MIR-29 MODULATES WNT SIGNALING IN HUMAN OSTEOBLASTS THROUGH A POSITIVE

FEEDBACK LOOP

Kristina Kapinas1, Catherine Kessler1, Tinisha Ricks1, Gloria Gronowicz2, Anne M. Delany1 1 Center for Molecular Medicine, 2Department of Surgery, University of Connecticut Health Center, 263

Farmington Ave, Farmington, CT 06030

Running head: miR-29/Wnt feedback loop in osteoblastic differentiation

Address correspondence to: Anne Delany, PhD, Center for Molecular Medicine, University of Connecticut Health Center, 263 Farmington Ave, Farmington, CT 06030; Ph: 860-679-8736; Fax: 860-679-7639; E-mail: adelany@uchc.edu

Differentiation of human mesenchymal stem cells into osteoblasts is controlled by extracellular cues. Canonical Wnt signaling is particularly important for maintenance of bone mass in humans. Post-transcriptional regulation of gene expression, mediated by microRNAs (miRNAs), plays an essential role in the control of osteoblast differentiation. Here, we find that miR-29a is necessary for human osteoblast differentiation, and miR-29a is increased during differentiation in the mesenchymal precursor cell line hFOB1.19 and in primary cultures of human osteoblasts. Further, the promoter of the expressed sequence tag containing the human miR-29a gene is induced by canonical Wnt signaling. This effect is mediated, at least in part, by two TCF/LEF binding sites within the proximal promoter. Further, we show that the negative regulators of Wnt signaling, Dikkopf-1 (Dkk1), Kremen2, and secreted frizzled related protein 2 (sFRP2), are direct targets of miR-29a. Endogenous protein levels for these Wnt antagonists are increased in cells transfected with synthetic miR-29a inhibitor. In contrast, transfection with miR-29a mimic decreases expression of these antagonists and potentiates Wnt signaling. Overall, we demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that can modulate osteoblast differentiation. Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a

potentiates Wnt signaling, contributing to a gene expression program important for osteoblast differentiation. This novel regulatory circuit provides additional insight into how miRNAs interact with signaling molecules during osteoblast differentiation, allowing for fine tuning of intricate cellular processes.

Human mesenchymal stem cells (hMSC) have the potential to differentiate into multiple cell lineages, including osteoblast, adipocyte, chondrocyte, and neural (1-4). Differentiation into a specific lineage depends on the extracellular cues received by the cell. Bone-anabolic agents such as bone morphogenetic protein (BMP) -2, parathyroid hormone (PTH), and canonical Wnts promote osteoblast differentiation through the stimulation of distinct second messenger pathways (5). In particular, canonical Wnt signaling is crucial for the regulation of bone mass in humans and for osteoblast differentiation (6-10).

The binding of a canonical Wnt member (i.e. Wnt3a) to a Frizzled receptor and a co-receptor, LRP5/6, initiates a signaling cascade that results in the release of cytoplasmic β-catenin from an inhibitory complex consisting of Axin and glycogen synthase kinase (GSK) 3. Upon de-phosphorylation and release, β-catenin can translocate to the nucleus, where it can interact with the T-cell factor/lymphoid enhancer factor-1 (TCF/LEF1) family of transcription factors, and activate transcription of genes necessary for osteoblast differentiation (11). Wnt signaling can be attenuated by several classes of negative regulators. For example, Dkk1 is a soluble factor that acts in conjunction with Kremen2, a decoy receptor, to inhibit Wnt signaling by

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http://www.jbc.org/cgi/doi/10.1074/jbc.M110.116137The latest version is at JBC Papers in Press. Published on June 15, 2010 as Manuscript M110.116137

Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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preventing the binding of Wnt proteins to the LRP5/6 co-receptor (12). There is a marked increase in bone mineral density in Dkk1 haploinsufficient mice and in Kremen-null mice (13, 14). Further, the chromosomal region that contains the Dkk1 locus has been linked to reduced bone mass in young osteoporotic men (15). In addition, the secreted frizzled-related protein (sFRP) family of proteins also binds extracellular Wnts, to prevent their binding to cell surface receptors. Wnts and their negative regulators have critical roles in bone development and/or maintenance, and their expression is modulated during the course of osteoblastic differentiation (16-18).

Canonical Wnt signaling increases bone mass by a number of mechanisms (19). During early differentiation, Wnt signaling promotes MSC proliferation (20-22). Canonical wnt signaling then drives the differentiation of osteo-chondral progenitors towards the osteoblastic lineage (23). In addition, Wnt signaling inhibits osteoblast and osteocyte apoptosis (19). However, it appears that canonical Wnt signaling regulates osteoblast differentiation in a dose- and time-dependent manner, and that pathway components must be tightly regulated for proper differentiation. For example, low doses of LiCl or Wnt3a stimulate proliferation of human bone marrow-derived MSCs (hBMSCs), but higher doses actually inhibit proliferation and initiate osteoblastic differentiation (20, 22). Indeed, the expression of two negative regulators of Wnt signaling, sFRP2 and Dkk1, is decreased in mature osteoblasts, providing a potential mechanism for increased Wnt signaling in more differentiated cells (18).

There is increasing evidence that post-transcriptional regulation of gene expression, mediated by microRNAs (miRNAs), plays an important role in the control of osteoblastic differentiation (24-28). miRNAs are 21-23 nucleotide, noncoding RNAs that negatively regulate gene expression at the post-transcriptional level (29). Mature miRNAs modulate gene expression by base pairing to complementary sequences within an mRNA target. Through their association with the RNA-induced silencing complex (RISC), miRNAs can facilitate degradation of the bound transcript or inhibit its translation. Since a single miRNA can

have many targets, it is possible that one miRNA could regulate families of structural molecules or could regulate distinct signaling molecules within a particular pathway (30-33). For example, we and others have demonstrated that the miR-29 family, consisting of miR-29a, miR-29c, miR-29b1 and miR-29b2, can down regulate the expression of fibrillar collagens (i.e. COL1A1, COL3A1, COL4A2) and the expression of osteonectin/SPARC (secreted protein acidic and rich in cysteine), which regulates collagen fibrillogenesis (24, 25, 34, 35).

In addition, miR-29 is important for murine osteoblast differentiation (24, 25). The expression of miR-29 family members increases during the progression of osteoblastic differentiation in primary cultures of murine calvarial osteoblasts. We demonstrated that expression of miR-29a and miR-29c is rapidly induced by canonical Wnt signaling in these osteoblasts (24). However, the mechanisms mediating this up-regulation and its consequences remain unexplored.

miR-29c and miR-29b2 are transcribed in tandem, in the same primary miRNA (pri-miRNA). This pri-miRNA is found within the last exon of an EST (expressed sequence tag) on human chromosome 1 (36). The pri-miRNA is processed to yield the two distinct miRNAs, miR-29c and miR-29b2. An interaction between the transcription factor Myc and the putative promoter region of the miR-29c-b2 gene was recently demonstrated, and Myc is thought to down regulate expression of the gene in lymphoma cells (36). Similarly, in muscle cells, the transcription factor YY1 interacts with the miR-29c-b2 gene promoter and represses its expression (33). Like miR-29c and miR-29b2, miR-29a and miR-29b1 are transcribed within the same pri-miRNA. However, mapping of the pri-miRNA structure demonstrated that the miR-29a-b1 pri-miRNA is contained within an intron of a spliced EST on chromosome 7. The transcription factor Myc has also been shown to interact with the putative promoter region of this EST (36).

Since canonical Wnt signaling rapidly induces miR-29 levels, we hypothesized that Wnt signaling induces miR-29 transcription. In addition, given that both miR-29 and Wnt signaling are important for osteoblastic differentiation, and that the expression of some negative regulators of Wnt signaling is decreased during osteogenesis, we hypothesized that miR-29 may negatively regulate inhibitors of Wnt signaling. We chose to test these

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hypotheses in human osteoblasts, focusing on the promoter for the miR-29a-b1 gene, since it is less well studied and because osteoblasts appear to have higher levels of miR-29a than miR-29c (24). Further, we determined whether miR-29a could decrease the expression of selected negative regulators of Wnt signaling.

Experimental Procedures Cell Culture- The human fetal osteoblastic 1.19 cell line (hFOB) was obtained from American Type Culture Collection (ATCC, Manassas, VA). The hFOB cell line carries a temperature sensitive SV40 T antigen transgene. Culture at the permissive temperature of 33.5°C allows these cells to proliferate, while culture at the restrictive temperature of 39.5°C slows proliferation and promotes differentiation (1). hFOB cells were maintained at 33.5°C in complete medium consisting of: 1:1 Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium without phenol red (Invitrogen, Fredrick, MD), supplemented with 10% fetal bovine serum (FBS) (Atlas Biologicals, Fort Collins, CO), 0.3 mg/mL G418/geneticin (Calbiochem, Gibbstown, NJ), and 1X Penicillin/Streptomycin (Pen/Strep) (Invitrogen). For in vitro osteoblastic differentiation, confluent cultures of hFOB cells were maintained in complete medium with the addition of differentiation cocktail: 100 µg/µL ascorbic acid, 10-8 M menadione, 5mM β-glycerolphosphate, and 10-7 M 1-25(OH)2-Vitamin D3 (all from Sigma, St Louis, MO) (1).

Primary cultures of human osteoblasts were derived from bone chips obtained from healthy individuals undergoing routine orthopedic procedures (37-42). Isolation of osteoblasts from these de-identified specimens was deemed exempt by the University of Connecticut Health Center Institutional Review Board. Experiments were performed on cells isolated from both males and females. Cells were maintained in 1:1 DMEM/Ham’s F-12 medium without phenol red, supplemented with 10% non-heat-inactivated FBS and 1X Pen/Strep. At confluence, culture medium was supplemented with 100 µg/µL ascorbic acid and 5 mM β-glycerolphosphate, to induce osteoblast differentiation. Primary human osteoblasts were

cultured for up to 4 weeks, with twice weekly change of medium. Alkaline phosphatase staining was performed with a kit from Sigma. Constructs- PCR and appropriate primer sets were used to amplify the first 2 kilobases (kb) and several deletion mutants of the human miR-29a putative promoter region (EU154353) using human genomic DNA as a template (36). Promoter fragments were subcloned into the luciferase reporter pGL4.10 (Promega, Madison, WI) using KpnI and EcoRV restriction enzyme sites. Site-directed mutagenesis of putative TCF/LEF-1 binding sites was performed using overlap extension (43).

PCR and appropriate primer sets were used to amplify SFRP2 (NM_003013), DKK1 (NM_012242), and KREMEN2 (NM_172229) 3’ untranslated regions (UTRs) of interest, using human genomic DNA as a template. UTR fragments were subcloned into the CMV promoter-luciferase reporter pMIR-REPORT (Ambion, Austin, TX) using MluI and SacI restriction enzyme sites. Transfections- hFOB cells were plated at 10,000 cells/cm2. After 24 hours, Fugene6 (Roche, Indianapolis, IN) (Fugene:DNA ratio 3 µL:1 µg) was used to co-transfect luciferase constructs and a constitutively expressed β-galactosidase (β-gal) construct (Clontech, Mountain View, CA; GenBank Accession No. U02451), as a control for transfection efficiency. 24 hours post-transfection, cultures were either maintained in complete medium at 33.5°C (“proliferative conditions”) or in osteoblastic differentiation medium at 39.5°C (“differentiation conditions”) for 48 hours. Cultures were serum-deprived for 24 hours prior to harvest in 1X Reporter Lysis Buffer (Promega), according to the manufacturer’s instructions. All transfection experiments were performed at least 3 times, using N=4 or 6 for each experiment, and at least 2 preparations of each plasmid were tested.

Co-transfection of hFOB cells with luciferase-UTR constructs, β–gal expression construct, and 70nm Anti-miR miRNA inhibitors (Ambion, cat# AM1700) or Pre-miR miRNA precursor mimics (Ambion, cat# AM17100), was accomplished using X-tremeGENE reagent (X-tremeGENE:nucleic acid ratio 5 µL:1 µg, Roche). 24 hours post-transfection, cells were serum-deprived for 24 hours and then harvested.

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Experiments utilizing miRNA inhibitors were performed at 39.5°C, except for Figure 8B. This experiment was performed at 33.5°C, in proliferating conditions, because recombinant Wnt3a was incapable of stimulating TOPFlash activity at 39.5°C (data not shown). Experiments utilizing miRNA mimics were performed at 33.5°C, except for the experiment shown in Figure 1D and E. This was performed in differentiating conditions because ALP and osteocalcin were not expressed in proliferating conditions (Supplementary Figure 1).

For some experiments, hFOB cells were transfected with the Wnt signaling luciferase reporter construct TOPFlash or its negative control, FOPFlash (Addgene, Cambridge, MA). Luciferase reporter and β–gal expression constructs were co-transfected using Fugene6 or X-tremeGENE as described above. Cells were maintained at 33.5°C for 48 hours post-transfection. Cells were serum-deprived for 24 hours prior to treatment with 5 mM-20 mM NaCl or LiCl (Sigma) or 0-100 ng/mL recombinant human Wnt3a (R&D Systems, Minneapolis, MN) for up to 6 hours. TOPFlash and FOPFlash were normalized to β–gal. Data are represented as normalized TOPFlash/FOPFlash values.

Equal aliquots of cell lysate were used to determine luciferase activity (Luciferase Assay System, Promega) and β–gal activity (Galacton chemiluminescent assay system, Tropix, Bedford, MA). To control for transfection efficiency, luciferase activity was normalized to that of β–gal.

Western Blotting- hFOB cells were cultured for 3 days post-confluence under proliferative or osteoblastic differentiation conditions. In selected experiments, hFOB cells were transfected with 50-150 nM miR-29a miRNA inhibitor or negative control inhibitor (scrambled) using Oligofectamine (Invitrogen) (Anti-miR miRNA inhibitors, Ambion), then cultured under osteoblastic differentiation conditions for 3 days. Cells were serum-deprived for the last 24 hours of culture.

Cell layers were lysed in 1X sample buffer (62.5 mM Tris pH 6.8, 10% glycerol, and 2% SDS) and homogenized. Protein content was measured using the BioRad (Hercules, CA)

DC Protein Assay Kit, according to the manufacturer’s instructions. Equal amounts of cell layer were subjected to electrophoresis through a 10.5% SDS-polyacrylamide gel under reducing conditions, and transferred to a PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with 5% nonfat milk powder in PBST (phosphate buffered saline, 0.1% Tween) and probed with goat anti-human sFRP2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-human Dkk1 (Millipore), mouse anti-human Kremen2 (Abcam, Cambridge, MA), mouse anti-human active (de-phosphorylated at Ser37 or Thr41) β-catenin (Millipore), or mouse anti-human total β-catenin (Millipore) primary antibody. Goat anti-mouse- or rabbit anti-goat-horseradish peroxidase (HRP) conjugated secondary antibody (Sigma) was used, as appropriate. Bands were visualized by chemiluminescence (Cell Signaling, Danvers, MA) and fluorography. Blots were stripped and re-probed with goat anti-rabbit actin antibody (Sigma). Relative band intensities in scanned images were analyzed with Image J software (http://rsbweb.nih.gov/ij/index.html). Western blot experiments were performed at least twice, with N=3-6 in each experiment.

Quantitative Real-Time PCR- RNA was isolated from cells using Trizol (Invitrogen) and quantified spectroscopically. Equal amounts of RNA were treated with RQ1 DNase I (Promega) prior to analysis, to exclude any signal from DNA contamination. To quantify mRNA levels in total RNA, DNased RNA was reverse transcribed with MMLV-Reverse Transcriptase (Invitrogen), followed by qPCR with iQ SYBR Green Supermix (BioRad) in a MiQ qPCR cycler (BioRad). The primer sets used are shown in Supplementary Table 1. RNA levels were determined using standard curves and were normalized to 18S rRNA. To quantify miRNA levels in total RNA, the mirVana qRT-PCR miRNA Detection Kit and primers (Ambion) were used, according to the manufacturer’s instructions. 50 ng aliquots were used for miR-29a and 5S detection. RNA levels were determined using standard curves and miR-29 levels were normalized to 5S rRNA (relative quantity). qRT-PCR experiments were performed at least twice, with N=3 for each experiment, and each sample analyzed in duplicate.

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Data Analysis- Data are presented as mean ± SEM. Data were analyzed by one-way ANOVA with Bonferroni post-hoc test or by Student’s t test (KaleidaGraph, Synergy Software, Reading, PA).

RESULTS

miR-29a expression in differentiating human osteoblasts. In confluent cultures of hFOB mesenchymal progenitor cells and in proliferating cultures maintained for 3 days post-confluence in the absence of differentiation cocktail, there was little alkaline phosphatase (ALP) activity, which is a marker of early osteoblastogenesis (44, 45). However, in cells cultured for 3 days post-confluence in osteoblastic differentiation medium, ALP staining was readily apparent (Supplemental Figure 1A). qRT-PCR confirmed the induction of ALP mRNA by the osteoblastic culture conditions, which was accompanied by increased expression of transcripts for Runx2, a transcription factor critical for osteoblastic differentiation, and osteocalcin, a mature osteoblast marker (41, 46-48) (Supplemental Figure 1 B-D). Importantly, miR-29a levels were also increased ~3 fold in these differentiating osteoblasts (Figure 1A), similar to our observations of miR-29 expression in mouse osteoblasts (24).

To determine the function of miR-29a in human osteoblastic differentiation, hFOBs were transiently transfected with miR-29a inhibitor, and then cultured under osteoblastic differentiation conditions. qRT-PCR showed that when miR-29a function is blocked, ALP (Figure 1B) and osteocalcin (Figure 1C) mRNA expression was significantly decreased compared to the scrambled control inhibitor. When cells were transfected with miR-29a mimic, ALP mRNA levels (Figure 1D) were unaffected, but osteocalcin (Figure 1E) mRNA expression was significantly increased compared to the scrambled control mimic. miR-29 is not predicted to directly interact with either ALP or osteocalcin mRNA. These data suggest that miR-29a promotes osteoblastic differentiation, and that loss of miR-29a function suppresses this process.

Next, we quantified miR-29a levels in differentiating cultures of primary human osteoblasts. We tested cells derived from bone chips obtained from two individuals: a 62 year old female, and a 54 year old male (Figure 2). Osteoblastic cells were grown to confluence and then cultured in the presence of ascorbic acid and β-glycerolphosphate for up to 4 weeks, promoting osteoblastic differentiation. qRT-PCR showed that, for the most part, Runx2 mRNA levels peaked in the early weeks of the differentiation process, and were subsequently down regulated (46) (Figure 2). In samples derived from females, osteocalcin mRNA peaked at 4 weeks of culture (Figure 2 and data not shown), whereas in the male sample, osteocalcin mRNA peaked after only one week of culture. In all samples tested, miR-29a expression gradually increased with time in culture, with highest levels after 3-4 weeks, suggesting that miR-29a may be a marker of differentiation. Since all three markers of osteoblastic differentiation are increased in both the hFOB cell line and primary human osteoblasts, we proceeded to use the hFOB model system to investigate miR-29a promoter activity and the effects of miR-29a on Wnt signaling.

Canonical Wnt signaling up-regulates miR-29a in human osteoblasts. Treatment of cells with LiCl mimics canonical Wnt signaling by inhibiting GSK3 phosphorylation of β-catenin (49, 50). To confirm this effect in our system, we treated hFOBs with 5-20 mM LiCl for 3 hours, and examined relative levels of active (de-phosphorylated) β-catenin using Western blot analysis (Figure 3A). 10mM LiCl increased active β-catenin compared to the NaCl control, whereas 5 mM and 20 mM LiCl did not. To document that LiCl activates canonical Wnt signaling in hFOBs, we used the Wnt signaling reporter construct TOPFlash. In this TOPFlash reporter, transcription of the luciferase gene is driven by 7 TCF/LEF binding sites, upstream of a minimal promoter. Its negative control, FOPFlash, is a similar construct in which the TCF/LEF binding sites are mutated. Treatment of transiently transfected hFOB cells with 10 mM LiCl for 6 hours increased TOPFlash activity 4-fold, suggesting accumulation of nuclear β-catenin and activation of canonical Wnt signaling (Figure 3B). Endogenous miR-29a expression was significantly increased in hFOBs treated with 10 mM LiCl for 1, 3 or 6 hours, with the greatest fold-increase apparent after 1 hour (Figure 3C). Further, both 25 and 50 ng/ml

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recombinant Wnt3a significantly increased miR-29a expression after 3 hours (Figure 3D). A time course study demonstrated that 50 ng/ml Wnt3a induced miR-29a expression at 1 and 3 hours, but not at 6 hours of treatment (Figure 3E). Together, these data showed canonical Wnt signaling induces mature miR-29a expression in human osteoblasts.

miR-29a promoter is activated by Wnt signaling. The transcription start site for the EST containing the miR-29a-b1 coding region was previously defined (36). PCR and appropriate primer sets were used to amplify the genomic region -2159 to -1 base pairs (bp) from this transcription start site. This ~2.1 kb promoter fragment and a series of 5’ deletion mutants were subcloned into a promoterless luciferase reporter construct (Figure 4A). When transiently transfected into hFOB cells, the activity of the -2159 to -1 promoter-luciferase construct was significantly greater than that of the promoterless control. However, transcriptional activity was greatly increased with subsequent 5’ deletions of the promoter, down to -688 bp. These data suggest the presence of negative regulatory elements between bp -2159 and -688 (Figure 4B). The activity of the construct containing -142 to -1 of promoter region was not significantly different from the promoterless control. Thus, the minimal promoter for the miR-29a-b1 containing EST may lie within the -688 to -1 base region. Analysis of the 2159 bp promoter region using the CHIP Bioinformatics Mapper suggested the presence of 3 putative binding sites for TCF/LEF, the transcription factors activated by nuclear β-catenin during canonical Wnt signaling (51, 52). Since the -982 to -1 promoter region showed high basal activity and had two putative TCF/LEF sites, we used transient transfection of this promoter construct to determine whether the miR-29a promoter was responsive to canonical Wnt signaling. Similar to endogenous miR-29a, miR-29a promoter-reporter gene expression was significantly increased in cells treated with 50 ng/mL Wnt3a after 6 hours (Figure 4C). Treatment of hFOBs with 10 mM LiCl for 3 hours also increased reporter gene expression (Figure 4D). A time course study showed that treatment of cells with 10 mM LiCl for more than 3 hours did not

enhance reporter activity (Figure 4E and data not shown). These data suggested that canonical Wnt signaling can rapidly induce miR-29a promoter activity. To determine whether the putative TCF/LEF binding sites are responsible for this effect, we generated constructs with mutations at these sites, within the context of the -982 to -1 promoter region (Figure 5A). Shown in Figure 5B is the sequence of the putative TCF/LEF binding sites within the miR-29 promoter (WT sequence), and the mutants we generated, which are predicted to abolish TCF/LEF binding. Basal level activity of the single TCF/LEF mutants was similar to that of the WT construct, however the double mutant (mut/mut) had significantly greater reporter gene expression, suggesting that these mutations relieved TCF/LEF-mediated transcriptional repression (Figure 5 C and D). Treatment of the wild type promoter construct with LiCl caused a significant increase in promoter activity, whereas this effect was abolished when either one or both TCF/LEF binding sites were mutated (Figure 5C). Similar effects were observed in transfected cells treated with 50 ng/mL Wnt3a (Figure 5D). These data suggested that both of the TCF/LEF sites are necessary for the induction of miR-29a promoter activity by canonical Wnt signaling.

miR-29a targets negative regulators of Wnt signaling. Canonical Wnt signaling and miR-29a expression promote osteoblast differentiation (Figure 2; 53). Further, miR-29 is increased during osteoblastic differentiation, whereas selected negative regulators of Wnt signaling are decreased (Figure 1; 18, 24). To test the hypothesis that miR-29a targets antagonists of canonical Wnt signaling, we used RNAHybrid to identify potential targets of miR-29a that are known inhibitors of canonical Wnt signaling (54). Based on the potential for interaction of miR-29a with the 3’ UTR of each transcript, 3 potential targets, Dkk1, Kremen2, and sFRP2, emerged from our search. Transcripts for Kremen2 and Dkk1 had 1 potential miR-29a binding site, and the sFRP2 transcript had 2 potential binding sites in the 3’UTR (Figure 6A). We first determined whether the endogenous mRNA and protein levels of these Wnt antagonists were regulated during osteoblastic differentiation in hFOB cells. qRT-PCR and Western blot analysis showed that, compared to proliferating cells, Dkk1 and sFRP2 mRNA and protein levels were significantly decreased in hFOB

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cells undergoing osteoblastic differentiation (Figure 6 B and D, E and G). Although Kremen2 transcripts were not decreased in osteogenic cells, Kremen2 protein levels were significantly reduced, indicating repression of translation (Figure 6 C and F).

To determine whether the 3’UTR of these selected Wnt antagonists could differentially regulate gene expression, we generated luciferase reporter constructs in which the 3’UTR for Dkk1, Kremen2, or sFRP2 was cloned into the multiple cloning site of the pMIR-REPORT vector (Figure 6A). The inserted 3’UTR acts as the 3’UTR for the luciferase gene, modulating luciferase transcript stability and/or translation. Constructs were transiently transfected into hFOB cells and cells were then cultured under proliferative or osteogenic conditions. The 3’ UTR of each Wnt antagonist, Dkk1, Kremen2, and sFRP2, mediated a significant decrease in gene expression in osteogenic cells compared to proliferative cells (Figure 7A).

Next we determined whether miR-29a could directly regulate the 3’ UTRs of the selected Wnt antagonists. Luciferase-3’UTR constructs were transiently transfected into hFOB cells along with scrambled or miR-29a inhibitors or mimics (Figure 7 B and C). Inhibitor-treated cells were cultured under osteogenic conditions, where endogenous miR-29a expression is high. Transfection with miR-29a inhibitor relieved 3’ UTR-mediated repression of luciferase activity of Dkk1, Kremen2 and sFRP2 3’UTR constructs. Cells transfected with the miRNA mimic were cultured under proliferating conditions, where miR-29a expression is low. Transfection with the miR-29a mimic decreased luciferase activity of Dkk1, Kremen2 and sFRP2 3’UTR constructs. Together, these data suggested that miR-29a acts directly on the 3’ UTRs of these Wnt signaling antagonists, to negatively regulate their expression.

We confirmed this by examining the endogenous protein levels of Dkk1, Kremen2, and sFRP2 when miR-29a function is blocked by transfection with the miR-29a inhibitor. Western blot analysis of transfected hFOB cells grown under osteogenic conditions showed that blocking miR-29a function significantly

increased Dkk1, Kremen2, and sFRP2 protein expression compared to the scrambled control (Figure 7D-F). These data demonstrated that miR-29a negatively regulates endogenous levels of Dkk1, Kremen2, and sFRP2 protein during osteogenic differentiation in human cells.

Since miR-29 negatively regulates Wnt antagonists, we hypothesized that miR-29 positively affects Wnt signaling activity. To test this hypothesis, hFOB cells were transiently transfected with the Wnt signaling reporter TOPFlash or negative control FOPFlash, along with miR-29a mimic or scrambled control. In cells transfected with the scrambled control, treatment with Wnt3a caused a modest increase in TOPFlash activity, as seen in the past. However, in cells treated with miR-29a mimic, Wnt3a increased TOPFlash activity by ~4-fold, indicating that Wnt signaling was potentiated (Figure 8A). In the converse experiment, when cells were transfected with miR-29a inhibitor, Wnt3a was unable to significantly increase TOPFlash activity (Figure 8B). These data demonstrate that miR-29a can enhance Wnt signaling activity, likely due, at least in part, to its inhibition of Wnt antagonists.

DISCUSSION

Both canonical Wnt signaling and miR-29 promote osteoblast differentiation, through a variety of mechanisms (24, 25, 55). Here, we provide evidence for a novel positive feedback loop involving canonical Wnt signaling and miR-29, likely contributing to human osteoblast differentiation (Figure 8C). Specifically, canonical Wnt signaling induces miR-29a transcription. The subsequent down regulation of key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, by miR-29a could potentiate Wnt signaling, and contribute to a gene expression program important for osteoblast differentiation.

miRNAs orchestrate osteoblast differentiation through their regulation of diverse signaling molecules and pathways (56). For example, miR-210 acts on transcripts for activin A receptor type 1B, down-regulating the receptor and promoting osteoblastic differentiation (57). In ST2 cells, BMP-2 induces expression of miR-2861, and miR-2861 down regulates histone deacetylase (HDAC) 5. Since HDAC5 enhances degradation of Runx2, the induction of miR-2861 promotes

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differentiation. Further, mice treated systemically with a miR-2861 inhibitor develop osteopenia (58).

Importantly, miRNAs can also inhibit osteoblast differentiation. Transgenic mice expressing miR-206 in osteoblasts develop a low bone mass phenotype due to impaired osteoblast differentiation, because miR-206 targets connexin43, which propagates signaling between osteoblasts to promote differentiation (27). Further, miR-204 and -211 target Runx2 in bone marrow stromal cells. Since Runx2 is required for the transcription of genes important for osteoblast differentiation, over expression of miR-204 represses osteoblast differentiation and promotes adipogenic differentiation (28). In addition, miR-26a inhibits osteoblast differentiation of human adipose-derived stem cells by down-regulating SMAD1, an important downstream mediator of BMP signaling (59). miR-141/-200a are highly expressed during differentiation and were recently found to inhibit osteoblast differentiation by targeting Dlx5 (distal-less homeobox 5), a transcription factor expressed in pre-osteoblasts (60). These studies highlight the wide range of signaling pathways modulated by miRNAs in osteoblasts.

There is a rapidly growing understanding of the mechanisms controlling the post-transcriptional processing of miRNAs, but there is less known about the regulation of miRNA gene transcription (61, 62). miRNAs can be transcribed by RNA polymerase II or III, and transcription can be modulated by the interaction of trans-acting factors with the promoter region (63-67). For example, MyoD, Mef2, and SRF transcription factors determine the heart-specific expression of miR-1, whereas BMP-2 induces miR-23b~27b~24-1 transcription, to promote adipogenesis of C3H10T1/2 cells (68, 69). Interestingly, c-Myc, activates the transcription of the miR-17-92 cluster (70), but represses transcription of several other miRNA genes (36).

Here, we show that the proximal promoter region of the miR-29a-b1 containing EST has strong promoter activity when transfected into human osteoblasts. The 982 bp

miR-29a promoter region contains two putative TCF/LEF sites. When both sites were mutated, there was a significant increase in basal promoter activity (Figure 5). This is likely due to the repressor function of TCF/LEF. When the accumulation of nuclear β-catenin is limited, TCF/LEF can repress transcription through its interaction with the Groucho/TLE (Transducin-like-Enhance of Split) family of repressor proteins (71-73). Interaction of the co-activator β-catenin with TCF/LEF results in the displacement of Groucho/TLE, and allows the induction of gene transcription (74). Interestingly, mutation of only one TCF/LEF site was not sufficient to relieve repression of the basal promoter activity. However, mutation of a single TFC/LEF site was sufficient to abolish induction of the promoter by Wnt signaling. This suggests that both sites are needed to induce transcription stimulated by Wnt.

We show that miR-29 promotes osteoblastic differentiation, and its ability to down-regulate the expression of key Wnt signaling inhibitors likely contributes to this effect by promoting Wnt signaling. Like bone, Wnt signaling is important in cardiomyocyte differentiation. During myogenesis, miR-29c expression is de-repressed, promoting myogenic differentiation (33). In cardiomyocytes, sFRP2 inhibits differentiation by inhibiting transcriptional activation of Wnt3a (75). It is possible that targeting of sFRP2 RNA by miR-29 is one mechanism contributing to myogenic differentiation. Since miR-29 is expressed in a wide array of tissues, it is likely that other miR-29 regulatory networks could exist (76-78).

miRNAs provide a mechanism for fine tuning intricate cellular processes. In combination with a variety of transcription factors and signaling molecules, miRNAs help to control the complex program of osteoblast differentiation. Our data demonstrate that miR-29 and Wnt signaling are involved in a regulatory circuit that modulates osteoblast differentiation, and likely impacts bone remodeling and the maintenance of bone mass. This novel regulatory circuit provides additional insight into how miRNAs interact with signaling molecules during osteoblast differentiation and has implications for the treatment of bone loss diseases.

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FOOTNOTES

This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, AR44877 (AM Delany). We thank Nathan Selsky for technical assistance.

FIGURE LEGENDS Fig. 1. miR-29a is necessary for osteoblast differentiation. (A) Relative quantity of miR-29a vs. 5S rRNA. * = significantly different from proliferative conditions p < 0.05. hFOB cells were transiently transfected with miR-29a inhibitor and then cultured for 3 days under osteoblast differentiation conditions. Relative quantity (RQ) of (B) alkaline phosphatase (ALP) and (C) osteocalcin mRNA expression, normalized to 18S rRNA. hFOB cells were transiently transfected with miR-29a mimic and then cultured for 3 days under osteoblast differentiation conditions. Relative quantity (RQ) of (D) ALP and (E) osteocalcin mRNA expression, normalized to 18S rRNA. * = significantly different from the scrambled negative control p < 0.05.

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Fig. 2. Characterization of miR-29a expression in primary cultures of human osteoblastic cells. Primary cells were cultured for up to 4 weeks post-confluence in osteoblast differentiation medium. Relative quantity (RQ) of Runx2 (●), osteocalcin (◘), and miR-29a (▲) RNA expression normalized to 18S rRNA or 5S rRNA, respectively. (A) 62-year old female, and (B) 54-year old male. * = significantly different from week 0 (confluence) p < 0.05. Fig. 3. Canonical Wnt signaling induces miR-29a expression. (A) Activated (de-phosphorylated) β-catenin protein normalized to total β-catenin protein in hFOB cells treated with 10 mM NaCl (control) or LiCl for 3 hours. * = significantly different from NaCl p < 0.05. (B) Relative luciferase activity (RLU) of TOPFlash/FOPFlash, in transiently transfected hFOB cells treated with 10 mM NaCl or LiCl for 6 hours. * = significantly different from NaCl p < 0.05. (C) Time course of miR-29a expression in response to 10 mM LiCl or NaCl. RQ = relative quantity vs. 5S rRNA. * = significantly different from NaCl p < 0.05. (D) Induction of miR-29a expression in hFOB cells treated with 0-100 ng/mL recombinant human Wnt3a for 3 hours. RQ = relative quantity vs. 5S rRNA. * = significantly different from 0 ng/mL Wnt3a (vehicle) p < 0.05. (E) Time course of miR-29a expression in hFOB cells treated with vehicle or 50 ng/mL Wnt3a for up to 6 hours. RQ = relative quantity vs. 5S rRNA. * = significantly different from vehicle at that time point p < 0.05. Fig. 4. Canonical Wnt signaling induces miR-29a transcription. (A) Diagram of miR-29a promoter constructs, indicating the number of bases from the transcription start site. Predicted Tcf/Lef sites are represented by black bars. (B) Luciferase activity of the promoter constructs in transiently transfected hFOB cells. * = significantly different from pGL4.10 (vector alone) p < 0.05. (C) Activity of the -982 to -1 miR-29a promoter construct in transiently transfected hFOB cells treated with 0-50 ng/mL recombinant human Wnt3a for 3 or 6 hours. * = significantly different from 0 ng/mL Wnt3a at that time point p < 0.05. (D) Activity of the -982 to -1 miR-29a promoter construct in hFOB cells treated with 5-20 mM NaCl or LiCl for 3 hours in hFOB cells. * = significantly different from NaCl p <0.05. (E) Activity of the -982 to -1 miR-29a promoter construct in hFOB cells treated with 10 mM NaCl or LiCl for 3 or 6 hours in hFOB cells. * = significantly different from NaCl p <0.05. Fig. 5. Canonical Wnt signaling induces miR-29a promoter activity through two Tcf/Lef sites. (A) Diagram of the -982 to -1 miR-29a promoter, and single or double mutants of the two predicted Tcf/Lef binding sites. A black box indicates a WT TCF/LEF site, and an X indicates a mutated site. (B) Consensus sequences for the Tcf/Lef binding site, the WT sequences in the endogenous -982 to -1 miR-29a promoter, and the sequences of the mutated binding sites (underlined). Activity of wild type and mutant -982 to -1 base constructs in transiently transfected hFOB cells treated with 10 mM NaCl or LiCl for 3 hours (C) or with vehicle or 50 ng/mL recombinant human Wnt3a for 6 hours (D) * = significantly different from NaCl or vehicle p < 0.05. # = significantly different from WT/WT p < 0.05. Fig. 6. Wnt signaling antagonists are down regulated during osteoblast differentiation. (A) Diagram of Kremen2, Dkk1, and sFRP2 3’UTRs and potential miR-29a binding sites. Relative quantity (RQ) of (B) Dkk1, (C) Kremen2, and (D) sFRP2 mRNA normalized to 18S rRNA, in hFOB cells. Relative expression of (E) Dkk1, (F) Kremen2, and (G) sFRP2 protein, normalized to actin, in hFOB cells. * = significantly different from proliferative conditions p < 0.05. Fig. 7. miR-29a negatively regulates Wnt antagonists. (A) Activity of luciferase-3’ UTR constructs in transiently transfected hFOB cells, under proliferating or differentiating conditions. * = significantly different from proliferative conditions p < 0.05. (B) Activity of luciferase-3’ UTR constructs in hFOB cells, transiently co-transfected with 70 nm miRNA inhibitor and cultured under differentiating conditions. * = significantly different from scrambled control. (C) Activity of luciferase-3’ UTR constructs in hFOB cells, transiently co-transfected with 70 nm miRNA mimic and cultured under proliferative conditions. * = significantly different from scrambled control p < 0.05. Protein levels for

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(D) Dkk1, (E) Kremen2, and (F) sFRP2 in differentiating hFOBs treated with 50-150 nm scrambled or miR-29a inhibitor. Protein expression was normalized to actin. * = significantly different from scrambled control p < 0.05. Fig. 8. Wnt signaling is regulated by a miR-29/Wnt positive feedback loop. Luciferase activity of TOPFlash/FOPFlash in hFOBs transiently co-transfected with (A) 70nm miRNA mimic or (B) 70nm miRNA inhibitor, cultured in proliferating conditions. (C) Proposed model. Canonical Wnt signaling and miR-29 promote osteoblast differentiation, through a variety of mechanisms (dotted lines). Canonical Wnt signaling induces miR-29a transcription. miR-29a subsequently down regulates key Wnt signaling antagonists, Dkk1, Kremen2, and sFRP2, potentiating Wnt signaling. These two actions promote a gene expression program necessary for osteoblast differentiation.

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DelanyKristina Kapinas, Catherine B. Kessler, Tinisha Ricks, Gloria Gronowicz and Anne M.

loopmiR-29 modulates WNT signaling in human osteoblasts through a positive feedback

published online June 15, 2010J. Biol. Chem. 

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