miR-34a blocks osteoporosis and bone metastasis by inhibiting osteoclastogenesis and Tgif2.

Bone-resorbing osteoclasts significantly contribute to osteoporosis and bone metastases of cancer. MicroRNAs play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how microRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is downregulated during osteoclast differentiation. Osteoclastic miR-34a-overexpressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify transforming growth factor-β-induced factor 2 (Tgif2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.

We examined the levels of several cancer-related miRNAs during a time course of bone marrow osteoclastogenesis assay (). While the expression of an osteoclast marker tartrate-resistant acid phosphatase (TRAP) was rapidly increased by RANKL and further elevated by rosiglitazone[7,8] (), miR-34a was rapidly down-regulated by RANKL and further diminished by rosiglitazone (). The levels of miR-34b/c, two other members in the miR-34 family, were unaffected and expressed at much lower levels than miR-34a ().

The sequence of miR-34a is evolutionally conserved and identical in mice and humans. Osteoclast differentiation from both mouse bone marrow precursors () and human peripheral blood mononuclear cells (hPBMN) () was inhibited by a miR-34a precursor (pre-miR-34a) but enhanced by an antisense miR-34a inhibitor (anti-miR-34a), indicating that miR-34a regulation of bone resorption in mice will likely translate to human pathophysiology.

We generated osteoclastic miR-34a transgenic mice using CAG34a mice () and Tie2-cre mice[7,8]. FACS and imaging showed that osteoclast progenitors from the 34a-Tie2-Tg (CAG34a+Cre+) mice were converted to GFP+LacZ- whereas the controls (CAG34a+Cre-) remained GFP-LacZ+ (). Northern blot confirmed the over-expression of mature-miR-34a in the bone marrow of 34a-Tie2-Tg mice ().

Osteoclast differentiation assay reveal that the higher levels of mature miR-34a in the 34a-Tie2-Tg cultures resulted in a lower induction of osteoclast markers, diminished number/size of mature osteoclasts, and reduced resorptive activity, whereas precursor proliferation or survival was unaltered (, ). Consequently, serum bone resorption marker CTX-1 (C-terminal telopeptides of Type I collagen) and osteoclast number were decreased, whereas osteoblast number, bone formation rate (BFR) and mineral apposition rate (MAR) were unaltered ().

μCT analysis of the proximal tibiae showed that 34a-Tie2-Tg mice had increased bone mass and decreased structure model index (SMI), which quantifies the relative amount of plates (SMI=0, strong) and rods (SMI=3, fragile) (). Cortical BV/TV was also higher (). Moreover, miR-34a transgenic mice generated by three other osteoclast-targeting cre drivers also exhibited a similar phenotype (). Thus, miR-34a in the osteoclast lineage augment bone mass by suppressing osteoclastogenesis and bone resorption.

To determine whether miR-34a is a physiologically relevant regulator of bone resorption, we next examined miR-34a knockout (34a-KO) and heterozygous (34a-Het) mice (). Northern blot confirmed the diminished levels of miR-34a in 34a-KO (). Consistent with prior reports[9,10], miR-34a deletion had no overt effect on mouse development. Osteoclast differentiation was augmented in 34a-Het and 34a-KO cultures, while precursor proliferation or survival was unaffected (). As a result, serum CTX-1 and osteoclast number were elevated (, ). μCT revealed that 34a-KO and 34a-Het mice exhibited a low-bone-mass with decreased connectivity density (Conn.D) and increased SMI (). Global miR-34a deletion also decreased bone formation as the serum marker P1NP (N-terminal propeptide of type I procollagen), osteoblast number, BFR and MAR were reduced (). The increased resorption in 34a-Het indicates that miR-34a function is haploinsufficient and sensitive to dosage reduction. The recently published miR-34abc triple KO (34abc-TKO)[10] and full miR-34a KO[9] also showed a similar phenotype (), which validate our miR-34a gene trap mice and strengthen the finding that miR-34a loss-of-function elevates bone resorption.

Bone marrow transplantation showed that WT mice receiving 34a-KO marrow also exhibited higher CTX-1 () compared to WT mice receiving WT marrow. Furthermore, osteoclastic miR-34a conditional KO mice (34a-Tie2-KO) also exhibited elevated osteoclast differentiation and bone resorption, but unaltered bone formation, leading to a decreased bone mass (). Thus, miR-34a deletion in the osteoclast lineage elevates bone resorption.

Our genetic findings prompt us to investigate whether pharmacological administration of a miR-34a mimic can attenuate postmenopausal osteoporosis using an ovariectomy (OVX) mouse model and a chitosan (CH) nanoparticle vehicle. Reduction of uterine weight in all ovariectomized mice indicated effective estrogen depletion (). Unaltered body weight indicates the absence of obvious toxicity from CH nanoparticles (). Compared to sham controls, OVX mice treated with miR-Ctrl-CH showed increased CTX-1 and decreased P1NP, whereas both effects were largely prevented in OVX mice treated with miR-34a-CH (). Consequently, OVX-induced bone loss was attenuated by miR-34a-CH (). miR-34a-CH also decreased bone resorption and increased bone formation in sham controls, leading to a higher bone mass (). Biodistribution analysis showed that miR-34a level in the bone marrow was the highest, and further increased by 5-fold by miR-34a-CH, indicating an efficient miR-34a delivery ().

In addition to acute systemic miR-34a treatment, we also examined the effects of chronic osteoclastic miR-34a over-expression. OVX-induced bone resorption and bone loss were also attenuated in 34a-Tie2-Tg mice without altering OVX effects on bone formation (). These results indicate that osteoclastic miR-34a over-expression is sufficient to impede osteoporosis, and osteoclast is a key site for miR-34a therapeutic benefit.

To determine if osteoclastic miR-34a confers protection from bone metastases, we employed two cancer-cell-cardiac-injection models. First, a human breast cancer cell line (MDA231-BoM-1833) was xenografted into female nude mice. This model allows us to assess cancer cells from human. Second, a mouse melanoma cell line (B16-F10) was allografted into immunocompetent male mice. This model took consideration of adaptive immunity. In both models, bone metastases were attenuated in 34a-Tie2-Tg and 34a-PT-Tg mice but exacerbated in 34a-KO and 34a-Het mice (). Since miR-34a remained intact in the exogenous cancer cells, the altered bone metastases resulted from the altered miR-34a in the bone microenvironment of the host.

Pharmacologically, we tested both a treatment protocol using the human breast cancer model and a prevention protocol using the mouse melanoma model. In both cases, bone metastases were diminished by miR-34a-CH (). Systemic miR-34a-CH delivery affected neither tumor growth nor metastasis to other organs such as lung (). Moreover, treating only the cancer cells with miR-34a-CH before injection had no effect (). Consistent with the published finding that miR-34abc deletion does not increase tumorigenesis[10], our 34a-KO mice also showed unaltered cancer susceptibility ().

Since systemic miR-34a-CH treatment not only decreases bone resorption but also increases bone formation, we examined the effects of miR-34a over-expression in osteoblasts. We bred the CAG34a mice with Osterix-CreER mice to generate 34a-Osx-Tg mice. Osteoblast differentiation was reduced for 34a-KO and 34a-Het mice, but increased for 34a-Osx-Tg mice (). Consequently, 34a-Osx-Tg mice exhibited a higher bone formation but unaltered bone resorption, leading to an increased bone mass (). Importantly however, the elevated bone formation alone in the 34a-Osx-Tg mice was insufficient to attenuate either OVX-induced bone loss or cancer bone metastases (). Together, our conditional miR-34a transgenic mouse models pinpointed the mechanisms underlying the therapeutic benefits of miR-34a by revealing that osteoclast, rather than cancer cell or osteoblast, is the critical and essential player.

To elucidate the mechanisms, we identified Tgif2 as a novel direct miR-34a target in the osteoclast lineage (). Tgif2 expression was suppressed by miR-34a gain-of-function, but increased by miR-34a loss-of-function, in both mouse and human osteoclast cultures (). The miR-34a seed region in Tgif2 3′UTR is evolutionally conserved in mammals (). Luciferase reporter assay showed that Tgif2 3′UTR is sufficient to confer miR-34a regulation (). Importantly, when the miR-34a seed region in the Tgif2 3′UTR was mutated, miR-34a regulation was abolished ().

Tgif2 expression was increased during WT osteoclast differentiation (). Tgif2-KO and Tgif2-Het mice had lower bone resorption and higher bone mass (). Tgif2 deletion reduced osteoclast differentiation, and abolished the anti-osteoclastogenic effects of miR-34a (). Moreover, Tgif2/miR-34a double knockout mice (DKO) could no longer increase osteoclast differentiation or bone resorption () compared to Tgif2-KO mice. These results indicate that Tgif2 is pro-osteoclastogenic and essential for miR-34a regulation.

We next investigated how Tgif2 potentiates RANKL signaling. Transfection assays revealed that NFATc1, c-fos and c-jun, also to a lesser extent NFκB (p65), could induce Tgif2 expression (). Response elements for NFATc1 and AP-1, but not NFκB, were identified in the Tgif2 promoter region (-5Kb to +5Kb). ChIP analysis in osteoclast cultures showed that NFATc1, c-jun and c-fos bound to these sites upon RANKL stimulation, leading to activated Tgif2 transcription shown by the elevated H3K4me3 level at the transcription start site (). This indicates that NFATc1 and AP-1 induce Tgif2 expression during osteoclastogenesis.

Luciferase reporter assay showed that Tgif2 augmented the activity of NFATc1, NFκB and c-Jun, but not c-fos (Fig. 6o). Consistently, the activity of endogenous NFATc1, NFκB and c-Jun, but not c-fos, was reduced in Tgif2-KO cultures and enhanced in 34a-KO cultures (). Furthermore, NFATc1 mRNA, c-Jun phosphorylation and IκBα degradation were decreased in Tgif2-KO cultures and increased in 34a-KO cultures (). Therefore, Tgif2 potentiates osteoclastogenesis via a positive feedback loop in which RANKL-induced transcription factors activate Tgif2 expression, and Tgif2 in turn promotes their activity. Collectively, these findings reveal Tgif2 as a novel yet critical regulator of osteoclastogenesis and bone resorption, as well as a key miR-34a direct target that is essential for miR-34a regulation ().

The roles of miRNAs in bone physiology have just begun to emerge. Recent studies show that osteoblast-specific gain-of-function of miR-34b/c decreases bone mass by suppressing osteoblastogenesis and bone formation[11,12]. Here we show that osteoclast-specific miR-34a gain-of-function protects bone by suppressing osteoclastogenesis and bone resorption. These findings uncover an interesting functional divergence among the miR-34 family members. Our study paves the road for future discovery of other miRNAs that may be regulated by RANKL and/or control Tgif2 expression, as well as future epidemiological and clinical studies to explore the pathological and therapeutic roles of this miR-34a-Tgif2 pathway in human.

Most systemically delivered drugs can target multiple tissues and cell types. We found that miR-34a also enhances bone formation; although miR-34a over-expression in osteoblast is neither sufficient nor essential for the therapeutic benefits of miR-34a in osteoporosis or bone metastasis, miR-34a may be a superior new therapy that exerts both anti-catabolic and anabolic effects compared to the current drugs that are solely anti-catabolic. Our identification of miR-34a, as well as the recent report of miR-141 and miR-219[13], opens an exciting avenue for the development of a whole new generation of RNA-based osteo-protective medicine.

Other miR-34a target genes have been reported in different biological context, such as SIRT1[14], SIRT6[15] and PNUTS[16]. Although miR-34a may also target genes other than Tgif2 in osteoclasts, our genetic rescue ex vivo and in vivo show that Tgif2 is the key miR-34a target, suggesting that other genes are likely secondary or functionally irrelevant to osteoclastogenesis.

miR-34abc are commonly deleted in human cancers[17]. In vitro studies suggest that miR-34abc may be critical mediators of p53 function and potential tumor suppressors[18]. Surprisingly, in vivo studies reveal that miR-34abc triple KO mice exhibit intact p53 function without increased tumorigenesis[10]. Nonetheless, systemic miR-34a administration can indeed attenuate cancer malignancy[19]. This raises the intriguing possibility that its anti-cancer effects may reside in other cells that constitute the tumor microenvironment such as the osteoclasts in the bone metastatic niche. Indeed, our findings illustrate that bone metastases are effectively blocked by miR-34a in osteoclasts, thus providing the first in vivo genetic evidence that miR-34a opposes malignant progression of cancer by disarming the metastatic niche.

METHODS SUMMARY

Conditional miR-34a transgenic mice were generated using the CAG-Z-EGFP vector. miR-34a knockout mice were generated using a gene-trap ES cell line.

METHODS

Mice

To generate cre-flox controlled conditional miR-34a transgenic mice (CAG34a), a 431 base-pair genomic sequence containing 168 base pairs 5′ and 161 base pairs 3′ of the pre-miR-34a sequence was inserted into the CAG-Z-EGFP vector[20]. Transgenic founders on pure C57BL/6J background were established by pronuclear injection at the UT Southwestern transgenic core. From 14 founders that carry the LacZ and GFP transgenes, we selected 6 founders that had the highest tail lacZ expression, and bred them to cre transgenic mice. Representative results from at least two independent founders are reported here. To establish osteoclastic miR-34a transgenic mice, CAG34a mice were bred with the previously described Tie2cre mice[7,8], PPARγ-tTA;TRE-cre (PT-cre) mice[21], lysozyme-cre (Lys-cre) mice[22] or Ctsk-cre mice[23]. To establish osteoblastic miR-34a transgenic mice, CAG34a mice were bred with the previously described Osx-CreER mice[24]. All conditional miR-34a transgenic mice were on pure C57BL/6J background, and compared to littermate controls that carry only the transgene allele or only the Cre allele; representative results for “transgene only” group is shown as “Ctrl” group; consistent with previous studies, these Cre lines alone do not exhibit bone phenotype. miR-34a knockout mice in a C57BL/6-129P2 mixed genetic background were generated using a mouse embryonic stem cell line (International Gene Trap Consortium clone YHA350) harboring a gene-trap integration in the miR-34a transcription unit, and backcrossed to C57BL/6J mice for at least five generations. In this gene-trap allele, a splice-acceptor followed by a β-geo cassette (fusion of β-galactosidase and neomycin transferase) was inserted between exon 2 and 3 of the mouse miR-34a gene, leading to a truncated and non-functional pri-miR-34a transcript. miR-34abc triple knockout mice and WT controls in a C57BL/6-129SvJae mixed background were provided by Dr. Andrea Ventura (Memorial Sloan-Kettering Cancer Center)[10]. miR-34a full knockout mice and WT controls on a pure C57BL/6 background were provided by Dr. Lin He (University of California at Berkeley)[9]. Tgif2-KO mice on a C57BL/6-129 mixed background were provided by Dr. David Wotton (University Of Virginia)[25]. miR-34a flox mice on a pure C57BL/6 background were from Jackson Laboratory[9]. Osteoclastic miR-34a conditional KO mice (34a-Tie2-KO) were generated by breeding miR-34a flox mice with Tie2-cre mice. Bone marrow transplantation was performed as described[7]. Briefly, bone marrow cells from 2-month-old male donor (WT or 34a-KO) were intravenously transplanted into five 2-month-old male C57BL/6J recipients that were irradiated at lethal dose (1000 roentgen); the mice were analyzed 3 month post transplantation. Ovariectomy or sham operation was performed on 10-20 week old female mice. miRNA-carrying chitosan (CH) nanoparticles[26] were delivered by intravenous injections at 5μg/mouse or 10μg/mouse twice per week for 4-5 weeks. Sample size estimate was based on power analyses performed using SAS 9.3 TS X64_7PRO platform. With the observed group differences which is of great biological value, and the relatively small variation of the in vivo measurements, a sample size of four per group (n=4) will provide higher than 90% power at type I error rate of 0.05 (two-sided test), and a sample size of three per group (n=3) will provide higher than 80% power at type I error rate of 0.05 (two-sided test). For example, on BV/TV measures with a mean difference of 0.12 between the WT and mutant groups (standard deviation of 0.035 and 0.03 for each of the two groups), 4 mice per group will yield 98% power and 3 mice per group will yield 83% power using two sample t-test. Samples were randomly allocated to each group. Analyses were conducted in a blind fashion to the operator. All experiments were conducted using littermates. All protocols for mouse experiments were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center.

Reagents

Mouse Tgif2 siRNA or control siRNA were from Santa Cruz Biotechnology. miR-34a precursor (pre-miR-34a) and negative control (pre-control), miR-34a inhibitor (anti-miR-34a) and negative control (anti-control) were from Life Technologies. All miRNA and siRNA were transfected with Lipofectamine™ RNAiMAX (Life Technologies) into bone marrow osteoclast progenitors. For in vivo miRNA delivery, HPLC-purified mirVana™ miR-34a mimic or negative control (Life Technologies) was packaged into chitosan nanoparticles as described[26]. RAW264.7 mouse macrophage cell line was from ATCC (TIB-71). Anti-Tgif2 antibody (catalog # 09-718) was from Millipore; anti-NFATc1 (7A6), anti c-Jun (H-79) anti IκBα (C-21) antibodies were from Santa Cruz Biotechnologies; anti-H3K4me3 (ab8580) and anti-c-fos (ab7963) antibodies were from Abcam; anti-p-c-Jun (Ser73) (catalog # 9164) was from Cell Signaling.

Bone Analyses

Micro-Computed Tomography (μCT) was performed to evaluate bone volume and architecture using a Scanco μCT-35 instrument (SCANCO Medical) as described[8]. Mouse tibiae were fixed in 70% ethanol and scanned at several resolutions for both overall tibial assessment (14 micron resolution) and the structural analysis of trabecular and cortical bone (7 micron resolution). Trabecular bone parameters were calculated using the Scanco software to analyze the bone scans from the trabecular region directly distal to the proximal tibial growth plate. As a bone resorption marker, serum CTX-1 was measured with the RatLapsTM EIA kit (Immunodiagnostic Systems)[27]. As a bone formation marker, serum amino-terminal propeptide of type I collagen (P1NP) was measured with the Rat/Mouse P1NP enzyme immunoassay kit (Immunodiagnostic Systems)[27]. Static and dynamic histomorphometry were performed with femurs and vertebrae as described[27]. Calcein (20mg/kg) were injected into 2 month old mice 2 and 10 days before bone collection.

Ex Vivo Osteoclast and Osteoblast Differentiation

Osteoclasts were differentiated from bone marrow cells as described[7,8]. Briefly, hematopoietic bone marrow cells were purified with 40μm cell strainer, and differentiated with 40ng/ml of mouse M-CSF (R&D Systems) in α-MEM containing 10% FBS for 3 days, then with 40ng/ml of mouse MCSF and 100ng/ml of mouse RANKL (R&D Systems) for 3-9 days, in the presence or absence of rosiglitazone (1μM). Mature osteoclasts were identified as multinucleated (>3 nuclei) TRAP+ cells. Osteoclast differentiation was quantified by the RNA expression of osteoclast marker genes using RT-QPCR analysis, as well as number and size of mature osteoclasts. For osteoclast resorptive function analyses, bone marrow osteoclast differentiation was conducted in OsteoAssay bone plates (Lonza), and osteoclast activity was quantified as calcium release from bone into culture medium using CalciFluo ELISA assay (Lonza). Osteoclast precursor proliferation was quantified using a bromodeoxyuridine (BrdU) cell proliferation assay kit (GE Healthcare) as described[27]. Osteoclast apoptosis was quantified using Annexin V: PE Apoptosis Detection Kit I (BD Biosciences). Human PBMN cells (ReachBio) were differentiated into osteoclasts in α-MEM containing 10% FBS, 25ng/ml MCSF, 50ng/ml hRANKL, 1μM Dexamethasome and 1μM rosiglitazone for 14 days; pre-miR or anti-miR were transfected on day 0 and day 6; human RANKL was added on day 7. Osteoblasts were differentiated from bone marrow cells as described[27].

Gene Expression Analyses

For mRNA expression, RNA was reverse transcribed into cDNA using an ABI High Capacity cDNA RT Kit (Life Technologies) and then analyzed using real-time quantitative PCR (SYBR Green) in triplicate. All mRNA expression was normalized by L19. For mature miRNA expression, RNA was reverse transcribed into cDNA using NCode VILO miRNA cDNA Synthesis Kit (Life Technologies) and then analyzed in triplicate using real-time quantitative PCR (SYBR Green) and a primer specific for the mature miRNA. All miRNA expression was normalized by sno251.

Identification of miR-34a Targets in the Osteoclast Lineage

To elucidate the molecular mechanisms for miR-34a inhibition of osteoclastogenessi and bone resorption, we identified key direct miR-34a target genes that are pro-osteoclastogenic. First, we used the TargetScan bioinformatic tool to predict all the miR-34a targets by searching for conserved 8mer or 7mer sites that match the miR-34a seed region. Second, we searched databases such as BioGPS to select secondary targets that are expressed in the macrophage-osteoclast lineage. Third, we performed RT-QPCR to select tertiary targets that can be inhibited by miR-34a during osteoclast differentiation. Fourth, we performed luciferase reporter assay to test if the 3′UTR of each tertiary target could directly suppress gene expression in response to miR-34a. To generate a CMV-Luc-3′UTR reporter, a ~300bp Tgif2 3′UTR region centering the miR-34a target sequence was cloned into the pMIR-REPORT™ vector (Life Technologies) downstream of the luciferase open reading frame. To generate a mutant reporter with miss-matched miR-34a binding site, the miR-34a target sequence was altered using QuikChange II XL site-directed mutagenesis kit (Stratagene). The reporters were co-transfected with CMV-β-gal (as an internal transfection control), together with pre-miR-34a or pre-miR-control, anti-miR-34a or anti-miR-control using FuGENE HD reagent (Roche). The transfection assay was conducted in human embryonic kidney 293 cells and CV-1 monkey kidney cells to assess the intrinsic properties of the 3′UTR in different cellular context, and representative results for 293 cells are shown. Luciferase activity was normalized by β-gal activity.

Bone Metastasis Analyses

Using a VisualSonics Vevo770 small animal ultrasound device, luciferase-labeled cancer cells were injected into the left cardiac ventricle so that they can bypass the lung and efficiently migrate to the bone[28]. Bone metastases were detected and quantified weekly post injection by bioluminescence imaging (BLI) using a Caliper Xenogen Spectrum instrument at UTSW small animal imaging core facility. The osteolytic metastatic lesions were imaged by radiography using Faxitron Cabinet X-ray System with the X-ray tube voltage fixed at 26 kVp and the exposure time at 15 s. The luciferase-labeled bone-metastasis-prone MDA-MB-231 human breast cancer cell sub-line (MDA231-BoM-1833)[29] was generously provided by Joan Massagué (a Howard Hughes Medical Institute Investigator at Memorial Sloan-Kettering Cancer Center) and injected into 6-week-old female nude mice (NCI) at 1×105 cells/mouse in 100μl PBS. The luciferase-labeled B16-F10 mouse melanoma cell line[30] was generously provided by Katherine Weilbaecher (Washington University) and injected into 8-week-old male C57BL/6J mice at 5×104/mouse in 100μl PBS.

Statistical Analyses

All statistical analyses were performed with Student's t-Test and represented as mean ± standard deviation (SD) unless noted otherwise. No animal or sample was excluded from the analysis. The p values were designated as: *, p<0.05; **, p<0.01; ***, p<0.005; ****, p<0.001; n.s. non-significant (p>0.05).