Differentiation 86 (2013) 65–74

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Differentiation

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Discovering small molecules that inhibit adipogenesis and promoteosteoblastogenesis: Unique screening and Oncostatin M-like activity

Katsuhiko Nawa a,n,1, Hirotaka Ikeno d,1, Norikazu Matsuhashi b,Tomomi Ogasawara b, Eri Otsuka c

a Frontier Research Laboratories, Daiichi Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japanb Medical Chemistry Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo 134-8630, Japanc Cardiovascular-Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo 140-8710, Japand Biological Research Department, Daiichi Sankyo RD Novare Co., Ltd., Tokyo 134-8630, Japan

a r t i c l e i n f o

Article history:Received 18 January 2013Received in revised form21 June 2013Accepted 23 July 2013

Keywords:Mesenchymal stem cellAdipogenesisOsteogenesisOncostatin MIsoxazole

81/$ - see front matter & 2013 International SInternational Society for Differentiation (wwx.doi.org/10.1016/j.diff.2013.07.005

viations: MSC, mesenchymal stem cell;kin-6; IL-11, Interleukin-11; LIF, leukemia inhe kinase; ALP, alkaline phosphatase; PPAR, ped receptor; Runx, runt-related transcription faprotein; Osx, Osterix; TCF, T-cell factor; LEF,esponding author. Tel.: +81 3 3492 3131; fax:ail address: nawa.katsuhiko.cs@daiichisankyo.ese two authors contributed equally to this w

a b s t r a c t

Oncostatin M (OSM), one of the IL-6 family cytokines, inhibits adipogenic differentiation and stimulatesosteoblastogenic differentiation from human bone marrow mesenchymal stem cells (hBMSCs). Thisfunctional study of OSM enabled us to develop a two-dimensional small-molecule screen that shiftshBMSC differentiation from adipocyte to osteoblast. Several structurally related compounds (isoxazoles)inhibited the accumulation of intracellular lipid droplets, whereas they promoted alkaline phosphataseactivity and extracellular matrix calcification. Isoxazoles also reduced the expression of adipogenictranscription factor PPARγ and increased the levels of osteogenic transcription factors Runx2 and Osterix.They also induced the expression of the Wnt/β-catenin downstream gene and TOPflash reporter;however, the dephosphorylated β-catenin-active form was not significantly increased. Interestingly,the slight modification of the active compound led to a complete reversion of the dual differentiationactivities. In summary, we have identified isoxazoles with anti-adipogenic and pro-osteogenic activitiesthat provide a potential new tool for exploring the lineage commitment of mesenchymal stem cells and apossible lead for therapeutic intervention in osteopenia and osteoporosis.

& 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

1. Introduction

Bone marrow mesenchymal stem cells (BMSCs) can give risenot only to osteoblasts, but also to a range of other cell typesincluding adipocytes, chondrocytes and myoblasts (Pittenger et al.,1999). Differentiation to osteoblasts and adipocytes is important inthe maintenance of normal bone homeostasis. The shift of BMSCdifferentiation to the adipocyte lineage may contribute to theprogressive increase in adipocyte formation and decrease inosteoblast numbers that coincide with age-related bone loss. Thisreciprocal relationship is suggested by the results that inductivefactors for adipogenesis inhibit osteoblast formation (Akune et al.,2004), and that promotive factors for osteoblastogenesis inhibitadipocyte differentiation (Ross et al., 2000; Suzawa et al., 2003;

ociety of Differentiation. Publishedw.isdifferentiation.org)

OSM, Oncostatin M; IL-6,ibitory factor; GSK, glycogenroxisome proliferator-ctor; C/EBP, CCAAT/enhancer-lymphoid enhancer factor+81 3 5740 3644.co.jp (K. Nawa).ork.

Ichida et al., 2004; Hong et al., 2005). Furthermore, the pathogenicstates associated with bone loss, including aging, glucocorticoidside-effects and osteoporosis, coincide with increased bonemarrow adiposity (Chan and Duque, 2002). Thus, successfullyidentifying the critical factors that shift adipogenesis to osteoblas-togenesis opens the potential to provide novel pharmacologictargets and to lead to more efficient interventions in osteopeniaand osteoporosis.

Genetic studies indicate that Wnt signaling is critical fornormal osteogenesis (Day et al., 2005; Hill et al., 2005). Enhance-ment of Wnt/β-catenin activity by Wnt overexpression (Bennettet al., 2007) or that deficiency of Wnt antagonists (Morvan et al.,2006; ten Dijke et al., 2008) is associated with increased boneformation in mice and humans. In vitro experiments suggest thatWnt ligands not only stimulate osteoblastogenesis but also inhibitadipogenesis in mesenchymal precursor cells (Kang et al., 2007).Inhibiting the Wnt pathway may be a potential therapeutic targetfor osteoporosis; however, activation of this pathway may causevarious side effects, including increased susceptibility to cancer.

In addition to Wnt, members of the interleukin-6 (IL-6) familyof cytokines are also implicated in adipocyte and osteoblastdifferentiation. These include IL-6, IL-11, Oncostatin M (OSM),leukemia inhibitory factor (LIF), cardiotrophin-1 and ciliary

by Elsevier B.V. All rights reserved.

K. Nawa et al. / Differentiation 86 (2013) 65–7466

neurotrophic factor, and share a common signal transducingreceptor, gp130 (Bravo and Heath, 2000). These cytokines havebeen shown to regulate the processes of bone formation and boneresorption, and to function in the pathogenesis of bone (Sims andWalsh, 2010). We have focused on OSM because of its robust anti-adipogenic activity (Miyaoka et al., 2006) and extensive boneformation in OSM transgenic mice (Malik et al., 1995). It has alsobeen reported that OSM is a factor that inhibits adipogenesis andpromotes osteoblastogenesis in adipose tissue-derived mesenchy-mal stem cells (Yanai and Obinata, 2001; Song et al., 2007).However, the precise role underlying OSM-induced modulationbetween adipogenesis and osteoblastogenesis remains to besolved. In the current study, we showed that OSM shifts adipogen-esis to osteoblastic differentiation via the JAK/STAT and MEK/ERKpathways.

Cell-based phenotypic assays of synthetic small molecules andnatural products have historically provided useful chemical toolsfor the study of cellular differentiation (Ding and Schltz, 2004). Forexample, the finding of 5-aza-C, which induces myogenic differ-entiation of C3H10T1/2 cells, led to the discovery of a mastertranscription factor, MyoD, responsible for skeletal myogenic fatedetermination (Lassar et al., 1986). In the current study, wefocused on several modulations of hBMSCs from adipogenicdifferentiation to osteoblastic differentiation. Significant findingsregarding OSM-induced phenotypic changes in hBMSCs enabledus to identify small compounds that have anti-adipogenic and pro-osteogenic activities by using OSM as an internal standard. Inter-estingly, a family of isoxazoles was identified from our chemicallibrary. Isoxazole ISX-1, as well as OSM, had the unique actionsof inhibiting adipogenesis and promoting osteoblastogenesisin hBMSCs. We have provided the first ever characterization ofisoxazole-induced modulation of lineage commitment.

2. Materials and methods

2.1. Materials

The materials and their suppliers were as follows: hBMSCs andAdipoRed dye, Lonza; α-MEM, Invitrogen; Oncostatin M, IL-6 andIL-11, R&D Systems; leukemia inhibitory factor, Millipore; JAKinhibitor, Calbiochem; Stat3 inhibitor (Stattic) and MEK inhibitor(U0126), Sigma. Antibody sources were as follows: anti-activeβ-catenin (dephosphorylated form) and anti-PPARgγ, Millipore;anti-β-catenin, Becton Dickinson; anti-β-actin, Cell SignalingTechnology; secondary antibody conjugated with peroxidase, GEhealthcare.

2.2. Cell culture for adipogenic differentiation

The hBMSCs were cultured with MSCBM (Lonza) containing10% fetal bovine serum (FBS) at 37 1C with 5% CO2 in Air. Afterconfluence, the cells were harvested with Trypsin/EDTA andrepeated at one-fourth of the confluent density for continuedpassage. All experiments were performed at passage numbers 3–5.For differentiation into adipocytes, hBMSCs were seeded in a 96-well plate at a density of 104 cells per well, and cultured for 24 h inα-MEM containing 10% FBS. The medium was changed to 100 μL ofan adipogenic differentiation medium [α-MEM containing 10% FBSand MDI (0.5 mM isobutyl methylxanthine, 1 μM dexamethasone,1 μM insulin)] or an adipogenic differentiation medium containing10 mM β-glycerophosphate and 50 μg/mL ascorbic acid (GA) andincubated for 3 days. Then, 100 μL of α-MEM containing 10% FCSand GA was added to each well and the cells were further culturedfor 7 days.

2.3. Assay for adipogenic and osteogenic differentiation

For lipid droplet staining, cells were washed with PBS andincubated in AdipoRed (1% solution in PBS) at room temperature.After 15 min, the fluorescence activity was determined on aFlexStation (Molecular Devices) according to the instruction man-ual. In some experiments, the images of intracellular lipid dropletswere photographed under fluorescence microscopy. To measurealkaline phosphatase (ALP) activity, cells were washed with PBSand solubilized in 50 μL of lysis buffer (Promega) for 30 min atroom temperature. Each lysate (5 μL) was transferred to anotherwhite plate and 50 μL of Lumi-Phos Plus reagent (Lumigen) wasadded as the substrate. After 5 min incubation at 37 1C, thechemical luminescence of each well was determined on an ARVOfluorescence luminometer (PerkinElmer). For calcium assay, cellswere washed twice with PBS and incubated overnight in 50 μL of1 M HCl. The supernatant was assayed for calcium concentrationusing a Calcium E-test kit (Wako).

2.4. Phenotypic change-based screen for small molecules

The experimental compounds, provided from Daiichi SankyoResearch Institute, were dissolved in dimethylsulfoxide (DMSO).For high throughput screening (HTS), hBMSCs were seeded in 384-well black/clear-bottom plates at a density of 2500 cells/well andcultured in α-MEM (25 μL) containing 10% FBS for 24 h, and thentreated with each compound (5 μM) in an adipogenic differentia-tion medium containing GA. After 3 days, 20 μL of the same freshmedium containing the compound was added and further incu-bated for 7 days. The accumulation of intracellular lipid dropletswas determined by AdipoRed staining as described above. Afterthe fluorescence intensity was measured on a FlexStation usingexcitation at 485 nm and measuring emission at 572 nm, theAdipoRed solution was completely removed from the well forALP assay. Cells were solubilized in 10 μL of lysis buffer (Promega),and 20 μL of ALP-substrate solution (AttoPhos, Promega) was thenadded directly to the same well for measurement of ALP activity.After the plates had been incubated for 2 min at room tempera-ture, the ALP reaction was terminated by adding 5% EDTA solution.Fluorescence activity was determined on a FlexStation usingexcitation at 435 nm and emission at 555 nm.

2.5. Real-time PCR

Total RNA was extracted from cultured cells with a CellAmpDirected RNA Prep Kit (Takara). cDNAs were synthesized withrandom primer and the objective mRNA was quantified by real-time qPCR on an ABI 7500 using a One Step SYBR Prime ScriptRT-PCR Kit and specific primers (Takara). The ribosomal proteinlarge P0 (RPLP0) was used as the reference gene.

2.6. Reporter assay

HEK293 cells were transfected with TOPflash or FOPflashreporter vector. The transfected cells were then subcultured inDMEM containing 10% FBS and 400 μg/mL G418 at 37 1C for2 weeks with 5% CO2 in air and established as a stable transfor-mant. The cells (2�104 cells/well) were plated into 96-well platescoated with Poly-D-Lysine and cultured overnight in DMEM con-taining 10% FBS. Test samples were added and the cells werefurther cultured for 24 h. Luciferase activity was measured usingthe Bright-Glo Assay System (Promega).

K. Nawa et al. / Differentiation 86 (2013) 65–74 67

2.7. Western blot analysis

Cells derived from one well of a 12-well plate were lysed in100 μL of LIPA lysis buffer (Santa Cruz). The whole cell lysate(20 μg) was subjected to SDS/PAGE on 10–20% polyacrylamide gel.After electrophoresis, the proteins were transferred to a PVDFmembrane (Millipore). The membrane was blocked with 5% non-fat dried skim milk in TBS and probed with primary antibodyfor 1 h at room temperature. After washing, the membrane wasincubated with the HRP-conjugated secondary antibody for 1 h atroom temperature. The specific signal was detected using an ECLPlus (GE Healthcare) and a FAS-1000 Lumino Imaging Analyzer(Toyobo). The intensities of protein bands were quantified bysoftware ImageQuant TL (GE Healthcare).

2.8. Statistical analyses

The significance of each set of values was assessed by theunpaired two-tailed and unpaired Student's t-test. Statistical sig-nificance was defined as po0.05.

3. Results

3.1. Effects of OSM on adipogenic and osteoblastogenicdifferentiation of hBMSCs

hBMSCs are capable of differentiating into osteoblasts oradipocytes. The induction protocol of hBMSCs to adipocytes hasbeen extensively studied, and we also confirmed their highdifferentiation potential to adipocytic lineage after the additionof MDI (0.5 mM isobutyl methylxanthine, 1 μM dexamethasoneand 1 μM insulin). However, a dual lineage differentiation assaysystem is suitable for discovering some molecules to regulate bothosteoblastic and adipocytic differentiation. The addition of GA(10 mM β-glycerophosphate and 50 μg/mL ascorbic acid) to MDIresulted in a slight enhancement of lipid droplet accumulationwithout ALP induction (Supplementary Fig. S1). When hBMSCswere induced to differentiate into adipocytes in the presence ofMDIGA, OSM inhibited the accumulation of lipid droplets in adose-dependent manner, showing maximal inhibition at 0.8 ng/mL (Fig. 1A and B). While a similar effect of OSM has been studiedin the mouse preadipocyte cell line 3T3-L1, mouse embryonicfibroblasts (Miyaoka et al., 2006) and human adipose tissue-derived stem cells (Song et al., 2007), we also confirmed OSM-attenuated adipogenic differentiation in hBMSCs. PPARγ, C/EBPα,C/EBPβ and FABP4 are adipogenic differentiation regulators/mar-kers. The expression levels of the factors were determined at3 days after adipogenic stimulation. In agreement with thesephenotypic changes, OSM dose-dependently prevented the induc-tion of PPARγ, C/EBPα and FABP4 (Fig. 1E). However, it had noeffect on C/EBPβ expression. These results suggest that OSMnegatively modulates the adipogenic differentiation of hBMSCsvia the PPARγ pathway.

Since the commitment of hBMSCs to adipocytes or osteoblastsis generally thought to be reciprocal, we examined the effect ofOSM on osteoblastic differentiation of hBMSCs under cultureconditions that favored adipogenic differentiation. First, we eval-uated the induction of ALP activity, an early marker of osteoblas-togenic differentiation. While OSM had minimal effect on theinduction of ALP activity in the absence of MDI, hBMSCs showed adose-dependent increase in ALP activity when exposed to adifferentiation medium containing MDIGA (Fig. 1C). Since dexa-methasone was more effective than MDI for stimulating ALPactivity, this OSM-induced ALP activity appears to be dependenton the dexamethasone included in the adipogenic differentiation

medium (Supplementary Fig. S2A). Since ALP induction shouldlead to eventual mineralization, calcium deposition was alsoassayed to confirm this early osteogenic effect. In agreement withthe results of ALP induction, OSM increased the calcification ofhBMSCs after the initial treatment with MDI (Fig. 1D). OSMappears to modulate hBMSCs from adipogenesis to osteoblastgen-esis during MDI treatment. We also examined the expression ofosteoblastic marker genes. In addition to ALP, Runx2 and Osterix,which have been identified as transcription factors essentialfor osteoblastgenesis, had increased at 3 days after exposure toMDI (Fig. 1F). These observations showed that OSM inhibitsadipogenesis and stimulates osteoblastogenesis under adipogenicdifferentiation.

To determine the critical phase of switching adipogenesis toosteoblastogenesis, hBMSCs were cultured with OSM for differentperiods after the addition of MDI. While pulse incubation ofhBMSCs with OSM during the early phase (day 0–3) had minimaleffect on ALP induction, the addition of OSM through Day 3–7 wassufficient to induce ALP activity with a similar effect in thepresence of OSM for 7 days (Supplementary Fig. S2B), suggestingthat OSM is required for ALP induction at a late phase rather thanan early phase. This result and the MDI-requirement for OSM-induced pro-osteogenic activity (Fig. 1C and D) suggest that somecommitment from hBMSCs to preadipocytes is required for OSM-reduced adipogenesis and OSM-induced osteoblastogenesis. Infact, human subcutaneous preadipocytes showed a significantresponse to OSM in the absence of MDI (Supplementary Fig.S2D). In contrast, MDI-induced adipogenesis was completelyinhibited by OSM in both the early phase and the late phase(Supplementary Fig. S2C).

3.2. Effects of IL-6 family cytokines on adipogenic andosteoblastogenic differentiation

Since OSM is a cytokine in the IL-6 family, we next compared theeffects of IL-11, LIF and IL-6 on the adipogenesis and osteogenesis ofhBMSCs. As shown in Supplementary Fig. S3A, accumulation of lipiddroplets was strongly inhibited by OSM and moderately by IL-11,whereas LIF and IL-6 had no significant effect. The potencies of thesecytokines to induce ALP activity were closely paralleled by their anti-adipogenic activities (Supplementary Fig. S3B). These results specifi-cally implicate OSM in the modulation of adipogenesis andosteoblastogenesis.

To investigate whether the effect of IL-6 family cytokines isspecific to hBMSCs, we examined these cytokines' effects on anothermesenchymal stem cell system. Mouse C3H10T1/2 cells showfibroblastic morphology in cell culture and are functionally similarto hBMSCs (Davis et al., 1987). The effects of these cytokines wereexamined on C3H10T1/2 cells after stimulation of adipogenic differ-entiation. While the differentiation capability of C3H10T1/2 cells toadipocyte lineage was low in the MDI-culture, the further addition ofβ-glycerophosphate and ascorbic acid (GA) increased the number ofintracellular lipid droplets (Supplementary Fig. S1). In agreementwith Supplementary Fig. S3, OSM showed higher anti-adipogenicactivity than the other cytokines (Supplementary Fig. S4A and B).Similar activities of LIF, IL-11 and IL-6 were observed; however, thesecytokines had less effect than OSM when inhibiting adipogenicdifferentiation of C3H10T1/2 cells. Next, we investigated pro-osteogenic changes under adipogenic conditions. Since MDI stronglyinduced ALP activity in C3H10T1/2 cells (Supplementary Fig. S1), theMDI-induced system was unsuitable for evaluating the cytokine-induced switching effect from adipogenesis to osteoblastogenesis.We thus focused on the fact that BMP4 can induce the commitmentof C3H10T1/2 cells to preadipocytes (Tang et al., 2004). AlthoughBMP4 alone did not promote adipogenesis, we were able to observedevelopment into adipocytes when β-glycerophosphate and ascorbic

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Fig. 1. OSM is a reciprocal modulator to adipogenesis and osteoblastogenesis of hBMSCs under adipogenic stimulation. (A) Cells were cultured for 10 days under theindicated conditions, and stained with AdipoRed to visualize intracellular lipid droplets. After nuclear staining with DAPI, the images were photographed in an invertedmicroscope. Bar¼100 mm. (B) Cells were cultured in adipogenic differentiation medium, including GA at different concentrations of OSM for 10 days. Lipid droplets inadipocytes were stained with AdipoRed and the fluorescence activity was determined. (–), no-induction. (C) Cells were treated with the indicated concentration of OSM inα-MEM containing MDIGA (closed bar) or α-MEM containing GA (open bar) for 7 days. ALP activity was determined with a fluorogenic substrate. (–), no induction. (D) Cellswere cultured in different concentrations of OSM in the presence (closed bar) or absence (open bar) of MDI for 4 days. The medium was replaced with osteogenic mediumcontaining 0.1 μM dexamethasone and GA, and OSM was further added at the same concentration. After 7 days, the cells were fixed in 1 N HCl and incubated overnight at4 1C. The supernatant was used for analysis of Ca-amount with colorimetric assay. (E and F) hBMSCs were treated with OSM (0, 0.8, 4.0, and 20 ng/mL) in the presence of MDIfor 3 days. The expression levels of adipogenic and osteogenic markers were analyzed by RT-qPCR. Results are means7S.D. (n¼4). ** po0.01 compared with OSM negativecontrol.

K. Nawa et al. / Differentiation 86 (2013) 65–7468

acid (GA) were further added to the differentiation medium (unpub-lished data). The effects of these cytokines were then examinedunder culture conditions that included BMP4 and GA (BMP4GA).Overall, similar effects of IL-6 family cytokines were obtained inagreement with the results shown in Supplementary Fig. S4A and B.BMP4GA-induced accumulation of lipid droplets was dose-depe-ndently inhibited by these cytokines (Supplementary Fig. S4C). LIF,IL-6 and IL-11 showed similar inhibition of adipogenesis, but wereless effective than OSM. Under the same adipogenic conditions, OSMdose-dependently induced ALP activity, whereas the bell-shapedinhibitory effect was observed at 20 ng/mL (Supplementary Fig.S4D). LIF and IL-11 were also similar to OSM when inducing ALPactivity, whereas IL-6 had minimal effect on ALP induction. OSM-reduced adipogenesis and OSM-induced osteoblastogenesis appear

to be observable irrespective of cell species or adipogenic stimulant.No OSM-induced effects were observed in cultures without BMP4GAstimulation (unpublished data).

3.3. Molecular mechanism involved in OSM-reduced adipogenesisand OSM-induced osteoblastogenesis

Two major intracellular signal transduction pathways, the Januskinase (JAK)/signal transducer and activator of transcription(STAT), and the extracellular signal-regulated kinase (ERK) path-ways are thought to mediate most of the biological effects of OSM(Heinrich et al., 2003). For analysis of the signaling mechanismleading to the OSM-reduced adipogenesis, hBMSCs were exposedto OSM along with JAK, Stat3 or MEK inhibitors under adipogenic

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Fig. 2. Characterization of two-dimensional high throughput screen with a small subset of the small-molecule library. A small subset of our small-molecule library (3200compounds) was screened. hBMSCs were cultured for 10 days in adipocyte differentiation medium including GA with assay compounds (5 μM). The intracellular lipiddroplets were measured after staining the cells with AdipoRed, and then ALP activity was measured after cell lysis. The percent stimulation values in ALP were normalized tothe percentage of 4 ng/mL OSM-positive control, and the percent inhibition values in AdipoRed staining were calculated using the values of adipocyte differentiation control(0% inhibition) and OSM-induced inhibition control (100% inhibition). A single plot corresponds to one compound. The threshold was set on 15% stimulation of ALP activityand 50% inhibition of AdipoRed staining (dotted lines). Two compounds (closed circles) in the upper right corner were identified as positive hits.

K. Nawa et al. / Differentiation 86 (2013) 65–74 69

conditions. The MEK inhibitor U0126 did not affect the accumula-tion of intracellular lipid droplets (Supplementary Fig. S5E).However, JAK and Stat3 inhibitors recovered the OSM-attenuatedstaining of lipid droplets (Supplementary Fig. S5A and C), suggest-ing that OSM-reduced adipogenesis is mediated by the JAK/STATpathway. Next, we examined the pathway required for OSM-stimulated osteoblastogenesis. OSM induced ALP activity at 7 daysafter adipogenic induction with MDI; however, JAK and MEKinhibitors dose-dependently attenuated OSM-promoted ALPinduction (Supplementary Fig. S5B and F). In contrast, treatmentof the cells with Stat3 inhibitor did not inhibit ALP induction(Supplementary Fig. S5D). These results show that the ERK path-way through JAK plays a key role in OSM-stimulated osteoblasto-genesis after adipogenic stimulation of hBMSCs.

3.4. Two-dimensional chemical compound screens efficientlyidentified isoxazole showing the anti-adipogenic and pro-osteogenicactivities

There are a few small compounds that both inhibit adipogenicdifferentiation and promote osteoblastic differentiation. However,these compounds' anti-adipogenic and osteoblastogenic activitieswere evaluated in separate systems that were optimized for eachtype of differentiation. The switching effects from adipogenesis toosteoblastogenesis had not been clarified in previous studies(Byun et al., 2012; Jang et al., 2012). In contrast, it is possible forour experimental system to examine the extent of both differ-entiations in an identical well under the same conditions. Wehypothesized that such unique molecules may be identified byusing phenotypic change-based screening and an appropriate

internal standard without complete knowledge of their targetmolecules. Since OSM is a promising modulator that possessesthe anti-adipogenic and osteoblastogenic activities describedabove, the application of OSM might make it possible to constructa unique screening system and to identify a novel small molecule.We developed a two-dimensional high-throughput screeningassay with a dual readout procedure in the same well. A typicalresult with a small subset of our library (3200 compounds) isshown in Fig. 2. While there were large numbers of compoundsthat inhibited lipid droplet formation, only three compoundsexhibited an ALP induction effect. Of these, one compound wasremoved from the final hit compounds due to low anti-adipogenicactivity. Similar to this, various types of low anti-adipogenic andlow osteoblastogenic compounds, including cell-toxic compounds,were selectively excluded, and a small number of compounds withanti-adipogenic and ALP-inducing activities were screened in thisfirst screening. As the result of screening a small molecule librarycontaining �100,000 compounds, we identified 17 confirmed hitsthat are structurally related, containing an identical scaffold,isoxazole. Of these, ISX-1 and ISX-2 were further characterizedfor anti-adipogenic and pro-osteogenic activities (Fig. 3B).

3.5. Characterization of isoxazole-mediated anti-adipogenic andpro-osteogenic differentiation of hBMSCs

To assess and compare the potency of compounds, dose-response analyses were performed in the same plate. ISX-1 andISX-2 dose-dependently inhibited the accumulation of intracellu-lar lipid droplets (Fig. 3A and C) and stimulated ALP activity(Fig. 3D) in agreement with the first screening results. The

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Fig. 3. Isoxazole-mediated anti-adipogenic and osteoblastogenic differentiations of hBMSCs. (A) hBMSCs were cultured for 10 days with or without the chemical compoundin adipogenic medium containing MDI. After staining with AdipoRed to visualize intracellular lipid droplets, the images were photographed in an inverted microscope.Bar¼200 μm. (B) Chemical structures of representative isoxazoles including ISX-1, ISX-2 and ISX-3. ISX-1, N-[3-(1H-imidazol-1-yl)propyl]-5-(thiophen-2-yl)-1,2-oxazole-3-carboxamide; ISX-2, N-[3-(1H-imidazol-1-yl)propyl]-5-phenyl-1,2-oxazole-3-carboxamide; ISX-3, 5-(3-chlorophenyl)-N-[3-(1H-imidazol-1-yl)propyl]-1,2-oxazole-3-car-boxamide. (C and D) hBMSCs (384 wells) were cultured for 10 days with the indicated concentrations of compounds in adipocyte differentiation medium including GA.The dual assays for intracellular lipid droplets (C) and ALP activity (D) were performed in the same well. (E) hBMSCs were treated with ISX-1 (0, 1.3, 6.5 and 33 μM) in thepresence of MDI for 3 days. The expression levels of adipogenic and osteogenic markers were analyzed by RT-qPCR. (F) hBMSCs were cultured with different concentrationsof ISX-1 in the presence (closed bar) or absence (open bar) of MDI for 3 days. The medium was replaced with osteogenic medium and further incubated with ISX-1. After7 days, the cells were fixed in 1 N HCl and incubated overnight at 4 1C. The supernatant was used for analysis of Ca-amount using a colorimetric assay. Results are means7S.D.(n¼4). * po0.05, ** po0.01 compared with ISX negative control.

K. Nawa et al. / Differentiation 86 (2013) 65–7470

concentration necessary for 50% inhibition of lipid droplet forma-tion (IC50) and the concentration required for 50% induction ofALP activity (EC50) were calculated on the basis of efficacy when4 ng/mL OSM was added as an internal standard. The IC50 valuesfor lipid droplet formation of ISX-1 and ISX-2 were 1.9 and 4.9 μM,respectively, and the EC50 values for ALP induction of ISX-1 andISX-2 were 1.2 and 1.8 μM, respectively. ISX-1-induced ALP activitywas observed in the presence of MDI but not in its absence,consistent with an OSM-induced effect (Supplementary Fig. S6A).

Furthermore, ISX-1 was similar to OSM with respect to anti-adipogenic and ALP-inducing activities (Supplementary Fig. S6B).

To confirm Isx-reduced adipogenesis and Isx-induced osteo-blastogenesis, the expression levels of the adipocyte and osteo-blast markers during adipogenic induction of hBMSCs weredetermined by RT-qPCR analysis (Fig. 3E). In agreement with thephenotypic change, ISX-1 inhibited the mRNA induction of PPARγand FABP4 genes under the adipogenic differentiation of hBMSCs.Unlike OSM, ISX-1 showed no effect on C/EBPα expression,

K. Nawa et al. / Differentiation 86 (2013) 65–74 71

suggesting that ISX-1-attenuated adipogenic differentiation isC/EBPα-independent. For analysis of osteoblastic differentiation,the treatment with ISX-1 increased the mRNA levels of ALP, Runx2and Osterix, whereas the increase in Runx2 was weak. In additionto these osteogenic markers, Col1A1, osteocalcin, and osteopontinare extracellular matrix proteins secreted from osteoblasts. Theexpressions of these bone matrix markers also increased bytreatment with ISX-1 (Supplementary Fig. S7). We also confirmedISX-1-induced osteoblastogenesis in terms of extracellular calcifi-cation, the final differentiation phenotype of osteoblasts. ISX-1 aswell as OSM increased the Ca2+-deposition after adipogenicstimulation, whereas this stimulating effect was not observed inMDI-free culture (Fig. 3F).

05

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β-catenin

Fig. 4. Isoxazole derivatives induce TCF/LEF-mediated transcription. (A) hBMSCs were trThe expression levels of the indicated genes were analyzed by RT-qPCR. (B) Stable transincubated in the presence or absence of LiCl (0, 20, 50 and 100 mM) or ISX-1 (0, 1.3, 6.5treated for 24 h with ISX-1 (6.5 and 33 mM, low and high, respectively) or LiCl (100 mM). Wtotal β-catenin or its activated forms were visualized with each specific antibody. Resultsto total β-catenin was determined by image analysis. Results are means7S.D. (n¼4). *

3.6. ISX-1 promotes TCF/LEF-mediated gene transcription

Wnt/β-catenin signaling induces osteoblastogenesis and inver-sely inhibits adipocyte differentiation (Kang et al., 2007). Thesesimilar activities of isoxazoles and Wnt/β-catenin signaling stimu-lants led us to hypothesize that isoxazoles modulate the differ-entiation of hBMSCs by stimulating the Wnt/β-catenin pathway.To determine whether ISX-1 activates Wnt/β-catenin signaling, weexamined the change in its down-stream gene expression. ISX-1increased mRNA levels of β-catenin and c-Myc (Fig. 4A), suggestingthat the compound activates the Wnt/β-catenin signaling pathwayunder adipogenic culture conditions. We then investigated the roleof ISX-1 using a TOPflash reporter assay, which is driven by T cell

47

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eated with the indicated concentrations of ISX-1 in the presence of MDI for 3 days.formant HEK 293 cells transfected with TOPflash or FOPflash reporter vector wereand 33 μM). Luciferase activity was determined after 24 h. (C) HEK 293 cells werehole cell lysates (20 μg) were prepared for western blot analysis. For development,

are representative of two independent experiments. (D) The ratio of activated formpo0.05, ** po0.01.

K. Nawa et al. / Differentiation 86 (2013) 65–7472

factor/lymphoid enhancer factor (TCF/LEF)-binding elements andis responsible for Wnt/β-catenin signaling. Due to low transfectionefficiency with hBMSCs, HEK 293 cells transfected with TOPflashreporter vector were used for this analysis. As expected, ISX-1increased the expression of TOPflash reporter with a similaractivity to LiCl, which activates β-catenin as the result of GSK3-inhibition (Fig. 4B), whereas it did not substantially affect theactivity of FOPflash, a negative control vector with mutated TCF/LEF-binding sequence. We also examined the active form of β-catenin in western blot analysis by using the specific antibodyagainst the dephosphorylated β-catenin (Fig. 4C). LiCl significantlyincreased both the amount of activated and total β-catenin.Although the effect of ISX-1 on the total amount of β-cateninwas similar to that of LiCl, ISX-1 was less effective than LiCl for theactivation of β-catenin as judged by the ratio of the activation formto total β-catenin (Fig. 4D). These results suggest that isoxazolesstimulate TCF/LEF-mediated gene transcription via GSK3-independent signaling pathways.

To investigate the potential role of ISX-1 in Wnt/β-cateninsignaling, the modulator activities of GSK3 inhibitor were alsoevaluated in a differentiation assay with hBMSCs. Although GSK3inhibitor dose-dependently inhibited MDI-promoted adipogenesisof hBMSCs (Supplementary Fig. S8B), it had lower activity thanOSM for ALP-induction (Supplementary Fig. S8A). Since the ALP-inducing activity of ISX-1 is similar to that of OSM (SupplementaryFig. S6A), ISX-1 appears to be distinct from GSK3 inhibitor and toinduce osteoblastogenesis via an unknown pathway.

3.7. The relationship between ISX-1 and ISX-3 Is reciprocal

ISX-2 converted from Thiophene to Phenyl, as well as ISX-1,inhibited the MDI-stimulated adipocytic differentiation of hBMSCsand induced ALP-activity (Fig. 3C and D) and calcification of thecells (unpublished data). Interestingly, ISX-3, a compound relatedto ISX-2, had a stimulatory effect on the accumulation of lipiddroplets and an inhibitory effect on ALP induction (Fig. 3A, Cand D), suggesting that the substitution of phenyl to the3-chlorophenyl group completely reversed the dual activities. Toinvestigate this functional differentiation, the expression level ofPPARγ was analyzed by western blotting when hBMSCs weretreated with ISX-3 in the presence of MDI (Fig. 5). While ISX-1as well as OSM decreased the induction of PPARγ, ISX-3 increasedthe PPARγ expression level as expected from Fig. 3C and E. ISX-3appears to further promote MDI-stimulated adipogenesis throughthe increase of PPARγ and to inhibit osteoblastogenesis. Theseresults, in which the slight modification of the parent compound(ISX-2) led to a complete reversion of the dual differentiationactivities, suggest that these compounds target the commonmolecule/pathway. Furthermore, the relationship of these two

PPARγ

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OSM

ISX-

1

ISX-

3

β-actin

Fig. 5. Effects of OSM, ISX-1 and ISX-3 on PPARγ expression. hBMSCs were treatedfor 2 days with OSM (20 ng/mL), ISX-1 or ISX-3 (50 μM) in the presence of MDI. Thecells were harvested and the whole cell lysate (20 μg) was subjected to SDS-PAGEand western blot analysis for PPARγ. Results are representative of two independentexperiments.

compounds appears to be as activator/inhibitor or agonist/antagonist.

4. Discussion

OSM has been shown to have a principal role in bone home-ostasis (Malik et al., 1995; Jay et al., 1996; Walker et al., 2010).While hBMSCs were not responsive to OSM in the resting statewithout stimulation, OSM induced ALP activity and ALP mRNAafter the MDI treatment. This might be due to the expression levelsof OSMR and gp130, since they are rapidly induced after MDIstimulation in mouse pre-adipocyte 3T3-L1 cells (Miyaoka et al.,2006). The binding of OSM to its receptor activates the JAK/STATsignaling pathway and leads to the ERK cascade (Heinrich et al.,2003). Pharmacological inhibition of STAT3 resulted in the inhibi-tion of OSM-reduced adipogenesis. In contrast, blocking the ERKsignaling pathway decreased OSM-induced ALP induction. Ourresults are consistent with a previous study which revealed thatJAK/STAT and ERK pathways are implicated in OSM-attenuatedadipogenesis and OSM-induced osteoblastogenesis, respectively(Song et al., 2007). Furthermore, the current study showed thatOSM promotes osteoblastogenesis and suppresses adipogenesis,not only in hBMSCs but also in mouse C3H10T1/2 cells. OSMappears to be a potential master regulator of MSC lineage alloca-tion, since it was able to shift MSCs from the adipogenic to theosteoblastic lineage, regardless of cell specificity or adipogenicstimulant.

Since OSM is a pro-inflammatory cytokine, it would be difficultto use its receptor agonist as a potential target for therapeuticintervention. Would it then be possible to mimic the OSM-dualeffects, using a small molecule, without activating the OSM/gp130receptor? Our evidence that OSM has both anti-adipogenic andpro-osteogenic activities in hBMSCs allowed us to identify smallmolecules that have these unique characteristics. Only isoxazolederivatives were isolated as positive hit compounds in thisinvestigation, suggesting that it is necessary and sufficient for thisscreening to acquire the intended compounds. ISX-1, one of thesecompounds, had dual activity not only in inhibiting hBMSCs fromdifferentiating into adipocytes, but also in promoting their osteo-blastic differentiation. These activities of ISX-1 were comparable tothose of OSM. However, ISX-1 does not seem to be the OSM/gp130receptor agonist, as it has no effect on the C/EBPα mRNA level(Fig. 3E).

Activation of Wnt/β-catenin signaling has also been shown tosuppress adipogenesis through inhibition of C/EBPα- and PPARγ-expression, and to promote osteoblastogenesis by directly stimu-lating Runx2 expression (Kang et al., 2007). Although ISX-1activated TCF/LEF-mediated gene transcription (Fig. 4B), it didnot significantly increase the β-catenin activation form (Fig. 4C andD). Isoxazole appears to increase Wnt/β-catenin signaling throughactivation of TCF/LEF transcription factors and/or epigenetic genemodifications. GSK3 inhibitor decreased both PPARγ- and C/EBPα-mRNA (Kang et al., 2007), whereas ISX-1 did not inhibit C/EBPαgene transcription (Fig. 3E). GSK3 inhibitor, which activates Wnt/β-catenin signaling, was also less effective than OSM and ISX-1 ininducing ALP activity (Supplementary Figs. S6 and S8). Theseresults suggest that ISX-1 is neither a GSK3 inhibitor nor a simpleactivator of the Wnt/β-catenin signaling pathway.

PPARγ is the master regulator of adipogenesis (Rosen andSpiegelman, 2000), and its ligand activation drives the differentia-tion of mesenchymal stem cells towards adipocytes in preferenceto osteoblasts (Lecka-Czernik et al., 2002). Furthermore, PPARγhaplo-insufficiency has been shown to enhance osteoblastogenesisin vitro and to increase bone mass in vivo (Akune et al., 2004).However, it is controversial as to whether PPARγ antagonism

K. Nawa et al. / Differentiation 86 (2013) 65–74 73

stimulates osteoblastic differentiation. Although suppression ofPPARγ by retroviral-mediated PPARγ knockdown is sufficientto stimulate osteoblastogenesis in mouse embryonic fibroblasts(Kang et al., 2007), PPARγ antagonists, bisphenol A diglycidyl ether(BADGE), GW9662, and lentiviral-mediated PPARγ knockdown didnot promote osteoblastogenesis of hBMSCs (Yu et al., 2012). Wealso confirmed that GW9662 was not effective for osteoblastogen-esis as assessed by induction of ALP activity (unpublished data).While ISX-1 reduced PPARγ expression in hBMSCs, it does notappear to be a simple PPARγ antagonist.

In the action mode of isoxazole, it was recently reported thatsome isoxazoles cause activation of ERK1/2 and increase theactivity of the histone acetyl transferase p300 through an ERK1/2-dependent mechanism (Dioum et al., 2011). In addition, p300increases the Runx2 half-life as well as its transcriptional activitythrough the acetylation of specific lysine residues (Jun et al., 2010),and also stimulates β-catenin transcriptional activity throughpreferential association of β-catenin and TCF/LEF (Levy et al.,2004). Exogenous Runx2 expression promotes osteoblastic differ-entiation (Ducy et al., 1997; Byers et al., 2002) and the Wnt ligandinhibits adipocyte differentiation (Kang et al., 2007). As a result,isoxazole appears to promote osteoblastogenesis and inhibitadipogenesis through modulating the ERK-p300 pathway. In con-trast, a specific inhibitor of the ERK1/2 signaling pathway resultedin reduced osteoblastogenic differentiation and promoted adipo-genic differentiation in hBMSCs (Jaiswal et al., 2000). Modulationof ERK1/2 activity may be critical for the lineage commitment toadipocytes or osteoblasts, although, how isoxazole could activateERK1/2 remains to be elucidated.

Interestingly, the small molecule SKL2001, which is a family ofisoxazoles and structurally related to ISX-1, was recently identifiedas an activator of the Wnt/β-catenin pathway (Gwak et al., 2012).This compound as well as ISX-1 promoted osteoblastogenesis andsuppressed adipocyte differentiation. Treatment of HEK 293 cellswith SKL2001 inhibited β-catenin phosphorylation via disruptionof the Axin/β-catenin interaction and resulted in upregulation ofβ-catenin, whereas ISX-1 stabilized β-catenin without significantinhibition of its phosphorylation (Fig. 4C and D). Since theisoxazole-activated ERK-p300 pathway can promote β-cateninacetylation and improve its stability (Ge et al., 2009; Dioumet al., 2011), inhibition of the β-catenin phosphorylation is notcritical for TCF/LEF mediated gene transcription (Fig. 4B). Althoughthere is minimal difference in the structure of SKL2001 and ISX-1,more careful consideration is needed regarding the functionalanalysis.

The transcriptional modulator TAZ has recently been reportedto act as a molecular modulator between adipogeneis and osteo-blastogenesis by inhibiting PPARγ activity and stimulating Runx2activity (Hong et al., 2005). It would be interesting to examine theeffects of isozazoles on Runx2-dependent transcription andPPARγ-dependent transcription. TM-25659, a TAZ modulator, hasbeen characterized as its nuclear translocation enhancer (Janget al., 2012), whereas its chemical structure proved to be differentfrom isoxazole active compounds and did not contain the iso-xazole scaffold. In another study, zinc finger protein 467 was alsoidentified as a novel co-factor that promotes adipocyte differentia-tion and suppresses osteoblast differentiation (Quach et al., 2010).We need to regard these molecules as candidates for the isoxazoletarget or its downstream mediator.

In conclusion, isoxazole is one of the few single molecules thatcan shift hBMSCs from adipogenesis to osteoblastogenesis(Supplementary Fig. S9). Furthermore, a small modification ofthe scaffold results in complete reciprocal modulation in theseactivities. A study to acquire more details on the molecularmechanism and a search for the isoxazole target molecule shouldenable us to identify the master regulator controlling the balance

between adipogenic and osteoblastogenic differentiations fromhBMSCs. ISX-2 and ISX-3 are potentially useful probes in studies todetermine the mechanism of commitment to adipogenesis andosteoblastogenesis. Isoxazole derivatives effective in nano-molarconcentrations are also promising compounds for therapeuticintervention in osteoporosis and osteopenia. ISX-1 and ISX-2 haveno significant effect on the proliferation and survival of hBMSCs at5 μM (Supplementary Table 1).

Acknowledgments

All the authors are employees of Daiichi Sankyo Co., Ltd., orDaiichi Sankyo RD Novare Co., Ltd. We thank Drs. F. Isono, O. Ando,K. Morishita, K. Sakano, Y. Kimura and H. Kudo for helpfuldiscussions and encouragement. We are also grateful to M. Fukuifor her skilled technical assistance and Dr. K. Aoki for preparationof this manuscript.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.diff.2013.07.005.

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