Effects of TGF‑β1 on the migration and morphology of RAW264.7 cells in vitro

Ueta,Miho; Takaoka,Kazuki; Yamamura,Michiyo; Maeda,Hanako; Tamaoka,Joji; Nakano,Yoshioro; Noguchi,Kazuma; Kishimoto,Hiromitsu

doi:10.3892/mmr.2019.10662

Effects of TGF‑β1 on the migration and morphology of RAW264.7 cells in vitro

Authors:
- Miho Ueta
- Kazuki Takaoka
- Michiyo Yamamura
- Hanako Maeda
- Joji Tamaoka
- Yoshioro Nakano
- Kazuma Noguchi
- Hiromitsu Kishimoto
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Affiliations: Department of Oral and Maxillofacial Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo 663‑8501, Japan, Department of Genetics, Hyogo College of Medicine, Nishinomiya, Hyogo 663‑8501, Japan
Published online on: September 10, 2019 https://doi.org/10.3892/mmr.2019.10662
Pages: 4331-4339

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Abstract

Osteoclasts (OCs) differentiate from monocyte/macrophage‑lineage hematopoietic precursor cells, which are known as OC precursors (OCPs). Several studies have investigated cell chemotaxis in the bone microenvironment; however, OCP migration ability in the bone microenvironment during OC differentiation is yet to be elucidated. As an initial investigation of this characteristic, the present study aimed to determine the effects of transforming growth factor (TGF)‑β1 on OCP migration in vitro. Pre‑osteoclastic RAW264.7 cells were cultured with and without TGF‑β1 (2, 5 or 20 ng/ml), receptor activator of NF‑κB ligand (RANKL; 50 ng/ml), and/or SB431542 (10 µM), a potent and specific inhibitor of TGF‑β1 receptor kinase activity. Cell proliferation was significantly inhibited in the presence of TGF‑β1 for 3 days, and the effect was reversed by SB431542. Tartrate‑resistant acid phosphatase (TRAP) activity in RAW264.7 cells was significantly increased by RANKL treatment, compared with TRAP activity in control cells on day 3. The highest TRAP activity in RAW264.7 cells was induced by the combined treatment with TGF‑β1 (2 ng/ml) and RANKL. When TGF‑β1 signaling was inhibited by addition of SB431542 to the medium during culture, OC differentiation was notably suppressed. These findings suggest that TGF‑β1 accelerates RANKL‑induced OC differentiation, but does not act in a dose‑dependent manner. The migration of RAW264.7 cells was promoted at 24 h, but was suppressed at 72 h, during RANKL‑induced osteoclast differentiation in the presence of TGF‑β1. These results were accompanied with the increased expression of small G‑proteins, RhoA and Rac, at 24 h, but their expression decreased at 72 h. RAW264.7 cells treated with TGF‑β1 for 24 h underwent morphological changes, from round to polygonal morphology. Furthermore, protrusions were completely lost and the cell morphology reverted from polygonal to round after TGF‑β1 treatment for 72 h. Therefore, our findings indicated that OCP migration may be modified by differentiation in vitro.

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