MicroRNA-194 reciprocally stimulates osteogenesis and inhibits adipogenesis via regulating COUP-TFII expression

Osteoblasts and adipocytes are differentiated from common mesenchymal stem cells (MSCs) in processes which are tightly controlled by various growth factors, signaling molecules, transcriptional factors and microRNAs. Recently, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was identified as a critical regulator of MSC fate. In the present study, we aimed to identify some microRNAs (miR), which target COUP-TFII, and to determine the effects on MSCs fate. During osteoblastic or adipocytic differentiation from MSCs lineage cells, miR-194 expression was found to be reversal. In the cultures of mesenchymal C3H10T1/2 and primary bone marrow stromal cells, osteogenic stimuli increased miR-194 expression with accompanying decreases in COUP-TFII expression, whereas adipogenic stimuli reduced miR-194 expression with accompanying increases in COUP-TFII expression. A luciferase assay with COUP-TFII 3′-untranslated region (UTR) reporter plasmid, including the miR-194 binding sequences, showed that the introduction of miR-194 reduced the luciferase activity. However, it did not affect the activity of mutated COUP-TFII 3′-UTR reporter. Enforced expression of miR-194 significantly enhanced osteoblast differentiation, but inhibited adipocyte differentiation by decreasing COUP-TFII mRNA and protein levels. In contrast, inhibition of the endogenous miR-194 reduced matrix mineralization in the MSCs cultures, promoting the formation of lipid droplets by rescuing COUP-TFII expression. Furthermore, overexpression of COUP-TFII reversed the effects of miR-194 on the cell fates. Taken together, our results showed that miR-194 acts as a critical regulator of COUP-TFII, and can determinate the fate of MSCs to differentiate into osteoblasts and adipocytes. This suggests that miR-194 and COUP-TFII may be good target molecules for controlling bone and metabolic diseases.

Mesenchymal stem cells (MSCs) are pluripotent progenitors with multilineage differentiation potential, capable of undergoing osteogenesis, adipogenesis and chondrogenesis. 1,2 General bone homeostasis relies on the inverse relation between osteogenesis and adipogenesis of MSCs. The imbalance between osteogenesis and adipogenesis is associated with pathologic conditions such as osteoporosis, which is accompanied by an increase in bone marrow adipocytes and a decrease in bone mass. 3,4 Bone and fat development are largely dependent on the key transcription factors runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptor gamma (PPARγ), respectively. 5,6 Moreover, Runx2 and PPARγ may also control the lineage commitment and differentiation of MSCs. Runx2 deficiency in chondrocytes causes enhanced adipogenic differentiation, 7 while PPARγ haploinsufficient mice show an increase in bone mass associated with a loss of adipose tissue volume. 8 In addition, transcriptional modulator TAZ interacts with Runx2 to promote osteogenesis, whereas binding of TAZ to PPARγ impairs adipogenesis. 9 Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2), a member of the orphan nuclear receptor superfamily, modulates mesenchymal cell commitment and differentiation to decide whether cells take part in osteogenesis, adipogenesis and chondrogenesis. 10,11 COUP-TFII activates PPARγ expression and simultaneously inhibits Runx2 activity, thus promoting the entry of mesenchymal progenitors to the adipocyte lineage, while impeding progression to alternative lineage pathways. 10 Conversely, ablation of COUP-TFII was found to increase bone formation and osteoblast differentiation, as well as reduce fat tissue and insulin sensitivity. 10 Haploinsufficiency of COUP-TFII also displayed impairment of white adipose tissue development and reduced body fat mass. 11 We previously demonstrated that COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation in mesenchymal C3H10T1/2 cells through the suppression of Runx2 activity. 12 In addition, we observed that BMP2 reduced the cellular levels of COUP-TFII protein with osteoblast differentiation. These findings indicated that COUP-TFII is a negative regulator of bone formation, and a positive regulator of adipogenesis.
MiRNAs are an abundant class of noncoding, small (about 20~25 nucleotides) RNAs, which have emerged as key regulators of gene expression through translational inhibition and mRNA stability, and have been implicated in the regulation of cell proliferation, determination of cell fate or differentiation and organ development. 13 Recently, several miRNAs were also discovered to be involved in the balance of osteogenesis and adipogenesis. For example, the increased expression of miR-204 and miR-637 were found to suppress osteoblast differentiation and enhance adipocyte differentiation by directly targeting Runx2 or Osterix. 14,15 Conversely, overexpression of miR-30e stimulated adipogenesis while inhibiting osteogenesis, by directly targeting low-density lipoprotein receptor-related protein 6. 16 These observations suggested that osteoblast and adipocyte differentiation from mesenchymal progenitor cells is tightly regulated by various miRNAs. However, the specific miRNAs for targeting COUP-TFII and subsequently controlling the differentiation of pluripotent mesenchymal cells have not yet been fully clarified.
In this study, we found that COUP-TFII has a critical role in determination of the fate of MSCs, and that miR-194 reciprocally regulates osteogenesis and adipogenesis by directly targeting the 3′-UTR of COUP-TFII mRNA.
These findings were consistent with other reports. 10 Consequently, the expression of miR − 194, − 195 and − 721 were upregulated when cells underwent differentiation toward osteogenic lineage (Figure 1b). In contrast, let-7d, miR − 24, − 194, − 195, − 298 and miR-721 expression was robustly decreased by adipogenic stimuli (Figure 1b). Among those detected, miR-194 showed the greatest changes in both osteogenesis and adipogenesis, revealing a twofold induction by osteogenic and a 50% reduction by AM treatment, respectively. Also, similar results were obtained from primary mouse bone marrow stromal cells (BMSCs) (Figures 1e-g). Collectively, these results revealed that COUP-TFII and miR-194 expression was reversible during osteogenesis and adipogenesis of MSCs, suggesting that miR-194 may be a regulator of COUP-TFII in these cells.
MiR-194 acts as an attenuator of COUP-TFII expression. The effects of miR-194 on COUP-TFII expression were further examined by gain-and loss-of-function experiments in C3H10T1/2 cells. Introduction of miR-194 or anti-miR-194 was confirmed by qRT-PCR (Figure 2a). The expression of COUP-TFII mRNA was reduced by enhanced expression of miR-194, whereas it was increased by inhibition of miR-194 ( Figure 2b). Furthermore, introduction of miR-194 led to a decrease in COUP-TFII protein level, whereas suppression of endogenous miR-194 increased in C3H10T1/2 cells (Figure 2c), suggesting that miR-194 targeted the COUP-TFII mRNA to suppress its protein expression.
To further determine whether miR-194 directly targets the 3′-UTR of COUP-TFII mRNA, sequences of wild type COUP-TFII-3′-UTR luciferase reporter plasmid (WT), containing the putative miR-194 binding site and mutant COUP-TFII-3′-UTR (MT), with a 4 bp mutation in the seed region, were synthesized ( Figure 2d). The WT-or MT-COUP-TFII 3′-UTR luciferase reporter plasmids were co-transfected with miR--194, control miRNA or anti-miR-194 into C3H10T1/2 cells, after which luciferase activity assays were performed. The cotransfection of miR-194 with a WT reporter resulted in a highly significant decrease in luciferase activity compared with the control-transfected group (Figure 2e). When the miR-194 was co-transfected with the empty vector or MT reporter, no decrease in luciferase activity was observed, confirming that the predicted site is a direct target of miR-194. Collectively, these results revealed that miR-194 negatively regulate the expression of COUP-TFII by directly binding to the COUP-TFII-3′-UTR.
MiR-194 promotes osteoblast differentiation. Because miR-194 was found to have an inhibitory effect on COUP-TFII expression, we investigated whether miR-194 controls osteoblast differentiation. The expression levels of miR-194 were first examined at different time-points of osteogenic differentiation in primary osteoblasts. The miR-194 expression increased following osteogenic differentiation, reaching a high on the 4th day (Figure 3a). At the time-points, expression of osteogenic markers such as Runx2 and osteocalcin (OC) were gradually increased with the deceased expression of COUP-TFII ( Figure 3b). To further verify whether miR-194 could regulate osteogenesis through the regulation of COUP-TFII, the expression of miR-194 was altered by transfecting C3H10T1/2 cells with a specific synthetic miRNA precursor or inhibitor (Supplementary Figure S1  In gain-or loss-of-function experiments, introduction of miR-194 elicited to decreasing expression of PPARγ, aP2 and adiponectin mRNA, whereas the inhibition of miR-194 increased them (Figures 4c and e,Supplementary Figure S2). Western blot analysis also revealed that miR-194 reduced PPARγ and COUP-TFII protein levels, and that anti-miR-194 increased them (Figures 4d and f). Consistently, overexpression of miR-194 reduced lipid droplet formation in 3T3-L1, and reversely anti-miR-194 enhanced it (Figures 4g and h), as evidenced by the Oil Red O staining. These results suggested that miR-194 negatively regulates adipocyte differentiation through suppressing COUP-TFII expression.
MiR-194 regulates lineage specification of mesenchymal cells via COUP-TFII. All the above results suggest that COUP-TFII may be a direct target of miR-194. To further confirm whether COUP-TFII directly mediates the miR-194 control of mesenchymal cell differentiation, we examined the effects of COUP-TFII overexpression on the miR-194 control of osteogenesis and adipogenesis. COUP-TFII expression vector, not including 3′-UTR, was co-transfected with miR-194 or control miRNA into mesenchymal C3H10T1/2 cells, after which qRT-PCR was performed (Supplementary Figure S3). The supplementation of COUP-TFII inhibited the promoting effect of miR-194 on mineralization with attenuating miR-194-induced Runx2 and OC expression (Figures 5a  and b). In addition, the supplementation relived the miR-194 inhibition of lipid droplet formation with the increased expression of aP2 and PPARγ in mesenchymal lineage cells (Figures 5c and d). These results suggest that miR-194 is critical for maintaining the balance of adipocyte and osteoblast differentiation from MSC through the regulation of COUP-TFII (Figure 5e).

Discussion
Osteoblasts and adipocytes arise from a common mesenchymal progenitor, and lineage allocation into osteoblasts and adipocytes is reciprocally exclusive. 17 Decreased bone formation which occurs with advanced age is usually accompanied by an accumulation of bone marrow adiposity. 18,19 Thus, the balance between osteogenesis and adipogenesis may be of great importance to prevent and treat metabolic disorders that involve bone loss and lipid accumulation. 20 The activation of multiple transcription factors has been identified to be associated with determination of the fate of mesenchymal cells. 21,22 These findings imply that targeting transcription factors may be one of strategy for controlling the commitment and differentiation of MSCs to a certain lineage.
COUP-TFII is one of critical transcription factors to determine the lineage specification of mesenchymal progenitors. 10 COUP-TFII activates PPARγ expression while inhibiting Runx2 activity, thus promoting the entry of progenitor cells into the adipocyte lineage, while impeding the access of MSCs to alternative lineages. 12,23 Recently, some miRNAs, especially targeting transcription factors, have emerged as one of the fate determinants of MSC. For example, miR-204 and miR-637 can control the MSC commitment through regulating Runx2 and OSX expression, which are major transcription factors for bone formation. 14,15 MiR-194 has been characterized as a regulatory mediator in liver fibrogenesis and metabolism of hepatic cholesterol and bile acid. 24 In addition, miR-194 inhibits chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5, 25 and suppresses osteosarcoma cell proliferation and metastasis in vitro and in vivo by targeting CDH2 and IGF1R. 26 These findings suggest that miR-194 might be a In the present study, we identified a novel function of miR-194 concerning the regulation of COUP-TFII-mediated osteogenesis and adipogenesis. The miR-194 is preferentially expressed in bone compared with other tissues in mice (Supplementary Figure S4), suggesting that it may also have a regulatory role in bone development and homeostasis. Furthermore, miR-194 expression was upregulated during the differentiation of MSCs toward an osteogenic lineage, while downregulated during adipogenesis. Overexpression of miR-194 also promoted osteoblast differentiation of primary osteoblasts and mesenchymal lineage cells with and inhibited adipocyte differentiation of 3T3-L1. In contrast, the knockdown of miR-194 produced the opposite effects. The miR-194 control of osteoblast and adipocyte differentiation was related to the expression of COUP-TFII, Runx2 and PPARγ (Figures 3  and 4). These results suggest that miR-194 might be a critical regulator in maintaining the balance between osteogenesis and adipogenesis.
MiRNAs control cellular activity by directly targeting the 3′-UTR of target mRNA with base-pair complementarity. 13 Bioinformatic analyses informed us that 3′-UTR of COUP-TFII mRNA has complementary sequence to miR-194, and luciferase assays provided evidence that miR-194 targets the COUP-TFII mRNA in cellular level (Figure 2). In addition, our results consistently showed that the levels of COUP-TFII mRNA and protein were regulated by miR-194 in  (Figures 3 and 4). In addition, inhibition of COUP-TFII by siRNA also produced the same phenomenon (Supplementary Figure S5), and COUP-TFII overexpression inhibited the effects of miR-194 on osteogenesis and adipogenesis. These results strongly support our previous reports that COUP-TFII is a negative regulator of osteoblast differentiation, 12 and also indicate that COUP-TFII may have a stimulatory role in adipogenesis.
Runx2 is involved in the lineage commitment and differentiation between osteoblasts and adipocytes, and also recently identified as a target molecule of COUP-TFII. 12 In the study, we also found that miR-194 overexpression was accompanied by decreased levels of COUP-TFII expression, whereas knockdown of miR-194 increased the COUP-TFII protein level (Figure 3). These findings provided evidence that COUP-TFII may regulate osteogenic differentiation by directly interacting with the Runx2 protein to suppress osteogenicrelated genes, which could be considered as one of the PPARγ is also considered as a crucial regulator between adipogenesis and osteogenesis. PPARγ repressed Runx2mediated OC transcription, 27 and its insufficiency enhances bone mass through osteoblast formation of bone marrow progenitors. 8,28 Adipocytes also directly modulate osteoblast function through paracrine effects of secretory adipokines, such as adiponectin and leptin, which are regulated by PPARγ. 29,30 Our study also revealed that miR-194 controlled adipogenesis as well as osteogenesis with alteration of COUP-TFII and PPARγ expression. Indeed, inhibition of miR-194 elicited to decreasing Runx2 expression and increasing PPARγ expression in mesenchymal cells under  Figure S6), suggesting inhibition of miR-194 may commit MSC into adipocytes over osteoblasts.
In conclusion, we demonstrated as a novel regulatory mechanism of mesenchymal progenitor cells that miR-194 stimulated osteogenesis and inhibited adipogenesis from MSC via regulating COUP-TFII expression (Figure 5e). Therefore, miR-194 may prove to be a promising therapeutic target for treatment of metabolic and bone disease, including osteoporosis or obesity.
Osteoblast and adipocyte differentiation. To induce osteoblast differentiation, cells were cultured with OM containing 10% FBS, 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate, in the presence or absence of 200 ng/ml of BMP2. The induction media was changed every 3 days. To induce adipocyte differentiation, cells were allowed to become confluent for 1 day, and then cultured with AM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 1 μg/ml insulin. After 2 days, the medium was replaced with fresh complete medium containing insulin, and the cells were incubated for an additional 2-day period. Thereafter, the cells were maintained in 10% FBS-DMEM with media changes every other day until they became fully differentiated.
DNA constructions. COUP-TFII expression vector without 3′-UTR, shCOUP-TFII and shLuc control vector was previously described. 12 By in silico analysis, COUP-TFII-3′-UTR was found to have a putative miR-194 binding site (UGUUACA; 92-98 bp far from stop codon; TargetScan). For functional analysis of miR-194, pmirGLO-COUP-TFII-3′-UTR-Luc constructs containing a partial fragment of COUP-TFII-3′-UTR and luciferase reporter genes were made. Amplification of wild-type and mutant plasmids was carried out under the same conditions. Cloning was carried out using specific forward and reverse primers (Supplementary Table 1). The fragments of 3′-UTR were sub-cloned between the SacI and XhoI restriction sites of the pmirGLO vector (Promega). The correct orientation and nucleotide sequence of the 3′-UTR fragments in the plasmid constructs were further confirmed by sequencing.
Transient transfection and luciferase assays. Cells were transfected transiently with the indicated plasmids, 20 nM miR-194 (or 20 nM miR-CTL) and 40 nM anti-miR-194 (or 40 nM anti-miR-CTL) using Lipofectamin RNAiMAX (Invitrogen). To measure promoter activity, cells were harvested 24-48 h after transfection, and the activity was measured with the luciferase reporter assay system (Promega). To confirm interaction between miR-194 and the COUP-TFII-3′-UTR region, cells were transfected with 100 ng of pmirGLO wild type (or 100 ng mutant type) or empty pmirGLO vector (100 ng) in the presence of 10 nM miR-194 precursor (or 10 nM miR-CTL) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Thereafter, firefly and renilla luciferase activities were determined using the Dual-Glo luciferase assay system (Promega).
RT-PCR and qRT-PCR. Total RNA or microRNAs (miRs) were isolated from the cultures using the TRIzol reagent (Invitrogen) or the mirVana miRNA isolation kit (Ambion). For quantitation of gene transcription, cDNA was generated with the Maxime RT premix kit (iNtRon, Sungnam, Korea), and then amplified on the StepOnePlus real-time PCR system (ABI, Abilene, TX, USA) using the QuantiTect SYBR PCR kit (Qiagen, Valencia, CA, USA) with specific primers (Supplementary Table 2). To quantify gene expression of adipogenc markers, qRT-PCR was performed using TaqMan probes with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster city, CA, USA) in the StepOnePlus real-time PCR System (Applied Biosystems). Unlabeled specific primers and the TaqMan probes were purchased from Applied Biosystems for detecting the aP2 gene (Assay ID: Mm00445878_m1), adiponectin gene (Assay ID: Mm00456425_m1) and PPARγ gene (Assay ID: Mm01184322_m1). All quantitation was normalized to an endogenous control 18S gene (Assay ID: Hs99999901_s1). MiRNAs were evaluated using the NCode VILO miRNA cDNA synthesis kit (Invitrogen) and the Express SYBR GreenER miRNA qRT-PCR kit with specific primers (Supplementary Table 3). The relative level of miRNA was quantified using the 2 − ΔΔCt method with sno234 RNA as an endogenous control.
Alizarin red staining. Cells were fixed with 70% ethanol for 1 h, rinsed with cold distilled water, and then treated with 40 mM AR-S solution at pH 4.2 for 10 min. After washing with phosphate-buffered saline for 15 min, the stained cultures were photographed. To quantify calcium deposition, cells were washed with distilled water, the dye was eluted with 10% cetylpyridinium chloride, and the absorbance was measured at 570 nM with a microplate reader (Bio-Rad).
Oil Red O staining. Cells were transfected with miR-194 precursors or anti-miR-302a using Lipofectamin RNAiMAX (Invitrogen), and then cultured in AM for 8 days. After washing with PBS, the cells were fixed with 4% formalin in PBS for 30 min, and then reacted with 60% saturated Oil Red O dye for 20 min. The Oil Red O-stained lipid droplets were photographed by LSM microscopy (Carl Zeiss, Oberkochen, Germany). To quantify the degree of lipid droplet formation, the stained cells were incubated with isopropanol to extract the dye from the lipid droplets, and the absorbance of reaction solution was then measured at 510 nM with a microplate reader (Bio-Rad).
Statistical analysis. All experiments were performed in triplicate on the same sample, and independently repeated at least three times. Results were expressed as mean ± S.D. Statistical analyses were performed using a Student's t-test or analysis of variance, followed by Duncan's multiple comparison tests. P-valueso0.05 were considered to be statistically significant.

Conflict of Interest
The authors declare no conflict of interest.