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Runx2 is essential for osteoblast differentiation and chondrocyte maturation. During osteoblast differentiation, Runx2 is weakly expressed in uncommitted mesenchymal cells, and its expression is upregulated in preosteoblasts, reaches the maximal level in immature osteoblasts, and is down-regulated in mature osteoblasts. Runx2 enhances the proliferation of osteoblast progenitors by directly regulating Fgfr2 and Fgfr3. Runx2 enhances the proliferation of suture mesenchymal cells and induces their commitment into osteoblast lineage cells through the direct regulation of hedgehog (Ihh, Gli1, and Ptch1), Fgf (Fgfr2 and Fgfr3), Wnt (Tcf7, Wnt10b, and Wnt1), and Pthlh (Pthr1) signaling pathway genes, and Dlx5. Runx2 heterozygous mutation causes open fontanelle and sutures because more than half of the Runx2 gene dosage is required for the induction of these genes in suture mesenchymal cells. Runx2 regulates the proliferation of osteoblast progenitors and their differentiation into osteoblasts via reciprocal regulation with hedgehog, Fgf, Wnt, and Pthlh signaling molecules, and transcription factors, including Dlx5 and Sp7. Runx2 induces the expression of major bone matrix protein genes, including Col1a1, Spp1, Ibsp, Bglap2, and Fn1, in vitro. However, the functions of Runx2 in differentiated osteoblasts in the expression of these genes in vivo require further investigation.

Runt-related transcription factor 2 (Runx2) is a fundamental transcription factor for bone development. In endochondral ossification, Runx2 induces chondrocyte maturation, enhances chondrocyte proliferation through Indian hedgehog (Ihh) induction, and induces the expression of vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) in the terminal hypertrophic chondrocytes. Runx2 inhibits the apoptosis of the terminal hypertrophic chondrocytes and induces their transdifferentiation into osteoblasts and osteoblast progenitors. The transdifferentiation is required for trabecular bone formation during embryonic and newborn stages but is dispensable for acquiring normal bone mass in young and adult mice. Runx2 enhances the proliferation of osteoblast progenitors and induces their commitment to osteoblast lineage cells through the direct regulation of the expressions of a hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathway genes and distal-less homeobox 5 (Dlx5), which all regulate Runx2 expression and/or protein activity. Runx2, Sp7, and Wnt signaling further induce osteoblast differentiation. In immature osteoblasts, Runx2 regulates the expression of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and induces osteoblast maturation. Osteocalcin (Bglap/Bglap2) is required for the alignment of apatite crystals parallel to the collagen fibers; however, it does not physiologically work as a hormone that regulates glucose metabolism, testosterone synthesis, or muscle mass. Thus, Runx2 exerts multiple functions essential for skeletal development.

Dental follicles are necessary for tooth eruption, surround the enamel organ and dental papilla, and regulate both the formation and resorption of alveolar bone. Dental follicle progenitor cells (DFPCs), which are stem cells found in dental follicles, differentiate into different kinds of cells that are necessary for tooth formation and eruption. Runt‐related transcription factor 2 (Runx2) is a transcription factor that is essential for osteoblasts and osteoclasts differentiation, as well as bone remodeling. Mutation of Runx2 causing cleidocranial dysplasia negatively affects osteogenesis and the osteoclastic ability of dental follicles, resulting in tooth eruption difficulties. Among a variety of cells and molecules, Nel-like molecule type 1 (Nell-1) plays an important role in neural crest-derived tissues and is strongly expressed in dental follicles. Nell-1 was originally identified in pathologically fused and fusing sutures of patients with unilateral coronal synostosis, and it plays indispensable roles in bone remodeling, including roles in osteoblast differentiation, bone formation and regeneration, craniofacial skeleton development, and the differentiation of many kinds of stem cells. Runx2 was proven to directly target the Nell-1 gene and regulate its expression. These studies suggested that Runx2/Nell-1 axis may play an important role in the process of tooth eruption by affecting DFPCs. Studies on short and long regulatory noncoding RNAs have revealed the complexity of RNA-mediated regulation of gene expression at the posttranscriptional level. This ceRNA network participates in the regulation of Runx2 and Nell-1 gene expression in a complex way. However, non-study indicated the potential connection between Runx2 and Nell-1, and further researches are still needed.

Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells.BACKGROUND/AIMSRunt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells.Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation.METHODSOverexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation.Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression.RESULTSOverexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression.We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.CONCLUSIONWe demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.

Protein phosphatase magnesium‐dependent 1A (PPM1A), serine/threonine protein phosphatase, in sera level was increased in patients with ankylosing spondylitis (AS). Preosteoblasts were differentiated actively to matured osteoblasts by intracellular PPM1A overexpression. However, it was unclear whether extracellular PPM1A contributes to the excessive bone‐forming activity in AS. Here, we confirmed that PPM1A and runt‐related transcription factor 2 (RUNX2) were increased in facet joints of AS. During osteoblasts differentiation, exogenous PPM1A treatment showed increased matrix mineralization in AS‐osteoprogenitor cells accompanied by induction of RUNX2 and factor forkhead box O1A (FOXO1A) protein expressions. Moreover, upon growth condition, exogenous PPM1A treatment showed an increase in RUNX2 and FOXO1A protein expression and a decrease in phosphorylation at ser256 of FOXO1A protein in AS‐osteoprogenitor cells, and positively regulated promoter activity of RUNX2 protein‐binding motif. Mechanically, exogenous PPM1A treatment induced the dephosphorylation of transcription factor FOXO1A protein and translocation of FOXO1A protein into the nucleus for RUNX2 upregulation. Taken together, our results suggest that high PPM1A concentration promotes matrix mineralization in AS via the FOXO1A‐RUNX2 pathway.

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM). Here we characterise a novel mechanism involving a combined activation of YAP targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD). Using collagen-coated polyacrylamide hydrogels of tuneable stiffness, we demonstrate that cardiac fibroblast proliferation, quantified using Edu incorporation, is enhanced by interaction with a stiff (50 kPa) ECM compared to a soft (0.5 kPa) ECM (7.02±0.77% on soft vs 33.10±0.52% on stiff; p<0.01). This is associated with activation of the transcriptional co-factor, YAP, indicated by reduced phosphorylation and increased nuclear localisation in cells cultured on stiff substrates. Reporter gene assays demonstrate that stiffness induced activation of YAP significantly enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Overexpression of an activate YAP mutant significantly enhanced TEAD and RUNX2 activity, whereas YAP silencing significantly reduced TEAD and RUNX2 activity. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation. Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

Background Osteoporosis (OP) is a progressive metabolic disorder that is difficult to cure clinically. The molecular mechanisms of OP urgently need to be further examined. This study was designed to explore the potential function of circ_0027885 during osteogenic differentiation, as well as the systematic interactions among circ_0027885, miR-203-3p and runt-related transcription factor 2 (RUNX2). Methods Relative levels of circ_0027885, miR-203-3p and RUNX2 were analyzed with RT-qPCR and western blotting. Alizarin red staining was performed to detect the mineralization ability under the control of circ_0027885 and miR-203-3p. Dual-luciferase reporter gene assay was conducted to examine the combination among circ_0027885, miR-203-3p and RUNX2. Results Our research demonstrated that circ_0027885 was significantly increased during hBMSCs differentiation. Overexpression of circ_0027885 notably facilitated osteogenic differentiation and upregulated RUNX2 expression, while knockdown of circ_0027885 reversed the above results. Through prediction on bioinformatics analysis, miR-203-3p was the target binding circ_0027885, and RUNX2 was the potential target of miR-203-3p. Subsequently, these changes induced by the overexpression of circ_0027885 were reversed upon addition of miR-203-3p mimic. Conclusions Circ_0027885 could sponge miR-203-3p to regulate RUNX2 expression and alleviate osteoporosis progression. Keywords: Circ_0027885, miR-203-3p, RUNX2, Osteoporosis, Osteogenic differentiation

Calcium deposition in vascular smooth muscle cells (VSMCs), a form of ectopic ossification in blood vessels, can result in rigidity of the vasculature and an increase in cardiac events. Here, we report that CCAAT/enhancer-binding protein beta (C/EBPβ) potentiates calcium deposition in VSMCs and mouse aorta induced by inorganic phosphate (Pi) or vitamin D3. Based on cDNA microarray and RNA sequencing data of Pi-treated rat VSMCs, C/EBPβ was found to be upregulated and thus selected for further evaluation. Quantitative RT-PCR and Western blot analysis confirmed that C/EBPβ was upregulated in Pi-treated A10 cells, a rat VSMC line, as well as vitamin D3-treated mouse aorta. The overexpression of C/EBPβ in A10 cells increased bone runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteopontin (OPN) mRNA in the presence of Pi, as well as potentiating the Pi-induced increase in calcium contents. The Runx2 expression was increased by C/EBPβ through Runx2 P2 promotor. Our results suggest that a Pi-induced increase in C/EBPβ is a critical step in vascular calcification.

Icariin is commonly used for the clinical treatment of osteonecrosis of the femoral head (ONFH). miR-23a-3p plays a vital role in regulating the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The present study aimed to investigate the roles of icariin and miR-23a-3p in the osteogenic differentiation of BMSCs and an ONFH model. BMSCs were isolated and cultured in vitro using icariin-containing serum at various concentrations, and BMSCs were also transfected with a miR-23a inhibitor. The alkaline phosphatase (ALP) activity and cell viability as well as BMP-2/Smad5/Runx2 and WNT/β-catenin pathway-related mRNA and protein expression were measured in BMSCs. Additionally, a dual-luciferase reporter assay and pathway inhibitors were used to verify the relationship of icariin treatment/miR-23a and the above pathways. An ONFH rat model was established in vivo, and a 28-day gavage treatment and lentivirus transfection of miR-23a-3p inhibitor were performed. Then, bone biochemical markers (ELISA kits) in serum, femoral head (HE staining and Digital Radiography, DR) and the above pathway-related proteins were detected. Our results revealed that icariin treatment/miR-23a knockdown promoted BMSC viability and osteogenic differentiation as well as increased the mRNA and protein expression of BMP-2, BMP-4, Runx2, p-Smad5, Wnt1 and β-catenin in BMSCs and ONFH model rats. In addition, icariin treatment/miR-23a knockdown increased bone biochemical markers (ACP-5, BAP, NTXI, CTXI and OC) and improved ONFH in ONFH model rats. In addition, a dual-luciferase reporter assay verified that Runx2 was a direct target of miR-23a-3p. These data indicated that icariin promotes BMSC viability and osteogenic differentiation as well as improves ONFH by decreasing miR-23a-3p levels and regulating the BMP-2/Smad5/Runx2 and WNT/β-catenin pathways.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.