A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.

MicroRNAs (miRNAs) interfere with translation of specific target mRNAs and are thought to thereby regulate many cellular processes. Recent studies have suggested that miRNAs might play a role in osteoblast differentiation and bone formation. Here, we identify a new miRNA (miR-2861) in primary mouse osteoblasts that promotes osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) expression at the post-transcriptional level. miR-2861 was found to be transcribed in ST2 stromal cells during bone morphogenetic protein 2-induced (BMP2-induced) osteogenesis, and overexpression of miR-2861 enhanced BMP2-induced osteoblastogenesis, whereas inhibition of miR-2861 expression attenuated it. HDAC5, an enhancer of runt-related transcription factor 2 (Runx2) degradation, was confirmed to be a target of miR-2861. In vivo silencing of miR-2861 in mice reduced Runx2 protein expression, inhibited bone formation, and decreased bone mass. Importantly, miR-2861 was found to be conserved in humans, and a homozygous mutation in pre-miR-2861 that blocked expression of miR-2861 was shown to cause primary osteoporosis in 2 related adolescents. Consistent with the mouse data, HDAC5 levels were increased and Runx2 levels decreased in bone samples from the 2 affected individuals. Thus, our studies show that miR-2861 plays an important physiological role in osteoblast differentiation and contributes to osteoporosis via its effect on osteoblasts.

[1]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[2]  O. Mäkitie,et al.  Heterozygous Mutations in the LDL Receptor‐Related Protein 5 (LRP5) Gene Are Associated With Primary Osteoporosis in Children , 2005, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[3]  Thomas Tuschl,et al.  Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. , 2004, RNA.

[4]  B. Cullen,et al.  Sequence requirements for micro RNA processing and function in human cells. , 2003, RNA.

[5]  T. Sugiyama,et al.  RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing , 2004, Nature Genetics.

[6]  Wei Yan,et al.  Cloning and expression profiling of testis-expressed piRNA-like RNAs. , 2007, RNA.

[7]  Di Chen,et al.  E3 Ubiquitin Ligase Smurf1 Mediates Core-binding Factor α1/Runx2 Degradation and Plays A Specific Role in Osteoblast Differentiation* , 2003, Journal of Biological Chemistry.

[8]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[9]  Y. Okazaki,et al.  miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. , 2008, Biochemical and biophysical research communications.

[10]  E. Sontheimer,et al.  Origins and Mechanisms of miRNAs and siRNAs , 2009, Cell.

[11]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[12]  James M. Tanner,et al.  Growth at Adolescence , 1956 .

[13]  E. Liao,et al.  Establishment of BMD reference plots and determination of peak BMD at multiple skeletal regions in mainland Chinese women and the diagnosis of osteoporosis , 2004, Osteoporosis International.

[14]  E. Miska,et al.  MicroRNA functions in animal development and human disease , 2005, Development.

[15]  R. Pochampally,et al.  Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression , 2008, Proceedings of the National Academy of Sciences.

[16]  G. Stein,et al.  Differential regulation of the two principal Runx2/Cbfa1 n-terminal isoforms in response to bone morphogenetic protein-2 during development of the osteoblast phenotype. , 2001, Endocrinology.

[17]  Muller Fabbri,et al.  A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. , 2005, The New England journal of medicine.

[18]  J. Steitz,et al.  Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5′ UTR as in the 3′ UTR , 2007, Proceedings of the National Academy of Sciences.

[19]  Yvonne Tay,et al.  MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation , 2009, Nature.

[20]  L. Ala‐Kokko,et al.  Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Barbara Jarzab,et al.  Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma , 2008, Proceedings of the National Academy of Sciences.

[22]  Yvonne Tay,et al.  A Pattern-Based Method for the Identification of MicroRNA Binding Sites and Their Corresponding Heteroduplexes , 2006, Cell.

[23]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[24]  Xiang-hang Luo,et al.  Establishment and evaluation of bone mineral density reference databases appropriate for diagnosis and evaluation of osteoporosis in Chinese women , 2003, Journal of Bone and Mineral Metabolism.

[25]  Brian D Athey,et al.  New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. , 2009, Genome research.

[26]  Peng Jin,et al.  Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA. , 2007, Human molecular genetics.

[27]  G. Stein,et al.  A microRNA signature for a BMP2-induced osteoblast lineage commitment program , 2008, Proceedings of the National Academy of Sciences.

[28]  E. Lai,et al.  microRNA control of cell-cell signaling during development and disease , 2008, Cell cycle.

[29]  Di Chen,et al.  Smurf1 Inhibits Osteoblast Differentiation and Bone Formation in Vitro and in Vivo* , 2004, Journal of Biological Chemistry.

[30]  V. Rosen,et al.  Effects of BMP-2, BMP-4, and BMP-6 on osteoblastic differentiation of bone marrow-derived stromal cell lines, ST2 and MC3T3-G2/PA6. , 1996, Biochemical and biophysical research communications.

[31]  R. Derynck,et al.  Repression of Runx2 function by TGF‐β through recruitment of class II histone deacetylases by Smad3 , 2005, The EMBO journal.

[32]  Minoru Yoshida,et al.  Bone Morphogenetic Protein-2 Stimulates Runx2 Acetylation* , 2006, Journal of Biological Chemistry.

[33]  C. Deng,et al.  Ubiquitin Ligase Smurf1 Controls Osteoblast Activity and Bone Homeostasis by Targeting MEKK2 for Degradation , 2005, Cell.

[34]  V. Kim,et al.  Biogenesis of small RNAs in animals , 2009, Nature Reviews Molecular Cell Biology.

[35]  D. Wei,et al.  In Vitro and In Vivo Synergistic Interactions Between the Runx2/Cbfa1 Transcription Factor and Bone Morphogenetic Protein‐2 in Stimulating Osteoblast Differentiation , 2003, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[36]  Stijn van Dongen,et al.  miRBase: microRNA sequences, targets and gene nomenclature , 2005, Nucleic Acids Res..

[37]  R. Mazess,et al.  Bilateral measurement of femoral bone mineral density. , 2000, Journal of clinical densitometry : the official journal of the International Society for Clinical Densitometry.

[38]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[39]  Sean R. Eddy,et al.  Rfam: annotating non-coding RNAs in complete genomes , 2004, Nucleic Acids Res..

[40]  N. Rajewsky,et al.  Silencing of microRNAs in vivo with ‘antagomirs’ , 2005, Nature.

[41]  T. Komori,et al.  Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. , 2000, Endocrine reviews.

[42]  R. Allshire,et al.  Hairpin RNAs and Retrotransposon LTRs Effect RNAi and Chromatin-Based Gene Silencing , 2003, Science.