MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2*

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.


MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this downmodulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.
Chondrogenesis, which is a prerequisite event for cartilage formation in the developing limb, involves mesenchymal cell recruitment/migration, condensation of progenitors, and chondrocyte differentiation and maturation (1,2). Precartilage condensation forms the cartilaginous template of the future skeleton; as such, it is a critical step in the initiation of cartilage differentiation. Condensation is mediated by the cell adhesion molecule, N-cadherin, along with various extracellular matrix molecules, such as fibronectin (FN), 2 the proteoglycans, and the collagens (3,4). These extracellular matrix molecules interact with cell adhesion molecules to activate focal adhesion kinase-mediated intracellular signaling pathways and to initiate the transition of chondroprogenitor cells into fully committed chondrocytes (3,5). The mechanisms of vertebrate limb development have been studied extensively in the chicken. In fact, our current understanding of vertebrate limb development has come largely from the chick model (6,7), and many of the cellto-cell interactions and genes known to be involved in limb formation were initially discovered using the chicken as a model system (8).
MicroRNAs (miRNAs) are 20 -25-nucleotide-long endogenous non-coding RNAs that are involved in multiple biological pathways in a variety of animals (9,10). miRNA can regulate target gene expression through translational repression and/or target mRNA degradation in a sequence-dependent manner (11). miRNAs have been shown to affect numerous biological processes in Caenorhabditis elegans and Drosophila melanogaster, including developmental timing (12)(13)(14), left/right asymmetrical neuronal cell fate determination (15), programmed cell death (12,16), muscle development (17,18), and fat metabolism (19). Furthermore, in mammals, miRNAs have been implicated in a broad range of processes, including hematopoietic lineage differentiation, developmental patterning, heart and skeletal muscle differentiation and function, insulin secretion, and immune function (20 -23).
A transcriptome analysis of almost 500,000 expressed sequence tags in the chicken previously identified 23 expressed miRNAs (24). Subsequent high throughput whole mount in situ hybridization of chick embryos showed that 21 of these miRNAs, including let-7a and miR-10b, -18b, and -363, were present in the limb primordia beginning around Hamburger-Hamilton stage 14. Among them, miR-10b showed high level expression in limb bud from stage 18 through at least stage 24 (25). The chicken ortholog of the C. elegans lin-41 gene was also identified as being expressed in the developing chick limb (26). Similar to C. elegans, includes known binding sites for the miRNAs, lin-4 and let-7, suggesting that these miRNAs could control the expression of chicken lin-41. Another miRNA, miR-196, is thought to be involved in specifying hind limb development (27). In addition, Kobayashi et al. (28), in studying dicer-null growth plates, recently showed that miRNA play important roles during cartilage development. Dicer deficiency in chondrocytes was found to reduce the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death in mice. However, although several recent reports have indicated that miRNAs are critical to the regulation of chondrocyte proliferation and differentiation during skeletal development (25,28), the precise roles of miRNAs in the chondrogenic differentiation of limb mesenchymal cells have not yet been fully established. In this study, we show that miR-221 is a key modulator in the chondrogenic differentiation This article has been withdrawn by the authors. The control day 4 image in

EXPERIMENTAL PROCEDURES
Cell Culture and Treatments-Mesenchymal cells derived from the distal tips of Hamburger-Hamilton stage 22/23 embryo leg buds of fertilized White Leghorn chicken eggs were micromass cultured as described previously (29). Briefly, the cells were suspended at a density of 2 ϫ 10 7 cells/ml in Ham's F-12 medium containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 g/ml streptomycin (Invitrogen). The cells were plated in 3 drops (15 l each) in 35-mm culture dishes or 19 drops (15 l each) in 60-mm culture dishes and incubated for 1 h at 37°C under 5% CO 2 to allow attachment. Thereafter, the cells were maintained in 1 ml of culture medium in the absence or presence of 5 M JNK inhibitor II (Calbiochem) for the entire culture period.
Analysis of Cell Condensation and Differentiation-Chondrogenic differentiation was measured by Alcian blue staining of sulfated cartilage glycosaminoglycans. To demonstrate the deposition of cartilage matrix proteoglycans, representative cultures were collected at day 5 of incubation and stained with 0.5% Alcian blue 8GX, pH 1.0. Alcian blue bound to sulfated glycosaminoglycan was extracted with 6 M guanidine HCl and quantified by measurement of absorbance at 600 nm. Binding of peanut agglutinin (PNA) was used as a specific marker for precartilage condensation. Briefly, cultures were rinsed twice with 0.02 M PBS, pH 7.2, fixed in methanol/acetone (1:1) for 1 min, air-dried, and then incubated with 100 g/ml biotinylated PNA (Sigma) for 1 h. Bound PNA was visualized using the Vectastain ABC and DAB substrate solution kit (Vector Laboratories Inc., Burlingame, CA).
Western Blot Analysis-Proteins (30 g) or conditioned media were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS and transferred to nitrocellulose membrane (Schleicher and Schuell). The membranes were incubated for 1 h at room temperature in blocking buffer (20 mM Tris-HCl, 137 mM NaCl, pH 8.0, containing 0.1% Tween and 3% nonfat dry milk) and probed with antibodies against the following: N-cadherin, FN, Slug, and Sox-9 (Calbiochem); Mdm2 and p53 (R&D Systems, Minneapolis, MN) and HSP70 (Stressgene, San Diego, CA). The blots were developed with a peroxidase-conjugated secondary antibody, and reacted proteins were visualized using an electrochemiluminescence (ECL) system (Pierce).
Cell Migration Assay-Cells were cultured on plates in growth medium for 10 h, and then an area on each plate was cleared using a pipette tip. The cultures were then incubated for 15 h in growth medium supplemented with the indicated chemicals and factors. Cultures were photographed at time 0 and at 15 h after clearing. Cell migration was determined by measuring the difference in cleared area before and after migration (area restored). This analysis was performed using the Image J software available (version 1.36b). Three independent experiments were performed.
Cell Proliferation Assay-Proliferation of mesenchymal cells was determined by directly counting cells from micromass cultures. Control and treated cultures were maintained for the indicated number of days, detached with trypsin/EDTA solution, and counted in triplicate using a hemacytometer.
FACS Analysis-Apoptosis was analyzed with a flow cytometer (FACSCalibur, BD Biosciences). To detect the extent of propidium iodide staining, cells were excited at 488 nm, and emission was observed at 585 nm.
RNA Preparation and miRNA Microarray Hybridization-Total RNA was isolated using the mirVana miRNA isolation kit (Ambion). For miRNA microarray analysis, the RNA samples were processed and hybridized to a PANArray miRNA microarray (Panagene), containing 200 miRNA probes. Data were analyzed using analysis of variance with multiple comparison-corrected p values less than 0.05 and a false discovery rate set to 0.05.
miRNA Quantification and Real-time Quantitative RT-PCR of mRNA-The expression levels of various miRNAs and mRNAs were independently quantified using the TaqMan microRNA and gene expression assays, respectively (Applied Biosystems) according to the manufacturer's protocols. miRNA expression was normalized with respect to that of the RNU43 small nuclear RNA (endogenous control). For assessment of mRNA, transcripts were quantified by real-time quantitative PCR (RT-PCR) and normalized with respect to ␤-actin expression.
PNA-based miRNA Inhibitor-mediated Knockdown and Pre-miR-221-mediated Up-regulation of miR-221-The PNAbased antisense oligonucleotides (ASOs), which included an O-linker at the N terminus of the PNA to improve solubility, were purchased from Panagene. A 200 nM concentration of the PNA-based ASO (PNA221, AGCUACAUUGUCUGCUG-GGUUUC) or a 50 nM concentration of pre-miR-221 (Ambion) was electroporated into isolated mesenchymal cells using a square wave generator (BTX-830, Gentronics, San Diego, CA) with 20 ms, 200 square pulses. A scrambled PNA-based ASO or miRNA was used as the negative control.
Reporter Vectors and DNA Constructs-The 3Ј-untranslated region (UTR) of mdm2 (1303 bp) was PCR-amplified and cloned downstream of the CMV-driven firefly luciferase cassette in the pMIR-Report vector (Ambion). A reporter vector containing a directly matched miRNA-binding site oligonucleotide (ϳ51 bp) for miR-221 was used as the positive control. For miRNA target validation, limb mesenchymal cells were electroporated using a square wave generator (with 20 ms, 200 square pulses; BTX-830, Gentronics) with 25-50 ng of each firefly luciferase reporter construct, 150 -175 ng of empty pcDNA3 vector, 200 ng of pcDNA3 harboring the Renilla luciferase gene (transfection control), and 30 pmol of pre-miR-221 or pre-miR-neg (Ambion). At 24 h post-transfection, the activities of firefly and Renilla luciferase were assayed (Promega). Normalized relative light units were used to represent the ratios of firefly luciferase activity versus Renilla luciferase activity.

Inhibition of JNK Signaling Suppresses Precartilage Condensation and
Chondrogenesis-Numerous protein kinases and pathways have been implicated in the chondrogenic process, including protein kinase A (PKA) (30), protein kinase C (PKC) (31), Rho kinase I and -II (ROCK I and -II) (32), and the Sma-and Mad-related protein (Smad) 15/8 (33) and -2/3 (34) pathways. Our laboratory previously showed that ERK and p38 MAPK regulate mesenchymal cell chondrogenesis in chick embryos by modulating the expression levels of various cell adhesion molecules, including N-cadherin, FN, and integrin ␣ 5 /␤ 1 (35,36). However, comparatively little is known about the potential functions of the c-Jun N-terminal kinase (JNK) pathways in the regulation of cartilage formation. Recently, a few studies have suggested that JNK signaling is involved in the differentiation of articular chondrocytes (37,38). However, the results from these studies are contradictory. In articular chondrocytes, Wnt-3a was found to cause dedifferentiation of chondrocytes by up-regulating c-Jun expression, increasing JNKmediated phosphorylation of c-Jun, activating the c-Jun/activator protein (38). In contrast, treatment with members of the transforming growth factor-␤ superfamily was reported to promote cartilage-specific gene expression during in vitro chondrogenic differentiation of mesenchymal progenitor cells from bone marrow and trabecular bone through activation of p38, ERK-1, and JNK (39). Thus, although JNK signaling appears to be involved in chondrogenic differentiation, the related pathways and interactions have not yet been fully elucidated.
In this present work, we observed that the phosphorylation of JNK-1 was continuously increased by day 3 of culture (Fig. 1A). To verify the effect of JNK signaling during chondrogenesis in our model system, we treated chick wing bud mesenchymal cells with a 5 M concentration of a JNK inhibitor and used PNA and Alcian blue staining to analyze precartilage condensation and chondrogenic differentiation, respectively. We found that the staining intensities of both PNA (Fig. 1B) and Alcian blue (Fig. 1C) decreased following JNK inhibitor treatment. In addition, JNK inhibitor treatment suppressed the expression of type II collagen, a typical marker for chondrogenic differentiation (Fig. 1D). These data suggest that blockage of JNK signaling suppressed chondrogenic differentiation in chick wing bud mesenchymal cells.
Chondrogenic differentiation may be regulated at three stages, namely during cell proliferation, precartilage condensation, and cartilage nodule formation (40,41). Condensation can result from either an increase in cell proliferation or through active cell movement that increases in cell packing density without an increase in cell proliferation (42,43). To examine the possible involvement of JNK signaling in the control of cell motility, we used a wound-healing assay to test the effects of the JNK inhibitor on the motility of chick wing bud mesenchymal cells. Our results revealed that inhibition of JNK signaling almost completely abrogated the motility of these cells (Fig. 1E). We then examined whether the JNK inhibitor also suppressed chondrogenesis by regulating cell proliferation. We found that exposure of cells to the JNK inhibitor increased apoptosis among chondrogenic cells, as determined by direct cell counting (Fig. 1F) and FACS analysis (Fig. 1G). This increase in cell death may be due to the JNK inhibitor-induced arrest of cells at the G 2 /M stage. Because prechondrogenic condensation begins with the aggregation of mesenchymal cells via migration and proliferation (35,43), the JNK inhibitor-mediated suppression of cell migration and stimulation of apoptosis is likely to suppress precartilage condensation.
miRNA-221 Regulates Proliferation of Chick Limb Mesenchymal Cells, Thereby Mediating Precartilage Condensation-MicroRNAs are known to modulate a variety of cellular pathways, including development, differentiation, cell proliferation, and apoptosis (20, 44 -46). In addition, miRNA signatures can be used to distinguish between tumoral and normal tissues, can help identify cancer types and tissues of origin, and may also be correlated with disease outcomes. We performed an miRNA array screen aimed at identifying miRNAs that were up-or down-regulated in JNK inhibitor-treated chick limb mesenchymal cells versus untreated controls. After cells were subjected to inhibitor treatment for 48 h, total RNA was extracted and used to determine the expression levels of 200 unique mouse and human miRNAs in their mature forms. When we used a p value of 0.01 as a cut-off for significance, we found that miR-221 was one of the miRNAs that was substantially induced by JNK inhibition (Fig. 2A). To confirm this finding, we converted a portion of the RNA to cDNA, which was then subjected to quantitative PCR. Consistent with our microarray findings, miR-221 was strongly induced in JNK inhibitor-treated cells (Fig. 2B). During limb development, the highest induction of miR-221 was detected in the limb buds of chick embryos at Hamburger-Hamilton stages 32 and 35 (Fig. 2C) and at later periods of culture (Fig.  2D). During this period, digits are formed through widespread and specific apoptotic cell death. Thus, our results suggest that miR-221 may be involved in the apoptotic death of limb mesenchymal cells during this critical developmental period.
To corroborate the involvement of miR-221 in the JNK inhibitor-mediated blockade of chondrogenic differentiation, we examined the effect of PNA-based ASO-mediated  knockdown of miR-221. As shown in Fig. 3A, miR-221 expression in the knockdown cells was 9-fold lower than that in mock-transfected cells, confirming the validity of our strategy. Furthermore, we found that the JNK inhibitor-induced decreases in PNA and Alcian blue staining intensity were recovered in cells that had been co-treated with the miR-221 inhibitor (Fig. 3B). The miR-221 inhibitor also overcame the ability of the JNK inhibitor to decrease the proliferation (Fig. 3C) and migration (Fig. 3D) of limb mesenchymal cells, as assessed by direct cell-counting and wound-healing assays, respectively.
Notably, treatment of chick limb mesenchymal cells with the miR-221 inhibitor alone significantly increased cell proliferation. Because condensation may result from either an increase in cell proliferation or active cell migration in the absence of cell proliferation, this miR-221 inhibitor-induced cell proliferation may account for the ability of the miR-221 inhibitor to block JNK inhibitor-mediated decreases in precartilage condensation.

miR-221 Inhibits Proliferation of Chondrogenic Progenitors by Inducing MDM2-mediated Ubiquitylation and Degradation
of Slug-To investigate the importance of miR-221 in regulating chondrogenesis-related gene expression patterns, we examined the protein expression levels of several target genes (e.g. N-cadherin, FN, and sox-9) in JNK-and miR-211 inhibitortreated chondroprogenitor cells (Fig. 4A). No significant differences were detected between control cells and those that had been exposed to the JNK or miR-221 inhibitors; in all three cases, the tested genes were up-regulated as chondrogenesis progressed. Because JNK signaling affects cell proliferation, we examined the protein levels of p53 and Mdm2, which are known to be involved in cell cycle arrest (Fig. 4B). We observed a significant reduction in Mdm2 protein expression among JNK inhibitor-treated chondroprogenitor cells, whereas these levels were recovered in cells that had been co-treated with the inhibitors against JNK and miR-221. Western blotting confirmed that Mdm2 protein expression was repressed in JNK inhibitor- treated cells and recovered in cells that had been co-treated with the miR-221 inhibitor (Fig. 4C). These findings suggest that miR-221 targets Mdm2 and represses its expression during JNK-mediated signaling in the developing chick limb bud.
A recent study suggested that Mdm2 can promote the ubiquitination and subsequent proteasomal degradation of the Slug protein and may repress cancer cell invasiveness through this mechanism (44). In the present study, we found that JNK inhibitor-treated cells showed down-regulation of the Mdm2 with concurrent up-regulation of Slug (Fig. 4C).
To further confirm that mdm2 is a target of miR-221, we cloned the entire 3Ј-UTR of mdm2 into a luciferase reporter vector, electroporated the vector into chondrogenic progenitor cells along with the precursor of miR-221 or a cognate nontargeting negative control, and assayed cell lysates for luciferase expression. We found that cells transfected with the mdm2 3Ј-UTR-driven vector plus miR-221 exhibited significantly less luciferase activity compared with cells that received the reporter plus the non-targeting negative control (Fig. 4D). Conversely, overexpression of the miR-221 precursor was found to suppress precartilage condensation, chondrogenic differentiation (Fig. 4E), and Mdm2 expression (Fig. 4F) in the tested cells.
We then examined whether mdm2 overexpression could change Slug protein levels in chondrogenic progenitor cells. Cells were electroporated with the mdm2-encoding expression vector and cultured in the presence of the JNK inhibitor, followed by treatment with MG132, a proteasome inhibitor, for 6 h before cells were harvested. In cells overexpressing mdm2, the JNK inhibitor-mediated increase in Slug protein expression was abrogated. Furthermore, the level of ubiquitinated Slug was increased (Fig. 4G, top), but no change was observed in the mRNA expression level of slug (Fig. 4G, bottom), indicating that the protein was increasingly tagged for proteasomal degradation. These findings suggest that the JNK inhibitor-mediated inhibition of Mdm2 via miR-221 suppresses the ubiquitination and subsequent degradation of Slug, leading to up-regulation of the Slug protein in these cells. To investigate whether overexpression of slug could inhibit the proliferation of chondroprogenitor cells, we electroporated cells with a slug-GFPencoding construct. Consistent with our findings with regard to JNK inhibition, slug overexpression was associated with a decrease in total cell numbers (Fig. 4H) and an increase in apoptosis (Fig. 4I).

DISCUSSION
miRNAs are known to be involved with various aspects of cellular biology, including the regulation of differentiation, proliferation, apoptosis, and development, and with clinically important diseases, such as cancer, cardiovascular diseases, neurological diseases, viral diseases, and metabolic disorders (44 -49).
Suomi et al. (50) showed that the expression of miR-199a was over 10-fold higher in chondroblasts than in undifferentiated mouse bone marrow stromal cells. Furthermore, miR-124a was found to be strongly up-regulated during chondrogenesis, whereas miR-96 was substantially down-regulated in the same cells. Another study showed that miR-140 was specifically expressed in the cartilage tissues of mouse embryos, where it plays important role in forming and/or maintaining cartilage by targeting histone deacetylase 4 (51). However, the precise roles of miRNAs in cartilage biology are relatively unknown, and many of the individual targets of miRNA within chondroblasts have not yet been identified. In the present study, we report the apparent involvement of miRNA-221 in the chondrogenic differentiation of chick limb mesenchymal cells. We found that that miR-221 was up-regulated in JNK inhibitor-treated chondroprogenitor cells, which showed decreased proliferation and precartilage condensation.
As is the case for many other miRNAs, the biological information available for miR-221 is largely limited to expression analysis. Here, we investigated the functional importance of miR-221 during chondrogenic differentiation of chick limb mesenchymal cells. Our results suggest that the chondro-inhibitory conditions created by JNK inhibition lead to up-regulation of miR-221 and down-regulation of chondroblast migration. In contrast, miR-221 knockdown reversed the JNK inhibitor-mediated decreases in chondroblast migration and cell proliferation.
One significant hurdle that has limited the interpretation of many miRNA-profiling studies is that it is relatively difficult to identify the specific targets of the various miRNAs. Previously, miR-221/222 was shown to modulate the expression of p27(Kip1) and c-kit (52,53) and to regulate proliferation or cell survival in various cell types, including smooth muscle cells (54) and mast cells (55). Here, we used a stringent strategy to identify genes that may be directly regulated by miR-221. We used four target prediction software programs and examined the molecules known to be involved in chondrogenic differentiation. Our findings indicated that Mdm2 is a target of miR-221. Mdm2 is an oncoprotein that has been shown to inhibit the activity of the p53 tumor suppressor protein; Mdm2 has E3 ubiquitin ligase activity and thus targets p53 for degradation by the proteasome (56). The ability of mdm2 to regulate p53 is essential for embryonic development (57). mdm2 can bind to many different proteins, including MDMX, Rb, E2F1, p73, TBP, and p300 (57)(58)(59). In addition, Mdm2 has been associated with many different cellular processes, including cell cycle progression, transcriptional regulation, and cell differentiation (57). A recent study showed that p53 can induce Mdm2-mediated degradation of the Slug protein, with concomitant up-regulation of E-cadherin (60). In addition, p53, Mdm2, and Slug appeared to form a complex that is important for Slug degradation (60). Here, we describe an apoptotic function of Mdm2 that is mediated through the Slug pathway during the chondrogenic differentiation of chick limb mesenchymal cells.
The members of the snail protein family were initially thought to be determinants of developmental processes (e.g. mesodermal induction), but more recent evidence has suggested that the Snail family members control developmental processes by regulating the genes involved in cell adhesion and migration. The Snail family proteins are zinc finger-containing transcriptional repressors that bind to DNA at the E-box motif(s) (CANNTG) of target gene promoters (61). Several recent reports have implicated Snail family members in tumor progression. Slug is a zinc finger protein that has been shown to be up-regulated in breast cancer and malignant mesothelioma (62). This occurs via repression of E-cadherin expression and the subsequent up-regulation of cell invasiveness and migration (63). Although the functions of Snail proteins during development and malignant progression are fairly well known, relatively little is known about their roles in normal developmental processes. Here, we report for the first time that the forced overexpression of Slug in chick limb mesenchymal cells can induce apoptosis. Based on our findings, it seems possible that the decreased migration seen in JNK inhibitor-treated cells may be due to apoptosis. This slug overexpression-induced apoptosis could be reversed by co-treatment with the miR-221 inhibitor, which decreased Slug levels and enhanced cell migration. In sum, our data collectively suggest that miR-221 is a negative modulator of chondrogenesis in chick limb mesenchymal cells and that this occurs via Mdm2 down-regulation and subsequent inhibition of Slug degradation.