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Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling

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Abstract

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors’ expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

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Fig. 1

adapted from Ikegame et al., 2019 [26]). The arms were closed to 5-mm distance of each other and then the end of each arm pierced the center of right or left parietal bone to apply 0.2-g tension to the sagittal suture of 4-day-old mouse calvaria. The control arms were fixed by a tape and attached to parietal bones. Lower: A schema of the frontal view of the sagittal suture. Bold arrows indicate the direction of tensile stress exerted by the tension device. Osteoblasts (OB) and preosteoblasts (POB) are alkaline phosphatase (ALP) positive (red line). The area surrounded by a green-dotted line is shown in the following pictures in C. B The gene expression levels of Vgll3 in mouse sagittal sutures cultured with (0.2 g) or without (0 g) tension for 3 h and 6 h, examined by real-time PCR. Values are expressed as means ± SEMs (n = 5). Student’s t test, ***P < 0.001 (6 h–0 g vs 6 h–0.2 g). C In the control groups cultured for 1 h (1 h–0 g) and 6 h (6 h–0 g), the end of parietal bone was surrounded by ALP-positive (red) osteoblasts (OB) and preosteoblasts (POB) (a, d), which were positive for VGLL3 immunostaining (green; b, c, e, f). In the tension group cultured for 6 h (6 h–0.2 g), the number of ALP-positive POB at the end of parietal bone was increased (g, arrow). These newly differentiated POB (arrow) and some fibroblastic cells (FB) in the middle of suture (arrowheads) were positive for VGLL3 staining, mainly in the nucleus (h, i). White-dotted lines indicate the boundary of parietal bones. Nuclear staining: methyl green (light green; a, d, g), DAPI (magenta, pseudo-color; c, f, i). Scale bar: 20 μm. All pictures are shown at the same magnification

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Data availability

The datasets of the current study are available from the corresponding authors on request.

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Acknowledgements

We would like to express our sincere gratitude to both Dr. Ikue Tosa, Okayama University (Professor Takarada Laboratory), for providing us with bone samples from Runx2-knockout mouse and Prof. Sadakazu Ejiri, Asahi University, for helpful discussions. We also thank Ms. Heriati Sitosari and Mr. Yilin Zheng for editing the manuscript.

Funding

This work was supported by JSPS KAKENHI grants JP2259203 and JP19K07269 to M.I.; a research grant from Koyanagi-Zaidan in 2020 to M.I.; and the National Natural Science Foundation of China grants 81870736 and 81801040 to B.Z. and Y.L., respectively.

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Authors and Affiliations

  1. Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Heilongjiang Provincial Key Laboratory of Hard Tissue Development and Regeneration, 246 Xuefu Road, Nangang, Harbin, 150001, Heilongjiang, People’s Republic of China

    Haoze Yuan, Ying Li & Bin Zhang

  2. Department of Oral Morphology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Haoze Yuan, Mika Ikegame, Yoko Fukuhara, Fumiko Takemoto, Yaqiong Yu, Yao Weng, Jiajie Guo & Hirohiko Okamura

  3. Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

    Fumiko Takemoto

  4. Department of Endodontics, School and Hospital of Stomatology, China Medical University, Liaoning Provincial Key Laboratory of Oral Diseases, Shenyang, P.R. China

    Yaqiong Yu & Jiajie Guo

  5. Department of Oral Function & Anatomy, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan

    Jumpei Teramachi

  6. Department of Regenerative Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University , Okayama, Japan

    Daisuke Yamada & Takeshi Takarada

  7. Heilongjiang Academy of Medical Sciences, Harbin, 150001, Heilongjiang, P.R. China

    Bin Zhang

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  1. Haoze Yuan
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  2. Mika Ikegame
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Contributions

HY contributed to validation, formal analysis, investigation, and visualization. MI contributed to conceptualization, methodology, writing of the original draft, visualization, supervision, and funding acquisition. YF contributed to formal analysis and statistics. FT performed investigation. YY contributed to formal analysis and investigation. JT contributed to methodology and investigation. YW contributed to investigation and editing of the manuscript. JG performed investigation. DY contributed to methodology. TT contributed to methodology and resources. YL contributed to project administration and funding acquisition. HO contributed to supervision, project administration, and editing of the manuscript. BZ contributed to project administration and funding acquisition.

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Correspondence to Mika Ikegame or Bin Zhang.

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Conflict of interest

Haoze Yuan, Mika Ikegame, Yoko Fukuhara, Fumiko Takemoto, Yaqiong Yu, Jumpei Teramachi, Yao Weng, Jiajie Guo, Daisuke Yamada, Takeshi Takarada, Ying Li, Hirohiko Okamura, and Bin Zhang declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

The protocol for the animal experiment was conducted according to the guidelines of the Animal Care and Use Committee, Okayama University (Approval No. OKU-2016114). These guidelines were established based on NIH guide for the care and use of Laboratory animals.

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Yuan, H., Ikegame, M., Fukuhara, Y. et al. Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling. Calcif Tissue Int 111, 331–344 (2022). https://doi.org/10.1007/s00223-022-00997-7

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