Osteoblast-specific Transcription Factor Research Articles

Loss of Function of the Retinoblastoma Gene Affects Gap Junctional Intercellular Communication and Cell Fate in Osteoblasts.

Loss of function of the Retinoblastoma gene (RB1) due to mutations is commonly seen in human osteosarcomas. One of the Rb1 gene functions is to facilitate cell fate from mesenchymal stem cells to osteoblasts and prevent adipocyte differentiations. In this study, we demonstrate that a stable reduction of Rb1 expression (RbKD) in murine osteoblasts causes them to express higher levels of PPAR-ɣ and other adipocyte-specific transcription factors while retaining high expression of osteoblast-specific transcription factors, Runx2/Cbfa1 and SP7/Osterix. Inhibition of gap junctional intercellular communication (GJIC) in osteoblasts is another mechanism that causes osteoblasts to transdifferentiate to adipocytes. We found that preosteoblasts exposed to osteoblast differentiating media (DP media) increased GJIC. RbKD cells showed reduced GJIC along with a reduction in expression of Cx43, the protein that mediates GJIC. Other membrane associated adhesion protein Cadherin 11 (Cad11) was also decreased. Since PPAR-ɣ is increased with Rb1 loss, we wondered if the reduction of this transcription factor would reverse the changes observed. Reduction of PPAR-ɣ in control osteoblasts slightly increased bone-specific expression and reduced adipocytic expression as expected along with an increase in Cad11 and Cx43 expression. GJIC remained high and was unaffected by a reduction in PPAR-ɣ in control cells. Knockdown of PPAR-ɣ in RbKD cells reduced adipocyte gene expression, while osteoblast-specific expression showed improvement. Cx43, Cad11 and GJIC remained unaffected by PPAR-ɣ reduction. Our observations suggest that increased PPAR-ɣ that happens with Rb1 loss only affects osteoblast-adipocyte-specific gene expression but does not completely reverse Cx43 gene expression or GJIC. Therefore, these effects may represent independent events triggered by Rb1loss and/or the differentiation process.

Osteoblastic Cell Behavior and Gene Expression Related to Bone Metabolism on Different Titanium Surfaces.

The surface topography of titanium dental implants has a great influence on osseointegration. In this work, we try to determine the osteoblastic behavior and gene expression of cells with different titanium surfaces and relate them to the physicochemical properties of the surface. For this purpose, we have used commercial titanium discs of grade 3: as-received corresponds to machined titanium without any surface treatment (MA), chemically acid etched (AE), treated via sand blasting with Al2O3 particles (SB) and a sand-blasting treatment with acid etching (SB+AE). The surfaces have been observed using scanning electron microscopy (SEM) and the roughness, wettability and surface energy with dispersive and polar components have been characterized. Osteoblastic cultures were performed with SaOS-2 osteoblastic cells determining cell viability as well as alkaline phosphatase levels for 3 and 21 days, and osteoblastic gene expression was determined. The roughness values of the MA discs was 0.02 μm, which increases to 0.3 μm with acid attack and becomes the maximum for the sand-blasted samples, reaching values of 1.2 μm for SB and SB+AE. The hydrophilic behavior of the MA and AE samples with contact angles of 63° and 65° is superior to that of the rougher samples, being 75° for SB and 82° for SB+AE. In all cases, they show good hydrophilicity. GB and GB+AE surfaces present a higher polar component in the surface energy values, 11.96 and 13.18 mJ/m2, respectively, than AE and MA, 6.64 and 9.79 mJ/m2, respectively. The osteoblastic cell viability values at three days do not show statistically significant differences between the four surfaces. However, the viability of the SB and SB+AE surfaces at 21 days is much higher than that of the AE and MA samples. From the alkaline phosphatase studies, higher values were observed for those treated with sand blasting with and without acid etching compared to the other two surfaces, indicating a greater activity in osteoblastic differentiation. In all cases except in the Osterix (Ostx) -osteoblast-specific transcription factor-a decrease in gene expression is observed in relation to the MA samples (control). The most important increase was observed for the SB+AE condition. A decrease in the gene expression of Osteoprotegerine (OPG), Runt-related transcription factor 2 (Runx2), Receptor Activator of NF-κB Ligand (RANKL) and Alkaline Phosphatase (Alp) genes was observed in the AE surface.

Bone-targeting polyphosphodiesters that promote osteoblastic differentiation.

Polymers for pharmaceutical use have been attractive in medical treatments because of the conjugation of multifunctional components and their long circulation time in the blood stream. Bone-targeted drug delivery systems are also no exceptional, and several polymers have been proposed for the treatment of bone diseases, such as cancer metastasis and osteoporosis. Herein, we report that polyphosphodiesters (PPDEs) have a potential to enhance osteoblastic differentiation, and they have a targeting ability to bone tissues in vivo. Two types of PPDEs, poly (ethylene sodium phosphate) (PEP•Na) and poly (propylene sodium phosphate) (PPP•Na), have been synthesized. Regardless of the alkylene structure in the main chain of PPDEs, the gene expression of osteoblast-specific transcription factors and differentiation markers of mouse osteoblastic-like cells (MC3T3-E1 cells) cultured in a differentiation medium was significantly upregulated by the addition of PPDEs. Moreover, it was also clarified that the signaling pathway related to cytoplasmic calcium ions was activated by PPDEs. The mineralization of MC3T3-E1 cells has a similar trend with its gene expression and is synergistically enhanced by PPDEs with β-glycerophosphate. The biodistribution of fluorescence-labeled PPDEs was also determined after intravenous injection in mice. PPDEs accumulated well in the bone through the blood stream, whereas polyphosphotriesters (PPTEs) tended to be excreted from the kidneys. Hydrophilic PEP•Na showed a superior bone affinity as compared with PPP•Na. PPDEs could be candidate polymers for the restoration of bone remodeling and bone-targeting drug delivery platforms.

Ginsenoside Rg1 interferes with the progression of diabetic osteoporosis by promoting type H angiogenesis modulating vasculogenic and osteogenic coupling.

Ginsenoside Rg1 (Rg1) has been demonstrated to have antidiabetic and antiosteoporotic activities. The aim of this study was to investigate the protective effect of Rg1 against diabetic osteoporosis and the underlying mechanism. In vitro, we found that Rg1 increased the number of osteoprogenitors and alleviated high glucose (HG) induced apoptosis of osteoprogenitors by MTT assays and flow cytometry. qRT‒PCR and western blot analysis suggested that Rg1 can also promote the secretion of vascular endothelial growth factor (VEGF) by osteoprogenitors and promote the coupling of osteogenesis and angiogenesis. Rg1 can also promote the proliferation of human umbilical vein endothelial cells (HUVECs) cultured in high glucose, enhance the angiogenic ability of endothelial cells, and activate the Notch pathway to promote endothelial cells to secrete the osteogenesis-related factor Noggin to regulate osteogenesis, providing further feedback coupling of angiogenesis and osteogenesis. Therefore, we speculated that Rg1 may have similar effects on type H vessels. We used the Goto-Kakizaki (GK) rat model to perform immunofluorescence staining analysis on two markers of type H vessels, Endomucin (Emcn) and CD31, and the osteoblast-specific transcription factor Osterix, and found that Rg1 stimulates type H angiogenesis and bone formation. In vivo experiments also demonstrated that Rg1 promotes VEGF secretion, activates the Noggin/Notch pathway, increases the level of coupling between type H vessels and osteogenesis, and improves the bone structure of GK rats. All of these data reveal that Rg1 is a promising candidate drug for treating diabetic osteoporosis as a potentially bioactive molecule that promotes angiogenesis and osteointegration coupling.

RUNX2 interacts with SCD1 and activates Wnt/β-catenin signaling pathway to promote the progression of clear cell renal cell carcinoma.

Previous studies have demonstrated that Runt-associated transcription factor 2 (RUNX2) serves as the main transcription factor for osteoblast differentiation and chondrocyte maturation. RUNX2 is related to a variety of tumors, particularly tumor invasion and metastasis, while the expression and molecular mechanisms of RUNX2 in clear cell renal cell carcinoma (ccRCC) keep to be determined. Stearyl CoA desaturase 1 (SCD1), an endoplasmic reticulum fatty acid desaturase, transfers saturated fatty acids to monounsaturated fatty acids, is expressed highly in numerous malignancies. The Cancer Genome Atlas (TCGA) datebase and Western blot was used to analyzed the mRNA and protein levels of the target gene in ccRCC tissues and adjacent tissues. The proliferation ability of ccRCC cells was tested by colony forming and EdU assay. The migration ability of cells was detected by transwell assay. Immunoprecipitation was utilized to detect protein-protein interaction. Cycloheximide chase assay was used to measure the half-life of SCD1 protein. In this study, the expressions of RUNX2 and SCD1 are increased in ccRCC tissues as well as ccRCC cell lines. Both RUNX2 and SCD1 could promote proliferation and migration in ccRCC cells. Furthermore, RUNX2 could physically interact with SCD1. In addition, the functional degradation and the inactivation of Wnt/β-catenin signaling pathway triggered by the downregulation of RUNX2 could be partly offset by the overexpression of SCD1. The findings indicate that the RUNX2/SCD1 axis may act as a potential therapeutic target via the Wnt/β-catenin signaling pathway of ccRCC.

Identification of kaempferol as an OSX upregulator by network pharmacology-based analysis of qianggu Capsule for osteoporosis.

Osteoporosis is the most common metabolic disease of skeleton with reduced bone density and weaker bone. Qianggu Capsule as a traditional chinese medicine has been widely used to treat osteoporosis. The potential pharmacological mechanism of its active ingredient Gusuibu is not well understood. The purpose of this work is to analyze the anti-osteoporosis function of Gusuibu based on network pharmacology, and further explore the potential mechanism of Qianggu Capsule. The active compounds and their corresponding targets of Gusuibu were obtained from TCMSP, TCMID, and BATMAN-TCM databases. Potential therapeutic targets for osteoporosis were obtained through DisGeNET, TTD, GeneCards, MalaCards, CTD, and OMIM databases. The overlapping targets of Gusuibu and osteoporosis were obtained. GO and KEGG pathway enrichment analysis were performed. The “Gusuibu-active compounds-target genes-osteoporosis” network and protein-protein interaction (PPI) network were constructed, and the top hub genes were screened by using the plug-in CytoHubba. Molecular docking was used to verify the binding activity of hub genes and key compounds. We identified 21 active compounds and 140 potential therapeutic targets that may be related to Gusuibu and 10 hub genes (AKT1, IL6, JUN, TNF, MAPK3, VEGFA, EGFR, MAPK1, CASP3, PTGS2). Molecular docking analysis demonstrated that four key active small molecules in Gusuibu (including Luteolin, Naringenin, Kaempferol, and Beta-sitosterol) have excellent binding affinity to the target proteins encoded by the top 10 hub genes. Our new findings indicated that one key active compound kaempferol activated the expression of osteoblast specific transcription factor OSX through JNK kinase pathway.

Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress

Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

Inactivation of Ihh in Sp7-Expressing Cells Inhibits Osteoblast Proliferation, Differentiation, and Bone Formation, Resulting in a Dwarfism Phenotype with Severe Skeletal Dysplasia in Mice.

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.

Tumor necrosis factor-α and osterix expression after the transplantation of a hydroxyapatite scaffold from crab shell (Portunus pelagicus) in the post-extraction socket of Cavia cobaya

Background: Socket preservation using bone graft is one way to minimise resorption and maximise the bone formation process. Tumor necrosis factor-α (TNF-α) is an inflammatory cytokine that affects bone regenerating osteoblast activity, while osterix (Osx) is an osteoblast-specific transcription factor that activates gene receptors during pre-osteoblast differentiation. The hydroxyapatite (HA) scaffold from crab shells (Portunus pelagicus) has osteoconduction properties. Purpose: To analyse the decrease of TNF-α expression and the increase of Osx expression and the correlation between these two in the post-extraction socket after the transplantation of a crab shell HA scaffold. Methods: The lower left incisors of Cavia cobaya (n = 24) were extracted and divided into four groups: the first and second groups were control groups on Day 7 and Day 14 (K7 and K14), the third and fourth groups were treatment groups (P7 and P14). The statistical analysis used was a multivariate analysis of variance (MANOVA) with a significance level of 0.05. Results: A MANOVA test showed that the use of crab shell HA scaffolds led to a significant difference (p < 0.05) in TNF-α expression (p = 0.01) and Osx expression (p = 0.01). A Pearson correlation test result showed a strong inverse correlation between TNF-α and Osx expressions (p = 0.00 and r = -0.78). Conclusion: The transplantation of HA scaffolds from crab shells can decrease TNF-α expression but increase Osx expression in the post-extraction socket of C. cobayas. Furthermore, an inverse correlation was found between TNF-α and Osx.

The Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathway in Osteoblasts.

Extracellular signal-regulated kinases (ERKs) are evolutionarily ancient signal transducers of the mitogen-activated protein kinase (MAPK) family that have long been linked to the regulation of osteoblast differentiation and bone formation. Here, we review the physiological functions, biochemistry, upstream activators, and downstream substrates of the ERK pathway. ERK is activated in skeletal progenitors and regulates osteoblast differentiation and skeletal mineralization, with ERK serving as a key regulator of Runt-related transcription factor 2, a critical transcription factor for osteoblast differentiation. However, new evidence highlights context-dependent changes in ERK MAPK pathway wiring and function, indicating a broader set of physiological roles associated with changes in ERK pathway components or substrates. Consistent with this importance, several human skeletal dysplasias are associated with dysregulation of the ERK MAPK pathway, including neurofibromatosis type 1 and Noonan syndrome. The continually broadening array of drugs targeting the ERK pathway for the treatment of cancer and other disorders makes it increasingly important to understand how interference with this pathway impacts bone metabolism, highlighting the importance of mouse studies to model the role of the ERK MAPK pathway in bone formation.

The Polymorphism at PLCB4 Promoter (rs6086746) Changes the Binding Affinity of RUNX2 and Affects Osteoporosis Susceptibility: An Analysis of Bioinformatics-Based Case-Control Study and Functional Validation.

PurposeGenome-wide association studies have identified numerous genetic variants that are associated with osteoporosis risk; however, most of them are present in the non-coding regions of the genome and the functional mechanisms are unknown. In this study, we aimed to investigate the potential variation in runt domain transcription factor 2 (RUNX2), which is an osteoblast-specific transcription factor that normally stimulates bone formation and osteoblast differentiation, regarding variants within RUNX2 binding sites and risk of osteoporosis in postmenopausal osteoporosis (PMOP).MethodsWe performed bioinformatics-based prediction by combining whole genome sequencing and chromatin immunoprecipitation sequencing to screen functional SNPs in the RUNX2 binding site using data from the database of Taiwan Biobank; Case-control studies with 651 postmenopausal women comprising 107 osteoporosis patients, 290 osteopenia patients, and 254 controls at Tri-Service General Hospital between 2015 and 2019 were included. The subjects were examined for bone mass density and classified into normal and those with osteopenia or osteoporosis by T-scoring with dual-energy X-ray absorptiometry. Furthermore, mRNA expression and luciferase reporter assay were used to provide additional evidence regarding the associations identified in the association analyses. Chi-square tests and logistic regression were mainly used for statistical assessment.ResultsThrough candidate gene approaches, 3 SNPs in the RUNX2 binding site were selected. A novel SNP rs6086746 in the PLCB4 promoter was identified to be associated with osteoporosis in Chinese populations. Patients with AA allele had higher risk of osteoporosis than those with GG+AG (adjusted OR = 6.89; 95% confidence intervals: 2.23–21.31, p = 0.001). Moreover, the AA genotype exhibited lower bone mass density (p < 0.05). Regarding mRNA expression, there were large differences in the correlation between PLCB4 and different RUNX2 alleles (Cohen’s q = 0.91). Functionally, the rs6086746 A allele reduces the RUNX2 binding affinity, thus enhancing the suppression of PLCB4 expression (p < 0.05).ConclusionsOur results provide further evidence to support the important role of the SNP rs6086746 in the etiology of osteopenia/osteoporosis, thereby enhancing the current understanding of the susceptibility to osteoporosis. We further studied the mechanism underlying osteoporosis regulation by PLCB4.

Biological properties of Zn–0.04Mg–2Ag: a new degradable zinc alloy scaffold for repairing large-scale bone defects

Treatment of large-scale bone defects remains a clinical challenge. In particular, it preferably requires an implantable material with porous, absorbable, biocompatible, and antibacterial activities. Here we report the mechanical and biological properties of a porous Zn–0.04Mg–2Ag (wt%) biodegradable zinc alloy scaffold, prepared by template replication technique. In the aspect of cytocompatibility, cytoskeleton staining of stem cells co-cultured with Zn–0.04Mg–2Ag showed that the outline of cells and nuclei was clearly visible, and the interwoven network of linear fibers was abundant in the cells, which revealed lower cytotoxicity and less effect on cell proliferation. In the aspect of antibacterial properties, Zn–0.04Mg–2Ag alloy had little antibacterial effect on Escherichia coli , but had obvious antibacterial ability against Staphylococcus aureus and Staphylococcus epidermidis . In the aspect of bone induction, alizarin red staining and the expression of alkaline phosphatase showed no obvious increase; However, Zn–0.04Mg–2Ag alloy could significantly up-regulate the mRNA expression of osteoblast-specific transcription factors, osteocalcin and osteopontin. Zn–0.04Mg–2Ag degradable zinc alloy scaffold had excellent biocompatibility, and has significant antibacterial activity and fine osteogenic induction, which was an ideal bone substitute material for repairing large-scale bone defects.

Identification of a Novel Splice Site Mutation in RUNX2 Gene in a Family with Rare Autosomal Dominant Cleidocranial Dysplasia.

Background:Pathogenic variants of RUNX2, a gene that encodes an osteoblast-specific transcription factor, have been shown as the cause of CCD, which is a rare hereditary skeletal and dental disorder with dominant mode of inheritance and a broad range of clinical variability. Due to the relative lack of clinical complications resulting in CCD, the medical diagnosis of this disorder is challenging, which leaves it underdiagnosed. Methods:In this study, nine healthy and affected members of an Iranian family were investigated. PCR and sequencing of all exons and exon-intron boundaries of RUNX2 (NM_001024630) gene was performed on proband. Co-segregation analysis was conducted in the other family members for the identified variant. Additionally, a cohort of 100 Iranian ethnicity-matched healthy controls was screened by ARMS-PCR method. Results:The novel splice site variant (c.860-2A>G), which was identified in the intron 6 of RUNX2 gene, co-segregated with the disease in the family, and it was absent in healthy controls. Pathogenicity of this variant was determined by several software, including HSF, which predicts the formation or disruption of splice donor sites, splice acceptor sites, exonic splicing silencer sites, and exonic splicing enhancer sites. In silico analysis predicted this novel variant to be disease causing. Conclusion:The identified variant is predicted to have an effect on splicing, which leads to exon skipping and producing a truncated protein via introducing a premature stop codon.

Recent Advances of Osterix Transcription Factor in Osteoblast Differentiation and Bone Formation.

With increasing life expectations, more and more patients suffer from fractures either induced by intensive sports or other bone-related diseases. The balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is the basis for maintaining bone health. Osterix (Osx) has long been known to be an essential transcription factor for the osteoblast differentiation and bone mineralization. Emerging evidence suggests that Osx not only plays an important role in intramembranous bone formation, but also affects endochondral ossification by participating in the terminal cartilage differentiation. Given its essentiality in skeletal development and bone formation, Osx has become a new research hotspot in recent years. In this review, we focus on the progress of Osx’s function and its regulation in osteoblast differentiation and bone mass. And the potential role of Osx in developing new therapeutic strategies for osteolytic diseases was discussed.

Runt-related transcription factor-2 (Runx2) is required for bone matrix protein gene expression in committed osteoblasts in mice.

Runt-related transcription factor-2 (Runx2) is an essential transcription factor for osteoblast differentiation. However, its functions after the commitment into osteoblasts are controversial and remain to be clarified. We generated enhanced green fluorescent protein (EGFP)-Cre transgenic mice driven by the 2.3-kilobase (kb) Col1a1 promoter, and Runx2 was deleted in osteoblasts and odontoblasts in Runx2fl/flCre mice. The sutures and fontanelles were more widely opened in Runx2fl/flCre newborns than in Runx2fl/fl newborns. Runx2fl/flCre mice exhibited dwarfism with shorter incisors and 37% had irregularly aligned incisors. The volume of trabecular bone in femurs and vertebrae and their bone mineral density (BMD), in addition to the cortical thickness and BMD were reduced in Runx2fl/flCre mice compared with Runx2fl/fl mice in both sexes. The bone formation of both trabecular and cortical bone, osteoblast number, osteoclast surface, osteoblast proliferation, and the serum levels of procollagen type 1 N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase 5b (TRAP5b), and C-terminal cross-linked telopeptide of type 1 collagen (CTX1) were reduced in Runx2fl/flCre mice. The expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap&Bglap2, and of Tnfsf11 was lower in Runx2fl/flCre mice than in Runx2fl/fl mice. The expression of Runx2 target genes, including Ihh, Fgfr1, Fgfr2, Fgfr3, Tcf7, Wnt10b, Pth1r, Sp7, and Dlx5, was also reduced. Osteoblasts in Runx2fl/fl mice were cuboidal and contained abundant type I collagen α1 (Col1a1), whereas those in Runx2fl/flCre mice were deflated and contained a small amount of Col1a1. Runx2 activated the reporter activity of the 2.3-kb Col1a1 promoter and bound the region around the Col1a1 transcription start site. The deletion of Runx2 by Cre-expressing adenovirus in Runx2fl/fl primary osteoblasts impaired osteoblast differentiation and the expression of genes encoding major bone matrix proteins, and osteoclastogenesis was inhibited due to the reduction of Tnfsf11 expression in the osteoblasts. This study demonstrated that Runx2 is required for the expression of the major bone matrix protein genes and Tnfsf11 after commitment into osteoblasts in mice. © 2021 American Society for Bone and Mineral Research (ASBMR).

Shengu'an exerts anti-osteoporotic effect in rats via TGFβ1-Smad2/3 signal pathway, and enhancement of bone and cartilage metabolism

Purpose: To study the anti-osteoporotic effect of Shengu'an in rats, and elucidate the mechanism of action involved.Methods: Forty healthy female SPF mice were randomly divided into control group, saline-treated group, TGFβRⅡ receptor inhibitor group, and shengu'an group. The expressions of type Ⅱ collagen (Co1-II) and platelet endothelial cell adhesion factor (CD-31) were determined. The expressions of transforming growth factor β1 (TGF-β1), p-smad2/3, matrix metalloproteinase-9 (MMP-9) and osteoblast specific transcription factor (osterix) were assayed by western blotting.Results: The expression of Co1-II in the vertebral body was significantly lower in model mice than in control mice, but was significantly higher in shengu'an mice when compared with model mice (p &lt; 0.05). In shengu'an mice, CoI-I was markedly upregulated, relative to model mice, and the expressions of CD31 in TGFβRⅡreceptor inhibitor group and shengu'an group were lower than in model group (p &lt; 0.05). There were significantly lower expressions of TGF-β1 and p-smad2/3 in the vertebral body of shengu'an group than in model mice, but osterix was upregulated relative to model mice (p &lt; 0.05).Conclusion: Shengu'an exerts anti-osteoporotic effect by downregulating TGFβ/smad signal pathway. There is thus a potential for its clinical application in the management of osteoporosis.&#x0D; Keywords: Shengu'an, TGFβ1-Smad2/3 signal, Bone cartilage metabolism, Osteoporosis

The Study of Signal Pathway Regulating the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

Osteogenesis of mesenchymal stem cells to differentiate between bone marrow by multiple signaling pathways that control, directly or indirectly affect small related transcription factor 2 (runt-related transcription factor 2, Runx2) and osteoblast specific transcription factor (osterix, Osx), the expression of osteogenesis key transcription factors, such as in the development and regeneration of the bone, bone repair has played a key role in the process of reconstruction. These pathways play their mechanism of action, but also intertwined associated constitute a complex signal transduction network, but due to the limitations of research methods, the osteogenic differentiation related signaling pathways of the specific mechanism is still unclear, if you can clarify these different signaling pathways play to the role of their relevant mechanism and the relationship between various pathways and the mechanism study of osteogenesis differentiation is of great importance. This article will review the progress of various signaling pathways in the regulation of osteogenic differentiation of bone marrow mesenchymal stem cells.

MgCl2 promotes mouse mesenchymal stem cell osteogenic differentiation by activating the p38/Osx/Runx2 signaling pathway.

Magnesium, an important inorganic mineral component in bones, enhances osteoblast adhesion and osteogenic gene expression. Mg2+-containing hydroxyapatite promotes mouse mesenchymal stem cell (MMSC) osteogenic differentiation. In the present study, MMSCs were cultured in media containing different concentrations of MgCl2 (0 and 20 mM) for different time periods. Western blotting and reverse transcription-quantitative PCR were performed to determine the expression levels of phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK), the osteoblast-specific transcription factor Osterix (Osx), runt-related transcription factor 2 (Runx2), and p38 downstream genes, such as 27 kDa heat shock protein (hsp27), activating transcription factor 4 (Atf4), myocyte enhancer factor 2C (Mef2c) and CCAAT/enhancer-binding protein homologous protein (Ddit3). The facilitatory effect of MgCl2 on MMSC osteogenic differentiation was assessed via Alizarin Red staining. The results suggested that MgCl2 increased p38 phosphorylation compared with the control group. Downstream genes of the p38 signaling pathway, including Osx and Runx2, as well as several osteogenesis-associated downstream target genes, including Hsp27, Atf4, Ddit3 and Mef2c, were significantly upregulated in the Mg2+-treated group compared with the control group. The increased osteogenic differentiation in the Mg2+-treated group was significantly attenuated in MMSCs treated with SB203580, a specific inhibitor of the p38 signaling pathway. The results suggested that appropriate concentrations of MgCl2 promoted MMSC osteogenic differentiation via regulation of the p38/Osx/Runx2 signaling pathway.

Hdac9 inhibits medial artery calcification through down-regulation of Osterix

BackgroundsMedial artery calcification (MAC) significantly contributes to the increased cardiovascular death in patients with chronic kidney disease (CKD). Previous genome-wide association studies have shown that various genetic variants of the histone deacetylase Hdac9 are associated with cardiovascular disease, but the role of Hdac9 in MAC under CKD conditions remains unclear. MethodsHigh phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD) were used in the present study. Alizarin red staining, calcium quantitative assay, qPCR, western blotting and histology were performed. ResultsHdac9 expression was significantly down-regulated during high phosphate-induced vascular smooth muscle cell (VSMC) calcification and MAC in mice administered with vitamin D3 (vD). Furthermore, high phosphate treatment inhibited phosphorylation of Akt, and pharmacological inhibition of Akt signaling reduced Hdac9 expression in cultured VSMCs. Knockdown of Hdac9 significantly enhanced calcium deposition in VSMCs. Conversely, adenovirus mediated-overexpression of Hdac9 inhibited high phosphate induced VSMC in vitro calcification. Our subsequent mechanistic studies revealed that the anti-calcific effect of Hdac9 was mediated through down-regulation of osteoblast-specific transcription factor Osterix. ConclusionThese data suggest that Hdac9 is a novel inhibitor of MAC and may represent a potential therapeutic target for MAC in CKD patients.

Delayed tooth movement in Runx2+/− mice associated with mTORC2 in stretch-induced bone formation

Runt-related transcription factor 2 (Runx2) is an essential transcription factor for osteoblast differentiation, and is activated by mechanical stress to promote osteoblast function. Cleidocranial dysplasia (CCD) is caused by mutations of RUNX2, and CCD patients exhibit malocclusion and often need orthodontic treatment. However, treatment is difficult because of impaired tooth movement, the reason of which has not been clarified. We examined the amount of experimental tooth movement in Runx2+/− mice, the animal model of CCD, and investigated bone formation on the tension side of experimental tooth movement in vivo. Continuous stretch was conducted to bone marrow stromal cells (BMSCs) as an in vitro model of the tension side of tooth movement. Compared to wild-type littermates the Runx2+/− mice exhibited delayed experimental tooth movement, and osteoid formation and osteocalcin (OSC) mRNA expression were impaired in osteoblasts on the tension side of tooth movement. Runx2 heterozygous deficiency delayed stretch-induced increase of DNA content in BMSCs, and also delayed and reduced stretch-induced alkaline phosphatase (ALP) activity, OSC mRNA expression, and calcium content of BMSCs in osteogenic medium. Furthermore Runx2+/− mice exhibited delayed and suppressed expression of mammalian target of rapamycin (mTOR) and rapamycin-insensitive companion of mTOR (Rictor), essential factors of mTORC2, which is regulated by Runx2 to phosphorylate Akt to regulate cell proliferation and differentiation, in osteoblasts on the tension side of tooth movement in vivo and in vitro. Loss of half Runx2 gene dosage inhibited stretch-induced PI3K dependent mTORC2/Akt activity to promote BMSCs proliferation. Furthermore, Runx2+/− BMSCs in osteogenic medium exhibited delayed and suppressed stretch-induced expression of mTOR and Rictor. mTORC2 regulated stretch-elevated Runx2 and ALP mRNA expression in BMSCs in osteogenic medium. We conclude that Runx2+/− mice present a useful model of CCD patients for elucidation of the molecular mechanisms in bone remodeling during tooth movement, and that Runx2 plays a role in stretch-induced proliferation and osteogenesis in BMSCs via mTORC2 activation.