Exosome-mediated small interfering RNA delivery inhibits aberrant osteoblast differentiation in Apert syndrome model mice.

OBJECTIVE: Apert syndrome, an autosomal dominant congenital disorder characterized by craniosynostosis, is caused by a missense mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Exosomes are naturally occurring carriers that deliver nucleic acids, including small interfering RNA (siRNA), to induce gene silencing. This study aimed to develop siRNA-loaded exosomes (Ex-siRNAFgfr2S252W) to silence the Fgfr2S252W gain-of-function mutation, thereby inhibiting the increased osteoblastic differentiation caused by the constitutive activation of FGFR2 signaling in calvarial osteoblastic cells isolated from Apert syndrome model mice. DESIGN: Primary calvarial osteoblast-like cells were isolated from the embryonic calvarial sutures of the Apert syndrome model (Fgfr2S252W/+) and littermate wild-type mice (Ap-Ob and Wt-Ob, respectively). Exosomes were extracted from the serum of wild-type mice, validated using biomarkers, and used to encapsulate siRNAs. After exosome-mediated siRNA transfection, cells were analyzed under a fluorescence microscope to validate the delivery of Ex-siRNAFgfr2S252W, followed by western blot and real-time reverse transcription polymerase chain reaction analyses. RESULTS: After 24 h of Ex-siRNAFgfr2S252W delivery in both Ap-Ob and Wt-Ob, siRNA-loaded exosome delivery was validated. Moreover, p44/42 mitogen-activated protein kinase (MAPK) phosphorylation, runt-related transcription factor 2 (Runx2), and collagen type 1 alpha 1 (Col1a1) mRNA expression, and alkaline phosphatase (ALP) activity were significantly increased in Ap-Ob. The levels of phospho-p44/42 protein, Runx2, Col1a1, and ALP were significantly decreased after Ex-siRNAFgfr2S252W transfection but did not affect Wt-Ob. CONCLUSIONS: These results indicate that exosome-mediated delivery of siRNA targeting Fgfr2S252W is a potential non-invasive treatment for aberrant FGF/FGFR signaling.

Changes in superoxide dismutase 3 (SOD3) expression in periodontal tissue during orthodontic tooth movement of rat molars and the effect of SOD3 on in vitro hypoxia-exposed rat periodontal ligament cells.

BACKGROUND/OBJECTIVES: Hypoxia during orthodontic tooth movement (OTM) induces reactive oxygen species (ROS) production in periodontal tissues. Superoxide dismutase 3 (SOD3) is an anti-inflammatory enzyme that protects cells from ROS. This study investigated the expression and function of SOD3 during rat OTM and in hypoxia-exposed rat periodontal ligament (PDL) cells. MATERIALS/METHODS: OTM of right maxillary first molars were performed in 8-week-old male Sprague-Dawley rats using closed-coil spring for 1 and 14 days (n = 6 per group). SOD3 and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression was evaluated by immunohistochemistry. The effects of SOD3 on cell viability and proliferation, ROS production, and mRNA expression of Hif1-α, receptor activator of nuclear factor kappa-Β ligand (Rankl), and osteoprotegerin (Opg) in PDL cells and osteoclast differentiation were investigated under normal and hypoxic conditions. RESULTS: SOD3 expression in PDL tissues significantly decreased on the compression side on day 1 and on both sides on day 14 of OTM. HIF-1α levels significantly increased on the compression side on day 14. Cell viability, cell proliferation, and Opg mRNA expression decreased, whereas ROS production and Hif1-α and Rankl mRNA expression increased in the PDL cells upon SOD3 silencing. Hypoxia reduced Sod3 and Opg mRNA expression and increased ROS, Rankl mRNA expression, and osteoclast formation; SOD3 treatment attenuated these effects. CONCLUSION/IMPLICATIONS: SOD3 plays a role in periodontal tissue remodelling during OTM and in hypoxia-exposed PDL cells through ROS, HIF-1α, and RANKL/OPG pathways. Moreover, SOD3 treatment could attenuate the negative effects of hypoxia on the PDL cells.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth.

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

Inhibitory effects of miRNAs in astrocytes on C6 glioma progression via connexin 43.

In many types of tumor cells, cell communication via gap junction is decreased or missing. Therefore, cancer cells acquire unique cytosolic environments that differ from those of normal cells. This study assessed the differences in microRNA (miRNA) expression between cancer and normal cells. MicroRNA microarray analysis revealed five miRNAs that were highly expressed in normal astrocytes compared with that in C6 gliomas. To determine whether these miRNAs could pass through gap junctions, connexin 43 was expressed in C6 glioma cells and co-cultured with normal astrocytes. The co-culture experiment showed the possibility that miR-152-3p and miR-143-3p propagate from normal astrocytes to C6 glioma in connexin 43-dependent and -independent manners, respectively. Moreover, we established C6 glioma cells that expressed miR-152-3p or miR-143-3p. Although the proliferation of these miRNA-expressing C6 glioma cells did not differ from that of empty vectors introduced in C6 glioma cells, cell migration and invasion were significantly decreased in C6 glioma cells expressing miR-152-3p or miR-143-3p. These results suggest the possibility that miRNA produced by normal cells attenuates tumor progression through connexin 43-dependent and -independent mechanisms.