miR-29 Modulates Wnt Signaling in Human Osteoblasts through a Positive Feedback Loop^*

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, and 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/Ham's F-12 medium without phenol red (Invitrogen), supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO), 0.3 mg/ml G418/geneticin (Calbiochem), and 1× penicillin/streptomycin (Invitrogen). For in vitro osteoblastic differentiation, confluent cultures of hFOB cells were maintained in complete medium with the addition of differentiation cocktail as follows: 100 μg/μl ascorbic acid, 10⁻⁸ m menadione, 5 mm β-glycerol phosphate, and 10⁻⁷ m 1–25(OH)₂-vitamin D₃ (all from Sigma) (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 Dulbecco's modified Eagle's medium/Ham's F-12 medium without phenol red, supplemented with 10% nonheat-inactivated fetal bovine serum and 1× penicillin/streptomycin. At confluence, culture medium was supplemented with 100 μg/μl ascorbic acid and 5 mm β-glycerol phosphate, 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 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 cytomegalovirus 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 h, FuGENE 6 (Roche Applied Science) (FuGENE/DNA ratio 3 μl to 1 μg) was used to co-transfect luciferase constructs and a constitutively expressed β-galactosidase construct (Clontech; GenBank^TM accession number U02451), as a control for transfection efficiency. 24 h 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 h. Cultures were serum-deprived for 24 h prior to harvest in 1× Reporter Lysis Buffer (Promega), according to the manufacturer's instructions. All transfection experiments were performed at least three times, using n = 4 or 6 for each experiment, and at least two preparations of each plasmid were tested.

Co-transfection of hFOB cells with luciferase-UTR constructs, β-galactosidase expression construct, and 70-nm anti-miR miRNA inhibitors (Ambion, catalog no. AM1700) or pre-miR miRNA precursor mimics (Ambion, catalog no. AM17100) was accomplished using X-tremeGENE reagent (X-tremeGENE/nucleic acid ratio 5 μl to 1 μg; Roche Applied Science). 24 h post-transfection, cells were serum-deprived for 24 h and then harvested. Experiments utilizing miRNA inhibitors were performed at 39.5 °C, except for Fig. 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 Fig. 1, D and E. This was performed in differentiating conditions because ALP and osteocalcin were not expressed in proliferating conditions (supplemental Fig. 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-galactosidase expression constructs were co-transfected using FuGENE 6 or X-tremeGENE as described above. Cells were maintained at 33.5 °C for 48 h post-transfection. Cells were serum-deprived for 24 h prior to treatment with 5–20 mm NaCl or LiCl (Sigma) or 0–100 ng/ml recombinant human Wnt3a (R & D Systems, Minneapolis, MN) for up to 6 h. TOPFlash and FOPFlash were normalized to β-galactosidase. 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-galactosidase activity (Galacton chemiluminescent assay system, Tropix, Bedford, MA). To control for transfection efficiency, luciferase activity was normalized to that of β-galactosidase.

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) and then cultured under osteoblastic differentiation conditions for 3 days. Cells were serum-deprived for the last 24 h of culture.

Cell layers were lysed in 1× sample buffer (62.5 mm Tris, pH 6.8, 10% glycerol, and 2% SDS) and homogenized. Protein content was measured using the Bio-Rad 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 polyvinylidene difluoride 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 Ser-37 or Thr-41) β-catenin (Millipore), or mouse anti-human total β-catenin (Millipore) primary antibody. Goat anti-mouse or rabbit anti-goat horseradish peroxidase-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 ImageJ software (rsbweb.nih.gov). 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 Moloney murine leukemia virus-reverse transcriptase (Invitrogen), followed by qPCR with iQ SYBR Green Supermix (Bio-Rad) in a MiQ qPCR cycler (Bio-Rad). The primer sets used are shown in supplemental Table 1. RNA levels were determined using standard curves and were normalized to 18 S 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 5 S detection. RNA levels were determined using standard curves, and miR-29 levels were normalized to 5 S rRNA (relative quantity). qRT-PCR experiments were performed at least twice, with n = 3 for each experiment, and each sample was analyzed in duplicate.

Data Analysis

Data are presented as mean ± S.E. Data were analyzed by one-way analysis of variance with Bonferroni post-hoc test or by Student's t test (KaleidaGraph, Synergy Software, Reading, PA).

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 mixture, there was little 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 Fig. 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 (supplemental Fig. 1, B–D) (41, 46–48). Importantly, miR-29a levels were also increased ∼3-fold in these differentiating osteoblasts (Fig. 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 (Fig. 1B) and osteocalcin (Fig. 1C) mRNA expression was significantly decreased compared with the scrambled control inhibitor. When cells were transfected with miR-29a mimic, ALP mRNA levels (Fig. 1D) were unaffected, but osteocalcin (Fig. 1E) mRNA expression was significantly increased compared with 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 (Fig. 2). Osteoblastic cells were grown to confluence and then cultured in the presence of ascorbic acid and β-glycerol phosphate 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 (Fig. 2) (46). In samples derived from females, osteocalcin mRNA peaked at 4 weeks of culture (Fig. 2 and data not shown), whereas in the male sample, osteocalcin mRNA peaked after only 1 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. Because 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 h and examined relative levels of active (de-phosphorylated) β-catenin using Western blot analysis (Fig. 3A). 10 mm LiCl increased active β-catenin compared with the NaCl control, whereas 5 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 h increased TOPFlash activity 4-fold, suggesting accumulation of nuclear β-catenin and activation of canonical Wnt signaling (Fig. 3B). Endogenous miR-29a expression was significantly increased in hFOBs treated with 10 mm LiCl for 1, 3, or 6 h, with the greatest fold increase apparent after 1 h (Fig. 3C). Furthermore, both 25 and 50 ng/ml recombinant Wnt3a significantly increased miR-29a expression after 3 h (Fig. 3D). A time course study demonstrated that 50 ng/ml Wnt3a induced miR-29a expression at 1 and 3 h but not at 6 h of treatment (Fig. 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 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 (Fig. 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 (Fig. 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 three putative binding sites for TCF/LEF, the transcription factors activated by nuclear β-catenin during canonical Wnt signaling (51, 52). Because 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 h (Fig. 4C). Treatment of hFOBs with 10 mm LiCl for 3 h also increased reporter gene expression (Fig. 4D). A time course study showed that treatment of cells with 10 mm LiCl for more than 3 h did not enhance reporter activity (Fig. 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 (Fig. 5A). Shown in Fig. 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 (Fig. 5, C and D). Treatment of the wild type promoter construct with LiCl caused a significant increase in promoter activity, although this effect was abolished when either one or both TCF/LEF-binding sites were mutated (Fig. 5C). Similar effects were observed in transfected cells treated with 50 ng/ml Wnt3a (Fig. 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 (Fig. 1) (53). Furthermore, miR-29 is increased during osteoblastic differentiation, whereas selected negative regulators of Wnt signaling are decreased (Fig. 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, three potential targets, Dkk1, Kremen2, and sFRP2, emerged from our search. Transcripts for Kremen2 and Dkk1 had one potential miR-29a-binding site, and the sFRP2 transcript had two potential binding sites in the 3′-UTR (Fig. 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 with proliferating cells, Dkk1 and sFRP2 mRNA and protein levels were significantly decreased in hFOB cells undergoing osteoblastic differentiation (Fig. 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 (Fig. 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 (Fig. 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 with proliferative cells (Fig. 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 (Fig. 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 with the scrambled control (Fig. 7D–F). These data demonstrated that miR-29a negatively regulates endogenous levels of Dkk1, Kremen2, and sFRP2 protein during osteogenic differentiation in human cells.

Because 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 (Fig. 8A). In the converse experiment, when cells were transfected with miR-29a inhibitor, Wnt3a was unable to significantly increase TOPFlash activity (Fig. 8B). These data demonstrate that miR-29a can enhance Wnt signaling activity, likely due, at least in part, to its inhibition of Wnt antagonists.