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Contents lists available at ScienceDirect

The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel

All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/␤-catenin signaling pathways

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Yang Liu a , Yingzi Liu b,c , Ranxi Zhang a,b , Xing Wang a,b , Fan Huang a , Zhengjian Yan a,b , Mao Nie a,b , Jun Huang b,c , Yuanzheng Wang a , Yang Wang a , Liang Chen a , Liangjun Yin a , Baicheng He b,c,∗∗ , Zhongliang Deng a,∗ a b c

Department of Orthopaedic Surgery, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing, China Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, China

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Article history: Received 4 July 2013 Received in revised form 8 November 2013 Accepted 23 November 2013 Available online xxx

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Keywords: Bone morphogenic protein 9 All-trans retinoic acid Preadipocytes Osteogenic differentiation Adipogenic differentiation

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1. Introduction

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It is known that excessive adipogenesis contributes to osteoporosis, suggesting that trans-differentiation of adipogenic committed preadipocytes into osteoblasts may be a potential therapeutical approach for osteoporosis. We explored whether bone morphogenic protein 9 (BMP9) could induce 3T3-L1 preadipocytes to trans-differentiate into osteoblasts. BMP9 effectively increased expression of osteogenic markers and promoted mineralization in preadipocytes. However, BMP9 also led to adipogenic differentiation of preadipocytes, as evidenced by increased lipid accumulation and up-regulation of adipogenic transcription factors. In order to regulate the switch between osetogenesis and adipogenesis, we evaluated the effect of all-trans retinoic acid (ATRA) on BMP9-induced differentiation of preadipocytes. We found that ATRA enhanced BMP9-induced osteogenic differentiation and blocked BMP9-induced adipogenic differentiation both in vitro and in vivo. Mechanistically, ATRA was shown to elevate BMP9 expression and activate BMP/Smad signaling. Additionally, BMP9 and ATRA exerted a synergistic effect on activation of Wnt/␤-catenin signaling. Knockdown of ␤-catenin abolished the stimulatory effect of ATRA on BMP9-induced alkaline phosphatase activity and reversed the inhibitory effect of ATRA on BMP9-induced adipogenesis in preadipocytes. Furthermore, ATRA and BMP9 synergistically repressed glycogen synthase kinase 3␤ (GSK3␤) activity and promoted Akt phosphorylation, and inhibited expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) that antagonizes phosphatidylinositol-3-kinase (PI3K) function, suggesting that Wnt/␤-catenin signaling was activated at least partly through PI3K/Akt/GSK3␤ pathway. Collectively, ATRA mediated BMP9-induced osteogenic or adipogenic differentiation of 3T3-L1 preadipocytes by BMP/Smad and Wnt/␤-catenin signaling. The combination of BMP9 and ATRA may be explored as an effective therapeutic strategy for osteoporosis. © 2013 Published by Elsevier Ltd.

The low bone mass and increased marrow adipose tissue can be simultaneously observed in conditions leading to osteoporosis (Wronski et al., 1986; Justesen et al., 2001; Sottile et al., 2004), suggesting that reduced bone formation is associated with excess fat

∗ Corresponding author at: No.76, Linjiang Road, Yuzhong District, Chongqing 400010, China. Tel.: +86 1360836 7586. ∗∗ Corresponding author at: No.1, Yixueyuan Road, Yuzhong District, Chongqing 400010, China. Tel.: +8613310218650. E-mail addresses: [email protected] (B. He), [email protected] (Z. Deng).

content in osteoporotic population (Verma et al., 2002). Adipocytes and osteoblasts share a common bone marrow progenitor – multipotential mesenchymal stem cells (MSCs) (Pittenger et al., 1999). Osteogenic or adipogenic differentiation of MSCs is influenced by specific groups of transcription factors. Runt-related transcription factor 2 (Runx2) and Osterix (Osx) are the main determinants of osteogenesis, while peroxisome proliferators activated receptor-␥ (PPAR␥) and CCAAT/enhancer binding proteins (C/EBPs) promote adipogenesis of MSCs (Zhang et al., 2012). This process is complex in vivo, suggesting that adipocytes may be generated at the expense of osteoblasts during the process of osteoporosis. This shift of MSCs differentiation to adipocyte lineage may contribute to the increase in adipogenesis and decrease in osteogenesis that coincide with bone loss. Given the close association between adipocytes

1357-2725/$ – see front matter © 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biocel.2013.11.018

Please cite this article in press as: Liu Y, et al. All-trans retinoic acid modulates bone morphogenic protein 9-induced osteogenesis and adipogenesis of preadipocytes through BMP/Smad and Wnt/␤-catenin signaling pathways. Int J Biochem Cell Biol (2013), http://dx.doi.org/10.1016/j.biocel.2013.11.018

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and osteoblasts, increasing adipogenesis in bone marrow may play an important role in the development and deterioration of osteoporosis. Differentiation of MSCs to adipocytes involves two phases consisted of the commitment of MSCs to preadipocytes and the process of terminal adipogenic differentiation during which preadipocytes mature into adipocytes. Therefore, transdifferentiation of preadipocytes into osteoblasts may be a potential therapeutic strategy of osteoporosis with which to both prevent excessive marrow adipogenesis and divert committed preadipocytes to become more osteoblastic with a resulting increase in bone cells. Currently, increasing evidences have indicated a large degree of plasticity between preadipocytes and osteoblasts. A previous study showed that bone marrow adipocytes can differentiate in the osteogenic direction, which primarily requires an intermediate step to induce morphological change into fibroblast-like preadipocytes (Park et al., 1999). It was also found that subcutaneous preadipocytes are able to differentiate into bone-forming osteoblasts (Justesen et al., 2004). However, the mechanism underlying trans-differentiation of preadipocytes into osteoblasts is complex and remains to be thoroughly elucidated. It has been reported that 3T3-F442A preadipocytes express bone morphogenetic protein (BMP) receptors and that BMP2 plays a critical role in the process during which 3T3-F442A preadipocytes undergo commitment to osteoblastic lineage (Ji et al., 2000; Skillington et al., 2002). Moreover, overexpression of Runx2, the downstream target of BMP2, is effective for trans-differentiation of preadipocytes into fully differentiated osteoblasts (Takahashi, 2011). Liu et al. also showed that platelet-rich plasma stimulates osteogenic differentiation of 3T3-L1 preadipocytes partially through BMP2 signaling (Liu et al., 2011). Taken together, these findings indicate that adipogenic progenitor cells can revert to osteoblast lineage in response to specific extracellular signal, such as BMP signaling. BMPs are growth factors that belong to the TGF␤ superfamiliy. Based on analyzing 14 types of BMPs, it has been found that BMP9 is one of the most potent BMPs in inducing osteogenic differentiation of MSCs (Cheng et al., 2003). However, little information is available on the effect of BMP9 on differentiation of preadipocytes. Recently, BMP9 was shown to promote adipogenic differentiation of human white preadipocytes (Lord et al., 2010), whereas it is still unclear whether preadipocytes can be converted into osteoblasts in response to BMP9. Given the strongest osteoinductive activity of BMP9, we hypothesize that BMP9 may also effectively induce osteogenic differentiation of preadipocytes. Therefore, we evaluated the effect of BMP9 on osteogenic differentiation of preadipocytes in this study. As a cell model, we used the well established 3T3-L1 preadipocyte cell line that is used extensively in studying adipocyte biology. Though BMP9 has the most potent osteoinductive activity, other signaling molecules are also required to enhance BMP9-induced bone formation. Retinoic acids (RAs) are derivatives of vitamin A. We have previously demonstrated the synergism between RA and BMP9 in inducing osteogenic differentiation of MSCs (Zhang et al., 2010). Moreover, RA was shown to be able to inhibit adipogenic differentiation and cooperate with BMP2 to induce osteogenic differentiation in preadipocytes (Sato et al., 1980; Skillington et al., 2002). These observations suggest the potential of RA to cooperate with BMP9 to promote osteogenesis and reverse BMP9-induced adipogenesis in preadipocytes. Thus, we also investigated the effect of all-trans-RA (ATRA), the abundant form of RA, on BMP9-induced osteogenic and adipogenic differentiation of preadipocytes. We found that BMP9 could simultaneously induce osteogenic and adipogenic differentiation of 3T3-L1 preadipocytes both in vitro and in vivo. ATRA potentiated BMP9induced osteogenic differentiation and blocked BMP9-induced

adipogenic differentiation in preadipocytes through activation of BMP/Smand and Wnt/␤-catenin pathways. Lastly, our data also suggested that Wnt/␤-catenin activation may be resulted from activation of phosphatidylinositol-3-kinase (PI3K)/Akt/glycogen synthase kinase 3␤ (GSK3␤) pathway by ATRA and BMP9.

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2. Materials and methods

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3T3-L1, HCT 116 and HEK293 cell lines were obtained from ATCC. Cell lines were maintained in the conditions described (Cheng et al., 2003; Zhang et al., 2010). ATRA was obtained from Sigma–Aldrich (Saint Louis, USA). ATRA was dissolved in DMSO and aliquots were stored in −80 ◦ C. DMSO was used as solvent control. For cell culture treated with ATRA, the medium was changed every 3 days. Unless indicated otherwise, all chemicals were purchased from Sigma–Aldrich. 2.2. Construction of recombinant adenoviruses Recombinant adenoviruses expressing BMP9 (AdBMP9) and small interference RNA (siRNA) targeted ␤-catenin (AdR-simBC) were generated previously using the AdEasy technology, as described (Tang et al., 2009; Chen et al., 2010a,b). AdBMP9 and AdR-simBC also respectively expressed GFP and RFP as a marker for monitoring infection efficiency. Adenoviruses expressing only GFP (AdGFP) and RFP (AdRFP) were used as controls. 2.3. Preparation of conditioned medium Subconfluent HCT116 cells were infected with an optimal titer of AdBMP9. At 24 h after infection, the culture medium was changed to serum-free DMEM. BMP9 conditioned medium (BMP9-CM) was collected at 48 h after infection and used immediately. 2.4. Alkaline phosphatase (ALP) assays ALP activity was assessed by a modified Great Escape SEAP Chemiluminescence assay (BD Clontech, Mountain View, CA) as described previously (Chen et al., 2010a,b), and/or histochemical staining assay performed with BCIP/NBT Alkaline phosphatase Color Development Kit (Beyotime, Jiangsu, China) according to manufacturer’s instructions. For the bioluminescence assays, each assay condition was performed in triplicate and the results were repeated in at least three independent experiments. ALP activity was normalized by total cellular protein concentrations among the samples. 2.5. Western blotting analysis For total protein level assay, cells were washed with cold PBS (4 ◦ C) and lysed in 300 ␮l lysis buffer. For nucleus fraction protein extraction, the protein was harvested with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Jiangsu, China) according to manufacturer’s instructions. Cell lysate was denatured by boiling and loaded onto a 10% gradient SDS–PAGE. After electrophoretic separation, proteins were transferred to polyvinylidene difluoride membrane (Millipore). Membrane was blocked with a solution containing 10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20 (TBS-T) and 5% non-fat dry milk for 4 h, and probed with the primary antibody at 4 ◦ C overnight, followed by incubation with a secondary antibody conjugated with horseradish peroxidase for 2 h. The proteins of interest were detected using SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Chemical, Rockford, IL). Primary antibodies were obtained from Santa


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Cruz, as follows: anti-osteopontin, anti-osteocalcin, anti-Runx2, anti-Osx, anti-Dlx5, anti-PPAR␥, anti-C/EBP␣, anti-C/EBP␤, antiphosphor-Smad1/5/8, anti-Smad1/5/8, anti-␤-catenin, anti-BMP9, anti-Histone H2A.X, anti-phosphor-GSK3␤ (Ser9), anti-GSK3␤, anti-phosphor-Akt (Ser473), anti-Akt, anti-PTEN and anti-GAPDH.

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For Alizarin Red S staining, infected cells were cultured in the presence of ascorbic acid (50 ␮g/mL) and ␤-glycerophosphate (10 mM). At 14 days after infection, cells were fixed with 0.05% (vol/vol) glutaraldehyde at room temperature for 10 min. After being washed with distilled water, fixed cells were incubated with 0.4% Alizarin Red S (Sigma–Aldrich) for 5 min, followed by washing with distilled water. For Oil Red O staining, cells were fixed with 10% formalin for 60 min and stained with Oil Red O solution. The stained Oil Red O was extracted with isopropanol and the amount was quantified by measuring the optical absorbance at 570 nm using a BioTek spectrophotometer.

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Briefly, cells were fixed with 4% formalin and washed with PBS. The fixed cells were permeabilized with 0.25% Triton X-100 and blocked with 10% goat serum, followed by incubation with an antiosteocalcin, osteopontin, or Smad1/5/8 antibody overnight. After washing, cells were incubated with biotin-labeled secondary antibody for 30 min, followed by incubating cells with streptavidin-HRP conjugate for 20 min at room temperature. The presence of the expected protein was visualized by DAB staining and examined under a microscope.

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Cells were seeded in 25-cm2 flasks and transfected with 2 ␮g per flask of BMP Smad-responsive luciferase reporter (Chen et al., 2010a,b), p12xSBE-Luc or ␤-catenin/Tcf4-responsive luciferase reporter (Tang et al., 2009), pTOP-Luc using Lipofectamine (Invitrogen). At 12 h after transfection, cells were replated to 24-well plates and infected with AdBMP9 in the presence or absence of ATRA (1 ␮M) at 4 h after replating. At 24 h after treatment, cells were lysed and cell lysates were collected for luciferase assays using Promega’s Luciferase Assay Kit.

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All animal experiments were approved by Institutional Animal Care and Use Committee of Chongqing Medical University. 3T3-L1 cells infected with AdBMP9 or AdGFP were cultured in the presence or absence of ATRA (1 ␮M) for 7 days in vitro, then cells were harvested for subcutaneous injection (5 × 106 /injection) into the flanks of athymic nude (nu/nu) mice (5 animals per group, 4–6 weeks old, female, Harlan Sprague Dawley). In order to maintain the effect of ATRA in vivo, animals were given with ATRA solution (30 mg/kg) or solvent control once a day by intragastric administration. At 5 weeks after implantation, animals were euthanized, and the implantation sites were retrieved for micro-computed tomography (␮CT) analysis and histological evaluation.

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Retrieved tissues were decalcified, fixed in 10% formalin overnight, and embedded in paraffin. Serial sections of the embedded specimens were stained with hematoxylin and eosin (H&E).

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Masson Trichrome or Alcian Blue staining was carried out as described (Tang et al., 2009; Chen et al., 2010a,b). 2.11. Micro-computed tomographic (CT) imaging analysis All specimens were scanned by a cone-beam-type desktop ␮CT system (Explore Locus SP, GE Healthcare, USA). All image data analysis was performed using Micview V2.1.2 (Vaddio, New Hope, MN, USA) three-dimensional reconstruction processing software, and the trabecular bone volume density (BV/TV, %) was measured. 2.12. Quantitative analysis of image data using NIH ImageJ The image analysis software ImageJ was used to analyze the regions of interest and/or intensity of the acquired image data. Random sampling of 10 fields was used routinely to determine mean ± SD values for statistical analysis. 2.13. Statistical analysis Results were reported as the mean ± SD. Differences between samples were compared by the two-tailed Student’s t test. For all quantitative assays, each assay condition was performed in triplicate, and the results were repeated in at least three independent experiments. A value of p < 0.05 was defined as statistically significance. 3. Results 3.1. ATRA and BMP9 act synergistically in inducing ALP activity in 3T3-L1 preadipocytes By now, the role of RA in osteogenesis remains controversial (Wan et al., 2006; Wang et al., 2008). Hence, we firstly tested the effect of ATRA on induction of the early osteogenic marker ALP activity in 3T3-L1 preadipocytes. We found that ATRA effectively induced ALP activity in a dose-dependent manner when cells were treated with low concentrations of ATRA (0.5 ␮M, 1 ␮M, 2 ␮M) (Fig. 1A), while ATRA-induced ALP activity seemingly maintained at a stable level with high concentrations (5 and 10 ␮M) (Fig. 1A). We next tested the ability of BMP9 to induce ALP activity of preadipocytes and whether this effect of BMP9 could be potentiated by ATRA. Preadipocytes were infected with AdBMP9 or AdGFP, AdBMP9 was shown to effectively up-regulate BMP9 expression in 3T3-L1 cells (Fig. 1B). As shown in Fig. 1C, BMP9 induced a significantly increase in ALP activity, which was markedly enhanced by ATRA. The histochemical staining of ALP activity showed a consistent result (Fig. 1D). However, high concentration (5 ␮M) of ATRA did not increase BMP9-induced ALP activity any more than low concentrations (1 ␮M and 2 ␮M)) of ATRA while the cause of this phenomenon remains to understand. Collectively, these results demonstrate that ATRA can not only induce the early osteogenic marker ALP activity but also significantly enhance BMP9-induced ALP activity in 3T3-L1 preadipocytes. 3.2. ATRA promotes BMP9-induced expression of late osteogenic markers and osteogenic transcription factors, and matrix mineralization in 3T3-L1 preadipocytes We further analyzed the effect of ATRA on the late osteogenic markers osteopontin (OPN) and osteocalcin (OC) induced by BMP9. Preadipocytes were infected with AdBMP9 or AdGFP in the presence or absence of ATRA (1 ␮M). At 10 days post treatment, cells were subjected to immunohistochemical staining using OPN and OC antibodies. Though ATRA alone failed to induce detectable OPN


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Fig. 1. ATRA and BMP9 act synergistically in inducing ALP activity in 3T3-L1 preadipocytes. (A) ATRA induces ALP activity in 3T3-L1 preadipocytes. 3T3-L1 cells were treated with varying concentrations of ATRA or solvent control. ALP activity was measured at the indicated time points. Each assay condition was carried out in triplicate. “*”, p < 0.05; “**”, p < 0.01, compared with control group. (B) AdBMP9 effectively transduces preadipocytes. 3T3-L1 cells were infected with AdBMP9 and AdGFP. GFP signal was recorded under a fluorescence microscope at 48 h after infection. With western blotting analysis using an anti-BMP9 antibody, AdBMP9 effectively increased BMP9 expression at 48 h after infection. BF, bright field; FF, fluorescence field. (C and D) ATRA potentiates BMP9-induced ALP activity. 3T3-L1 cells were infected with AdBMP9 or AdGFP, followed by treatment with varying concentrations of ATRA or solvent control. ALP activity was measured at the indicated time points (C) or histochemically stained at 8 days after infection (D). Each assay condition was carried out in triplicate. “ ”, p < 0.01, compared with control group; “## , p < 0.01, compared with BMP9/DMSO group; “**”, p < 0.01, compared with corresponding GFP/ATRA group.

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or OC expression, cells stimulated with both BMP9 and ATRA exhibited a stronger expression of OPN or OC than BMP9-stimulated cells (Fig. 2A and B). Using western blotting analysis, we showed a consistent result (Fig. 2D). Next, we examined the effect of ATRA on BMP9-induced matrix mineralization and found that ATRA effectively promoted BMP9-induced in vitro mineralization in 3T3-L1 cells (Fig. 2C). Furthermore, cells stimulated by the combination of ATRA and BMP9 exhibited higher expression levels in osteogenic transcription factors, such as Runx2, Osx and Dlx5, than cells stimulated by separate treatment with ATRA or BMP9 (Fig. 2E). These findings strongly suggest that though ATRA has very limited ability to induce the late stage of bone formation, it is capable of significantly promoting BMP9-induced late stage of osteogenic differentiation in vitro via up-regulating expression of osteogenic transcription factors in preadipocytes. 3.3. ATRA inhibits BMP9-induced adipogenic differentiation of 3T3-L1 cells BMP9 is also an effective inducer of adipogenic differentiation of MSCs (Kang et al., 2009), hence we examined the effect of BMP9 on induction of adipogenic differentiation of preadipocytes and whether ATRA could reverse this process. 3T3-L1 cells were infected with AdBMP9 or AdGFP and then treated with ATRA (1 ␮M). At 8 days post treatment, cells were subjected to Oil Red O staining. As shown in Fig. 3A, BMP9 resulted in accumulation of a large amount of lipid in 3T3-L1 preadipocytes. As expected, a combination of BMP9 and ATRA treatment showed a less number of Oil Red O positive cells (Fig. 3A and B). Accordingly, our data revealed

that BMP9-induced expression of adipogenic transcription factors, including CEBP/␣, CEBP/␤ and PPAR␥, were effectively suppressed by ATRA (Fig. 3C). These data indicate the strong inhibitory effect of ATRA on BMP9-induced adipogenic differentiation of 3T3-L1 preadipocytes. 3.4. ATRA enhances BMP9-induced ectopic bone formation from 3T3-L1 preadipocytes We then explored whether preadipocytes can form osteoid tissue in vivo under the stimulation of BMP9 and ATRA. 3T3L1 preadipocytes infected with AdBMP9 or AdGFP were cultured in vitro in the presence or absence of ATRA (1 ␮M) for 7 days, and then cells were harvested and injected subcutaneously into athymic nude mice. To maintain the effect of ATRA in vivo, animals were given with ATRA (30 mg/kg) or solvent control once a day by intragastric administration. At week 5, the BMP9-transduced cells, either treated with ATRA or not, formed detectable bony masses (Fig. 4A), which were further confirmed by ␮CT imaging analysis (Fig. 4B). The ␮CT scanning data revealed that cells transduced with only BMP9 formed less relative bone volume (BV/TV) than cells stimulated with both BMP9 and ATRA (Fig. 4B). However, preadipocytes infected with AdGFP did not form detectable bone masses. On histologic examination, compared with BMP9 group, H&E histologic analysis indicated that preadipocytes stimulated with both BMP9 and ATRA led to significantly more mature and fully mineralized bone matrix, with minimal undifferentiated preadipocytes and mature adipocytes (Fig. 4C and F). These results were further confirmed by Masson’s Trichrome staining as


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Fig. 2. Augmentation of BMP9-induced late osteogenic markers and osteogenic transcription factors by ATRA. (A, B) Immunohistochemical staining of OPN and OC. 3T3-L1 cells were infected with AdBMP9 or AdGFP, and then treated with ATRA (1 ␮M) or not. Expression of OPN (A) or OC (B) was assessed by immunohistochemical staining analysis at 10 days after infection using anti-OPN or anti-OC antibody. (C) Alizarin Red S staining of matrix mineralization. 3T3-L1 cells were infected with AdBMP9 or AdGFP in the presence or absence of ATRA (1 ␮M). At 14 days after treatment, cells were stained with Alizarin Red S solution. (D) Western blotting analysis of OPN and OC expression. 3T3-L1 cells were infected with AdBMP9 or AdGFP, and then treated with ATRA (1 ␮M) or not. At day 10, cells were lysed and subjected to Western blotting analysis using anti-OPN or anti-OC antibody. Anti-GAPDH antibody was used to demonstrate equal loading of all samples. (E) Verification of ATRA and BMP9 induced expression of osteogenic transcription factors. 3T3-L1 cells were infected with AdBMP9, and then treated with ATRA (1 ␮M) or not. At 3 days postinfection, western blotting analysis was performed to assess the expression of Runx2, Osx and Dlx5.

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more mature and mineralized osteoid matrices were found in the specimens induced by both BMP9 and ATRA (Fig. 4D). However, Alcian blue staining showed that chondrogenesis by BMP9 was not markedly affected by ATRA (Fig. 4E). These in vivo results further demonstrate that ATRA can not only potentiate BMP9-induced ostegenesis but also inhibit BMP9-induced adipogenesis in 3T3-L1 preadipocytes. 3.5. ATRA acts synergistically with BMP9 in activating Smad signaling activity We next sought to explore the possible mechanism by which ATRA enhanced BMP9-induced osteogenic differentiation of 3T3L1 cells. Smad1, 5, and 8 (Smad1/5/8) are classic mediators for BMP9 osteoinductive signaling, we thus examined whether ATRA could affect BMP9 function at the Smad signaling level. We showed that ATRA alone did not dramatically induce nuclear translocation of Smad1/5/8 whereas BMP9-induced nuclear translocation of Smad1/5/8 was augmented by ATRA (Fig. 5A). Similarly, luciferase reporter assay showed that though ATRA only mildly activated BMPR-Smad reporter activity, it enhanced BMP9-induced BMPRSmad reporter activity (Fig. 5B). Using western blotting analysis, we obtained a consistent result that BMP9-induced phosphorylation of Smad1/5/8 was enhanced by ATRA (Fig. 5C). RA was reported to be able to induce expression of BMPs in various cell types (Hatakeyama et al., 1996; Li et al., 2003; Zhang et al., 2010), hence we assessed

whether ATRA potentiated BMP9-activated BMPR-Smad signaling through up-regulating BMP9 expression. It was found that ATRA significantly elevated BMP9 expression in preadipocytes (Fig. 5D). To sum up, these data suggest that ATRA may exhibit its stimulatory effect on BMP9 by up-regulating BMP9 expression thereby leading to increased BMPR-Smad transcriptional activity. 3.6. ATRA modulates BMP9-induced osteogenic and adipogenic differentiation of preadipocytes via Wnt/ˇ-catenin signaling BMP/Smad signaling also contributes to adipogenesis (Huang et al., 2009). Based on the opposite effects of ATRA on BMP9induced osteogenesis and adipogenesis, it is possible that ATRA may crosstalk with BMP9 through another signaling that has opposite effects on osteogenesis and adipogenesis. Given that Wnt/␤-catenin signaling could not only inhibit adipogenesis but also synergize with BMPs in promoting osteogenesis (Ross et al., 2000; Mbalaviele et al., 2005), and that either RA or BMP signaling could activate Wnt/␤-catenin signaling in various cell types (Liu et al., 2002; Chen et al., 2007; Zhang et al., 2009; Yasuhara et al., 2010; Papathanasiou et al., 2012), we examined the effect of BMP9 and ATRA on Wnt/␤-catenin signaling. We found that the protein level of ␤-catenin was increased in both cytoplasm and nucleus in response to BMP9 and ATRA. Notably, the increase in ␤-catenin content achieved a greater extent in cells receiving both ATRA and BMP9 stimulation (Fig. 6A). Accordingly, either BMP9 or ATRA


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Fig. 3. Inhibition of BMP9-induced adipogenic differentiation of 3T3-L1 pradipocytes by ATRA. (A, B) Lipid droplet formation. 3T3-L1 cells were infected with AdBMP9 or AdGFP in the presence or absence of ATRA (1 ␮M). At 8 days postinfection, cells were fixed and stained with Oil Red O solution (A). Oil Red O stained lipid droplets were quantified by measuring absorbance in spectrophotometer at 570 nm (B). (C) Verification of ATRA and BMP9 induced expression of adipogenic transcription factors. 3T3-L1 cells were infected with AdBMP9 in the presence or absence of ATRA (1 ␮M). At 3 days postinfection, western blotting analysis was performed to assess the expression of PPAR␥, C/EBP␣ and C/EBP␤.

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was shown to effectively induce luciferase activities of pTOPLuc reporter, which was further increased by the combination of BMP9 and ATRA (Fig. 6B). Next, we examined the potential role of Wnt/␤-catenin signaling in the effect of ATRA on BMP9-induced osteogenic and adipogenic differentiation of preadipocytes. 3T3L1 cells were infected with AdBMP9, AdRFP and/or AdR-simBC (Fig. 6C), in the presence or absence of ATRA (1 ␮M). AdR-simBC was shown to effectively inhibit the expression of endogenous ␤-catenin, as well as to inhibit ␤-catenin/Tcf4 transcriptional activation induced by BMP9 and ATRA (Fig. 6B and C). Knockdown of ␤-catenin effectively inhibited BMP9 activity and ATRA potentiation effect on BMP9 in induction of ALP activity and blocked the inhibitory effect of ATRA on BMP9-induced adipogenic differentiation of preadipocytes (Fig. 6D and E). These results suggest that ATRA may regulate BMP9-induced osteogenesis and adipogenesis, at least partly, through Wnt/␤-catenin signaling in preadipocytes. Lastly, we attempted to clarify the mechanism by which BMP9 and ATRA activated Wnt/␤-catenin signaling. As shown in Fig. 6F, the activity of GSK3␤, a negative regulator of Wnt/␤-catenin signaling, was significantly repressed in response to BMP9 and ATRA, as evidenced by increased phosphorylation level of GSK3␤ (Bikkavilli et al., 2008). Given that PI3K/Akt activation inhibits GSK3␤ by phosphorylation (Paez and Sellers, 2003), that PI3K/Akt signaling plays a critical role in BMP9-induce osteogenesis (Chen et al., 2010a,b), and that RA is shown to be able to activate PI3K/Akt signaling (López-Carballo et al., 2002), the inactivation of GSK3␤ may be a result of activation of PI3K/Akt signaling. As expected, ATRA and BMP9 synergistically enhanced Akt phosphorylation in preadipocytes (Fig. 6F). In addition, we also found that BMP9 and ATRA exerted an inhibitory effect on expression of PTEN, a lipid phosphatase that antagonizes PI3K function and consequently inhibits downstream signaling through Akt (Paez and Sellers, 2003). These data suggest that BMP9 and ATRA cooperate to elevate the level of ␤-catenin through PI3K/Akt/GSK3␤ pathway.

4. Discussion Considerable evidence support that excessive adipogenesis in bone marrow contributes to imbalance in bone formation/resorption and ultimately results in bone loss, it is therefore possible that modulation of adipose tissue volume in bone marrow via trans-differentiation of preadipocytes, the adipogenic committed cells, into osteoblasts could provide a potential therapeutic intervention for osteoporosis. Here, we provide compelling evidence that BMP9 promoted both ostogenesis and adipogenesis in 3T3-L1 preadipocytes. The dual role of BMPs in osteogenic and adipogenic differentiation of MSCs has been well demonstrated (Kang et al., 2009), while the bidirectional effect of BMPs on preadipocytes is indeterminate. It was reported that BMP2 increases the ALP activity in 3T3-F442A preadipocytes and also leads to a low level of adipogenic conversion (Skillington et al., 2002). Similarly, Ji et al. reported that BMP2 not only induces osteogenic differentiation but also promotes adipogenic differentiation in 3T3-F442A cells (Ji et al., 2000). However, another study showed that BMP2 alone fails to induce adipogenic differentiation of 3T3-L1 preadipocytes, though it strongly stimulates adipogenesis in the presence of PPAR␥ ligand thiazolidinedione (Sottile and Seuwen, 2000). A recent study has revealed that BMP2 or BMP9 can increase the adipogenic differentiation of human white preadipocytes in serum-free medium containing ciglitazone, another PPAR␥ ligand (Lord et al., 2010). These findings suggest that the ability of BMPs to induce adipogenic differentiation may require specific cytokine, such as PPAR␥. In this study, our data revealed that BMP9 alone was sufficient to induce both osteogenic and adipogenic differentiation of 3T3-L1 preadipocytes, suggesting that BMP9 may be used as a treatment for osteoporosis when it acts synergistically with appropriate agent that can modulate the balance between BMP9-induced osteogenesis and adipogenesis in preadipocytes.


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Fig. 4. ATRA enhances BMP9-induced ectopic bone formation. (A) Macrographic images of ectopic bone masses. Bony masses were found in BMP9 and BMP9 + ATRA stimulated cell groups at 5 weeks. No masses of any kind were formed in the cells infected with AdGFP, either stimulated with ATRA or not. (B) 3D reconstruction of bone masses by CT analysis. The retrieved masses were subjected to ␮CT scanning to obtain 3D surface images and bone volume density (BV/TV, %). (C–E) Histological stains of retrieved samples. Retrieved tissues were decalcified, fixed in 10% formalin overnight, and embedded in paraffin. Serial sections of the embedded specimens were stained with H&E, Masson’s Trichrome stain and Alcian Blue stain. (F) Quantitative analysis of percent trabecular area over total area was done by using ImageJ. At least 10 samples (with ×40 magnification) from each group were randomly selected and analyzed using ImageJ software. AC, adipocyte; OB, osteoblast; OC, osteocyte; MBM, mineralized bone matrix; BM, bone matrix; UPA, undifferentiated preadipocytes; CM, cartilage matrix.

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It has been described by various studies that the reciprocal relationship between BMP-induced osteogenesis and adipogenesis could be regulated by RA. For example, RA was shown to repress BMP2-induced adipogenesis and cooperate with BMP2 to induce osteogenesis in adipose-derived stromal cells (Wan et al., 2006). It was also reported that RA cooperates with BMP2 to repress adipogenesis and promote osteoblastic differentiation of 3T3-F442A preadipocytes (Skillington et al., 2002). Similarly, RA was shown to stimulate osteogenic differentiation through the BMP2-Smad-Runx2/Msx2 pathway and inhibit BMP2-induced adipogenic differentiation via down-regulation of PPAR␥ and C/EBPs (Hisada et al., 2013). These findings are partially supported by a recent study in which ATRA blocks BMP2-induced adipogenic differentiation of MEFs, whereas no synergism between ATRA and BMP2 in stimulating ALP activity was observed (Sun et al., 2009). Interestingly, Oki et al. evidenced that ATRA can up-regulate BMP2 expression in dedifferentiated fat cells and thereby resulting in trans-differentiation of these cells into osteoblasts, suggesting that the synergism between ATRA and BMP2 is involved in this process (Oki et al., 2008). In addition to BMP2, RA also exerts an inhibitory effect on the BMP4 induction of C3H10T1/2 adipogenic commitment (Lee et al., 2011). Furthermore, we have previously demonstrated that RA can potentiate BMP9-induced osteogenesis in MSCs (Zhang et al., 2010). Overall, though the effect of RA

on BMPs-induced osteogenesis and adipogenesis and the mechanism by which RA functions are diverse, these above studies agree on the notion that RA plays a positive role in BMPs-induced osteogenesis and exerts an inhibitory effect on BMPs-induced adipogenesis. In the current study, we found that ATRA showed a similar effect on BMP9-induced osteogenesis and adipogenesis in preadipocytes. Perplexingly, other studies using mouse embryonic palate mesenchymal cells and rat bone marrow stromal cells obtained opposite results that ATRA and BMP signaling cooperate to promote adipogenesis and inhibit osteogenesis (Wang et al., 2008; Chen et al., 2010a,b), suggesting that the synergistic action of RA and BMPs my be cell-type dependent. BMP9 typically signal through Smad pathways. Thus, we examined the effect of ATRA on BMP/Smad signaling to investigate the mechanism by which ATRA interacted with BMP9. Our data indicated that ATRA elevated BMP9 expression and enhanced BMP9-activated Smad signaling in preadipocytes. These results are consistent with our previous study (Zhang et al., 2010). Additionally, ATRA was also shown to increase the phosphorylation level of Smad1/5/8 and activate Smad signaling in osteosarcoma cells (Yang et al., 2012). Similarly, Chen et al. revealed that ATRA significantly prolongs activation of Smad1/5 in mouse embryonic stem cells (Chen et al., 2012). It was also demonstrated that RA could stimulate chondrocyte differentiation and enhance BMP


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Fig. 5. ATRA induces BMP9 expression and activates BMP/Smad pathway. (A) ATRA promotes BMP9-induced nuclear localization of Smad1/5/8. 3T3-L1 cells were infected with AdBMP9 in the presence or absence of ATRA (1 ␮M) for 24 h, and then cells were subjected to immunohistochemical staining using an anti-Smad1/5/8 antibody. Positive nuclear staining is indicated by arrows. (B) Effect of ATRA on BMP9-induced BMPR-Smad reporter activity. 3T3-L1 cells were transfected with BMPR-Samd-responsive luciferase reporter p12xSBE-Luc and infected with AdBMP9 in the presence or absence of ATRA (1 ␮M). At 12 h after infection, cells were collected for luciferase assay using Promega’s Luciferase Assay Kit. (C) ATRA promotes BMP9-induced Smad1/5/8 phosphorylation. 3T3-L1 cells were pretreated with ATRA (1 ␮M), and then treated with BMP9-CM. At 30 min post treatment, total amount and phosphorylated forms of Smad1/5/8 were analyzed by western blotting. (D) ATRA induces BMP9 expression in 3T3-L1 cells. 3T3-L1 cells were stimulated with ATRA (1 ␮M). At the indicated time points, western blotting analysis was performed to assess the expression of BMP9.

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effects through induction of Smad1/5 (Hatakeyama et al., 1996). However, results from other studies have indicated that RA may also act as an inhibitor of Smad signaling (Yu and Xing, 2006; Sheng et al., 2010). Sheng et al. proposed that RA enhances the interaction between phosphorylated Smad1 and its ubiquitin E3 ligases, thereby promoting phosphorylated Smad1 ubiquitination and proteasomal degradation, suggesting a mechanism by which RA suppresses BMP/Smad signaling (Sheng et al., 2010). Up to now, these conflicting observations can not be explained satisfactory, while it can be speculated that the interaction between RA and BMP/Smad signaling may be cell type-specific and/or cell contextdependent. Since BMP/Smad signaling also promotes adipogenesis, the mechanism underlying the inhibitory effect of ATRA on BMP9induced adipogenic differentiation of preadipocytes remains to be determined. A recent study revealed that RA inhibits adipogenesis of preadipocytes via activation of Wnt/␤-catenin signaling that is required in BMP9-induced osteogenesis (Tang et al., 2009; Kim et al., 2013), we therefore explored whether Wnt/␤-catenin signaling was involved in the interaction between ATRA and BMP9. In this study, we found that ATRA and BMP9 exhibited a synergism in activation of Wnt/␤-catenin signaling. These results are consistent with several previous studies that have evidenced that either RA or BMP signaling is able to activate canonical Wnt/␤catenin signaling in various cell types (Liu et al., 2002; Chen et al., 2007; Zhang et al., 2009; Yasuhara et al., 2010; Papathanasiou et al., 2012). Furthermore, knockdown of ␤-catenin by siRNA diminished the stimulatory effect of ATRA on BMP9-induced ALP activity and blocked the inhibitory effect of ATRA on BMP9-induced adipogenesis in preadipocytes. Thus, our data demonstrate that ATRA may modulate BMP9-induced osteogenic and adipogenic differentiation of preadipocytes via activating Wnt/␤-catenin signaling.

Though ATRA and BMP9 increased the protein level of ␤-catenin, there was no change in the mRNA expression of ␤-catenin (data not show), suggesting that ATRA and BMP9 cooperate to activate Wnt signaling through indirect targeting of ␤-catenin. The activity of canonical Wnt signaling is mediated through GSK3␤ that promotes phosphorylation of ␤-catenin and ultimately results in degradation of ␤-catenin and inactivation of Wnt signaling. In addition, inhibition of GSK-3␤ was evidenced to promote osteogenic differentiation (Gambardella et al., 2011). Hence, we tested whether activation of Wnt signaling by ATRA and BMP9 was associated with inhibition of GSK3␤ activity. We revealed that the inactivated (phosphorylated) form of GSK3␤ was significantly increased in response to ATRA and BMP9. Given that GSK3␤ is the downstream substrate of PI3K/Akt signaling that plays a critical role in BMP9-induced osteogenic differentiation of MSCs (Chen et al., 2010a,b), and that RA and BMPs were reported to be capable of activating PI3K/Akt signaling (López-Carballo et al., 2002; Fong et al., 2008; Lee et al., 2009), we speculated that the inhibition of GSK3␤ maybe resulted from activation of PI3K/Akt. As expected, ATRA and BMP9 were shown to significantly increase Akt phosphorylation in preadipocytes. Additionally, we have shown here that ATRA and BMP9 repressed the expression of PTEN, an inhibitor of PI3K/Akt signaling (Paez and Sellers, 2003), which further suggested the positive role of ATRA and BMP9 in PI3K/Akt signaling. These results are supported by previous studies that PI3K/Akt-dependent signaling phophorylates GSK3␤ and suppresses the activity of GSK3␤ (Frame and Cohen, 2001; Hay and Sonenberg, 2004). Therefore, our data sheds light on the potential role of PI3K/Akt signaling in synergistic effect of ATRA and BMP9 on canonical Wnt activation. In summary, we indicated that RA signaling can control the balance between BMP9-induced osteogenesis and adipogenesis in 3T3-L1 preadipocytes. This study provided experimental support


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Fig. 6. ATRA mediates BMP9-induced osteogenesis and adipogenesis through Wnt/␤-catenin signaling in preadipocytes. (A) Western blotting analysis for ␤-catenin in the nucleus, cytoplasm and the whole cell. 3T3-L1 cells were infected with AdBMP9 in the presence or absence of ATRA (1 ␮M). At 3 days after infection, western blotting analysis was performed to assess the expression of ␤-catenin. N, nucleus; C, cytoplasm; W, whole cell. (B) Synergistic effect between ATRA and BMP-9 on luciferase activities of pTOP-Luc reporter. 3T3-L1 cells were transfected with pTOP-Luc reporter and infected with AdBMP9 and/or AdR-simBC in the presence or absence of ATRA (1 ␮M). At 24 h after infection, cells were collected for luciferase assay. (C) Efficient transduction of 3T3-L1 cells via coinfection and verification of the function of AdR-simBC. Cells were coinfected with AdBMP9 and AdR-simBC or other combinations. Fluorescence images were recorded at 24 h after infection. With western blotting analysis using an anti-␤catenin antibody, AdR-simBC effectively inhibited the endogenous ␤-catenin expression at 24 h after infection. (D and E) Knockdown of ␤-catenin inhibits the stimulatory effect of ATRA on BMP9-induced ALP activity and reverses the inhibitory effect of ATRA on BMP9-induced lipid accumulation. 3T3-L1 cells were infected with AdBMP9, AdRFP and/or AdR-simBC in the presence or absence of ATRA (1 ␮M). Quantitative assessment of ALP activities and Oil Red O staining were performed at the indicated time points. (F) Effect of ATRA and BMP9 on PI3K/Akt/GSK3␤ pathway in preadipocytes. 3T3-L1 cells were treated with BMP9-CM in the presence or absence of ATRA (1 ␮M). At 12 h post treatment, total GSK3␤, GSK3␤ phosphorylated at Ser9 (p-GSK3␤), total Akt, Akt phosphorylated at Ser473 (p-Akt) and total PTEN were analyzed by western blotting.

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for the potential application of the combination of BMP9 and ATRA for treating osteoporosis in the clinical settings.

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Conflict of interest

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recombinant adenoviruses and pTOP-luc reporter plasmid. This work was supported in part by research grants from the Natural Science Foundation of China (NSFC, 81071462 to BCH; NSFC, 81272005 to ZLD; NSFC, 31000434 to LC) and Chongqing Science & Technology Commission (CSTC, 2011BB5129 to BCH).

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All authors have no conflicts of interest. References

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Acknowledgements We thank Dr. Di Chen (University of Rochester Medical Center, USA) for providing p12 × SBE-Luc reporter plasmid and Dr. T.-C. He (University of Chicago Medical Center, USA) for providing

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Frizzled2 signaling regulates growth of high-risk neuroblastomas by interfering with β-catenin-dependent and β-catenin-independent signaling pathways.

β-catenin, p38MAPK, NFκB, and PPARγ signaling pathways.

β-catenin signaling pathways orchestrate early tooth development.

β-catenin and Hippo signaling pathways: a brief review.

β-catenin signaling pathway and estrogen signaling pathways.

β-Catenin Signaling in Skin.

β-catenin signaling.

β-catenin signaling.

Signaling Pathways in Melanogenesis.

SIGNALING PATHWAYS AS BIOMARKERS.

Akt Signaling Pathways.

β-catenin signaling pathways in MC3T3-E1 cells.

β-catenin and STAT3 signaling pathways.

Gli signaling pathways in colon cancer and implications for therapy.

β-catenin signaling pathways.

Caspase-3 Signaling Pathways.

PCP signaling pathways.

β-catenin signaling pathways promote the invasion of hypoxic gastric cancer cells.

Modulation of β-catenin signaling by the inhibitors of MAP kinase, tyrosine kinase, and PI3-kinase pathways.

Smad signaling pathways in an in vitro model of amyotrophic lateral sclerosis.

Inactivation of CK1α in multiple myeloma empowers drug cytotoxicity by affecting AKT and β-catenin survival signaling pathways.

Toll-like receptor signaling pathways.

Cancer stem cell signaling pathways.

Aberrant signaling pathways in glioma.