piR-368 promotes odontoblastic differentiation of dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

PURPOSE: The important role of non-coding RNAs in odontoblastic differentiation of dental tissue-derived stem cells has been widely demonstrated; however, whether piRNA (a subclass of non-coding RNA) involved in the course of odontoblastic differentiation is not yet available. This study aimed to investigate the expression profile of piRNA during odontogenic differentiation of mDPCs and the potential molecular mechanism in vitro. MATERIALS AND METHODS: The primary mouse dental papilla cells (mDPCs) were isolated from the first molars of 1-day postnatal Kunming mice. Then, they were cultured in odontogenic medium for 9 days. The expression profile of piRNA was detected by Small RNA sequencing. RT-qPCR was used to verify the elevation of piR-368. The mRNA and protein levels of mineralization markers were examined by qRT-PCR and Western blot analysis. Alkaline phosphatase (ALP) activity and alizarin red S staining were conducted to assess the odontoblastic differentiation ability. RESULTS: We validated piR-368 was significantly upregulated and interference with piR-368 markedly inhibited the odontogenic differentiation of mDPCs. In addition, the relationship between Smad1/5 signaling pathway and piR-368-induced odontoblastic differentiation has been discovered. Finally, we demonstrated Smurf1 as a target gene of piR-368 using dual-luciferase assays. CONCLUSION: This study was the first to illustrate the participation of piRNA in odontoblastic differentiation. We proved that piR-368 promoted odontoblastic differentiation of mouse dental papilla cells via the Smad1/5 signaling pathway by targeting Smurf1.

IPO7 promotes lipopolysaccharide-induced inflammatory responses in human dental pulp cells via p38 MAPK and NF-κB signaling pathways.

Pulpitis is a chronic inflammatory process that greatly affects the physical, mental health and life quality of patients. Human dental pulp cells (hDPCs) are essential components of dental pulp tissue and play a significant role in pulpitis. Lipopolysaccharide (LPS) is an initiator of pulpitis and can induce the production of inflammatory cytokines in hDPCs by activating p38 MAPK and NF-κB signaling pathways. Importin7 (IPO7), a member of the importin-ß family, is widely expressed in many tissues. Previous studies have shown that IPO7 mediated nuclear translocation of p-p38 after stimulation, and IPO7 homologous protein IPO8 participated in human dental pulp inflammation. This research aims to investigate whether IPO7 is involved in pulpitis and explore its underlying mechanisms. In the current study, we found the expression of IPO7 was increased in pulpitis tissue. In vitro, hDPCs treated with LPS to mimic the inflammatory environment, the expression of IPO7 was increased. Knockdown of IPO7 significantly inhibited the production of inflammatory cytokines and suppressed the p38 MAPK and NF-κB signaling pathways. Activating the p38 MAPK and NF-κB signaling pathways by the p38 activator and p65 activator reversed the inflammatory responses. IPO7 interacted with p-p38 under LPS stimulation in hDPCs. In addition, the increased binding between IPO7 and p-p38 is associated with the decreased binding ability of IPO7 to Sirt2. In conclusion, we found that IPO7 was highly expressed in pulpitis and played a vital role in modulating human dental pulp inflammation.

The regulation of Msx1 by BMP4/pSmad1/5 signaling is mediated by importin7 in dental mesenchymal cells.

Msx1 is essential for the maintenance of the odontogenic fate of dental mesenchymal cells, and is regulated by BMP/Smad1/5 signaling in a Smad4-independent manner. However, the exact co-factors that assist pSmad1/5 entering the nucleus to regulate Msx1 in dental mesenchymal cells are still unknown. Importin7 (IPO7) is one of the important members of importin ß-superfamily, which is mainly responsible for nucleocytoplasmic shuttling of RNAs and proteins, including transcription factors. This study aims to investigate whether IPO7 participates in the nuclear translocation of pSmad1/5 activated by BMP4 to regulate Msx1 expression in mouse dental mesenchymal cells. In the current study, we found that IPO7 was strongly expressed in the mouse dental mesenchymal cells at postnatal day 1 (PN1) both in vitro and in vivo. With BMP4 stimulation, IPO7 showed a translocation from the cytoplasm to the nucleus. Knockdown of IPO7 with siRNA inhibited the nuclear accumulation of pSmad1/5 in response to BMP4 stimulation. Furthermore, the co-immunoprecipitation assay showed pSmad1/5 was a nuclear import cargo of IPO7. Next, knockdown of IPO7 abolished the upregulation of Msx1 induced by BMP4, while overexpression of Smad1 was able to rescue the Msx1 expression. Finally, ChIP and Re-ChIP assay showed IPO7 facilitated the recruitment of pSmad1/5 to the Msx1 promoter. Taken together, our data demonstrated that the regulation of Msx1 by BMP4/pSmad1/5 signaling is mediated by importin7 in mouse dental mesenchymal cells.