(+)-Cholesten-3-one induces osteogenic differentiation of bone marrow mesenchymal stem cells by activating vitamin D receptor

Abstract

In our previous reports, it was revealed that steroids in traditional Chinese medicine (TCM) have the therapeutic potential to treat bone disease. In the present study, an in vitro model of a vitamin D receptor response element (VDRE) reporter gene assay in mesenchymal stem cells (MSCs) was used to identify steroids that enhanced osteogenic differentiation of MSCs. (+)-cholesten-3-one (CN), which possesses a ketone group that is modified in cholesterol and cholesterol myristate, effectively promoted the activity of the VDRE promoter. Phenotypic cellular analysis indicated that CN induced differentiation of MSCs into osteogenic cells and increased expression of specific osteogenesis markers, including alkaline phosphatase, collagen II and Runt-related transcription factor 2. Furthermore, CN significantly increased the expression of osteopontin, the target of the vitamin D receptor (VDR), which indicated that CN may activate vitamin D receptor signaling. Over-expression of VDR or knockdown studies with VDR-small interfering RNA revealed that the pro-differentiation effects induced by CN required VDR. Furthermore, the present study determined that the C-terminal region of the VDR is responsible for the action of CN. Taken together, the present findings demonstrated that CN induced osteogenic differentiation of MSCs by activating VDR. The present study explored the regulation of stem cells by using a series of similar steroids and provided evidence to support a potential strategy for the screening of novel drugs to treat bone disease in the future.

Profiling the miRNA-mRNA-lncRNA interaction network in MSC osteoblast differentiation induced by (+)-cholesten-3-one

“…Understanding the molecular mechanisms underlying MSC osteogenic differentiation is important for therapeutic purposes. Our previous reports [ 5 , 6 ] indicated that CN has the potential to promote the osteogenic differentiation of MSCs. However, the role of epigenetic regulation in the CN-induced osteogenic differentiation of MSCs remains poorly understood.…”

“…Bioinformatic analysis of the miRNA-mRNA-lncRNA interaction network identified 16 core genes, including 6 mRNAs (Vdr, Mgp, Fabp3, Fst, Cd38, and Col1a1), 5 miRNAs (miR-483, miR-298, miR-361, miR-92b and miR-155), and 5 lncRNAs (NR_046246.1, NR_046239.1, XR_086062.1, XR_145872.1 and XR_146737.1). Vdr, a ligand-activated transcription factor, has been reported to be a core gene during MSC osteogenic differentiation [ 5 , 6 , 50 ], and matrix gla protein (Mgp) has been recognized as a potent calcification inhibitor and regulator of bone morphogenetic protein-2 (BMP-2) [ 51 ]. Wang et al [ 52 ] indicated that overexpression of fatty acid binding protein 3 (Fabp3) inhibited MSC growth and proliferation via negatively regulating the cell cycle and MSC growth factors and enhancing cell survival under hypoxic or ischaemic conditions, which indicated that the Fabp3 gene potentially promotes MSC osteogenic differentiation.…”

“…Subsequent bioinformatics analysis revealed that miR-298 interacted closely with Vdr. Coincidentally, our previous study showed that Vdr positively regulates osteoblast differentiation induced by CN [ 5 , 6 ]. We then further examined the function of miR-298 in MSC osteogenic differentiation using transfection methods and found that miR-298 significantly inhibited MSC osteogenic differentiation.…”

“…Sprague dawley rats were used in this study and euthanised by cervical dislocation. MSCs were isolated and cultured as described in our previous reports [ 5 , 6 ] (Supplement 1). The initial culture medium was then replaced with fresh medium supplemented with 30 μg/mL CN, 10% FBS, and 1% penicillin-streptomycin to induce MSC osteoblast differentiation for 7 days and refreshed every 3 days thereafter.…”

“…Though it has great potential for clinical applications [ 2 ], the osteogenic differentiation of MSCs is precisely regulated and coordinated by mechanical and molecular signals from the extracellular environment and involves complex pathways at the transcriptional and post-transcriptional levels [ 3 , 4 ] that remain largely unexplored. Steroids, especially (+)-cholesten-3-one (CN), have therapeutic potential to enhance the osteoblast differentiation of MSCs and increase the expression of osteogenesis-specific factors, as reported in our previous study [ 5 , 6 ]. However, previous studies were focused on the transcriptional, post-transcriptional and epigenetic levels, and the exact molecular mechanisms involved in osteogenesis and pre-transcriptional levels require further study.…”