Mir24-2-5p suppresses the osteogenic differentiation with Gnai3 inhibition presenting a direct target via inactivating JNK-p38 MAPK signaling axis.

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.

Trio cooperates with Myh9 to regulate neural crest-derived craniofacial development.

Trio is a unique member of the Rho-GEF family that has three catalytic domains and is vital for various cellular processes in both physiological and developmental settings. TRIO mutations in humans are involved in craniofacial abnormalities, in which patients present with mandibular retrusion. However, little is known about the molecular mechanisms of Trio in neural crest cell (NCC)-derived craniofacial development, and there is still a lack of direct evidence to assign a functional role to Trio in NCC-induced craniofacial abnormalities. Methods:In vivo, we used zebrafish and NCC-specific knockout mouse models to investigate the phenotype and dynamics of NCC development in Trio morphants. In vitro, iTRAQ, GST pull-down assays, and proximity ligation assay (PLA) were used to explore the role of Trio and its potential downstream mediators in NCC migration and differentiation. Results: In zebrafish and mouse models, disruption of Trio elicited a migration deficit and impaired the differentiation of NCC derivatives, leading to craniofacial growth deficiency and mandibular retrusion. Moreover, Trio positively regulated Myh9 expression and directly interacted with Myh9 to coregulate downstream cellular signaling in NCCs. We further demonstrated that disruption of Trio or Myh9 inhibited Rac1 and Cdc42 activity, specifically affecting the nuclear export of ß-catenin and NCC polarization. Remarkably, craniofacial abnormalities caused by trio deficiency in zebrafish could be partially rescued by the injection of mRNA encoding myh9, ca-Rac1, or ca-Cdc42. Conclusions: Here, we identified that Trio, interacting mostly with Myh9, acts as a key regulator of NCC migration and differentiation during craniofacial development. Our results indicate that trio morphant zebrafish and Wnt1-cre;Triofl/fl mice offer potential model systems to facilitate the study of the pathogenic mechanisms of Trio mutations causing craniofacial abnormalities.

microRNA-1 Regulates NCC Migration and Differentiation by Targeting sec63.

Background/Aims: Neural crest cells play a vital role in craniofacial development, microRNA-1 (miR-1) is essential in development and disease of the cardiac and skeletal muscle, the objective of our study is to investigate effects of miR-1 on neural crest cell in the craniofacial development and its molecular mechanism. Methods: We knocked down miR-1 in zebrafish by miR-1 morpholino (MO) microinjection and observed phenotype of neural crest derivatives. We detected neural crest cell migration by time-lapse. Whole-mount in situ hybridization was used to monitor the expressions of genes involved in neural crest cell induction, specification, migration and differentiation. We performed a quantitative proteomics study (iTRAQ) and bioinformatics prediction to identify the targets of miR-1 and validate the relationship between miR-1 and its target gene sec63. Results: We found defects in the tissues derived from neural crest cells: a severely reduced lower jaw and delayed appearance of pigment cells. miR-1 MO injection also disrupted neural crest cell migration. At 24 hours post fertilization (hpf), reduced expression of tfap2a, dlx2, dlx3b, ngn1 and crestin indicated that miR-1 deficiency affected neural crest cell differentiation. iTRAQ and luciferase reporter assay identified SEC63 as a direct target gene of miR-1. The defects of miR-1 deficiency could be reversed, at least in part, by specific suppression of sec63 expression. Conclusion: miR-1 is involved in the regulation of neural crest cell development, and that it acts, at least partially, by targeting sec63 expression.

Class A Scavenger Receptor Exacerbates Osteoclastogenesis by an Interleukin-6-Mediated Mechanism through ERK and JNK Signaling Pathways.

Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, which are important for bone health. Class A scavenger receptor (SR-A) is a multifunctional molecule that functions during differentiation of monocyte into macrophages and osteoclasts. To further characterize the role of SR-A in osteoclasts, we used the murine tooth movement model (TM) and the murine anterior cruciate ligament transection model of osteoarthritis (ACLT OA). In these two models the bones involved are of different origin and have different properties. Bone resorption was decreased in SR-A-/- mice compared to SR-A+/+ mice. Further evaluation showed that the number of multinucleated osteoclasts in SR-A-/- mice, compared to SR-A+/+ mice, was significantly decreased both in vivo and in vitro. The levels of interleukin-6 (IL-6) produced by osteoclasts were reduced in SR-A-/- mice compared to SR-A+/+ mice. In the in vitro marrow-derived osteoclast formation assay and in both mouse models, osteoclastogenesis was restored to normal in SR-A-/- mice by administration of recombinant murine IL-6. Moreover, neutralization of IL-6 reduced the number of osteoclasts formed in SR-A+/+ mice of TM model. Both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38, signaling pathways were downregulated in receptor activator of nuclear factor-κB ligand (RANKL)-stimulated SR-A-/- osteoclasts. Importantly, when treated with either ERK or JNK inhibitor, the numbers of osteoclasts generated from RANKL-induced bone marrow derived-macrophages of SR-A+/+ mice, and their IL-6 production, were significantly decreased. This suggests that SR-A activates the ERK and JNK signaling pathways, and promotes production of IL-6 by osteoclasts to further stimulate osteoclast formation.

Histologic investigation of gingival epithelium implantation and the nonincision placement of miniscrews.

PURPOSE: This study investigated whether the nonincision placement of miniscrews could lead to ectopic implantation of epithelium at the bone-implant interface and, if so, whether the epithelial cells could survive. The fate of grafted epithelial cells was also investigated. MATERIALS AND METHODS: Ten healthy male beagle dogs were included in the study and were divided into three groups. Group 1 dogs (n = 3) were implanted with miniscrews without any other procedures. In group 2, epithelial cells were collected, infected by adeno-associated virus-2 containing green fluorescent protein (GFP), and injected into pilot holes drilled before miniscrew implantation in three dogs, and one dog was injected with epithelial cells as a negative control. In group 3, tiny gingival tissue pieces were injected into pilot holes drilled before miniscrew implantation. In group 1, hematoxylin-eosin and fluorescent immunohistochemical (IHC) staining of cytokeratin were performed on both undecalcified and decalcified slices. In groups 2 and 3, IHC staining of cytokeratin and apoptotic testing were performed. RESULTS: No ectopically implanted epithelial cells were found at the bone-implant interface of the miniscrews in group 1. In group 2, fluorescence emitted by cytokeratin antibody and by GFP was observed in specimens at 0 days, had decreased by 3 days, and had disappeared by 7 days. In group 3, areas of fluorescent cytokeratin-positive tissues were much smaller at 7 days than at 0 days, and none were found at 28 days. In groups 2 and 3, there were many apoptotic epithelial cells at the bone-implant interface at 3 days and 7 days, respectively. CONCLUSION: There was little possibility of implantation of epithelium into alveolar bone though placement of miniscrews with a nonincision procedure. Furthermore, even when epithelial cells or tissue was ectopically implanted, it was soon cleared through an apoptotic process.