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.

Endochondral bone formation, including chondrocyte proliferation, maturation, terminal differentiation and apoptosis, is one major type of bone formation in the vertebrate skeleton. Numerous signaling molecules, cell cycle regulatory proteins, and transcription factors precisely control the balance between chondrocyte proliferation and differentiation.Runx2 is a critical transcription factor that promotes chondrocyte maturation. In Runx2-knockout (Runx2 -/-) mice, the formation of hypertrophic chondrocytes is severely impaired in some skeletal elements including the femur and the humerus (Inada et al., 1999). Targeted expression of Runx2 in non-hypertrophic Col2a1-expressing chondrocytes accelerates chondrocyte differentiation and rescues the chondrocyte phenotype in Runx2 -/-mice (Takeda et al., 2001;Ueta et al., 2001). By contrast, over expression of a dominantnegative Runx2 in Col2a1-expressing chondrocytes inhibits chondrocyte maturation (Takeda et al., 2001;Ueta et al., 2001). These results indicate that Runx2 plays an important role in chondrocyte maturation and also suggests that Runx2 acts not only in hypertrophic chondrocytes but also in Col2a1-expressing proliferating chondrocytes.Runx3, which also belongs to the Runt-domain family of transcription factors, is crucial for gastric epithelial cell growth, neurogenesis of the dorsal root ganglia and CD8-lineage T cell differentiation (Li et al., 2002;Levanon et al., 2002;Taniuchi et al., 2002;Woolf et al., 2003). In addition, Runx3 also plays an In chondrocytes, PTHrP maintains them in a proliferative state and prevents premature hypertrophy. The mechanism by which PTHrP does this is not fully understood. Both Runx2 and Runx3 are required for chondrocyte maturation. We recently demonstrated that cyclin D1 induces Runx2 protein phosphorylation and degradation. In the present studies, we tested the hypothesis that PTHrP regulates both Runx2 and Runx3 protein stability through cyclin D1. We analyzed the effects of cyclin D1 on Runx3 protein stability and function using COS cells, osteoprogenitor C3H10T1/2 cells and chondrogenic RCJ3.1C5.18 cells. We found that cyclin D1 induced Runx3 degradation in a dose-dependent manner and that both Myctagged Runx3 and endogenous Runx3 interact directly with CDK4 in COS and RCJ3.1C5.18 cells. A conserved CDK recognition site was identified in the C-terminal region of Runx3 by sequence analysis (residues 356-359). Pulse-chase experiments showed that the mutation of Runx3 at Ser356 to alanine (SA-Runx3) increased the half-life of Runx3. By contrast, the mutation at the same serine residue to glutamic acid (SE-Runx3) accelerated Runx3 degradation. In addition, SA-Runx3 was resistant to cyclin D1-induced degradation. GSTRunx3 was strongly phosphorylated by CDK4 in vitro. By contrast, CDK4 had no effect on the phosphorylation of SARunx3. Although both wild-type and SE-Runx3 were ubiquitylated, this was not the case for SA-Runx3. Runx3 degradation by cyclin D1 was completely blocked by the proteasome inhibitor PS1. In C3H10T1/2...

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.

Cell growth control by interferons (IFNs) involves upregulation of the tumor suppressor interferon regulatory factor 1 (IRF1). To exert its anti-proliferative effects, this factor must ultimately control transcription of several key genes that regulate cell cycle progression. Here we show that the G 1 /S phase-related cyclindependent kinase 2 (CDK2) gene is a novel proliferationrelated downstream target of IRF1. We find that IRF1, but not IRF2, IRF3, or IRF7, selectively represses CDK2 gene transcription in a dose-and time-dependent manner. We delineate the IRF1-responsive repressor element between nt ؊68 to ؊31 of the CDK2 promoter. For comparison, the tumor suppressor p53 represses CDK2 promoter activity independently of IRF1 through sequences upstream of nt ؊68, and the CDP/cut/Cux1 homeodomain protein represses transcription downstream of ؊31. Thus, IRF1 repression represents one of three distinct mechanisms to attenuate CDK2 levels. The ؊68/؊31 segment lacks a canonical IRF responsive element but contains a single SP1 binding site. Mutation of this element abrogates SP1-dependent enhancement of CDK2 promoter activity as expected but also abolishes IRF1-mediated repression. Forced elevation of SP1 levels increases endogenous CDK2 levels, whereas IRF1 reduces both endogenous SP1 and CDK2 protein levels. Hence, IRF1 represses CDK2 gene expression by interfering with SP1-dependent transcriptional activation. Our findings establish a causal series of events that functionally connect the anti-proliferative effects of interferons with the IRF1-dependent suppression of the CDK2 gene, which encodes a key regulator of the G 1 /S phase transition.Interferon regulatory factors (IRFs) 1 are activated by the anti-proliferative actions of interferons through JAK/STATmediated signaling mechanisms to inhibit cell growth (1-3).Genetic evidence suggests IRF1, the prototypical member of the IRF class of transcription factors, functions as a tumor suppressor (4, 5) presumably by regulating cell growth-related target genes (6). There are few experimentally validated IRF1 target genes, and only a subset of these may contribute to the cell growth inhibitory potential of IRF1 (1, 6 -10). To clarify the biological functions of IRF1 as a tumor suppressor, it is necessary to define additional cell growth regulatory genes that are IRF1-responsive.Our laboratory has shown that IRF1, as well as the closely related protein IRF2 (also known as histone nuclear factor-M (HiNF-M)), can function in the activation of histone H4 gene transcription at the G 1 /S phase transition through a phylogenetically conserved cell cycle regulatory element (9 -16), which encompasses a canonical IRF consensus sequence (17). A characteristic N-terminal DNA binding domain spanning a winged helix-turn-helix motif (18 -20) mediates the interaction of IRF factors with their cognate sites. The C-terminal region supports transcriptional enhancement or repression (21-23). The transcriptional activity of IRF factors is influenced by posttranslational modifications, ...

ADP-ribosylation of proteins plays key roles in multiple biological processes, including DNA damage repair. Recent evidence suggests that serine is an important acceptor for ADP-ribosylation, and that serine ADP-ribosylation is hydrolyzed by ADP-ribosylhydrolase 3 (ARH3 or ADPRHL2). However, the structural details in ARH3-mediated hydrolysis remain elusive. Here, we determined the structure of ARH3 in a complex with ADP-ribose (ADPR). Our analyses revealed a group of acidic residues in ARH3 that keep two Mg ions at the catalytic center for hydrolysis of Ser-linked ADP-ribosyl group. In particular, dynamic conformational changes involving Glu were observed in the catalytic center. Our observations suggest that Mg ions together with Glu and water351 are likely to mediate the cleavage of the glycosidic bond in the serine-ADPR substrate. Moreover, we found that ADPR is buried in a groove and forms multiple hydrogen bonds with the main chain and side chains of ARH3 residues. On the basis of these structural findings, we used site-directed mutagenesis to examine the functional roles of key residues in the catalytic pocket of ARH3 in mediating the hydrolysis of ADP-ribosyl from serine and DNA damage repair. Moreover, we noted that ADPR recognition is essential for the recruitment of ARH3 to DNA lesions. Taken together, our study provides structural and functional insights into the molecular mechanism by which ARH3 hydrolyzes the ADP-ribosyl group from serine and contributes to DNA damage repair.