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FOXQ1 Promotes the Osteogenic Differentiation of Bone

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FOXQ1 Promotes the Osteogenic Differentiation of Bone Xiang et al. Stem Cell Research & Therapy (2020) 11:403 https://doi.org/10.1186/s13287-020-01928-9 RESEARCH Open Access FOXQ1 promotes the osteogenic differentiation of bone mesenchymal stem cells via Wnt/β-catenin signaling by binding with ANXA2 Lusai Xiang1*† , Junming Zheng2†, Mengdan Zhang1, Tingting Ai1 and Bin Cai1 Abstract Background: This study investigated the role of Forkhead box Q1 (FOXQ1) in the osteogenic differentiation of bone mesenchymal stem cells. Methods: Mouse bone mesenchymal stem cells (mBMSCs) were transfected with lentivirus to generate Foxq1- overexpressing mBMSCs, Foxq1-suppressed mBMSCs, and mBMSC controls. The activity of osteogenic differentiation was evaluated with alizarin red staining, alkaline phosphatase activity assay, and RT-qPCR. Wnt/β-catenin signaling activities were compared among groups by TOPFlash/FOPFlash assay, immunofluorescence staining, and western blot assay of beta-catenin (CTNNB1). Coimmunoprecipitation mass spectrometry was also carried out to identify proteins binding with FOXQ1. Results: Our data showed that FOXQ1 expression was positively correlated with the osteogenic differentiation of the mBMSCs. FOXQ1 also promoted the nuclear translocation of CTNNB1 in the mBMSCs, enhancing Wnt/β-catenin signaling, which was also shown to be essential for the osteogenic differentiation-promoting effect of FOXQ1 in the mBMSCs. Annexin A2 (ANXA2) was bound with FOXQ1, and its depletion reversed the promoting effect of FOXQ1 on Wnt/β-catenin signaling. Conclusion: These results showed that FOXQ1 binds with ANXA2, promoting Wnt/β-catenin signaling in bone mesenchymal stem cells, which subsequently promotes osteogenic differentiation. Keywords: Forkhead box Q1, Bone mesenchymal stem cells, Osteogenic differentiation, Wnt/β-catenin, Annexin A2 Background catenin [3] pathway. The Wnt/β-catenin signaling path- Mesenchymal stem cells constitute a group of multipotent way plays an important role in mesenchymal stem cell cells capable of differentiating into various types of cells, stimulation and differentiation regulation [4, 5]. The main including osteoblasts, adipocytes, and various other types event in Wnt/β-catenin signaling involves the stabilization of cells. This process is modulated by various signaling and nuclear translocation of beta-catenin (CTNNB1), pathways, including BMP [1], TGF-β [2], and the Wnt/β- which then forms a complex with TCF/LEF and initiates downstream gene transcription [6]. Similar to other major * Correspondence: [email protected] β † signaling pathways, Wnt/ -catenin is regulated by mul- Lusai Xiang and Junming Zheng contributed equally to this work. tiple factors [7]. 1Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, No. 56 Forkhead box Q1 (FOXQ1, also known as HFH1) is a Lingyuan west Road, Guangzhou 510055, Guangdong, China member of the forkhead box (FOX) family of proteins. It Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xiang et al. Stem Cell Research & Therapy (2020) 11:403 Page 2 of 10 was first identified as a regulator of hair follicle develop- Cell culture ment [8]. Later identified as an oncogene, FOXQ1 is Commercially available mouse bone mesenchymal stem highly expressed in colorectal cancer, breast cancer, liver cells (mBMSCs) derived from the bone marrow of Balb/ cancer, and various other cancers [9], and multiple stud- c mice (MUCMX-01001; Cyagen Biosciences; Guang- ies have demonstrated a close relationship between dong, China) were purchased and cultured with alpha- FOXQ1 and Wnt/β-catenin in cancer cells [10, 11]. modified Eagle medium (⍺-MEM; Life Technologies, FOXQ1 also regulates various physiological processes, CA, USA) supplemented with 10% fetal bovine serum including the survival [12] and proliferation of stem cells (FBS, Life Technologies, CA, USA), 100 U/mL penicillin [13]; however, its role in osteogenic differentiation re- (Sigma, MO, USA), and 100 mg/mL streptomycin mains to be elucidated. (Sigma, MO, USA). After the 3rd passage, the cells were In the current study, we aimed to investigate the influ- used for experiments. ence of FOXQ1 on the osteogenic differentiation of mes- enchymal stem cells and to elucidate the underlying FOXQ1 expression manipulation molecular mechanisms. Our findings suggested that Full-length Foxq1 cDNA was amplified with Flag-tagged FOXQ1 promotes osteogenic differentiation of mouse primers from total RNA and then cloned into a bone mesenchymal stem cells via the Wnt/β-catenin sig- pCDNA3.1 vector (V79020, Thermo Fisher Scientific, naling pathway. MA, USA) to produce the bait, Flag-tagged FOXQ1 pro- tein, for the coimmunoprecipitation study. Full-length Methods Foxq1 cDNA was also cloned from total RNA and Animal study and ethical approval of the protocol inserted into a pGLV5 vector (GenePharma, Shanghai, To obtain mouse embryo and alveolar bone tissue for China). PGLV3 lentivirus containing the Foxq1 shRNA histological evaluation, pregnant Chinese Kunming sequence and lentivirus particles with an empty pGLV3 (KM) mice (4 weeks old) and Chinese Kunming mice (7 vector and with a pGLV5 plasmid were purchased from days old) were purchased from Sun Yat-sen University. GenePharma (GenePharma, Shanghai, China). MBMSCs Chinese Kunming mice (7 days old) came from the same were then transfected with lentiviral particles containing pregnant mouse to minimize the genetic difference be- Foxq1-sh pGLV3, Foxq1-overexpressing pGLV5, an tween groups. The study protocol was approved by the empty pGLV3, or an empty pGLV5 vector, creating 4 Ethics Committee of the Hospital of Stomatology, Sun groups of cells denoted as Foxq1-sh mBMSCs, Foxq1- Yat-sen University (ERC-2013-15; Guangzhou, China). over mBMSCs, lv3 mBMSCs, and lv5 mBMSCs, respect- ively. Real-time quantitative reverse transcription- polymerase chain reaction (RT-qPCR) and western blot Tissue preparation and histology evaluation assay were carried out to assess Foxq1 expression in each To observe FOXQ1 expression in alveolar bone tissue, a group of mBMSCs. pregnant KM mouse was sacrificed to obtain 3 mouse embryos at embryonic day 15.5 (E15.5), and 3 KM mice were sacrificed at each time point (postnatal day 7 (P7) Osteogenic differentiation, alizarin red staining, and and postnatal day 11 (P11)), whose mandibles were iso- alkaline phosphatase activity assay lated surgically. The whole embryos of E15.5 mouse and Mouse bone mesenchymal stem cells were seeded into the mandibles from P7 and P11 mice were fixed with 4% 6-well plates at a density of 1.0 × 106 cells per well. An paraformaldehyde at room temperature for 72 h. Then, osteogenic induction medium was prepared according to the samples were dehydrated with graded solutions of al- previous studies [14]. The cells were cultured in the in- cohol and embedded. Anti-FOXQ1 polyclonal antibody duction medium for 14 days, during which period the (5 μg/mL; MBS9408074; My BioSource, Inc., San Diego, medium was changed every 3 days, and then evaluated. USA) was used as the primary antibody. Alizarin red staining (ARS) was conducted to visualize Immunofluorescence analyses were carried out to the mineral deposition in each group after osteogenic in- evaluate the transnucleation of CTNNB1. Cells were in- duction. The cells were first fixed with cold methanol at cubated overnight with anti-CTNNB1 polyclonal anti- room temperature for 10 min, rinsed twice with deionized body (5 μg/mL; ab2365; Abcam, Cambridge, UK) at 4 °C. water, and stained with alizarin red (10 μL/mL, 130-22-3; Then, the sections were incubated with secondary anti- Sigma, MO, USA) at room temperature for 30 min. Then, body (1:1000 dilution; A-21206; Invitrogen, CA, USA) the excess dye was removed with deionized water. For for 1 h in a dark chamber. Finally, the sections were quantification of ARS, stain was desorbed with 10% cetyl- counterstained with 4′6-diamidino-2-phenylindole pyridinium chloride (CPC) in PBS, pH 7.0, for 15 min at (DAPI; 0.5 μg/mL; Thermo Fisher Scientific, MA, USA) room temperature. Then, ARS concentration was deter- for 15 min for nuclear labeling. mined by absorbance at 560 nm with a spectrometer. Xiang et al. Stem Cell Research & Therapy (2020) 11:403 Page 3 of 10 Alkaline phosphatase activity assay was also carried Table 1 Primer for RT-qPCR analysis out to evaluate the osteogenic differentiation of Gene Sequence mBMSCs from 4 groups. Cultured
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