Downstream Target Of Runx2 Research Articles

The role of lysophosphatidylcholine acyltransferase 2 in osteoblastic differentiation of C2C12 cells.

Glycerophospholipids, a primary component of cellular membranes, play important structural and functional roles in cells. In the remodelling pathway (Lands' cycle), the concerted actions of phospholipase As and lysophospholipid acyltransferases (LPLATs) contribute to the incorporation of diverse fatty acids in glycerophospholipids in an asymmetric manner, which differ between cell types. In this study, the role of LPLATs in osteoblastic differentiation of C2C12 cells was investigated. Gene and protein expression levels of lysophosphatidylcholine acyltransferase 2 (LPCAT2), one of the LPLATs, increased during osteoblastic differentiation in C2C12 cells. LPCAT2 knockdown in C2C12 cells downregulated the expression of osteoblastic differentiation markers and the number and size of lipid droplets (LDs) and suppressed the phosphorylation of Smad1/5/9. In addition, LPCAT2 knockdown inhibited Snail1 and the downstream target of Runx2 and vitamin D receptor (VDR). These results suggest that LPCAT2 modulates osteoblastic differentiation in C2C12 cells through the bone morphogenetic protein (BMP)/Smad signalling pathway.

SOX9 is a key component of RUNX2-regulated transcriptional circuitry in osteosarcoma

BackgroundThe absence of prominent, actionable genetic alternations in osteosarcomas (OS) implies that transcriptional and epigenetic mechanisms significantly contribute to the progression of this life-threatening form of cancer. Therefore, the identification of potential transcriptional events that promote the survival of OS cells could be key in devising targeted therapeutic approaches for OS. We have previously shown that RUNX2 is a transcription factor (TF) essential for OS cell survival. Unfortunately, the transcriptional network or circuitry regulated by RUNX2 in OS cells is still largely unknown.MethodsThe TFs that are in the RUNX2 transcriptional circuitry were identified by analyzing RNAseq and ChIPseq datasets of RUNX2. To evaluate the effect of SOX9 knockdown on the survival of osteosarcoma cells in vitro, we employed cleaved caspase-3 immunoblotting and propidium iodide staining techniques. The impact of SOX9 and JMJD1C depletion on OS tumor growth was examined in vivo using xenografts and immunohistochemistry. Downstream targets of SOX9 were identified and dissected using RNAseq, pathway analysis, and gene set enrichment analysis. Furthermore, the interactome of SOX9 was identified using BioID and validated by PLA.ResultOur findings demonstrate that SOX9 is a critical TF that is induced by RUNX2. Both in vitro and in vivo experiments revealed that SOX9 plays a pivotal role in the survival of OS. RNAseq analysis revealed that SOX9 activates the transcription of MYC, a downstream target of RUNX2. Mechanistically, our results suggest a transcriptional network involving SOX9, RUNX2, and MYC, with SOX9 binding to RUNX2. Moreover, we discovered that JMJD1C, a chromatin factor, is a novel binding partner of SOX9, and depletion of JMJD1C impairs OS tumor growth.ConclusionThe findings of this study represent a significant advancement in our understanding of the transcriptional network present in OS cells, providing valuable insights that may contribute to the development of targeted therapies for OS.

The transcription factor RUNX2 fuels YAP1 signaling and gastric cancer tumorigenesis

Despite considerable efforts in the detection and treatment of gastric cancer (GC), the underlying mechanism of the progression of GC remains unknown. Our previous work has demonstrated the remarkable role of Runt‐related transcription factor 2 (RUNX2), in fueling the invasion and metastasis of GC. The present study aimed to elucidate the role of RUNX2 in tumorigenesis of GC. We assessed Runx2 expression and its clinical significance via bioinformatic analysis of the Cancer Genome Atlas and Gene Expression Omnibus databases. Roles for Runx2 in self‐renewal and tumorigenesis were examined in vitro and in vivo. Further bioinformatic analysis was applied to study the mechanism of GC progression. We found that Runx2 was highly expressed in the early stage of GC and positively correlated with a poor clinical outcome of patients. Runx2 was also significantly correlated with clinicopathological features, such as Hp infection, new neoplastic events, primary therapeutic outcome, ethnicity, race, and tumor stage. Multivariate analysis revealed that together with Runx2, age, cancer status, M stage, and T stage were independent prognostic factors for the outcome of GC patients. RUNX2 overexpression induced increased anchorage‐independent colony formation, sphere formation, and tumorigenesis in GC cells in vitro and in vivo. Mechanistically, bioinformatic analysis indicated that yes1 associated transcriptional regulator (YAP1) might be a downstream target of RUNX2. Specific knockdown of YAP1 reduced the tumor‐initiating ability of GC cells induced by ectopic Runx2 expression. Our findings support the hypothesis that RUNX2 exerts oncogenic properties via YAP1 regulation, highlighting essential roles for RUNX2 and YAP1 in gastric carcinogenesis and suggesting potential therapeutic targets.

RUNX2/miR‑31/SATB2 pathway in nickel‑induced BEAS‑2B cell transformation.

Nickel (Ni) compounds are classified as Group 1 carcinogens by the International Agency for Research on Cancer (IARC) and are known to be carcinogenic to the lungs. In our previous study, special AT‑rich sequence‑binding protein2 (SATB2) was required for Ni‑induced BEAS‑2B cell transformation. In the present study, a pathway that regulates the expression of SATB2 protein was investigated in Ni‑transformed BEAS‑2B cells using western blotting and RT‑qPCR for expression, and soft agar, migration and invasion assays for cell transformation. Runt‑related transcription factor2 (RUNX2), a master regulator of osteogenesis and an oncogene, was identified as an upstream regulator for SATB2. Ni induced RUNX2 expression and initiated BEAS‑2B transformation and metastatic potential. Previously, miRNA‑31 was identified as a negative regulator of SATB2 during arsenic‑induced cell transformation, and in the present study it was identified as a downstream target of RUNX2 during carcinogenesis. miR‑31 expression was reduced in Ni‑transformed BEAS‑2B cells, which was required to maintain cancer hallmarks. The expression level of miR‑31 was suppressed by RUNX2 in BEAS‑2B cells, and this increased the expression level of SATB2, initiating cell transformation. Ni caused the repression of miR‑31 by placing repressive marks at its promoter, which in turn increased the expression level of SATB2, leading to cell transformation.

Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM.

ABSTRACTProgenitor cells are crucial in controlling organ morphogenesis. Tooth development is a well‐established model for investigating the molecular and cellular mechanisms that regulate organogenesis. Despite advances in our understanding of how tooth crown formation is regulated, we have limited understanding of tooth root development. Runt‐related transcription factor 2 (RUNX2) is a well‐known transcription factor in osteogenic differentiation and early tooth development. However, the function of RUNX2 during tooth root formation remains unknown. We revealed in this study that RUNX2 is expressed in a subpopulation of GLI1+ root progenitor cells, and that loss of Runx2 in these GLI1+ progenitor cells and their progeny results in root developmental defects. Our results provide in vivo evidence that Runx2 plays a crucial role in tooth root development and in regulating the differentiation of root progenitor cells. Furthermore, we identified that Gli1, Pcp4, NOTUM, and Sfrp2 are downstream targets of Runx2 by integrating bulk and single‐cell RNA sequencing analyses. Specifically, ablation of Runx2 results in downregulation of WNT inhibitor NOTUM and upregulation of canonical WNT signaling in the odontoblastic site, which disturbs normal odontoblastic differentiation. Significantly, exogenous NOTUM partially rescues the impaired root development in Runx2 mutant molars. Collectively, our studies elucidate how Runx2 achieves functional specificity in regulating the development of diverse organs and yields new insights into the network that regulates tooth root development. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Hypoxia Inhibits Osteogenesis in Human Mesenchymal Stem Cells through Direct Regulation of RUNX2 by TWIST

BackgroundBone loss induced by hypoxia is associated with various pathophysiological conditions, however, little is known about the effects of hypoxia and related signaling pathways on osteoblast differentiation and bone formation. Because bone marrow-derived mesenchymal stem cells (MSCs) survive under hypoxic conditions and readily differentiate into osteoblasts by standard induction protocols, they are a good in vitro model to study the effects of hypoxia on osteoblast differentiation.Methodology/Principle FindingsUsing human MSCs, we discovered TWIST, a downstream target of HIF-1α, was induced under hypoxia and acted as a transcription repressor of RUNX2 through binding to the E-box located on the promoter of type 1 RUNX2. Suppression of type 1 RUNX2 by TWIST under hypoxia further inhibited the expression of BMP2, type 2 RUNX2 and downstream targets of RUNX2 in MSCs.Conclusions/SignificanceOur findings point to the important role of hypoxia-mediated signalling in osteogenic differentiation in MSCs through direct regulation of RUNX2 by TWIST, and provide a method for modifying MSC osteogenesis upon application of these cells in fracture healing and bone reconstruction.

Zfp64 participates in Notch signaling and regulates differentiation in mesenchymal cells

Notch signaling is required for multiple aspects of tissue and cell differentiation. In this study, we identified zinc finger protein 64 (Zfp64) as a novel coactivator of Notch1. Zfp64 is associated with the intracellular domain of Notch1, recruited to the promoters of the Notch target genes Hes1 and Hey1, and transactivates them. Zfp64 expression is under the control of Runx2, and is upregulated by direct transactivation of its promoter. Zfp64 suppresses the myogenic differentiation of C2C12 cells and promotes their osteoblastic differentiation. Our data demonstrate two functions of Zfp64: (1) it is a downstream target of Runx2 and, (2) its cognate protein acts as a coactivator of Notch1, which suggests that Zfp64 mediates mesenchymal cell differentiation by modulating Notch signaling.

Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2

We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5' flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2(-/-) calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis.

The Runx2 Osteogenic Transcription Factor Regulates Matrix Metalloproteinase 9 in Bone Metastatic Cancer Cells and Controls Cell Invasion

The Runx2 (Cbfa1/AML3) transcription factor and matrix metalloproteinase 9 (MMP9) are key regulators of growth plate maturation and bone formation. The genes for both proteins are characteristic markers of breast and prostate cancer cells that metastasize to bone. Here we experimentally addressed the compelling question of whether Runx2 and MMP are functionally linked. By cDNA expression array analysis, we identified MMP9 as a novel downstream target of Runx2. Like that of MMP13, MMP9 expression is nearly depleted in Runx2 mutant mice. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the recruitment of Runx2 to the MMP9 promoter. We show by mutational analysis that the Runx2 site mediates transactivation of the MMP9 promoter in osteoblasts (MC3T3-E1) and nonosseous (HeLa) cells. The overexpression of Runx2 by adenovirus delivery in nonmetastatic (MCF-7) and metastatic breast (MDA-MB-231) and prostate (PC3) cancer cell lines significantly increases the endogenous levels of MMP9. The knockdown of Runx2 by RNA interference decreases MMP9 expression, as well as that of other Runx2 target genes, including the genes for MMP13 and vascular endothelial growth factor. Importantly, we have demonstrated using a cell invasion assay that Runx2-regulated MMP9 levels are functionally related to the invasion properties of cancer cells. These results are consistent with Runx2 control of multiple genes that contribute to the metastatic properties of cancer cells and their activity in the bone microenvironment.

BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2

BMP-2 stimulates the expression of three osteogenic master transcription factors: Runx2, Dlx5, and Osterix (Osx). However, the hierarchical regulatory relationships among them are not yet clearly understood. Osx was commonly stimulated in osteogenic and non-osteogenic cells in response to BMP-signaling, as Dlx5 was in our previous report. A cycloheximide experiment indicated that Osx expression by BMP-2 requires new protein synthesis. Even if Osx has been suggested as a downstream target of Runx2, the results of this study indicated that Osx expression was still induced by BMP-2 treatment in Runx2 null cells, but not induced by Runx2 overexpression in myogenic C2C12 cells. Instead, Osx expression by BMP-2 was completely abrogated by the antisense blocking of Dlx5. Depending upon the coincident expression pattern of Osx and Dlx5, and the blocking of Osx expression by the antisense Dlx5, BMP-2-induced Osx expression is mainly mediated not by Runx2, but by Dlx5.