Although bone morphogenetic protein 2 (BMP2) can induce chondrogenic differentiation of mesenchymal stem cells (MSCs), its induction of endochondral ossification limits the application of BMP2-based cartilage regeneration. Here, we clarified the mechanisms of BMP2-induced endochondral ossification of MSCs. In vitro and in vivo chondrogenic, osteogenic, and angiogenic differentiation models of MSCs were constructed. The expression of target genes was identified at both protein and mRNA levels. RNA sequencing, molecular docking, co-immunoprecipitation, and chromatin immunoprecipitation followed by sequencing were applied to investigate the molecular mechanisms. We found that BMP2 up-regulated the expression of Notch receptors and ligands in MSCs. Notch1 signaling activation was related to inhibition of chondrogenic differentiation, promotion of osteogenic and angiogenic differentiation. In vivo ectopic stem cell implantation identified that Notch1 signaling activation blocked BMP2-induced chondrogenesis and facilitated endochondral ossification of MSCs. Mechanistically, we elucidated Notch1 intracellular domain (NICD1)-RBPjk complex binding to SRY-box transcription factor 9 (Sox9) and vascular endothelial growth factor A (VEGFA) promoters to decrease Sox9 expression and increase VEGFA expression. These findings suggest that Notch1 signaling can regulate BMP2-induced endochondral ossification by promoting RBPjk-mediated Sox9 inactivation and VEGFA expression. It is conceivable that targeting Notch1 signaling mediated endochondral ossification would benefit BMP2-based cartilage regeneration.

Recent advances in deep sequencing technologies have revealed that, while less than 2% of the human genome is transcribed into mRNA for protein synthesis, over 80% of the genome is transcribed, leading to the production of large amounts of noncoding RNAs (ncRNAs). It has been shown that ncRNAs, especially long non-coding RNAs (lncRNAs), may play crucial regulatory roles in gene expression. As one of the first isolated and reported lncRNAs, H19 has gained much attention due to its essential roles in regulating many physiological and/or pathological processes including embryogenesis, development, tumorigenesis, osteogenesis, and metabolism. Mechanistically, H19 mediates diverse regulatory functions by serving as competing endogenous RNAs (CeRNAs), Igf2/H19 imprinted tandem gene, modular scaffold, cooperating with H19 antisense, and acting directly with other mRNAs or lncRNAs. Here, we summarized the current understanding of H19 in embryogenesis and development, cancer development and progression, mesenchymal stem cell lineage-specific differentiation, and metabolic diseases. We discussed the potential regulatory mechanisms underlying H19's functions in those processes although more in-depth studies are warranted to delineate the exact molecular, cellular, epigenetic, and genomic regulatory mechanisms underlying the physiological and pathological roles of H19. Ultimately, these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.

BMP9 mediated osteogenic differentiation mechanisms of MSCs were widely explored, however, mechanisms of BMP9-induced angiogenesis still need to be clarified. We previously characterized that Notch1 promoted BMP9-induced osteogenesis–angiogenesis coupling process in mesenchymal stem cells (MSCs). Here, we explored the underlying mechanisms of lncRNA H19 (H19) mediated regulation of BMP9-induced angiogenesis through activating Notch1 signaling. We demonstrated that basal expression level of H19 was high in MSCs, and silencing H19 attenuates BMP9-induced osteogenesis and angiogenesis of MSCs both in vitro and in vivo. Meanwhile, we identified that BMP9-induced production of CD31⁺ cells was indispensable for BMP9-induced bone formation, and silencing H19 dramatically blocked BMP9-induced production of CD31⁺ cells. In addition, we found that down-regulation of H19 inhibited BMP9 mediated blood vessel formation and followed subsequent bone formation in vivo. Mechanistically, we clarified that H19 promoted p53 phosphorylation by direct interacting and phosphorylating binding, and phosphorylated p53 potentiated Notch1 expression and activation of Notch1 targeting genes by binding on the promoter area of Notch1 gene. These findings suggested that H19 regulated BMP9-induced angiogenesis of MSCs by promoting the p53-Notch1 angiogenic signaling axis.