-β1 on bone matrix production was reversed. Fourthly, there was constantly low expression of Wnt3amRNA but progressively increasing that of its endogenous antagonist Dkk-1mRNA throughout. Conclusions Together these results suggest that TGF-β1 requires the active Wnt/β-catenin signaling pathway to exert its inhibitory effects on osteogenic differentiation of AD-MSCs.

Background: Due to the contradictory and temporally variable effects of transforming growth factor-β (TGF-β) and the Wnt/β-catenin pathways on osteogenic differentiation in different stem cell types, we sought to examine the activity of these pathways as well as their interaction during the osteogenic differentiation of the osteo-induced adiposederived mesenchymal stem cells (AD-MSCs). Methods: The osteo-induced AD-MSCs were treated with TGF-β1 (1 ng/mL) either alone or together with its antagonist SB- 431542 (10 μM) or that of the Wnt antagonist, inhibitor of Wnt production 2 (IWP2) (3 μM), every 3 days for 21 days. Cells were then analyzed for calcium deposit, bone matrix production, and the osteogenic markers gene expression. Results: Our results showed firstly that, either of the pathways is active since the mRNA expressions of their respective target genes, PAI-1 and Cyclin D1 were detectable although the latter was at a very low level. Secondly that, treatment with TGF-β1 decreased levels of calcium deposit, bone matrix production and the osteogenic markers gene expression. Accordingly, osteogenesis was induced in those treated with SB either alone or together with the TGF-β1, pointing to inhibitory effect of TGF-β pathway on osteogenic differentiation. Thirdly that following treatment with IWP2 and TGF-β1, the inhibitory effect of TGF-β1 on bone matrix production was reversed. Fourthly, there was constantly low expression of Wnt3amRNA but progressively increasing that of its endogenous antagonist Dkk-1mRNA throughout. Conclusions Together these results suggest that TGF-β1 requires the active Wnt/β-catenin signaling pathway to exert its inhibitory effects on osteogenic differentiation of AD-MSCs.