Examinando por Autor "Stein, Gary S."
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Ítem Enhancer of zeste homolog 2 inhibition stimulates bone formation and mitigates bone loss caused by ovariectomy in skeletally mature mice(American Society for Biochemistry and Molecular Biology Inc. CODEN JBCHA, 2016-11) Dudakovic, Amel; Camilleri, Emily T.; Riester, Scott M.; Paradise, Christopher R.; Gluscevic, Martina; O'Toole, Thomas M.; Thaler, Roman; Evans, Jared M.; Yan, Huihuang; Subramaniam, Malayannan; Hawse, John R.; Stein, Gary S.; Montecino, Martin A.; McGee-Lawrence, Meghan E.; Westendorf, Jennifer J.; Van Wijnen, Andre J.Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults.Ítem Epigenetic control of skeletal development by the histone methyltransferase Ezh2(American Society for Biochemistry and Molecular Biology Inc., 2015-12) Dudakovic, Amel; Camilleri, Emily T.; Xu, Fuhua; Riester, Scott M.; McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Paradise, Christopher R.; Lewallen, Eric A.; Thaler, Roman; Deyle, David R.; Larson, A. Noelle; Lewallen, David G.; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Westendorf, Jennifer J.; Van Wijnen, Andre J.Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) resultsinmultiple defectsinskeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.Ítem Epigenetic control of the bone-master Runx2 gene during osteoblast-lineage commitment by the histone demethylase JARID1B/KDM5B(American Society for Biochemistry and Molecular Biology Inc., 2015-12) Rojas, Adriana; Aguilar, Rodrigo; Henriquez, Berta; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; Van Wijnen, Andre J.; Van Zundert, Brigitte; Allende, Miguel L.; Montecino, MartinTranscription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform) is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.Ítem Epigenetic pathways regulating bone homeostasis:Potential targeting for intervention of skeletal disorders(Current Medicine Group LLC 1, 2014) Gordon, Jonathan A.R.; Montecino, Martin A.; Aqeilan, Rami I.; Stein, Janet L.; Stein, Gary S.; Lian, Jane B.Epigenetic regulation utilizes different mechanisms to convey heritable traits to progeny cells that are independent of DNA sequence, including DNA silencing, posttranslational modifications of histone proteins, and the posttranscriptional modulation of RNA transcript levels by noncoding RNAs.Although long non-coding RNAs have recently emerged as important regulators of gene imprinting, their functions during osteogenesis are as yet unexplored.In contrast, microRNAs (miRNAs) are well characterized for their control of osteogenic and osteoclastic pathways; thus, further defining how gene regulatory networks essential for skeleton functions are coordinated and finely tuned through the activities of miRNAs.Roles of miRNAs are constantly expanding as new studies uncover associations with skeletal disorders.The distinct functions of epigenetic regulators and evidence for integrating their activities to control normal bone gene expression and bone disease will be presented.In addition, potential for using “signature miRNAs” to identify, manage, and therapeutically treat osteosarcoma will be discussed in this review. © Springer Science+Business Media New York 2014.Ítem Epigenetic regulators controlling osteogenic lineage commitment and bone formation(Elsevier Inc., 2024) Dashti, Parisa; Lewallen, Eric A.; Gordon, Jonathan A.R.; Montecino, Martin A.; Davie, James R.; Davie J.R.; Stein, Gary S.; van Leeuwen, Johannes P.T.M.; van der Eerden, Bram C.J.; van Wijnen, Andre J.Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineagespecific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). Thisnarrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for selfrenewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesisÍtem Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2(Wiley-Liss Inc., 2018-11) Araya, Héctor F.; Sepúlveda, Hugo; Lizama, Carlos O.; Vega, Oscar A.; Jerez, Sofía; Briceño, Pedro F.; Thaler, Romane; Riester, Scott M.; Antonelli, Marcelo; Salazar-Onfray, Flavio; Rodríguez, Juan Pablo; Moreno, Ricardo D.; Montecino, Martín; Charbonneau, Martín; Dubois, Claire M.; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario A.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. © 2018 Wiley Periodicals, Inc.Ítem Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells(Wiley-Liss Inc., 2016-05) Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, MarioEpigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that “bookmark” the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. J. Cell. Physiol. 231: 1001–1014, 2016. © 2015 Wiley Periodicals, Inc.Ítem Profiling of human epigenetic regulators using a semi-automated real-time qPCR platform validated by next generation sequencing(Elsevier B.V., 2017-04) Dudakovic, Amel; Gluscevic, Martina; Paradise, Christopher R.; Dudakovic, Halil; Khani, Farzaneh; Thaler, Roman; Ahmed, Farah S.; Li, Xiaodong; Dietz, Allan B.; Stein, Gary S.; Montecino, Martin A.; Deyle, David R.; Westendorf, Jennifer J.Epigenetic mechanisms control phenotypic commitment of mesenchymal stromal/stem cells (MSCs) into osteogenic, chondrogenic or adipogenic lineages. To investigate enzymes and chromatin binding proteins controlling the epigenome, we developed a hybrid expression screening strategy that combines semi-automated real-time qPCR (RT-qPCR), next generation RNA sequencing (RNA-seq), and a novel data management application (FileMerge). This strategy was used to interrogate expression of a large cohort (n > 300) of human epigenetic regulators (EpiRegs) that generate, interpret and/or edit the histone code. We find that EpiRegs with similar enzymatic functions are variably expressed and specific isoforms dominate over others in human MSCs. This principle is exemplified by analysis of key histone acetyl transferases (HATs) and deacetylases (HDACs), H3 lysine methyltransferases (e.g., EHMTs) and demethylases (KDMs), as well as bromodomain (BRDs) and chromobox (CBX) proteins. Our results show gender-specific expression of H3 lysine 9 [H3K9] demethylases (e.g., KDM5D and UTY) as expected and upregulation of distinct EpiRegs (n > 30) during osteogenic differentiation of MSCs (e.g., HDAC5 and HDAC7). The functional significance of HDACs in osteogenic lineage commitment of MSCs was functionally validated using panobinostat (LBH-589). This pan-deacetylase inhibitor suppresses osteoblastic differentiation as evidenced by reductions in bone-specific mRNA markers (e.g., ALPL), alkaline phosphatase activity and calcium deposition (i.e., Alizarin Red staining). Thus, our RT-qPCR platform identifies candidate EpiRegs by expression screening, predicts biological outcomes of their corresponding inhibitors, and enables manipulation of the human epigenome using molecular or pharmacological approaches to control stem cell differentiation. © 2017 Elsevier B.V.Ítem Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation(Elsevier Inc., 2023-12) Dashti, Parisa; Lewallen, Eric A.; Gordon, Jonathan A. R.; Montecino, Martin A.; van Leeuwen, Johannes P. T. M.; Stein, Gary S.; van der Eerden, Bram C. J.; Davie, James R.; van Wijnen, Andre J.Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.Ítem The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization(Elsevier B.V., 2023-01-30) Dashti, Parisa; van de Peppel, Jeroen; Thaler, Roman; Paradise, Christopher R.; Stein, Gary S.; Montecino, Martin A.; van Leeuwen, Johannes P.T.M.; van der Eerden, Bram J.; Dudakovic, Amel; van Wijnen, Andre J.Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation. © 2022 Elsevier B.V.