Lineage progression in osteoblasts and chondrocytes is stringently controlled by the cell-fate-determining transcription factor Runx2. In this study, we directly addressed whether microRNAs (miRNAs) can control the osteogenic activity of Runx2 and affect osteoblast maturation. A panel of 11 Runx2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-137, miR-204, miR-205, miR-217, miR-218, and miR-338) is expressed in a lineage-related pattern in mesenchymal cell types. During both osteogenic and chondrogenic differentiation, these miRNAs, in general, are inversely expressed relative to Runx2. Based on 3′UTR luciferase reporter, immunoblot, and mRNA stability assays, each miRNA directly attenuates Runx2 protein accumulation. Runx2-targeting miRNAs differentially inhibit Runx2 protein expression in osteoblasts and chondrocytes and display different efficacies. Thus, cellular context contributes to miRNA-mediated regulation of Runx2. All Runx2-targeting miRNAs (except miR-218) significantly impede osteoblast differentiation, and their effects can be reversed by the corresponding anti-miRNAs. These findings demonstrate that osteoblastogenesis is limited by an elaborate network of functionally tested miRNAs that directly target the osteogenic master regulator Runx2.osteogenesis | chondrogenesis | post-transcriptional regulation C ell-fate determination and subsequent lineage progression of phenotype-committed cells are mediated by master regulatory transcription factors that integrate multiple cell-signaling inputs and generate epigenetic changes in chromatin to modulate gene expression. Transcription factors are components of positive and negative feedback loops that initiate or maintain the acquisition of distinct biological states. Epigenomic mechanisms, including attenuation of mRNA and protein expression by small noncoding microRNAs (miRNAs) (1), permit effective control of gene expression beyond genomic interactions between transcription factors and their cognate elements in gene promoters. The biological potency of miRNAs, which are generated by the RNA processing enzyme Dicer, is based on their ability to control mRNA accumulation and/or protein synthesis through specific interactions with the 3′UTRs of target genes (1). Gene regulatory networks involving transcription factors and miRNAs may mutually reinforce cell fates and support phenotypic maturation of lineage-committed cells.Osteogenic differentiation provides an effective cell model in which to define both epigenetic and epigenomic mechanisms required for cell-fate determination and phenotypic differentiation. Differentiation of multipotent mesenchymal stem cells into the osteoblast lineage and maturation of osteoprogenitors are controlled by multiple extracellular ligands [e.g., BMPs, WNTs, and FGFs] (2-4) that direct the activities of key transcription factors, including Runx2, Osterix, and different classes of homeodomain proteins (5-8). Runx2 is a critical regulator of the osteogenic lineage, and its epigenetic functions modulate ...

SummaryThe BMP signaling pathway has a crucial role in chondrocyte proliferation and maturation during endochondral bone development. To investigate the specific function of the Bmp2 and Bmp4 genes in growth plate chondrocytes during cartilage development, we generated chondrocyte-specific Bmp2 and Bmp4 conditional knockout (cKO) mice and Bmp2,Bmp4 double knockout (dKO) mice. We found that deletion of Bmp2 and Bmp4 genes or the Bmp2 gene alone results in a severe chondrodysplasia phenotype, whereas deletion of the Bmp4 gene alone produces a minor cartilage phenotype. Both dKO and Bmp2 cKO mice exhibit severe disorganization of chondrocytes within the growth plate region and display profound defects in chondrocyte proliferation, differentiation and apoptosis. To understand the mechanism by which BMP2 regulates these processes, we explored the specific relationship between BMP2 and Runx2, a key regulator of chondrocyte differentiation. We found that BMP2 induces Runx2 expression at both the transcriptional and post-transcriptional levels. BMP2 enhances Runx2 protein levels through inhibition of CDK4 and subsequent prevention of Runx2 ubiquitylation and proteasomal degradation. Our studies provide novel insights into the genetic control and molecular mechanism of BMP signaling during cartilage development.Key words: Bmp2, Bmp4, Chondrocyte, Endochondral bone formation Introduction During skeletal development, the majority of the bones in the body are established by the endochondral bone formation process, which is initiated by mesenchymal cell condensation and subsequent mesenchymal cell differentiation into chondrocytes and surrounding perichondrial cells. The committed chondrocytes proliferate rapidly forming the cartilage growth plate where cells are arranged in columns of proliferating, differentiating and terminally hypertrophic chondrocytes. Chondrocytes near the center of the cartilage elements exit the cell cycle initiating the process of hypertrophic differentiation to generate a calcified cartilage matrix. Eventually, the local vasculature, perichondrial osteoblasts and various other types of cells invade the calcified cartilage, replacing the terminally mature chondrocytes with marrow components and trabecular bone matrix. Primary ossification occurs with osteoblast-mediated bone formation, which initially occurs on the calcified cartilage template. Chondrocyte maturation and the endochondral bone development process is tightly regulated by a series of growth factors and transcription factors, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), indian hedgehog (Ihh),

At the G 1 /S phase cell cycle transition, multiple histone genes are expressed to ensure that newly synthesized DNA is immediately packaged as chromatin. Here we have purified and functionally characterized the critical transcription factor HiNF-P, which is required for E2F-independent activation of the histone H4 multigene family. Using chromatin immunoprecipitation analysis and ligation-mediated PCR-assisted genomic sequencing, we show that HiNF-P interacts with conserved H4 cell cycle regulatory sequences in vivo. Antisense inhibition of HiNF-P reduces endogenous histone H4 gene expression. Furthermore, we find that HiNF-P utilizes NPAT/p220, a substrate of the cyclin E/cyclin-dependent kinase 2 (CDK2) kinase complex, as a key coactivator to enhance histone H4 gene transcription. The biological role of HiNF-P is reflected by impeded cell cycle progression into S phase upon antisense-mediated reduction of HiNF-P levels. Our results establish that HiNF-P is the ultimate link in a linear signaling pathway that is initiated with the growth factor-dependent induction of cyclin E/CDK2 kinase activity at the restriction point and culminates in the activation of histone H4 genes through HiNF-P at the G 1 /S phase transition.The G 1 /S phase transition represents the critical stage during the somatic cell cycle that defines cellular commitment to replicate the genome and to progress towards mitotic division. Passage beyond the G 1 /S boundary depends initially on the activation of a cyclin/cyclin-dependent kinase (CDK) cascade by growth factors and induction of the cyclin E/CDK2 kinase complex at the restriction (R) point (9,14,29). Subsequently, at the onset of S phase, de novo synthesis of histone proteins is required to package nascent DNA into chromatin immediately upon initiation of DNA synthesis (28,35,36). The exquisitely stringent coupling between histone biosynthesis and DNA replication necessitates the transcriptional activation of the 14 distinct human genes encoding histone H4, the most highly conserved nucleosomal protein (1,13,19,28).Control of histone genes provides a paradigm for gene expression that is temporally and functionally linked to DNA synthesis. It has long been postulated but not yet experimentally validated that the multiple histone H4 genes are coordinately regulated by a single histone H4 gene subtype-specific factor. However, the identity of this protein has never been established. We and others have previously demonstrated that histone H4 genes are regulated by multiple elements and cognate DNA binding activities (6,7,16,30,31,44). The histone gene proximal promoter element site II interacts with three factors (HiNF-M, -D, and -P) and mediates cell cycle control of transcription at the onset of S phase (3,6,7,16,30,31,36,(43)(44)(45)(46)(47). Site II encompasses the H4 subtype-specific element that is phylogenetically conserved among multiple histone H4 genes in metazoan species. The cell cycle regulatory mechanisms operative at site II at the onset of S phase function independently of...

To investigate the role of Wnt–β-catenin signaling in bone remodeling, we analyzed the bone phenotype of female Axin2-lacZ knockout (KO) mice. We found that trabecular bone mass was significantly increased in 6- and 12-month-old Axin2 KO mice and that bone formation rates were also significantly increased in 6-month-old Axin2 KO mice compared with wild-type (WT) littermates. In vitro studies were performed using bone marrow stromal (BMS) cells isolated from 6-month-old WT and Axin2 KO mice. Osteoblast proliferation and differentiation were significantly increased and osteoclast formation was significantly reduced in Axin2 KO mice. Nuclear β-catenin protein levels were significantly increased in BMS cells derived from Axin2 KO mice. In vitro deletion of the β-catenin gene under Axin2 KO background significantly reversed the increased alkaline phosphatase activity and the expression of osteoblast marker genes observed in Axin2 KO BMS cells. We also found that mRNA expression of Bmp2 and Bmp4 and phosphorylated Smad1/5 protein levels were significantly increased in BMS cells derived from Axin2 KO mice. The chemical compound BIO, an inhibitor of glycogen synthase kinase 3β, was utilized for in vitro signaling studies in which upregulated Bmp2 and Bmp4 expression was measured in primary calvarial osteoblasts. Primary calvarial osteoblasts were isolated from Bmp2fx/fx;Bmp4fx/fx mice and infected with adenovirus-expressing Cre recombinase. BIO induced Osx, Col1, Alp and Oc mRNA expression in WT cells and these effects were significantly inhibited in Bmp2/4-deleted osteoblasts, suggesting that BIO-induced Osx and marker gene expression were Bmp2/4-dependent. We further demonstrated that BIO-induced osteoblast marker gene expression was significantly inhibited by Osx siRNA. Taken together, our findings demonstrate that Axin2 is a key negative regulator in bone remodeling in adult mice and regulates osteoblast differentiation through the β-catenin–BMP2/4–Osx signaling pathway in osteoblasts.

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP-2 acts synergistically with β-catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross-talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3-E1 cells to investigate the effect of BMP-2 on β-catenin signaling. We found that BMP-2 stimulates Lrp5 expression and inhibits the expression of β-TrCP, the F-box E3 ligase responsible for β-catenin degradation and subsequently increases β-catenin protein levels in osteoblasts. In vitro deletion of the β-catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP-2 treatment. These findings suggest that BMP-2 may regulate osteoblast function in part through modulation of the β-catenin signaling.