Runx2 Transcriptional Activity Research Articles

The correlated evolution of Runx2 tandem repeats, transcriptional activity, and facial length in Carnivora

To assess the ability of protein-coding mutations to contribute to subtle, inter-specific morphologic evolution, here, we test the hypothesis that mutations within the protein-coding region of runt-related transcription factor 2 (Runx2) have played a role in facial evolution in 30 species from a naturally evolving group, the mammalian order Carnivora. Consistent with this hypothesis, we find significant correlations between changes in Runx2 glutamine-alanine tandem-repeat ratio, and both Runx2 transcriptional activity and carnivoran facial length. Furthermore, we identify a potential evolutionary mechanism for the correlation between Runx2 tandem repeat ratio and facial length. Specifically, our results are consistent with the Runx2 tandem repeat system providing a flexible genetic mechanism to rapidly change the timing of ossification. These heterochronic changes, in turn, potentially act on existing allometric variation in carnivoran facial length to generate the disparity in adult facial lengths observed among carnivoran species. Our results suggest that despite potentially great pleiotropic effects, changes to the protein-coding regions of genes such as Runx2 do occur and have the potential to affect subtle morphologic evolution across a diverse array of species in naturally evolving lineages.

P68 (Ddx5) interacts with Runx2 and regulates osteoblast differentiation

Runx2 is an essential transcription factor for osteoblast development from mesenchymal progenitors. Runx2 regulates gene expression by interacting with numerous transcription factors and co-activators to integrate signaling events within the nucleus. In this study we used affinity purification and proteomic techniques to identify novel Runx2 interacting proteins. One of these proteins is the DEAD box RNA helicase, p68 (Ddx5). p68 regulates many aspects of RNA expression, including transcription and splicing. p68 co-localized with Runx2 in punctate foci within the nucleus. In transcription assays, p68 functioned as a co-activator of Runx2, but its helicase activity was not essential for co-activation. In accordance, Runx2 transcriptional activity was muted in p68-suppressed cells. Surprisingly, osteoblast differentiation of the multipotent progenitor C2C12 cell line was accelerated by p68 suppression and Runx2 suppressed p68 expression in calvarial progenitor cells. Together these data demonstrate that p68 is a novel co-activator for Runx2, but it inhibits osteogenic differentiation of progenitor cells. Moreover Runx2 has an active role in regulating p68 levels in osteoblast precursors. Thus, crosstalk between Runx2 and p68 controls osteoblast specification and maturation at multiple levels.

Inductive effects of dexamethasone on the mineralization and the osteoblastic gene expressions in mature osteoblast-like ROS17/2.8 cells

We examined the effects of dexamethasone (Dex), a synthetic glucocorticoid, on the formation of mineralized bone nodules and the gene expressions of the late osteoblastic markers, bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) in mature osteoblast ROS17/2.8 cells. Treatment of ROS17/2.8 cells with Dex resulted in the induction of mineralization accompanied with increasing BSP and OC expressions. Previous reports have demonstrated that BSP and OC expressions are regulated by Runx2. Then, we hypothesized that Dex might promote osteoblastic differentiation and mineralization on ROS17/2.8 by Runx2. In this study, no effect was observed in mRNA and protein expression of Runx2. However, the transcriptional activity of Runx2 was enhanced by Dex treatment. Furthermore, the Dex-induced BSP and OC expressions decreased after the transfection of Runx2 small-interfering RNAs (siRNAs). These results suggested that the enhancement of Runx2 transcriptional activity by Dex treatment may be followed by the activation of osteoblast marker genes, such as BSP and OC to thereby produce a bone-specific matrix that subsequently becomes mineralized.

O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2

O-Linked β- N-acetylglucosamine ( O-GlcNAc) modification, a reversible post-translational modification, has been implicated in the regulation of protein stability, subcellular localization of proteins and protein–protein interaction. Here, we demonstrate that O-GlcNAc modification regulates the expression of osteocalcin, an osteoblast-specific marker, via Runx2 transcriptional activity in osteoblastic differentiation. Protein-associated O-GlcNAc was increased during osteoblastic differentiation in MC3T3-E1 preosteoblasts. In addition, PUGNAc, an inhibitor of O-GlcNAcase, potentiated the expression of osteocalcin caused by ascorbic acid, parathyroid hormone (PTH) and forskolin. By conducting activity assays of the osteocalcin promoter and transcription factor, we found that the OSE2 site in the osteocalcin promoter and Runx2 were important for increased osteocalcin promoter activity by PUGNAc. Furthermore, PUGNAc led to increased O-GlcNAc modification of Runx2, which regulated the transcription of its target gene osteocalcin. Thus, these data provide evidence that O-GlcNAc modification may be a new mode of osteoblastic differentiation regulation.

Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development

The extracellular signal–regulated kinase (ERK)–mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/− animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/− mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK–MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

Osteoblast response to titanium regulates transcriptional activity of Runx2 through MAPK pathway

Bone formation around an implant surface is indispensable for osseointegration of dental implants. Transcriptional factor Runx2 is essential for osteoblast differentiation and bone formation. However, little is known about the involvement of Runx2 in osseointegration. The purpose of this study was to investigate molecular interactions between Runx2 and implant titanium materials at the transcriptional level. Primary osteoblasts isolated from mouse calvaria and C3H10T1/2 cells were cultured on titanium plates or in the presence of titanium particles. Transcriptional activity of Runx2 was measured by reporter assay using osteocalcin gene promoter. We found significant increase (p < 0.05) in Runx2 transcriptional activity in cells cultured on titanium plates compared to plastic plates. Titanium particles also upregulated Runx2 transcriptional activity for over 2-folds compared to control and this effect was abolished by the specific inhibitor for ERK1/2, PD98059. Moreover, treatment with PD98059 clearly suppressed osteoblast mineralization cultured on titanium plates. These data suggest that osteoblast attachment to titanium enhanced Runx2 transcriptional activity and bone formation via MAPK pathway during the osseointegration of dental implants.

The mechanisms of uremic serum-induced expression of bone matrix proteins in bovine vascular smooth muscle cells

We have previously found that uremic human serum upregulates RUNX2 in vascular smooth muscle cells (VSMCs), and that RUNX2 is upregulated in areas of vascular calcification in vivo. To confirm the role of RUNX2, we transiently transfected a dominant-negative RUNX2 (DeltaRUNX2) construct in bovine vascular smooth muscle cells (BVSMCs). Blocking RUNX2 transcriptional activity significantly decreased uremic serum induced alkaline phosphatase (ALP) activity (268+/-34 vs 188+/-9.5 U/g protein, P<0.05) and osteocalcin expression (172+/-17 vs 125+/-9 ODU, P<0.05). To determine the mechanism by which uremic serum upregulates RUNX2, we examined cell signaling pathways. BVSMCs were incubated in the presence or absence of inhibitors and RUNX2 expression and ALP activity were determined. The results demonstrate that the cyclic AMP (cAMP)/protein kinase A (PKA), but not protein kinase C, signaling pathway is involved in uremic serum-induced RUNX2 expression and ALP activity in BVSMCs. To examine potential uremic 'toxins', we measured bone morphogenetic protein (BMP)-2 concentration and found that uremic serum contained increased BMP-2 (uremic serum=169+/-33 pg/ml, normal serum=117+/-15 pg/ml, P<0.05). The incubation of BVSMCs with noggin, an inhibitor of BMP, decreased RUNX2 expression. In addition, BMP-2 secretion progressively increased during calcification and uremic serum enhanced its secretion compared to normal serum. In conclusion, this study demonstrates that RUNX2 transcriptional activity is critical in uremic serum-induced bone matrix protein expression in BVSMCs and that the cAMP/PKA pathway is involved. BMP-2 is also increased in uremic serum and can upregulate RUNX2 and calcification in vitro in VSMCs.

Histone Deacetylase Inhibitors Promote Osteoblast Maturation

HDIs are potential therapeutic agents for cancer and neurological diseases because of their abilities to alter gene expression, induce growth arrest or apoptosis of tumors cells, and stimulate differentiation. In this report, we show that several HDIs promote osteoblast maturation in vitro and in calvarial organ cultures. Histone deacetylase inhibitors (HDIs) are currently in phase I and II clinical trials as anticancer agents. Some HDIs are also commonly prescribed treatments for epilepsy and bipolar disorders. Although administered systemically, the effects of HDIs on osteoblasts and bone formation have not been extensively examined. In this study, we investigated the effect of histone deacetylase inhibition on osteoblast proliferation and differentiation. MC3T3-E1 cells, calvarial-derived primary osteoblasts, and calvarial organ cultures were treated with various commercially available HDIs (trichostatin A [TSA], sodium butyrate [NaB], valproic acid [VPA], or MS-275). The effects of these inhibitors on cell proliferation, viability, cell cycle progression, Runx2 transcriptional activity, alkaline phosphatase production, and matrix mineralization were determined. Expression levels of osteoblast maturation genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in response to TSA were measured by quantitative PCR. Concentrations of HDIs that caused hyperacetylation of histone H3 induced transient increases in osteoblast proliferation and viability but did not alter cell cycle profiles. These concentrations of HDIs also increased the transcriptional activity of Runx2. TSA accelerated alkaline phosphatase production in MC3T3-E1 cells and calvarial organ cultures. In addition, TSA accelerated matrix mineralization and the expression of osteoblast genes, type I collagen, osteopontin, bone sialoprotein, and osteocalcin in MC3T3-E1 cells. These studies show that histone deacetylase activity regulates osteoblast differentiation and bone formation at least in part by enhancing Runx2-dependent transcriptional activation. Therefore, HDIs are a potentially new class of bone anabolic agents that may be useful in the treatment of diseases that are associated with bone loss such as osteoporosis and cancer.

Mutations in NOTCH1 cause aortic valve disease

Calcification of the aortic valve is the third leading cause of heart disease in adults. The incidence increases with age, and it is often associated with a bicuspid aortic valve present in 1-2% of the population. Despite the frequency, neither the mechanisms of valve calcification nor the developmental origin of a two, rather than three, leaflet aortic valve is known. Here, we show that mutations in the signalling and transcriptional regulator NOTCH1 cause a spectrum of developmental aortic valve anomalies and severe valve calcification in non-syndromic autosomal-dominant human pedigrees. Consistent with the valve calcification phenotype, Notch1 transcripts were most abundant in the developing aortic valve of mice, and Notch1 repressed the activity of Runx2, a central transcriptional regulator of osteoblast cell fate. The hairy-related family of transcriptional repressors (Hrt), which are activated by Notch1 signalling, physically interacted with Runx2 and repressed Runx2 transcriptional activity independent of histone deacetylase activity. These results suggest that NOTCH1 mutations cause an early developmental defect in the aortic valve and a later de-repression of calcium deposition that causes progressive aortic valve disease.

Menin Is Required for Bone Morphogenetic Protein 2- and Transforming Growth Factor β-regulated Osteoblastic Differentiation through Interaction with Smads and Runx2

Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) gene, is required for commitment of multipotential mesenchymal stem cells to the osteoblast lineage, however, it inhibits their later differentiation (Sowa, H., Kaji, H., Canaff, L., Hendy, G.N., Tsukamoto, T., Yamaguchi, T., Miyazono, K., Sugimoto, T., and Chihara, K. (2003) J. Biol. Chem. 278, 21058-21069). Here, we have examined the mechanism of action of menin in regulating osteoblast differentiation using the mouse bone marrow stromal ST2 and osteoblast MC3T3-E1 cell lines. In ST2 cells, reduced menin expression achieved by transfection of menin antisense DNA (AS) antagonized bone morphogenetic protein (BMP)-2-induced alkaline phosphatase activity and osteocalcin and Runx2 mRNA expression. Menin was co-immunoprecipitated with Smad1/5 in ST2 and MC3T3-E1 cells, and inactivation of menin antagonized BMP-2-induced transcriptional activity of Smad1/5 in ST2 cells, but not MC3T3-E1 cells. Menin was co-immunoprecipitated with the key osteoblast regulator, Runx2, and AS antagonized Runx2 transcriptional activity and the ability of Runx2 to stimulate alkaline phosphatase activity only in ST2 cells but not in MC3T3-E1 cells. In the osteoblast MC3T3-E1 cells, transforming growth factor-beta and its signaling molecule, Smad3, negatively regulated Runx2 transcriptional activity. Menin and Smad3 were co-immunoprecipitated, and combined menin and Smad3 overexpression antagonized, whereas menin and the dominant-negative Smad3DeltaC together enhanced BMP-2-induced transcriptional activity of Smad1/5 and Runx2. Smad3 alone had no effect. Therefore, menin interacts physically and functionally with Runx2 in uncommitted mesenchymal stem cells, but not in well differentiated osteoblasts. In osteoblasts the interaction of menin and the transforming growth factor-beta/Smad3 pathway negatively regulates the BMP-2/Smad1/5- and Runx2-induced transcriptional activities leading to inhibition of late-stage differentiation.

Coordinated Activation of Notch, Wnt, and Transforming Growth Factor-β Signaling Pathways in Bone Morphogenic Protein 2-induced Osteogenesis: Notch TARGET GENE Hey1 INHIBITS MINERALIZATION AND Runx2 TRANSCRIPTIONAL ACTIVITY

To examine early events in osteoblast differentiation, we analyzed the expression of about 9,400 genes in the murine MC3T3 cell line, whose robust differentiation was documented cytochemically and molecularly. The cells were stimulated for 1 and 3 days with the osteogenic stimulus containing bone morphogenic protein 2. Total RNA was extracted and analyzed by Affymetrix GeneChip oligonucleotide arrays. A regulated expression of 394 known genes and 295 expressed sequence tags was detected. The sensitivity and reliability of detection by microarrays was shown by confirming the expression pattern for 20 genes by radioactive quantitative reverse transcription-PCR. Functional classification of regulated genes was performed, defining the groups of regulated growth factors, receptors, and transcription factors. The most interesting finding was concomitant activation of transforming growth factor-beta, Wnt, and Notch signaling pathways, confirmed by strong up-regulation of their target genes by PCR. The transforming growth factor-beta pathway is activated by stimulated production of the growth factor itself, while the exact mechanism of Wnt and Notch activation remains elusive. We showed that bone morphogenic protein 2 stimulated expression of Hey1, a direct Notch target gene, in mouse MC3T3 and C2C12 cells, in human mesenchymal cells, and in mouse calvaria. Small interfering RNA-mediated inhibition of Hey1 induction led to an increase in osteoblast matrix mineralization, suggesting that Hey1 is a negative regulator of osteoblast maturation. This negative regulation is apparently achieved via interaction with Runx2: Hey1 completely abrogated Runx2 transcriptional activity. These findings identify the Notch-Hey1 pathway as a negative regulator of osteoblast differentiation/maturation, which is a completely novel aspect of osteogenesis and could point to possible new targets for bone anabolic agents.

Establishment and characterization of a stable cell line to evaluate cellular Runx2 activity.

Runx2 is an essential transcription factor for osteoblast differentiation from early commitment step to final differentiation. Based on its crucial role in osteoblast differentiation, the transcriptional activity of Runx2 protein implies more valuable information for osteoblast differentiation than any other parameters, such as Runx2 mRNA or protein level. Thus, a sensitive, specific, and consistent method to determine the Runx2 transcriptional activity has long been expected. Here we suggest a stable cell line that carries 6xOSE2-Luciferase reporter vector would be a good evaluation system to determine biological Runx2 transcriptional activity. The proliferation rate, cell shape, and the myogenic differentiation potential of the cloned cell line were similar to those of parental premyoblastic C2C12 cells. The cells specifically responded to Runx2 modulating agent such as FGF2. The stable cell line responded 5-6 folds more sensitively than the transiently transfected cells with Runx2. Though overexpression of any Runx gene stimulated the luciferase activity, Runx2 enhanced the reporter activity the highest. Collectively, the 6xOSE2-luc stable cells would be a good biological evaluation system to assess the activity of extracellular Runx2 modulating stimulations as well as the signal transduction pathways involved in the stimulations.

Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription.

Src/Yes tyrosine kinase signaling contributes to the regulation of bone homeostasis and inhibits osteoblast activity. Here we show that the endogenous Yes-associated protein (YAP), a mediator of Src/Yes signaling, interacts with the native Runx2 protein, an osteoblast-related transcription factor, and suppresses Runx2 transcriptional activity in a dose-dependent manner. Runx2, through its PY motif, recruits YAP to subnuclear domains in situ and to the osteocalcin (OC) gene promoter in vivo. Inhibition of Src/Yes kinase blocks tyrosine phosphorylation of YAP and dissociates endogenous Runx2-YAP complexes. Consequently, recruitment of the YAP co-repressor to subnuclear domains is abrogated and expression of the endogenous OC gene is induced. Our results suggest that Src/Yes signals are integrated through organization of Runx2-YAP transcriptional complexes at subnuclear sites to attenuate skeletal gene expression.

Effect of Rehmannia glutinosa Libosch extracts on bone metabolism

Background: Rehmannia glutinosa Libosch extracts (RGX) were investigated to determine if they play roles in bone metabolism. Methods: The effects on osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) mRNA expression of bone-related proteins, (4) transcriptional activity of Runx2, and (5) osteoprotegerin (OPG) secretion. The effects on the osteoclasts were investigated by measuring (1) tartrate-resistant acid phosphatase-positive [TRAP(+)] multinucleated cell (MNC) formation and (2) resorption areas after culturing osteoclast precursors. Bone mineral density (BMD) measurements and histological observations on rats were also carried out. Results: RGX treatment showed a significant increase in both the proliferation and ALP activity of osteoblasts. RGX increased the expression of the bone-related genes. OPG secretion was markedly increased after RGX treatment. In addition, RGX treatment decreased the number of TRAP(+) MNCs and the resorption areas. In vivo studies using ovariectomy-induced osteoporotic rats revealed that RGX alleviated the decrease in the trabecular BMD, and increased the cortical bone thickness and trabeculation of the bone marrow spaces. Conclusions: RGX stimulates the proliferation and activities of osteoblasts, while inhibiting the generation and resorptive activities of osteoclasts. It also shows preventive effects on osteoporotic bone loss induced by an ovariectomy. Although the active substances have not yet been identified, it is believed that the RGX seems to contain active components that have a potential to enhance the bone metabolism in osteoporosis.

Lymphoid Enhancer Factor-1 and ॆ-Catenin Inhibit Runx2-dependent Transcriptional Activation of the Osteocalcin Promoter

Functional control of the transcription factor Runx2 is crucial for normal bone formation. Runx2 is detectable throughout osteoblast development and maturation and temporally regulates several bone-specific genes. In this study, we identified a novel post-translational mechanism regulating Runx2-dependent activation of the osteocalcin promoter. A functional binding site for the high mobility group protein lymphoid enhancer-binding factor 1 (LEF1) was found adjacent to the proximal Runx2-binding site in the osteocalcin promoter. In transcription assays, LEF1 repressed Runx2-induced activation of the mouse osteocalcin 2 promoter in several osteoblast lineage cell lines. Mutations in the LEF1-binding site increased the basal activity of the osteocalcin promoter; however, the LEF1 recognition site in the osteocalcin promoter was surprisingly not required for LEF1 repression. A novel interaction between the DNA-binding domains of Runx2 and LEF1 was identified and found crucial for LEF1-mediated repression of Runx2. LEF1 is a nuclear effector of the Wnt/LRP5/beta-catenin signaling pathway, which is also essential for osteoblast proliferation and normal skeletal development. A constitutively active beta-catenin enhanced LEF1-dependent repression of Runx2. These data identify a novel mechanism of regulating Runx2 activity in osteoblasts and link Runx2 transcriptional activity to beta-catenin signaling.