Bone-related Transcription Factor Research Articles

Suppression of Bone-related Transcription Factors by TNF-α and IL-6 Reduced Alkaline Phosphatase Activities in Osteoblast-like Saos2 Cells

Suppression of Bone-related Transcription Factors by TNF-α and IL-6 Reduced Alkaline Phosphatase Activities in Osteoblast-like Saos2 Cells

A Review on the Enhancement of Calcium Phosphate Cement with Biological Materials in Bone Defect Healing.

Calcium phosphate cement (CPC) is a promising material used in the treatment of bone defects due to its profitable features of self-setting capability, osteoconductivity, injectability, mouldability, and biocompatibility. However, the major limitations of CPC, such as the brittleness, lack of osteogenic property, and poor washout resistance, remain to be resolved. Thus, significant research effort has been committed to modify and reinforce CPC. The mixture of CPC with various biological materials, defined as the materials produced by living organisms, have been fabricated by researchers and their characteristics have been investigated in vitro and in vivo. This present review aimed to provide a comprehensive overview enabling the readers to compare the physical, mechanical, and biological properties of CPC upon the incorporation of different biological materials. By mixing the bone-related transcription factors, proteins, and/or polysaccharides with CPC, researchers have demonstrated that these combinations not only resolved the lack of mechanical strength and osteogenic effects of CPC but also further improve its own functional properties. However, exceptions were seen in CPC incorporated with certain proteins (such as elastin-like polypeptide and calcitonin gene-related peptide) as well as blood components. In conclusion, the addition of biological materials potentially improves CPC features, which vary depending on the types of materials embedded into it. The significant enhancement of CPC seen in vitro and in vivo requires further verification in human trials for its clinical application.

Effects of a Novel Bioactive Glass Composition on Biological Properties of Human Dental Pulp Stem Cells.

Functional reconstruction of bone defects represents a clinical challenge in the regenerative medicine field, which targets tissue repair following traumatic injuries and disease-related bone deficiencies. In this regard, the optimal biomaterial should be safe, biocompatible and tailored in order to promote the activation of host progenitor cells towards bone repair. Bioactive glasses might be suitable biomaterials due to their composition being able to induce the host healing response and, eventually, anti-bacterial properties. In this study we investigated whether and how an innovative bioactive glass composition, called BGMS10, may affect cell adhesion, morphology, proliferation, immunomodulation and osteogenic differentiation of human dental pulp stem cells (hDPSCs). When cultured on BGMS10, hDPSCs maintained their proliferation rate and typical fibroblast-like morphology, showing the expression of stemness markers STRO-1 and c-Kit. Moreover, the expression of FasL, a key molecule in mediating immunomodulation effects of hDPSCs, was maintained. BGMS10 also proved to trigger osteogenic commitment of hDPSCs, as confirmed by the activation of bone-related transcription factors RUNX2 and Osx and the ongoing deposition of extracellular matrix supported by the expression of OPN and OCN. Our findings suggest that BGMS10 not only maintains the typical biological and immunomodulatory properties of hDPSCs but also favors the osteogenic commitment.

An osteoinductive effect of phytol on mouse mesenchymal stem cells (C3H10T1/2) towards osteoblasts

In recent years, phytochemicals have been widely researched and utilized for the treatment of various medical conditions such as cancer, cardiovascular diseases, age-related problems and are also said to have bone regenerative effects. In this study, phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), an acyclic unsaturated diterpene alcohol and a secondary metabolite derived from aromatic plants was investigated for its effect on osteogenesis. Phytol was found to be nontoxic in mouse mesenchymal stem cells (C3H10T1/2). At the cellular level, phytol-treatment promoted osteoblast differentiation, as seen by the increased calcium deposits. At the molecular level, phytol-treatment stimulated the expression of Runx2 (a bone-related transcription factor) and other osteogenic marker genes. MicroRNAs (miRNAs) play an essential role in controlling bone metabolism by targeting genes at the post-transcriptional level. Upon phytol-treatment in C3H10T1/2 cells, mir-21a and Smad7 levels were increased and decreased, respectively. It was previously reported that mir-21a targets Smad7 (an antagonist of TGF-beta1 signaling) and thus, protects Runx2 from its degradation. Thus, based on our results, we suggest that phytol-treatment promoted osteoblast differentiation in C3H10T1/2 cells via Runx2 due to downregulation of Smad7 by mir-21a. Henceforth, phytol was identified to bolster osteoblast differentiation, which in turn may be used for bone regeneration.

Regulatory Effects of O-GlcNAcylation in Vascular Smooth Muscle Cells on Diabetic Vasculopathy

Vascular complications from uncontrolled hyperglycemia are the leading cause of death in patients with diabetes mellitus. Previous reports have shown a strong correlation between hyperglycemia and vascular calcification, which increases mortality and morbidity in individuals with diabetes. However, the precise underlying molecular mechanisms of hyperglycemia-induced vascular calcification remain largely unknown. Transdifferentiation of vascular smooth muscle cells (VSMC) into osteoblast-like cells is a known culprit underlying the development of vascular calcification in the diabetic vasculature. Pathological conditions such as high glucose levels and oxidative stress are linked to enhanced osteogenic differentiation of VSMC both in vivo and in vitro. It has been demonstrated that increased expression of runt-related transcription factor 2 (Runx2), a bone-related transcription factor, in VSMC is necessary and sufficient for the induction of VSMC calcification. Addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety to the serine/threonine residues of target proteins (O-GlcNAcylation) has been observed in the arteries of diabetic patients, as well as in animal models in association with the enhanced expression of Runx2 and aggravated vascular calcification. O-GlcNAcylation is a dynamic and tightly regulated process, that is mediated by 2 enzymes, O-GlcNAc transferase and O-GlcNAcase. Glucose is metabolized into UDP-β-D-N-acetylglucosamine, an active sugar donor of O-GlcNAcylation via the hexosamine biosynthetic pathway. Overall increases in the O-GlcNAcylation of cellular proteins have been closely associated with cardiovascular complications of diabetes. In this review, the authors provide molecular insights into cardiovascular complications, including diabetic vasculopathy, that feature increased O-GlcNAcylation in people with diabetes.

The epigenetic reader Brd4 is required for osteoblast differentiation.

Transcription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain-containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA-sequencing (RNA-seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow-up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent "bromodomain and extra terminal domain" (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast-related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone-related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast-specific genes and may cooperate with bone-related transcription factors to promote osteoblast lineage commitment and maturation.

Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Role of Protein Phosphatase 2A in Osteoblast Differentiation and Function

The reversible phosphorylation of proteins plays hugely important roles in a variety of cellular processes, such as differentiation, proliferation, and apoptosis. These processes are strictly controlled by protein kinases (phosphorylation) and phosphatases (de-phosphorylation). Here we provide a brief history of the study of protein phosphorylation, including a summary of different types of protein kinases and phosphatases. One of the most physiologically important serine/threonine phosphatases is PP2A. This review provides a description of the phenotypes of various PP2A transgenic mice and further focuses on the known functions of PP2A in bone formation, including its role in osteoblast differentiation and function. A reduction in PP2A promotes bone formation and osteoblast differentiation through the regulation of bone-related transcription factors such as Osterix. Interestingly, downregulation of PP2A also stimulates adipocyte differentiation from undifferentiated mesenchymal cells under the appropriate adipogenic differentiation conditions. In osteoblasts, PP2A is also involved in the ability to control osteoclastogenesis as well as in the proliferation and metastasis of osteosarcoma cells. Thus, PP2A is considered to be a comprehensive factor in controlling the differentiation and function of cells derived from mesenchymal cells such as osteoblasts and adipocytes.

MicroRNA-466 inhibits tumor growth and bone metastasis in prostate cancer by direct regulation of osteogenic transcription factor RUNX2

MicroRNAs (miRNAs) have emerged as key players in cancer progression and metastatic initiation yet their importance in regulating prostate cancer (PCa) metastasis to bone has begun to be appreciated. We employed multimodal strategy based on in-house PCa clinical samples, publicly available TCGA cohorts, a panel of cell lines, in silico analyses, and a series of in vitro and in vivo assays to investigate the role of miR-466 in PCa. Expression analyses revealed that miR-466 is under-expressed in PCa compared to normal tissues. Reconstitution of miR-466 in metastatic PCa cell lines impaired their oncogenic functions such as cell proliferation, migration/invasion and induced cell cycle arrest, and apoptosis compared to control miRNA. Conversely, attenuation of miR-466 in normal prostate cells induced tumorigenic characteristics. miR-466 suppressed PCa growth and metastasis through direct targeting of bone-related transcription factor RUNX2. Overexpression of miR-466 caused a marked downregulation of integrated network of RUNX2 target genes such as osteopontin, osteocalcin, ANGPTs, MMP11 including Fyn, pAkt, FAK and vimentin that are known to be involved in migration, invasion, angiogenesis, EMT and metastasis. Xenograft models indicate that miR-466 inhibits primary orthotopic tumor growth and spontaneous metastasis to bone. Receiver operating curve and Kaplan–Meier analyses show that miR-466 expression can discriminate between malignant and normal prostate tissues; and can predict biochemical relapse. In conclusion, our data strongly suggests miR-466-mediated attenuation of RUNX2 as a novel therapeutic approach to regulate PCa growth, particularly metastasis to bone. This study is the first report documenting the anti-bone metastatic role and clinical significance of miR-466 in prostate cancer.

Histone H4 Methyltransferase Suv420h2 Maintains Fidelity of Osteoblast Differentiation.

Osteogenic lineage commitment and progression is controlled by multiple signaling pathways (e.g., WNT, BMP, FGF) that converge on bone-related transcription factors. Access of osteogenic transcription factors to chromatin is controlled by epigenetic regulators that generate post-translational modifications of histones ("histone code"), as well as read, edit and/or erase these modifications. Our understanding of the biological role of epigenetic regulators in osteoblast differentiation remains limited. Therefore, we performed next-generation RNA sequencing (RNA-seq) and established which chromatin-related proteins are robustly expressed in mouse bone tissues (e.g., fracture callus, calvarial bone). These studies also revealed that cells with increased osteogenic potential have higher levels of the H4K20 methyl transferase Suv420h2 compared to other methyl transferases (e.g., Suv39h1, Suv39h2, Suv420h1, Ezh1, Ezh2). We find that all six epigenetic regulators are transiently expressed at different stages of osteoblast differentiation in culture, with maximal mRNAs levels of Suv39h1 and Suv39h2 (at day 3) preceding maximal expression of Suv420h1 and Suv420h2 (at day 7) and developmental stages that reflect, respectively, early and later collagen matrix deposition. Loss of function analysis of Suv420h2 by siRNA depletion shows loss of H4K20 methylation and decreased expression of bone biomarkers (e.g., alkaline phosphatase/Alpl) and osteogenic transcription factors (e.g., Sp7/Osterix). Furthermore, Suv420h2 is required for matrix mineralization during osteoblast differentiation. We conclude that Suv420h2 controls the H4K20 methylome of osteoblasts and is critical for normal progression of osteoblastogenesis. J. Cell. Biochem. 118: 1262-1272, 2017. © 2016 Wiley Periodicals, Inc.

MiR-5100 promotes osteogenic differentiation by targeting Tob2.

MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.

Osterix represses adipogenesis by negatively regulating PPARγ transcriptional activity.

Osterix is a novel bone-related transcription factor involved in osteoblast differentiation, and bone maturation. Because a reciprocal relationship exists between adipocyte and osteoblast differentiation of bone marrow derived mesenchymal stem cells, we hypothesized that Osterix might have a role in adipogenesis. Ablation of Osterix enhanced adipogenesis in 3T3-L1 cells, whereas overexpression suppressed this process and inhibited the expression of adipogenic markers including CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Further studies indicated that Osterix significantly decreased PPARγ-induced transcriptional activity. Using co-immunoprecipitation and GST-pull down analysis, we found that Osterix directly interacts with PPARγ. The ligand-binding domain (LBD) of PPARγ was responsible for this interaction, which was followed by repression of PPARγ-induced transcriptional activity, even in the presence of rosiglitazone. Taken together, we identified the Osterix has an important regulatory role on PPARγ activity, which contributed to the mechanism of adipogenesis.

Histone deacetylase 3 is required for maintenance of bone mass during aging

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKOOCN mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKOOCN mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKOOCN mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKOOCN mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging.

Biomechanical stimulation of osteoblast gene expression requires phosphorylation of the RUNX2 transcription factor.

Bone can adapt its structure in response to mechanical stimuli. At the cellular level, this involves changes in chromatin organization, gene expression, and differentiation, but the underlying mechanisms are poorly understood. Here we report on the involvement of RUNX2, a bone-related transcription factor, in this process. Fluid flow shear stress loading of preosteoblasts stimulated translocation of extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) to the nucleus where it phosphorylated RUNX2 on the chromatin of target genes, and increased histone acetylation and gene expression. MAPK signaling and two RUNX2 phosphoacceptor sites, S301 and S319, were critical for this response. Similarly, in vivo loading of mouse ulnae dramatically increased ERK and RUNX2 phosphorylation as well as expression of osteoblast-related genes. These findings establish ERK/MAPK-mediated phosphorylation of RUNX2 as a critical step in the response of preosteoblasts to dynamic loading and define a novel mechanism to explain how mechanical signals induce gene expression in bone.

Skeletal stem cell physiology on functionally distinct titania nanotopographies

Functionalisation of the surface of orthopaedic implants with nanotopographies that could stimulate in situ osteogenic differentiation of the patient’s stem or osteoprogenitor cells would have significant therapeutic potential. Mesenchymal stem cell (MSC) responses to titanium substrates patterned with nanopillar structures were investigated in this study. Focal adhesions were quantified in S-phase cells, the bone-related transcription factor Runx2 was examined, osteocalcin production was noted, and Haralick computational analysis was used to assess the relatedness of the cell responses to each of the titanium substrata based on cytoskeletal textural features. Metabolomics was used as a novel means of assessing cellular responses to the biomaterial substrates by analysing the global metabolite profile of the cells on the substrata, and shows promise as a technique with high data yield for evaluating cell interactions with materials of different surface chemistry or topography. The cell response to 15nm high nanopillars was distinct, consistent with a transition from a more quiescent phenotype on the planar substrate, to an ‘active’ phenotype on the pillars. These studies illustrate the potential for clinically relevant titania nanopillared substrata to modulate MSCs, with implications for orthopaedic device design and application.

Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility

IntroductionMetastatic breast cancer cells frequently and ectopically express the transcription factor RUNX2, which normally attenuates proliferation and promotes maturation of osteoblasts. RUNX2 expression is inversely regulated with respect to cell growth in osteoblasts and deregulated in osteosarcoma cells.MethodsHere, we addressed whether the functional relationship between cell growth and RUNX2 gene expression is maintained in breast cancer cells. We also investigated whether the aberrant expression of RUNX2 is linked to phenotypic parameters that could provide a selective advantage to cells during breast cancer progression.ResultsWe find that, similar to its regulation in osteoblasts, RUNX2 expression in MDA-MB-231 breast adenocarcinoma cells is enhanced upon growth factor deprivation, as well as upon deactivation of the mitogen-dependent MEK-Erk pathway or EGFR signaling. Reduction of RUNX2 levels by RNAi has only marginal effects on cell growth and expression of proliferation markers in MDA-MB-231 breast cancer cells. Thus, RUNX2 is not a critical regulator of cell proliferation in this cell type. However, siRNA depletion of RUNX2 in MDA-MB-231 cells reduces cell motility, while forced exogenous expression of RUNX2 in MCF7 cells increases cell motility.ConclusionsOur results support the emerging concept that the osteogenic transcription factor RUNX2 functions as a metastasis-related oncoprotein in non-osseous cancer cells.

Synergism between Wnt3a and Heparin Enhances Osteogenesis via a Phosphoinositide 3-Kinase/Akt/RUNX2 Pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells

Osterix is a bone-related transcription factor that functions genetically downstream of Runx2, which controls both growth and differentiation in osteoblasts. Here we assessed the biological function of Osterix in mesenchymal cells that are not normally committed to the osteogenic lineage. Stably transfected NIH3T3 fibroblasts that express exogenous Osterix were examined for their ability to convert into osteoblastic cells by analyzing gene expression profiles of bone phenotype related markers, as well as by measuring bone nodule formation and cell proliferation. Forced expression of Osterix stimulates osteopontin gene expression but not the expression or activity of other bone-related markers, including collagen type I, alkaline phosphatase, osteocalcin, or osteonectin. Moreover, cells stably expressing Osterix do not induce bone nodule formation. Strikingly, both polyclonal and monoclonal cells expressing Osterix exhibit enhanced proliferation. Collectively, these results indicate that Osterix is insufficient to establish osteogenic lineage commitment, perhaps due to the ability of Osterix to promote cell growth. We propose that regulatory pathways operating upstream of or in parallel with Osterix are required for osteogenic conversion of uncommitted mesenchymal cells.

Canonical WNT Signaling Promotes Osteogenesis by Directly Stimulating Runx2 Gene Expression

Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (-97 to -93). By chromatin immunoprecipitation, recruitment of beta-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of beta-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-beta signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.

Parathyroid hormone-related peptide (PTHrP) inhibits Runx2 expression through the PKA signaling pathway

The bone-related transcription factor Runx2 (Cbfa1) has been extensively shown to regulate osteoblast differentiation and function. Recent studies demonstrate that Runx2 is also a positive regulator of chondrocyte maturation and vascular invasion in cartilage. Runx2 activity can be modulated in several ways, including direct stimulation of gene expression, post-translational modification, and protein–protein interactions. We have previously reported cooperative effects between BMP and RA downstream signaling involving Smad proteins and Runx2. Furthermore, our previous studies showed that PTHrP inhibits chondrocyte maturation primarily through CREB and AP-1 signaling pathways. In the present study, we investigated the effect of PTHrP on Runx2 expression in chick upper sternal chondrocytes (USCs). We further determined the signaling pathways through which PTHrP regulates Runx2 transcription. Our results show that PTHrP inhibits Runx2 expression at both the mRNA and protein levels concomitant with a PTHrP-mediated suppression of the phenotypic marker of hypertrophy, type X collagen. We further determined potential signaling pathways through which PTHrP inhibits Runx2 expression using protein kinase inhibitors, H89 (PKA inhibitor): Go-6976 (PKC inhibitor): SB203850 (p38 MAPK inhibitor), and U0126 (MEK inhibitor). We show that pretreatment with PKA and, to a lesser extent, PKC inhibitors significantly blocked PTHrP suppression of Runx2, while p38 MAPK and MEK inhibitors had no significant effect. Furthermore, PTHrP suppression of Runx2 mRNA was partially blocked in USCs infected with RCAS-A-CREB, a dominant negative reagent that abrogates CREB activity. Overall, our results demonstrate that PTHrP downregulates Runx2 expression primarily through the PKA signaling pathway.