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Toshihisa Komori - One of the best experts on this subject based on the ideXlab platform.
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Requisite roles of RUNX2 and Cbfb in skeletal development.
Journal of Bone and Mineral Metabolism, 2020Co-Authors: Toshihisa KomoriAbstract:Each Runx (runt-related gene) protein exerts a fundamental role in different cell lineages. RUNX2 is essential for osteoblast differentiation and plays an important role in chondrocyte maturation. RUNX2 determines the lineage of osteoblastic cells from multipotent mesenchymal cells, enhances osteoblast differentiation at an early stage, and inhibits osteoblast differentiation at a late stage. In addition, RUNX2 is involved in the production of bone matrix proteins. Further, RUNX2 is a positive regulator of chondrocyte maturation and is involved in vascular invasion into the cartilage. Core binding factor β (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb is required for the functions of Runx1 and RUNX2. Thus, RUNX2/Cbfb heterodimers play essential roles in skeletal development.
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Regulation of Osteoblast and Odontoblast Differentiation by RUNX2
Journal of Oral Biosciences, 2020Co-Authors: Toshihisa KomoriAbstract:Abstract Runx 2-deficient mice completely lack osteoblasts and bone formation. Overexpression of Runx 2 in osteoblasts inhibits osteoblast maturation, leading to immature bone, which is easily resorbed, while the expression of dominant-negative Runx 2 in osteoblasts increases the volume of trabecular bone by promoting the formation of mature bone, which is relatively resistant to bone resorption. Thus, RUNX2 directs mesenchymal stem cells to the osteoblast lineage, and supplies immature osteoblasts to form immature bone, but RUNX2 has to be down-regulated for terminal differentiation into mature osteoblasts, which form mature bone. Thus, the level of RUNX2 determines the maturational stage of osteoblasts, bone maturity, and the bone turnover rate. Endogenous RUNX2 protein is detected in the nuclei of preodontoblasts, immature odontoblasts, and mesenchymal cells in the dental sac at 3 days of age, and transiently detected in ameloblasts in the coronal regions at one week of age. Overexpression of Runx 2 in odontoblasts inhibited their terminal differentiation. Further, dentin sialophosphoprotein (Dspp) expression was lost and nestin (NES) expression was markedly decreased, while the expressions of bone gamma -carboxyglutamate (gla) protein 1/osteocalcin (BGLAP), osteopontin (SPP1), and dentin matrix protein 1 (DMP1) were up-regulated in the odontoblasts, resulting in the formation of a bone structure. These findings indicate that RUNX2 is able to induce the transdifferentiation of odontoblasts into osteoblasts, and that RUNX2 expression has to be down-regulated during odontoblast differentiation to achieve full differentiation for dentinogenesis.
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Molecular Mechanism of RUNX2-Dependent Bone Development.
Molecules and Cells, 2020Co-Authors: Toshihisa KomoriAbstract:: RUNX2 is an essential transcription factor for skeletal development. It is expressed in multipotent mesenchymal cells, osteoblast-lineage cells, and chondrocytes. RUNX2 plays a major role in chondrocyte maturation, and Runx3 is partly involved. RUNX2 regulates chondrocyte proliferation by directly regulating Ihh expression. It also determines whether chondrocytes become those that form transient cartilage or permanent cartilage, and functions in the pathogenesis of osteoarthritis. RUNX2 is essential for osteoblast differentiation and is required for the proliferation of osteoprogenitors. Ihh is required for RUNX2 expression in osteoprogenitors, and hedgehog signaling and RUNX2 induce the differentiation of osteoprogenitors to preosteoblasts in endochondral bone. RUNX2 induces Sp7 expression, and RUNX2, Sp7, and canonical Wnt signaling are required for the differentiation of preosteoblasts to immature osteoblasts. It also induces the proliferation of osteoprogenitors by directly regulating the expression of Fgfr2 and Fgfr3. Furthermore, RUNX2 induces the proliferation of mesenchymal cells and their commitment into osteoblast-lineage cells through the induction of hedgehog (Gli1, Ptch1, Ihh), Fgf (Fgfr2, Fgfr3), Wnt (Tcf7, Wnt10b), and Pthlh (Pth1r) signaling pathway gene expression in calvaria, and more than a half-dosage of RUNX2 is required for their expression. This is a major cause of cleidocranial dysplasia, which is caused by heterozygous mutation of RUNX2. Cbfb, which is a co-transcription factor that forms a heterodimer with RUNX2, enhances DNA binding of RUNX2 and stabilizes RUNX2 protein by inhibiting its ubiquitination. Thus, RUNX2/Cbfb regulates the proliferation and differentiation of chondrocytes and osteoblast-lineage cells by activating multiple signaling pathways and via their reciprocal regulation.
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RUNX2, an inducer of osteoblast and chondrocyte differentiation
Histochemistry and Cell Biology, 2018Co-Authors: Toshihisa KomoriAbstract:RUNX2 is a transcription factor that is essential for osteoblast differentiation and chondrocyte maturation. Ihh, expressed in prehypertrophic and hypertrophic chondrocytes, is required for the specification of RUNX2^+ osteoprogenitors in endochondral bone development. RUNX2 induces Sp7, an essential transcription factor for osteoblast differentiation. Canonical Wnt signaling is also required for osteoblast differentiation. RUNX2^+ osteoprogenitors retain the capacity to differentiate into chondrocytes, and Sp7 and canonical Wnt signaling direct cells to osteoblasts, thereby inhibiting chondrocyte differentiation. The function of RUNX2 after the commitment to osteoblasts remains controversial. Runx3 has a redundant function with RUNX2 in chondrocyte maturation. RUNX2 regulates the expression of Ihh, Col10a1, Spp1, Ibsp, Mmp13 , and Vegfa in the respective layers in growth plates. RUNX2 enhances chondrocyte proliferation through the induction of Ihh . Ihh induces Pthlh, which inhibits RUNX2 and chondrocyte maturation, forming a negative feedback loop for chondrocyte maturation. RUNX2 is one of the genes responsible for the pathogenesis of osteoarthritis (OA) because RUNX2 is up-regulated in chondrocytes in OA cartilage and a germline haplodeficiency or deletion of RUNX2 in articular chondrocytes decelerates OA progression. RUNX2 plays an important role in the bone metastasis of breast and prostate cancers by up-regulating Spp1, Ibsp, Mmp9, Mmp13, Vegfa, Tnfsf11 , and Ihh expression and down-regulating Tnfrsf11b expression. Cbfb forms a heterodimer with RUNX2 and is required for the efficient DNA binding of RUNX2. Cbfb stabilizes Runx proteins at different levels among Runx family proteins by inhibiting their ubiquitination-mediated degradation. Cbfb plays more important roles in endochondral ossification than in intramembranous ossification.
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Roles of RUNX2 in Skeletal Development.
Advances in Experimental Medicine and Biology, 2017Co-Authors: Toshihisa KomoriAbstract:RUNX2 is the most upstream transcription factor essential for osteoblast differentiation. It regulates the expression of Sp7, the protein of which is a crucial transcription factor for osteoblast differentiation, as well as that of bone matrix genes including Spp1, Ibsp, and Bglap2. RUNX2 is also required for chondrocyte maturation, and Runx3 has a redundant function with RUNX2 in chondrocyte maturation. RUNX2 regulates the expression of Col10a1, Spp1, Ibsp, and Mmp13 in chondrocytes. It also inhibits chondrocytes from acquiring the phenotypes of permanent cartilage chondrocytes. It regulates chondrocyte proliferation through the regulation of Ihh expression. RUNX2 enhances osteoclastogenesis by regulating Rankl. Cbfb, which is a co-transcription factor for Runx family proteins, plays an important role in skeletal development by stabilizing Runx family proteins. In Cbfb isoforms, Cbfb1 is more potent than Cbfb2 in RUNX2-dependent transcriptional regulation; however, the expression level of Cbfb2 is three-fold higher than that of Cbfb1, demonstrating the requirement of Cbfb2 in skeletal development. The expression of RUNX2 in osteoblasts is regulated by a 343-bp enhancer located upstream of the P1 promoter. This enhancer is activated by an enhanceosome composed of Dlx5/6, Mef2, Tcf7, Ctnnb1, Sox5/6, Smad1, and Sp7. Thus, RUNX2 is a multifunctional transcription factor that is essential for skeletal development, and Cbfb regulates skeletal development by modulating the stability and transcriptional activity of Runx family proteins.
Jane B. Lian - One of the best experts on this subject based on the ideXlab platform.
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The Thyroid Hormone Receptor-RUNX2 Axis: A Novel Tumor Suppressive Pathway in Breast Cancer
Hormones and Cancer, 2019Co-Authors: Eric L. Bolf, Jane B. Lian, Janet L. Stein, Gary S Stein, Noelle E. Gillis, Michael S. Barnum, Caitlin M. Beaudet, Grace Y. Yu, Jennifer A. Tomczak, Frances E. CarrAbstract:Metastatic breast cancer is refractory to conventional therapies and is an end-stage disease. RUNX2 is a transcription factor that becomes oncogenic when aberrantly expressed in multiple tumor types, including breast cancer, supporting tumor progression and metastases. Our previous work demonstrated that the thyroid hormone receptor beta (TRβ) inhibits RUNX2 expression and tumorigenic characteristics in thyroid cells. As TRβ is a tumor suppressor, we investigated the compelling question whether TRβ also regulates RUNX2 in breast cancer. The Cancer Genome Atlas indicates that TRβ expression is decreased in the most aggressive basal-like subtype of breast cancer. We established that modulated levels of TRβ results in corresponding changes in the high levels of RUNX2 expression in metastatic, basal-like breast cells. The MDA-MB-231 triple-negative breast cancer cell line exhibits low expression of TRβ and high levels of RUNX2. Increased expression of TRβ decreased RUNX2 levels. The thyroid hormone-mediated suppression of RUNX2 is TRβ specific as TRα overexpression failed to alter RUNX2 expression. Consistent with these findings, knockdown of TRβ in non-tumor MCF10A mammary epithelial-like cells results in an increase in RUNX2 and RUNX2 target genes. Mechanistically, TRβ directly interacts with the proximal promoter of RUNX2 through a thyroid hormone response element to reduce promoter activity. The TRβ suppression of the oncogene RUNX2 is a signaling pathway shared by thyroid and breast cancers. Our findings provide a novel mechanism for TRβ-mediated tumor suppression in breast cancers. This pathway may be common to many solid tumors and impact treatment for metastatic cancers.
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Expression of the IL-11 Gene in Metastatic Cells Is Supported by RUNX2-Smad and RUNX2-cJun Complexes Induced by TGFβ1
Journal of Cellular Biochemistry, 2015Co-Authors: Xuhui Zhang, Deli Hong, Gary S Stein, Jacqueline Akech, Hai Wu, Jason R. Dobson, Gillian Browne, Lucia R. Languino, Jane B. LianAbstract:In tumor cells, two factors are abnormally increased that contribute to metastatic bone disease: RUNX2, a transcription factor that promotes expression of metastasis related and osteolytic genes; and IL-11, a secreted osteolytic cytokine. Here, we addressed a compelling question: Does RUNX2 regulate IL-11 gene expression? We find a positive correlation between RUNX2, IL-11 and TGFβ1, a driver of the vicious cycle of metastatic bone disease, in prostate cancer (PC) cell lines representing early (LNCaP) and late (PC3) stage disease. Further, like RUNX2 knockdown, IL-11 knockdown significantly reduced expression of several osteolytic factors. Modulation of RUNX2 expression results in corresponding changes in IL-11 expression. The IL-11 gene has RUNX2, AP-1 sites and Smad binding elements located on the IL-11 promoter. Here, we demonstrated that RUNX2-c-Jun as well as RUNX2-Smad complexes upregulate IL-11 expression. Functional studies identified a significant loss of IL-11 expression in PC3 cells in the presence of the RUNX2-HTY mutant protein, a mutation that disrupts RUNX2-Smad signaling. In response to TGFβ1 and in the presence of RUNX2, we observed a 30-fold induction of IL-11 expression, accompanied by increased c-Jun binding to the IL-11 promoter. Immunoprecipitation and in situ co-localization studies demonstrated that RUNX2 and c-Jun form nuclear complexes in PC3 cells. Thus, TGFβ1 signaling induces two independent transcriptional pathways - AP-1 and RUNX2. These transcriptional activators converge on IL-11 as a result of RUNX2-Smad and RUNX2-c-Jun interactions to amplify IL-11 gene expression that, together with RUNX2, supports the osteolytic pathology of cancer induced bone disease.
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Pin1-mediated RUNX2 modification is critical for skeletal development
Journal of Cellular Physiology, 2013Co-Authors: Wonjoon Yoon, Andre J. Van Wijnen, Janet L. Stein, Toshihisa Komori, Jeonghwa Baek, Rabia Islam, Takafumi Uchida, Jane B. LianAbstract:RUNX2 is the master transcription factor for bone formation. Haploinsufficiency of RUNX2 is the genetic cause of cleidocranial dysplasia (CCD) that is characterized by hypoplastic clavicles and open fontanels. In this study, we found that Pin1, peptidyl prolyl cis-trans isomerase, is a critical regulator of RUNX2 in vivo and in vitro. Pin1 mutant mice developed CCD-like phenotypes with hypoplastic clavicles and open fontanels as found in the RUNX2+/− mice. In addition RUNX2 protein level was significantly reduced in Pin1 mutant mice. Moreover Pin1 directly interacts with the RUNX2 protein in a phosphorylation-dependent manner and subsequently stabilizes RUNX2 protein. In the absence of Pin1, RUNX2 is rapidly degraded by the ubiquitin-dependent protein degradation pathway. However, Pin1 overexpression strongly attenuated uniquitin-dependent RUNX2 degradation. Collectively conformational change of RUNX2 by Pin1 is essential for its protein stability and possibly enhances the level of active RUNX2 in vivo.
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Genomic occupancy of HLH, AP1 and RUNX2 motifs within a nuclease sensitive site of the RUNX2 gene
Journal of Cellular Physiology, 2012Co-Authors: Hayk Hovhannisyan, Jane B. Lian, Janet L. Stein, Gary S Stein, Martin Montecino, Mohammad Q Hassan, Ying Zhang, Hai Wu, Carlotta A. Glackin, Andre J. Van WijnenAbstract:Runt-related transcription factors are principal developmental regulators that control lineage commitment and cell type-specificity in diverse species. Null mutations in each of the three known runt-related transcription factor (Runx) genes in mouse causes dramatic tissue-specific phenotypes. Mutations and/or deregulation of the corresponding human genes are linked to familial diseases and genetic predispositions related to cancer and distinct abnormalities in tissue-formation (Blyth et al., 2005; Ito, 2008). While Runx proteins may functionally compensate each other, the unique phenotypes of mice with Runx null mutations are attributable to tissue- and developmental stage-specific activation of transcription. The RUNX2 gene is prominently transcribed in the mesenchymal lineage to support normal development of bone and cartilage in vivo which accounts for the observed skeletal phenotypes of mice with RUNX2 mutations that render mesenchymal cells RUNX2 deficient (Komori et al., 1997; Otto et al., 1997; Choi et al., 2001; Lengner et al., 2002; Jeong et al., 2008; Lou et al., 2009; Zhang et al., 2009a; Liu et al., 2011). RUNX2 transcription, as the initial rate-limiting step in its expression, is exquisitely regulated by a multitude of developmental signals, regulatory promoter elements and cognate transcription factors (Lian et al., 2006; Franceschi et al., 2007; Marie, 2008; Long, 2012). The RUNX2 gene is expressed from two promoters (P1 and P2), and the upstream P1 promoter supports osteoblast-specific gene transcription. Of note, polymorphisms in the P1 and P2 promoter have been correlated with bone mineral density and viral integration sites are capable of ectopic activation of RUNX2 gene transcription in non-osseous cell types (Stewart et al., 2002; Doecke et al., 2006; Lee et al., 2009). The P1 promoter is autoregulated through at least seven Runx binding sites (Drissi et al., 2000, 2002b) and responds to steroid hormones (Tou et al., 2001; Drissi et al., 2002a), BMPs (Xiao et al., 2001; Tou et al., 2003; Lee et al., 2005) and WNTs (Gaur et al., 2005). The P1 promoter is controlled by several homeodomain proteins including AP1 (Drissi et al., 2002a), Dlx5 (Gaur et al., 2005), Nkx3.2 (Lengner et al., 2005), HoxA10 (Hassan et al., 2007), SP1 and Ets proteins (Zhang et al., 2009b), Hif2α (Tamiya et al., 2008), C/EBPβ (Wiper-Bergeron et al., 2007; Henriquez et al., 2011) and NF-1 related proteins (Zambotti et al., 2002). Regulation of RUNX2 gene transcription by this cohort of primary DNA binding proteins occurs within the context of nucleosomal organization and higher order chromatin structure that together modulate accessibility of transcription factors to gene promoters. The RUNX2 gene, which is in throughout the osteogenic lineage, and the osteocalcin (OC) gene, which is transcriptionally activated at the maturation stage of osteoblast differentiation, together represent two versatile and intensively studied models for understanding transcriptional control during osteogenesis (Lian et al., 2004; Montecino et al., 2008). Activation of OC gene expression occurs concomitant with creation of nuclease hypersensitive sites, increased acetylation of histone H3 and H4, as well as specific binding of multiple transcription factors including RUNX2 (Javed et al., 1999; Shen et al., 2002, 2003; Hassan et al., 2004). Studies using rat osteosarcoma cells and trans-differentiated mouse myoblasts have revealed SWI/SNF dependent changes in the chromatin organization of the RUNX2 P1 promoter (Cruzat et al., 2009). In this study, we established key parameters of chromatin fine-structure of the RUNX2 gene promoter in mouse osteoblasts in which the RUNX2 P1 promoter is naturally activated. We define histone modifications, nuclease hypersensitive sites and two genomic protein/DNA interactions that mediate transcriptional regulation of RUNX2 gene expression. Our key finding is that the RUNX2 P1 promoter contains two stable genomic protein-DNA interaction domains that may transcriptionally control the multiple physiological activities of RUNX2 during skeletal development and bone formation in vivo.
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genomic promoter occupancy of runt related transcription factor RUNX2 in osteosarcoma cells identifies genes involved in cell adhesion and motility
Journal of Biological Chemistry, 2012Co-Authors: Margaretha Van Der Deen, Daniel W. Young, Jane B. Lian, Janet L. Stein, Jacqueline Akech, David S Lapointe, Sneha Gupta, Martin Montecino, Mario Galindo, Gary S SteinAbstract:Abstract Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5′-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFβ, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.
Hyunmo Ryoo - One of the best experts on this subject based on the ideXlab platform.
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Control of tooth morphogenesis by RUNX2.
Critical Reviews in Eukaryotic Gene Expression, 2020Co-Authors: Hyunmo Ryoo, Xiu-ping WangAbstract:: Odontogenesis is a complex process in which the interplay of signaling cascades of the epithelium and mesenchyme is critical. Evidence for the involvement of RUNX2--a well-known osteogenic master transcription factor--in odontogenesis, has been accumulating. Haploinsufficiency of RUNX2 in humans results in cleido-cranial dysplasia (CCD), characterized by supernumerary teeth; in RUNX2-/- mice, molar odontogenesis does not proceed beyond the late bud stage. In this article, we discuss the role of RUNX2 in tooth development, specifically in the signaling interplay of the epithelium and mesenchyme during the transition from bud stage to cap stage. RUNX2 is an important molecule of the dental mesenchyme; its expression is induced by epithelial fibroblast growth factor (FGF), and it is involved in regulating the induction of mesenchymal signaling back to the dental epithelium for epithelial morphogenesis. In addition, we discuss the role of the two major isoforms of RUNX2, and other Runx family genes, in odontogenic processes.
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Post-Translational Regulations of Transcriptional Activity of RUNX2.
Molecules and Cells, 2020Co-Authors: Hyunmo RyooAbstract:: Runt-related transcription factor 2 (RUNX2) is a key transcription factor for bone formation and osteoblast differentiation. Various signaling pathways and mechanisms that regulate the expression and transcriptional activity of RUNX2 have been thoroughly investigated since the involvement of RUNX2 was first reported in bone formation. As the regulation of RUNX2 expression by extracellular signals has recently been reviewed, this review focuses on the regulation of post-translational RUNX2 activity. Transcriptional activity of RUNX2 is regulated at the post-translational level by various enzymes including kinases, acetyl transferases, deacetylases, ubiquitin E3 ligases, and prolyl isomerases. We describe a sequential and linear causality between post-translational modifications of RUNX2 by these enzymes. RUNX2 is one of the most important osteogenic transcription factors; however, it is not a suitable drug target. Here, we suggest enzymes that directly regulate the stability and/or transcriptional activity of RUNX2 at a post-translational level as effective drug targets for treating bone diseases.
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bmp2 activated erk map kinase stabilizes RUNX2 by increasing p300 levels and histone acetyltransferase activity
Journal of Biological Chemistry, 2010Co-Authors: Wonjoon Yoon, Hyunmo Ryoo, Jeonghwa BaekAbstract:RUNX2 is a critical transcription factor for osteoblast differentiation. Regulation of RUNX2 expression levels and transcriptional activity is important for bone morphogenetic protein (BMP)-induced osteoblast differentiation. Previous studies have shown that extracellular signal-regulated kinase (Erk) activation enhances the transcriptional activity of RUNX2 and that BMP-induced RUNX2 acetylation increases RUNX2 stability and transcriptional activity. Because BMP signaling induces Erk activation in osteoblasts, we sought to investigate whether BMP-induced Erk signaling regulates RUNX2 acetylation and stability. Erk activation by overexpression of constitutively active MEK1 increased RUNX2 transcriptional activity, whereas U0126, an inhibitor of MEK1/2, suppressed basal RUNX2 transcriptional activity and BMP-induced RUNX2 acetylation and stabilization. Overexpression of constitutively active MEK1 stabilized RUNX2 protein via up-regulation of acetylation and down-regulation of ubiquitination. Erk activation increased p300 protein levels and histone acetyltransferase activity. Knockdown of p300 using siRNA diminished Erk-induced RUNX2 stabilization. Overexpression of Smad5 increased RUNX2 acetylation and stabilization. Erk activation further increased Smad-induced RUNX2 acetylation and stabilization, whereas U0126 suppressed these functions. On the other hand, knockdown of Smad1 and Smad5 by siRNA suppressed both basal and Erk-induced RUNX2 protein levels. Erk activation enhanced the association of RUNX2 with p300 and Smad1. Taken together these results indicate that Erk signaling increases RUNX2 stability and transcriptional activity, partly via increasing p300 protein levels and histone acetyltransferase activity and subsequently increasing RUNX2 acetylation by p300. In addition to the canonical Smad pathway, a BMP-induced non-Smad Erk signaling pathway cooperatively regulates osteoblast differentiation partly via increasing the stability and transcriptional activity of RUNX2.
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Bone morphogenetic protein-2 stimulates RUNX2 acetylation
Journal of Biological Chemistry, 2006Co-Authors: Eun Joo Jeon, Kwang Youl Lee, Nam Sook Choi, Mi Hye Lee, Yun Hye Jin, Hyun Nam Kim, Hyunmo Ryoo, Je-yong Choi, Minoru Yoshida, Norikazu NishinoAbstract:RUNX2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. RUNX2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that RUNX2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and RUNX2, in BMP signaling. BMP-2 signaling stimulates p300-mediated RUNX2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of RUNX2. HDAC4 and HDAC5 dea-cetylate RUNX2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases RUNX2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of RUNX2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and RUNX2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.
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dlx5 specifically regulates RUNX2 type ii expression by binding to homeodomain response elements in the RUNX2 distal promoter
Journal of Biological Chemistry, 2005Co-Authors: Wonjoon Yoon, Je-yong Choi, John M Wozney, Kang-young Choi, Yooseok Hwang, Hyunmo RyooAbstract:Abstract Two major isoforms of the RUNX2 gene are expressed by alternative promoter usage: RUNX2 type I (RUNX2-I) is derived from the proximal promoter (P2), and RUNX2 type II (RUNX2-II) is produced by the distal promoter (P1). Our previous results indicate that Dlx5 mediates BMP-2-induced RUNX2 expression and osteoblast differentiation (Lee, M.-H., Kim, Y-J., Kim, H-J., Park, H-D., Kang, A-R., Kyung, H.-M., Sung, J-H., Wozney, J. M., Kim, H-J., and Ryoo, H-M. (2003) J. Biol. Chem. 278, 34387-34394). However, little is known of the molecular mechanisms by which Dlx5 up-regulates RUNX2 expression in BMP-2 signaling. Here, RUNX2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and RUNX2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and RUNX2-I and Msx2 in the sutural mesenchyme. Furthermore, RUNX2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas RUNX2 P2 promoter activity was not. RUNX2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between -756 and -342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the RUNX2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the RUNX2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.
Gary S Stein - One of the best experts on this subject based on the ideXlab platform.
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The Thyroid Hormone Receptor-RUNX2 Axis: A Novel Tumor Suppressive Pathway in Breast Cancer
Hormones and Cancer, 2019Co-Authors: Eric L. Bolf, Jane B. Lian, Janet L. Stein, Gary S Stein, Noelle E. Gillis, Michael S. Barnum, Caitlin M. Beaudet, Grace Y. Yu, Jennifer A. Tomczak, Frances E. CarrAbstract:Metastatic breast cancer is refractory to conventional therapies and is an end-stage disease. RUNX2 is a transcription factor that becomes oncogenic when aberrantly expressed in multiple tumor types, including breast cancer, supporting tumor progression and metastases. Our previous work demonstrated that the thyroid hormone receptor beta (TRβ) inhibits RUNX2 expression and tumorigenic characteristics in thyroid cells. As TRβ is a tumor suppressor, we investigated the compelling question whether TRβ also regulates RUNX2 in breast cancer. The Cancer Genome Atlas indicates that TRβ expression is decreased in the most aggressive basal-like subtype of breast cancer. We established that modulated levels of TRβ results in corresponding changes in the high levels of RUNX2 expression in metastatic, basal-like breast cells. The MDA-MB-231 triple-negative breast cancer cell line exhibits low expression of TRβ and high levels of RUNX2. Increased expression of TRβ decreased RUNX2 levels. The thyroid hormone-mediated suppression of RUNX2 is TRβ specific as TRα overexpression failed to alter RUNX2 expression. Consistent with these findings, knockdown of TRβ in non-tumor MCF10A mammary epithelial-like cells results in an increase in RUNX2 and RUNX2 target genes. Mechanistically, TRβ directly interacts with the proximal promoter of RUNX2 through a thyroid hormone response element to reduce promoter activity. The TRβ suppression of the oncogene RUNX2 is a signaling pathway shared by thyroid and breast cancers. Our findings provide a novel mechanism for TRβ-mediated tumor suppression in breast cancers. This pathway may be common to many solid tumors and impact treatment for metastatic cancers.
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Expression of the IL-11 Gene in Metastatic Cells Is Supported by RUNX2-Smad and RUNX2-cJun Complexes Induced by TGFβ1
Journal of Cellular Biochemistry, 2015Co-Authors: Xuhui Zhang, Deli Hong, Gary S Stein, Jacqueline Akech, Hai Wu, Jason R. Dobson, Gillian Browne, Lucia R. Languino, Jane B. LianAbstract:In tumor cells, two factors are abnormally increased that contribute to metastatic bone disease: RUNX2, a transcription factor that promotes expression of metastasis related and osteolytic genes; and IL-11, a secreted osteolytic cytokine. Here, we addressed a compelling question: Does RUNX2 regulate IL-11 gene expression? We find a positive correlation between RUNX2, IL-11 and TGFβ1, a driver of the vicious cycle of metastatic bone disease, in prostate cancer (PC) cell lines representing early (LNCaP) and late (PC3) stage disease. Further, like RUNX2 knockdown, IL-11 knockdown significantly reduced expression of several osteolytic factors. Modulation of RUNX2 expression results in corresponding changes in IL-11 expression. The IL-11 gene has RUNX2, AP-1 sites and Smad binding elements located on the IL-11 promoter. Here, we demonstrated that RUNX2-c-Jun as well as RUNX2-Smad complexes upregulate IL-11 expression. Functional studies identified a significant loss of IL-11 expression in PC3 cells in the presence of the RUNX2-HTY mutant protein, a mutation that disrupts RUNX2-Smad signaling. In response to TGFβ1 and in the presence of RUNX2, we observed a 30-fold induction of IL-11 expression, accompanied by increased c-Jun binding to the IL-11 promoter. Immunoprecipitation and in situ co-localization studies demonstrated that RUNX2 and c-Jun form nuclear complexes in PC3 cells. Thus, TGFβ1 signaling induces two independent transcriptional pathways - AP-1 and RUNX2. These transcriptional activators converge on IL-11 as a result of RUNX2-Smad and RUNX2-c-Jun interactions to amplify IL-11 gene expression that, together with RUNX2, supports the osteolytic pathology of cancer induced bone disease.
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the cancer related transcription factor RUNX2 modulates cell proliferation in human osteosarcoma cell lines
Journal of Cellular Physiology, 2013Co-Authors: Claudia M J Lucero, Andre J. Van Wijnen, Gary S Stein, Oscar A Vega, Mariana Osorio, Julio C Tapia, Marcelo Antonelli, Mario GalindoAbstract:Osteosarcoma is the most common bone tumor in children and adolescents (Young and Miller, 1975). The highest incidence of osteosarcoma is in the second decade of life, which suggests a relationship between bone growth and tumor development (Fraumeni, 1967; Cotterill et al., 2004). One of the critical steps for normal skeletal development and bone formation is the proliferative expansion of mesenchymal cells, osteoprogenitors, and immature osteoblasts. Cell growth and differentiation of normal osteoprogenitors and pre-osteoblasts is tightly regulated by RUNX2, which favors a quiescent state (Pratap et al., 2003; Galindo et al., 2005). The growth suppressive potential of RUNX2 is controlled by modulation of its protein levels during the cell cycle (Galindo et al., 2005, 2007). Cell cycle dependent changes of RUNX2 levels occur with respect to G1 progression at a cell cycle stage when normal osteoblasts monitor extra-cellular cues for competency to initiate cell cycle progression beyond the G1/S phase transition. Accordingly, transient RUNX2 overexpression in synchronized cells delays cell cycle entry into S phase and significantly decreases cell proliferation in the MC3T3 pre-osteoblasts, RUNX2 null calvarian osteoprogenitors, C2C12 pluripotent mesenchymal, and IMR-90 fibroblasts cell lines (Pratap et al., 2003; Galindo et al., 2005; Young et al., 2007a; Teplyuk et al., 2008, 2009a). The function of RUNX2 as a negative regulator of cell proliferation is also reflected by linkage of RUNX2 deficiency to cell immortalization and tumorigenesis (Kilbey et al., 2007; Zaidi et al., 2007a). Apart from the growth suppressive potential that is evident during late G1 in osteoblasts (Pratap et al., 2003; Galindo et al., 2005), RUNX2 may have mitogenic potential in early G1 (Teplyuk et al., 2008). Several studies indicate that RUNX2-dependent control of proliferation is cell type-specific. RUNX2 inhibits proliferation of osteoprogenitors and committed osteoblasts (Pratap et al., 2003; Galindo et al., 2005), but it may have distinct biological roles in chondrocytes (Galindo et al., 2005; Hinoi et al., 2006; Komori, 2008) and endothelial cells (Inman and Shore, 2003; Qiao et al., 2006). While immature osteoblasts from mice with RUNX2 null mutations show accelerated proliferative potential, chondrocyte proliferation seems to be decreased in RUNX2 null mice (Pratap et al., 2003; Yoshida et al., 2004), suggesting that RUNX2 would also have opposites roles in different bone cell types. Moreover, ectopic expression of RUNX2 in aortic endothelial cells increases cell proliferation (Sun et al., 2004), whereas RUNX2 depletion inhibits cell proliferation in human marrow endothelial cells (Qiao et al., 2006). These findings support the concept that RUNX2 protein can function as either a bona fide tumor suppressor or a classical oncoprotein depending on the cellular context (Blyth et al., 2005). Current evidence indicates that RUNX2 expression is a key pathological factor in osteosarcoma (Martin et al., 2011) by controlling a number of cancer-related genes (van der Deen et al., 2012). Moreover, osteosarcoma development may be associated with RUNX2 overexpression and defects in osteogenic differentiation (Wagner et al., 2011). Over-expression of RUNX2 in transgenic mice within the osteoblast lineage inhibits osteoblast maturation, increases bone resorption, and causes osteopenia with multiple fractures (Liu et al., 2001; Geoffroy et al., 2002). RUNX2 is also clearly detected in clinical osteosarcoma samples (Andela et al., 2005; Lu et al., 2008; Sadikovic et al., 2009; Won et al., 2009; Kurek et al., 2010). Analysis of genomic DNA from osteosarcoma patients with amplication of the 6p12#x02013;p21 chromosomal interval, which spans the RUNX2 locus, increases the RUNX2 gene copy number and aberrantly elevates RUNX2 expression (Lau et al., 2004; Lu et al., 2008; Sadikovic et al., 2009). Increased expression of RUNX2 in osteosarcoma biopsies has been associated to increased tumorigenicity, tumor progression, metastases, lower survival, and poor prognosis (Won et al., 2009; Kurek et al., 2010; Sadikovic et al., 2010). Interestingly, osteosarcoma cell culture models may exhibit a similar variability of RUNX2 gene expression, because RUNX2 is expressed at different levels in a number of human osteosarcoma cell lines (Thomas et al., 2004; Lu et al., 2008; Luo et al., 2008; Kurek et al., 2010; Shapovalov et al., 2010). A subset of patient-derived osteosarcoma cell lines exhibit high levels of RUNX2, whereas others show decreased RUNX2 expression in accordance with the findings of Thomas and colleagues who suggested that RUNX2 protein levels are negatively regulated in some types of osteosarcoma (Thomas et al., 2004; Nathan et al., 2009; Pereira et al., 2009; San Martin et al., 2009; Won et al., 2009; Kurek et al., 2010; Sadikovic et al., 2010). Recently, we presented data indicating that cell cycle control of RUNX2, which is readily observed in osteoblasts, is deregulated in osteosarcoma cells (Galindo et al., 2005; San Martin et al., 2009). RUNX2 is constitutively expressed throughout the cell cycle in at least two osteosarcoma (human SaOS and rat ROS) cell lines (Young et al., 2007b; San Martin et al., 2009). Hence, the transcriptional and post-transcriptional mechanisms that mediate cell cycle control of RUNX2 gene expression in osteoblasts could be compromised in osteosarcoma cells. The latter may occur in conjunction with abrogation of other molecular mechanisms cells that mediate normal osteoblast proliferation and that may bypass the growth suppressive properties of RUNX2 in bone cancer cells (Nathan et al., 2009). In this article, we systematically examined human osteosarcoma cell lines with respect to RUNX2 gene expression and cell cycle regulation to understand the biological functions of RUNX2 in osteosarcoma cell proliferation. Our main finding is that forced expression of RUNX2 suppresses growth in all cell lines, indicating that stimulation of RUNX2 beyond its preestablished levels in osteosarcoma cells remains capable of triggering an anti-proliferative response. We propose that osteosarcoma cells in which RUNX2 is present must balance prooncogenic functions of RUNX2 with the requirement to maintain RUNX2 at levels that avoid tumor suppression.
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Genomic occupancy of HLH, AP1 and RUNX2 motifs within a nuclease sensitive site of the RUNX2 gene
Journal of Cellular Physiology, 2012Co-Authors: Hayk Hovhannisyan, Jane B. Lian, Janet L. Stein, Gary S Stein, Martin Montecino, Mohammad Q Hassan, Ying Zhang, Hai Wu, Carlotta A. Glackin, Andre J. Van WijnenAbstract:Runt-related transcription factors are principal developmental regulators that control lineage commitment and cell type-specificity in diverse species. Null mutations in each of the three known runt-related transcription factor (Runx) genes in mouse causes dramatic tissue-specific phenotypes. Mutations and/or deregulation of the corresponding human genes are linked to familial diseases and genetic predispositions related to cancer and distinct abnormalities in tissue-formation (Blyth et al., 2005; Ito, 2008). While Runx proteins may functionally compensate each other, the unique phenotypes of mice with Runx null mutations are attributable to tissue- and developmental stage-specific activation of transcription. The RUNX2 gene is prominently transcribed in the mesenchymal lineage to support normal development of bone and cartilage in vivo which accounts for the observed skeletal phenotypes of mice with RUNX2 mutations that render mesenchymal cells RUNX2 deficient (Komori et al., 1997; Otto et al., 1997; Choi et al., 2001; Lengner et al., 2002; Jeong et al., 2008; Lou et al., 2009; Zhang et al., 2009a; Liu et al., 2011). RUNX2 transcription, as the initial rate-limiting step in its expression, is exquisitely regulated by a multitude of developmental signals, regulatory promoter elements and cognate transcription factors (Lian et al., 2006; Franceschi et al., 2007; Marie, 2008; Long, 2012). The RUNX2 gene is expressed from two promoters (P1 and P2), and the upstream P1 promoter supports osteoblast-specific gene transcription. Of note, polymorphisms in the P1 and P2 promoter have been correlated with bone mineral density and viral integration sites are capable of ectopic activation of RUNX2 gene transcription in non-osseous cell types (Stewart et al., 2002; Doecke et al., 2006; Lee et al., 2009). The P1 promoter is autoregulated through at least seven Runx binding sites (Drissi et al., 2000, 2002b) and responds to steroid hormones (Tou et al., 2001; Drissi et al., 2002a), BMPs (Xiao et al., 2001; Tou et al., 2003; Lee et al., 2005) and WNTs (Gaur et al., 2005). The P1 promoter is controlled by several homeodomain proteins including AP1 (Drissi et al., 2002a), Dlx5 (Gaur et al., 2005), Nkx3.2 (Lengner et al., 2005), HoxA10 (Hassan et al., 2007), SP1 and Ets proteins (Zhang et al., 2009b), Hif2α (Tamiya et al., 2008), C/EBPβ (Wiper-Bergeron et al., 2007; Henriquez et al., 2011) and NF-1 related proteins (Zambotti et al., 2002). Regulation of RUNX2 gene transcription by this cohort of primary DNA binding proteins occurs within the context of nucleosomal organization and higher order chromatin structure that together modulate accessibility of transcription factors to gene promoters. The RUNX2 gene, which is in throughout the osteogenic lineage, and the osteocalcin (OC) gene, which is transcriptionally activated at the maturation stage of osteoblast differentiation, together represent two versatile and intensively studied models for understanding transcriptional control during osteogenesis (Lian et al., 2004; Montecino et al., 2008). Activation of OC gene expression occurs concomitant with creation of nuclease hypersensitive sites, increased acetylation of histone H3 and H4, as well as specific binding of multiple transcription factors including RUNX2 (Javed et al., 1999; Shen et al., 2002, 2003; Hassan et al., 2004). Studies using rat osteosarcoma cells and trans-differentiated mouse myoblasts have revealed SWI/SNF dependent changes in the chromatin organization of the RUNX2 P1 promoter (Cruzat et al., 2009). In this study, we established key parameters of chromatin fine-structure of the RUNX2 gene promoter in mouse osteoblasts in which the RUNX2 P1 promoter is naturally activated. We define histone modifications, nuclease hypersensitive sites and two genomic protein/DNA interactions that mediate transcriptional regulation of RUNX2 gene expression. Our key finding is that the RUNX2 P1 promoter contains two stable genomic protein-DNA interaction domains that may transcriptionally control the multiple physiological activities of RUNX2 during skeletal development and bone formation in vivo.
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genomic promoter occupancy of runt related transcription factor RUNX2 in osteosarcoma cells identifies genes involved in cell adhesion and motility
Journal of Biological Chemistry, 2012Co-Authors: Margaretha Van Der Deen, Daniel W. Young, Jane B. Lian, Janet L. Stein, Jacqueline Akech, David S Lapointe, Sneha Gupta, Martin Montecino, Mario Galindo, Gary S SteinAbstract:Abstract Runt-related transcription factors (RUNX1, RUNX2, and RUNX3) are key lineage-specific regulators of progenitor cell growth and differentiation but also function pathologically as cancer genes that contribute to tumorigenesis. RUNX2 attenuates growth and stimulates maturation of osteoblasts during bone formation but is also robustly expressed in a subset of osteosarcomas, as well as in metastatic breast and prostate tumors. To assess the biological function of RUNX2 in osteosarcoma cells, we examined human genomic promoter interactions for RUNX2 using chromatin immunoprecipitation (ChIP)-microarray analysis in SAOS-2 cells. Promoter binding of both RUNX2 and RNA polymerase II was compared with gene expression profiles of cells in which RUNX2 was depleted by RNA interference. Many RUNX2-bound loci (1550 of 2339 total) exhibit promoter occupancy by RNA polymerase II and contain the RUNX consensus motif 5′-((T/A/C)G(T/A/C)GG(T/G). Gene ontology analysis indicates that RUNX2 controls components of multiple signaling pathways (e.g. WNT, TGFβ, TNFα, and interleukins), as well as genes linked to cell motility and adhesion (e.g. the focal adhesion-related genes FAK/PTK2 and TLN1). Our results reveal that siRNA depletion of RUNX2, PTK2, or TLN1 diminishes motility of U2OS osteosarcoma cells. Thus, RUNX2 binding to diverse gene loci may support the biological properties of osteosarcoma cells.
Amjad Javed - One of the best experts on this subject based on the ideXlab platform.
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A RUNX2 threshold for the cleidocranial dysplasia phenotype
Human Molecular Genetics, 2008Co-Authors: Amjad Javed, Andre J. Van Wijnen, Jitesh Pratap, Tripti Gaur, Sadiq Hussain, Jennifer L. Colby, Dana Frederick, Stephen N. JonesAbstract:Cleidocranial dysplasia (CCD) in humans is an autosomal-dominant skeletal disease that results from mutations in the bone-specific transcription factor RUNX2 (CBFA1/AML3). However, distinct RUNX2 mutations in CCD do not correlate with the severity of the disease. Here we generated a new mouse model with a hypomorphic RUNX2 mutant allele (RUNX2 neo7 ), in which only part of the transcript is processed to full-length (wild-type) RUNX2 mRNA. Homozygous RUNX2 neo7/neo7 mice express a reduced level of wild-type RUNX2 mRNA (55‐70%) and protein. This mouse model allowed us to establish the minimal requirement of functional RUNX2 for normal bone development. RUNX2 neo7/neo7 mice have grossly normal skeletons with no abnormalities observed in the growth plate, but do exhibit developmental defects in calvaria and clavicles that persist through post-natal growth. Clavicle defects are caused by disrupted endochondral bone formation during embryogenesis. These hypomorphic mice have altered calvarial bone volume, as observed by histology and microCT imaging, and decreased expression of osteoblast marker genes. The bone phenotype of the heterozygous mice, which have 79‐84% of wild-type RUNX2 mRNA, is normal. These results show there is a critical gene dosage requirement of functional RUNX2 for the formation of intramembranous bone tissues during embryogenesis. A decrease to 70% of wild-type RUNX2 levels results in the CCD syndrome, whereas levels >79% produce a normal skeleton. Our findings suggest that the range of bone phenotypes in CCD patients is attributable to quantitative reduction in the functional activity of RUNX2.
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structural coupling of smad and RUNX2 for execution of the bmp2 osteogenic signal
Journal of Biological Chemistry, 2008Co-Authors: Amjad Javed, Andre J. Van Wijnen, Janet L. Stein, Jitesh Pratap, Gary S Stein, Sayyed K Zaidi, Faiza Afzal, Soraya E Gutierrez, Jane B. LianAbstract:Two regulatory pathways, bone morphogenetic protein (BMP)/transforming growth factor-β (TGFβ) and the transcription factor RUNX2, are required for bone formation in vivo. Here we show the interdependent requirement of these pathways to induce an osteogenic program. A panel of RUNX2 deletion and point mutants was used to examine RUNX2-SMAD protein-protein interaction and the biological consequences on BMP2-induced osteogenic signaling determined in RUNX2 null cells. These cells do not respond to BMP2 signal in the absence of RUNX2. We established that a triple mutation in the C-terminal domain of RUNX2, HTY (426-428), disrupts the RUNX2-SMAD interaction, is deficient in its ability to integrate the BMP2/TGFβ signal on promoter reporter assays, and is only marginally functional in promoting early stages of osteoblast differentiation. Furthermore, the HTY mutation overlaps the unique nuclear matrix targeting signal of Runx factors and exhibits reduced subnuclear targeting. Thus, formation of a RUNX2-SMAD osteogenic complex and subnuclear targeting are structurally and functionally inseparable. Our results establish the critical residues of RUNX2 for execution and completion of BMP2 signaling for osteoblastogenesis through a mechanism that requires RUNX2-SMAD transcriptional activity.
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mitotic retention of gene expression patterns by the cell fate determining transcription factor RUNX2
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Daniel W. Young, Amjad Javed, Mario Galindo, Mohammad Q Hassan, Sayyed K Zaidi, Xiaoqing Yang, Paul S Furcinitti, David S LapointeAbstract:During cell division, cessation of transcription is coupled with mitotic chromosome condensation. A fundamental biological question is how gene expression patterns are retained during mitosis to ensure the phenotype of progeny cells. We suggest that cell fate-determining transcription factors provide an epigenetic mechanism for the retention of gene expression patterns during cell division. Runx proteins are lineage-specific transcription factors that are essential for hematopoietic, neuronal, gastrointestinal, and osteogenic cell fates. Here we show that RUNX2 protein is stable during cell division and remains associated with chromosomes during mitosis through sequence-specific DNA binding. Using siRNA-mediated silencing, mitotic cell synchronization, and expression profiling, we identify RUNX2-regulated genes that are modulated postmitotically. Novel target genes involved in cell growth and differentiation were validated by chromatin immunoprecipitation. Importantly, we find that during mitosis, when transcription is shut down, RUNX2 selectively occupies target gene promoters, and RUNX2 deficiency alters mitotic histone modifications. We conclude that Runx proteins have an active role in retaining phenotype during cell division to support lineage-specific control of gene expression in progeny cells.
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Reconstitution of RUNX2/Cbfa1-null cells identifies a requirement for BMP2 signaling through a RUNX2 functional domain during osteoblast differentiation.
Journal of Cellular Biochemistry, 2007Co-Authors: Soraya E Gutierrez, Jane B. Lian, Andre J. Van Wijnen, Janet L. Stein, Jitesh Pratap, Gary S Stein, Radhika Narla, Rajitha Devados, Amjad JavedAbstract:The RUNX2/Cbfa1 transcription factor is a scaffolding protein that promotes osteoblast differentiation; however, the specific RUNX2-functional domains required for induction of the osteogenic lineage remain to be identified. We approached this question using a TERT-immortalized cell line derived from calvaria of RUNX2-null mice by reconstituting the osteogenic activity with wild-type and deletion mutants of RUNX2. The presence or absence of osteogenic media (β-glycerol phosphate and ascorbic acid) and/or with BMP2 did not stimulate osteoblastic gene expression in the RUNX2-null cells. However, cells infected with wild-type RUNX2 adenovirus showed a robust temporal increase in the expression of osteoblast marker genes and were competent to respond to BMP2. Early markers (i.e., collagen type-1, alkaline phosphatase) were induced (four- to eightfold) at Days 4 and 8 of culture. Genes representing mature osteoblasts (e.g., RUNX2, osteopontin, bone sialoprotein, osteocalcin) were temporally expressed and induced from 18- to 36-fold at Days 8 and 12. Interestingly, TGFβ and Vitamin D-mediated transcription of osteoblast genes (except for osteopontin) required the presence of RUNX2. RUNX2 lacking the C-terminal 96 amino acids (RUNX2 Δ432) showed a pattern of gene expression similar to wild-type protein, demonstrating the Groucho interaction and part of the activation domain are dispensable for RUNX2 osteogenic activity. Upon further deletion of the RUNX2 C-terminus containing the nuclear matrix targeting signal and Smad-interacting domain (Δ391), we find none of the osteoblast markers are expressed. Therefore, the RUNX2 391-432 domain is essential for execution of the BMP2 osteogenic signal. J. Cell. Biochem. 100: 434–449, 2007. © 2006 Wiley-Liss, Inc.
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mitotic occupancy and lineage specific transcriptional control of rrna genes by RUNX2
Nature, 2007Co-Authors: Daniel W. Young, Amjad Javed, Jitesh Pratap, Mario Galindo, Mohammad Q Hassan, Sayyed K Zaidi, Xiaoqing YangAbstract:Regulation of ribosomal RNA genes is a fundamental process that supports the growth of cells and is tightly coupled with cell differentiation. Although rRNA transcriptional control by RNA polymerase I (Pol I) and associated factors is well studied, the lineage-specific mechanisms governing rRNA expression remain elusive1. Runt-related transcription factors Runx1, RUNX2 and Runx3 establish and maintain cell identity2, and convey phenotypic information through successive cell divisions for regulatory events that determine cell cycle progression or exit in progeny cells3. Here we establish that mammalian RUNX2 not only controls lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also acts as a repressor of RNA Pol I mediated rRNA synthesis. Within the condensed mitotic chromosomes we find that RUNX2 is retained in large discrete foci at nucleolar organizing regions where rRNA genes reside. These RUNX2 chromosomal foci are associated with open chromatin, co-localize with the RNA Pol I transcription factor UBF1, and undergo transition into nucleoli at sites of rRNA synthesis during interphase. Ribosomal RNA transcription and protein synthesis are enhanced by RUNX2 deficiency that results from gene ablation or RNA interference, whereas induction of RUNX2 specifically and directly represses rDNA promoter activity. RUNX2 forms complexes containing the RNA Pol I transcription factors UBF1 and SL1, co-occupies the rRNA gene promoter with these factors in vivo, and affects local chromatin histone modifications at rDNA regulatory regions. Thus RUNX2 is a critical mechanistic link between cell fate, proliferation and growth control. Our results suggest that lineage-specific control of ribosomal biogenesis may be a fundamental function of transcription factors that govern cell fate.
