Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42.

Many bone diseases result from abnormal bone resorption by osteoclasts (OCs). Studying OC related regulatory genes is necessary for the development of new therapeutic strategies. Rho GTPases have been proven to regulate OC differentiation and function and only mature OCs can carry out bone resorption. Here we demonstrate that Rac1 and Cdc42 exchange factor Triple functional domain (Trio) is critical for bone resorption caused by OCs. In this study, we created LysM-Cre;Triofl/fl conditional knockout mice in which Trio was conditionally ablated in monocytes. LysM-Cre;Triofl/fl mice showed increased bone mass due to impaired bone resorption caused by OCs. Furthermore, our in vitro analysis indicated that Trio conditional deficiency significantly suppressed OC differentiation and function. At the molecular level, Trio deficiency significantly inhibited the expression of genes critical for osteoclastogenesis and OC function. Mechanistically, our researches suggested that perturbed Rac1/Cdc42-PAK1-ERK/p38 signaling could be used to explain the lower ability of bone resorption in CKO mice. Taken together, this study indicates that Trio is a regulator of OCs. Studying the role of Trio in OCs provides a potential new insight for the treatment of OC related bone diseases.

Pax2 is essential for proliferation and osteogenic differentiation of mouse mesenchymal stem cells via Runx2.

Mesenchymal stem cells (MSCs) have been widely studied in the field of regenerative medicine with the potential to solve osteoporosis. Paired box 2 (Pax2), as a transcription factor, is the master regulator of embryogenesis and oncogenesis. However, the function of Pax2 in osteogenesis is unknown. Here, we reported for the first time that the expression of Pax2 gradually increased during osteogenic differentiation of mouse MSCs, and osteoprogenitor cells. However, detected in osteoblastic cells of mouse tibia, the expression of Pax2 in the embryonic stage was higher than that in adulthood. In C3H/10/T1/2 cells and compact bone-derived mouse MSCs (mMSCs), Pax2 knock-down inhibited the proliferation of these cells, down-regulated the expression of osteogenic marker genes, as well as repressed the ALP activity and mineralization. In addition, Pax2 enhanced the transcriptional activity of Runx2, and activated the MAPK pathway genes (ERK, JNK and p38). Furthermore, knock-down of Pax2 repressed the mMSCs-mediated bone regeneration in an ectopic bone formation model. In conclusion, Pax2 promotes osteogenesis of mouse MSCs, suggesting that Pax2 has a role in the pathophysiology of bone related diseases, and has potential application in bone tissue regeneration.

The role of Rho-GEF Trio in regulating tooth root development through the p38 MAPK pathway.

Trio, the Rho guanine nucleotide exchange factor (Rho-GEF), plays diverse roles in cell migration, cell axon guidance and cytoskeleton reorganization. Conserved during evolution, Trio encodes two guanine nucleotide exchange factor domains (GEFs) and activates small GTPases. The Rho-family small GTPases RhoA and Rac1, which are target molecules of Trio, have been described to engage in craniofacial development and tooth formation. However, the exact role of Trio in tooth development remains elusive. In this study, we generated Wnt1-cre;Triofl/fl mice to address the potential function of Trio in tooth development. Wnt1-cre;Triofl/fl mice showed short root deformity as well as decreased expression of odontogenic makers such as RUNX2, OSX, OCN, and OPN. In vitro, Trio was silenced in human stem cells of dental papilla (SCAPs). Compared with the control group, the proliferation and migration ability in the experimental group was disrupted. After knocking down Trio in SCAPs, the cells showed phenotypes of poor odontogenic differentiation and weak mineralized nodules. To study the underlying mechanism, we investigated the p38 MAPK pathway and found that loss of Trio blocked the cascade transduction of p38 MAPK signaling. In conclusion, we identified Trio as a novel coordinator in regulating root development and clarified its relevant molecular events.

Integrated Cascade Nanozyme Remodels Chondrocyte Inflammatory Microenvironment in Temporomandibular Joint Osteoarthritis via Inhibiting ROS-NF-κB and MAPK Pathways.

Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative joint disease with no complete cure at present. Notably, the inflammatory microenvironment in TMJ OA is modulated by oxidative stress, which impacts cartilage metabolism, chondrocyte apoptosis, inflammatory cytokine release, and extracellular matrix (ECM) synthesis. Thus, it is reasoned that reducing excess reactive oxygen species (ROS) in the chondrocyte microenvironment may be an effective therapeutic strategy for TMJ OA. Recently, cascade nanozymes, including Pt@PCN222-Mn, have been exploited to treat ROS-associated diseases. Nevertheless, cascade nanozymes are not employed for TMJ OA therapy. To fill this gap, it is explored whether the Pt@PCN222-Mn cascade nanozyme could be applied to the treatment of TMJ OA. The in vitro results demonstrate that the Pt@PCN222-Mn nanozyme can inhibit the production of inflammatory factors, the degradation of ECM, and the apoptosis of chondrocytes by inhibiting the ROS-nuclear factor kappa-B (NF-κB_ and mitogen-activated protein kinase signaling pathways. The in vivo results further demonstrate that the Pt@PCN222-Mn nanozyme can delay the progression of TMJ OA in the rat unilateral anterior crossbite model. It is believed that insightful perspectives on the application of nanozymes in TMJ OA will be provided here.

Major vault protein (MVP) suppresses aging- and estrogen deficiency-related bone loss through Fas-mediated apoptosis in osteoclasts.

Osteoclasts (OCs), derived from monocyte/macrophage lineage, are key orchestrators in bone remodeling. Targeting osteoclast apoptosis is a promising approach to cut down excessive osteoclast numbers, and thus slow down the rate of bone mass loss that inevitably occurs during aging. However, the therapeutic target of apoptosis in osteoclasts has not been fully studied. Our previous work generated Mvpf/fLyz2-Cre mice, conditionally depleting major vault protein (MVP) in monocyte lineage, and identified MVP as a bone protector for its negative role in osteoclastogenesis in vivo and in vitro. Here, we observed a notable decline of MVP in osteoclasts with aging in mice, encouraging us to further investigate the regulatory role of osteoclast MVP. Then, Mvpf/fLyz2-Cre mice were exploited in two osteoporosis contexts, aging and abrupt loss of estrogen, and we revealed that conditional knockout of MVP inhibited osteoclast apoptosis in vivo and in vitro. Moreover, we reported the interaction between MVP and death receptor Fas, and MVP-Fas signaling cascade was identified to positively regulate the apoptosis of osteoclasts, thus preventing osteoporosis. Collectively, our comprehensive discovery of MVP's regulatory role in osteoclasts provides new insight into osteoclast biology and therapeutic targets for osteoporosis.

Effect of bone-shaped nanotube-hydrogel drug delivery system for enhanced osseointegration.

Anodic titanium dioxide nanotubes (TNT) have a range of beneficial theranostic properties. However, a lack of effective osseointegration is a problem frequently associated with the titanium dental implant surface. Here, we investigated whether bone-shaped nanotube titanium implants could enhance osseointegration via promoting initial release of vascular endothelial growth factor 165 (VEGF165) and dual release of recombinant human bone morphogenetic protein-2 (rhBMP-2). Thus, we generated cylindrical-shaped nanotubes (TNT1) and bone-shaped nanotubes (TNT2) through voltage-varying and time-varying electrochemical anodization methods, respectively. Additionally, we prepared rhBMP-2-loaded cylindrical-shaped nanotubes/VEGF165-loaded hydrogel (TNT-F1) and rhBMP-2-loaded bone-shaped nanotubes/VEGF165-loaded hydrogel (TNT-F2) drug delivery systems. We evaluated the characteristics and release kinetics of the drug delivery systems, and then analyzed the cytocompatibility and osteogenic differentiation of these specimens with mesenchymal stem cells (MSCs) in vitro. Finally, we utilized a rat femur defect model to test the bone formation capacity of nanotube-hydrogel drug delivery system in vivo. Among these different nanotubes structures, the bone-shaped one was the optimum structure for growth factor release.

Biocompatible Nanotube-Strontium/polydopamine-arginine-glycine-aspartic acid coating on Ti6Al4V enhances osteogenic properties for biomedical applications.

Titanium (Ti) alloys, particularly Ti6 Al4 V, are the most commonly used biomedical implant material. Ti alloys are biologically inert, so there have been continuous efforts to improve their osteogenic properties and clinical performance. Since TiO2 nanotubes (NT) appear to be excellent drug platforms, and strontium reportedly enhances osteogenesis, we constructed a TiO2 nanotube coating on the surface of Ti6 Al4 V and immersed it in Sr (OH)2 solution in order to incorporate Sr into TiO2 nanotubes (NT-Sr). The results of field emission scanning electron microscope and X-ray diffraction analysis verified the fabrication of NT-Sr. We next added polydopamine (PDA) and cyclo- (arginine-glycine-aspartic acid-phenylalanine-cysteine) [c(RGDfC)] peptides to further promote biocompatibility of the implant. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy confirmed the existence of PDA and c(RGDfC). Mesenchymal stem cells (MSCs) were planted on Ti, NT, NT-Sr, NT-Sr/PDA, and NT-Sr/PDA-RGD surfaces. The adhesion and differentiation of MSCs on different surfaces were evaluated. The mRNA expression of alkaline phosphatase, runt-related transcription factor 2 (Runx2) and type I collagen (Col I) of different groups were also tested. Finally, we observed that the NT-Sr/PDA-RGD group showed significantly better performance than other groups in terms of the differentiation and osteogenesis-related gene expression of MSCs. Thus, the NT-Sr/PDA-RGD complex may be an important modification strategy for Ti, as it shows excellent osteogenic potential.

Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoA.

Osteosarcoma (OS) is the most common primary bone tumor. Its high mortality rate and metastasis rate seriously threaten human health. Currently, the treatment has reached a plateau, hence we urgently need to explore new therapeutic directions. In this paper, we found that Trio was highly expressed in osteosarcoma than normal tissues and promoted the proliferation, migration, and invasion of osteosarcoma cells. Furthermore, Trio inhibited osteosarcoma cells' osteogenic differentiation in vitro and accelerated the growth of osteosarcoma in vivo. Given Trio contains two GEF domains, which have been reported as the regulators of RhoGTPases, we further discovered that Trio could regulate osteosarcoma progression and osteogenic differentiation through activating RhoGTPases. In summary, all our preliminary results showed that Trio could be a potential target and prognostic marker of osteosarcoma.

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 affects the development of the neural crest and craniofacial skeleton via the mitochondrial apoptosis pathway.

The neural crest is one of the key features of craniofacial development. MicroRNA-1 (miR-1) is a single-stranded noncoding RNA that serves an important role in embryonic development. However, the function of miR-1 in neural crest cells (NCCs) is unknown. Therefore, to evaluate the role of miR-1 in NCC development, a miR-1 mutant zebrafish was generated in the current study. Mouse NCCs were isolated from the first branchial arch of embryos at gestational day E9.5, and miR-1 was silenced using a miR-1 inhibitor. To the best of our knowledge, the present study was the first to report that homozygous zebrafish lacking miR-1 exhibited developmental defects in NCC-derived craniofacial bones, heart, melanocytes and iridophores. These defects may be caused by an increase in apoptosis of NCCs during their migration and differentiation in embryonic development. Moreover, the apoptosis analysis and western blotting results demonstrated that this effect was modulated via the mitochondrial apoptosis pathway, and miR-1 inhibited NCC apoptosis by modulating this pathway. These results collectively suggested that miR-1 in NCCs may be essential for craniofacial development.

Major vault protein (MVP) negatively regulates osteoclastogenesis via calcineurin-NFATc1 pathway inhibition.

Rationale: Bone homeostasis is maintained by a balanced interplay of osteoblasts and osteoclasts. Osteoclasts are derived from monocyte/macrophage lineage. Major vault protein (MVP) is known to promote apoptosis and prevent metabolic diseases in macrophage. However, whether MVP is involved in osteoclastogenesis is unknown. Here, we identified an important function of MVP as a negative regulator of osteoclastogenesis and its therapeutic potential in preventing bone loss. Methods: Expression of MVP in osteoclasts was investigated in human tumor tissues with immunohistochemical staining. Next, we generated total body (Mvp-/- ) and monocyte-specific (Mvpf/fLyz2-Cre) MVP gene knockout mice to observe bone phenotype and osteoclastogenesis using micro-CT and bone histomorphometry. Moreover, we examined the effects of MVP on osteoclast differentiation, bone resorption, NFATc1 activation and calcium oscillations in vitro. Finally, we explored the clinical potential of targeting MVP in two osteoporosis mouse models and used an adeno-associated virus (AAV) gene to overexpress MVP locally in mice. Results: We found that Mvp-/- and Mvpf/fLyz2-Cre mice both exhibited osteoporosis-like phenotypes. MVP-deficiency also enhanced calcineurin-NFATc1 signaling and promoted NFATc1 activity, which led to enhanced osteoclastogenesis and bone resorption. Calcineurin inhibition using the small molecule inhibitor FK506 corrected the enhanced osteoclastogenesis in Mvpf/fLyz2-Cre group. Additionally, MVP reexpression in Mvpf/fLyz2-Cre group rescued calcineurin expression. MVP overexpression in wild-type mice prevented pathologic bone loss in mouse models of ovariectomized (OVX) and calvaria-adjacent lipopolysaccharide (LPS)-injected. Conclusions: Our data suggested that MVP negatively regulates osteoclast differentiation and bone resorption via inhibition of calcineurin-NFATc1 signaling. In osteoclast-related bone diseases such as osteoporosis, manipulation of MVP activity may be an attractive therapeutic target.

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.

Guanine and nucleotide binding protein 3 promotes odonto/osteogenic differentiation of apical papilla stem cells via JNK and ERK signaling pathways.

Odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs) is a key process in tooth root formation and development. However, the molecular mechanisms underlying this process remain largely unknown. In the present study, it was identified that guanine and nucleotide binding protein 3 (GNAI3) was at least in part responsible for the odonto/osteogenic differentiation of SCAPs. GNAI3 was markedly induced in mouse tooth root development in vivo and in human SCAPs mineralization in vitro. Notably, knockdown of GNAI3 by lentiviral vectors expressing short­hairpin RNAs against GNAI3 significantly inhibited the proliferation, cell cycle progression and migration of SCAPs, as well as odonto/osteogenic differentiation of SCAPs in vitro, suggesting that GNAI3 may play an essential role in tooth root development. The promotive role of GNAI3 in odonto/osteogenic differentiation was further confirmed by downregulation of odonto/osteogenic makers in GNAI3­deficient SCAPs. In addition, knockdown of GNAI3 effectively suppressed activity of c­Jun N­terminal kinase (JNK) and extracellular­signal regulated kinase (ERK) signaling pathways that was induced during SCAPs differentiation, suggesting that GNAI3 promotes SCAPs mineralization at least partially via JNK/ERK signaling. Taken together, the present results implicate GNAI3 as a critical regulator of odonto/osteogenic differentiation of SCAPs in tooth root development, and suggest a possible role of GNAI3 in regeneration processes in dentin or other tissues.

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/ß-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted ß-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/ß-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.