SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice

PLOS ONE, Dec 2019

RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx2−/− calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter.

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SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice

Citation: Yoshida CA, Komori H, Maruyama Z, Miyazaki T, Kawasaki K, et al. ( SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice Carolina A. Yoshida 0 Hisato Komori 0 Zenjiro Maruyama 0 Toshihiro Miyazaki 0 Keishi Kawasaki 0 Tatsuya Furuichi 0 Ryo Fukuyama 0 Masako Mori 0 Kei Yamana 0 Kouhei Nakamura 0 Wenguang Liu 0 Satoru Toyosawa 0 Takeshi Moriishi 0 Hiroshi Kawaguchi 0 Kenji Takada 0 Toshihisa Komori 0 Andre Van Wijnen, University of Massachusetts Medical, United States of America 0 1 Department of Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, 2 Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan, 3 Department of Orthodontics and Dentofacial Orthopedics, Graduate School of Dentistry, Osaka University , Suita, Japan , 4 Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Hiroshima International University , Kure , Japan , 5 Teijin Institute for Biomedical Research , Teijin, Tokyo , Japan , 6 Department of Periodontology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan, 7 Department of Oral Pathology, Graduate School of Dentistry, Osaka University , Suita , Japan RUNX2 and SP7 are essential transcription factors for osteoblast differentiation at an early stage. Although RUNX2 inhibits osteoblast differentiation at a late stage, the function of SP7 at the late stage of osteoblast differentiation is not fully elucidated. Thus, we pursued the function of SP7 in osteoblast differentiation. RUNX2 induced Sp7 expression in Runx22/2 calvarial cells. Adenoviral transfer of sh-Sp7 into primary osteoblasts reduced the expression of Alpl, Col1a1, and Bglap2 and mineralization, whereas that of Sp7 reduced Bglap2 expression and mineralization at a late stage of osteoblast differentiation. Sp7 transgenic mice under the control of 2.3 kb Col1a1 promoter showed osteopenia and woven-bone like structure in the cortical bone, which was thin and less mineralized, in a dose-dependent manner. Further, the number of processes in the osteoblasts and osteocytes was reduced. Although the osteoblast density was increased, the bone formation was reduced. The frequency of BrdU incorporation was increased in the osteoblastic cells, while the expression of Col1a1, Spp1, Ibsp, and Bglap2 was reduced. Further, the osteopenia in Sp7 or Runx2 transgenic mice was worsened in Sp7/Runx2 double transgenic mice and the expression of Col1a1 and Bglap2 was reduced. The expression of Sp7 and Runx2 was not increased in Runx2 and Sp7 transgenic mice, respectively. The expression of endogenous Sp7 was increased in Sp7 transgenic mice and Sp7-transduced cells; the introduction of Sp7 activated and sh-Sp7 inhibited Sp7 promoter; and ChIP assay showed the binding of endogenous SP7 in the proximal region of Sp7 promoter. These findings suggest that SP7 and RUNX2 inhibit osteoblast differentiation at a late stage in a manner independent of RUNX2 and SP7, respectively, and SP7 positively regulates its own promoter. - Funding: This work was supported by a grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, the Uehara Memorial Foundation, and the Presidents Discretionary Fund of Nagasaki University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Kei Yamana, who is employed by a commercial company (Teijin, Tokyo, Japan), declares that neither himself nor Teijin have competing interests relating to employment, consultancy, patents, products in development or marketed products etc. Kei Yamanas authorship on the paper is based on the fact that he participated in the acquisition and analysis of data in a scientific fashion, free of any bias. The affiliation of Kei Yamana to a commercial company, does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. . These authors contributed equally to this work. After multipotent mesenchymal cells commit to the osteoblastic lineage, preosteoblasts differentiate into osteoblasts, which express bone matrix proteins including COL1A1, COL1A2, SPP1 (osteopontin), IBSP (bone sialoprotein), and BGLAP2 (osteocalcin) [1]. RUNX2, SP7/Osterix, and canonical Wnt signaling are essential for the commitment of mesenchymal cells to the osteoblastic lineage [2], [3]. The osteoblasts then express bone matrix protein genes at different levels depending on the maturity of the cells. Mesenchymal cells and preosteoblasts weakly express Col1a1 and Col1a2, but osteoblasts showed increased levels. Immature osteoblasts express Spp1 and then Ibsp, and mature osteoblasts strongly express Bglap2 [1]. Mature osteoblasts are embedded into the bone matrix and become osteocytes. Osteocytes, the most abundant cells in mature bone, are extensively interconnected with other osteocytes and osteoblasts through processes [4]. SP7 is a zinc finger- (...truncated)


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Carolina A. Yoshida, Hisato Komori, Zenjiro Maruyama, Toshihiro Miyazaki, Keishi Kawasaki, Tatsuya Furuichi, Ryo Fukuyama, Masako Mori, Kei Yamana, Kouhei Nakamura, Wenguang Liu, Satoru Toyosawa, Takeshi Moriishi, Hiroshi Kawaguchi, Kenji Takada, Toshihisa Komori. SP7 Inhibits Osteoblast Differentiation at a Late Stage in Mice, PLOS ONE, 2012, Volume 7, Issue 3, DOI: 10.1371/journal.pone.0032364