Limei Wang1,2, Jin Zhang3, Cunwei Wang4, Yuping Qi5, Mi Du1, Wenhua Liu1, Chengzhe Yang6, Pishan Yang1,2. 1. Department of Periodontology, School of Stomatology, Shandong University, Jinan, Shandong, China. 2. Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan, Shandong, China. 3. Department of Endodontics, School of Stomatology, Shandong University, Jinan, Shandong, China. 4. Department of Prosthodontics, School of Stomatology, Shandong University, Jinan, Shandong, China. 5. Department of Stomatology, Qilu Hospital, and Institute of Stomatology, Shandong University, Jinan, Shandong, China. 6. Department of Oral & Maxillofacial Surgery, Qilu Hospital, and Institute of Stomatology, Shandong University, Jinan, Shandong, China.
Abstract
OBJECTIVES: Low concentrations of tumour necrosis factor-alpha (TNF-α) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved. MATERIALS AND METHODS: MC3T3-E1 murine preosteoblasts were treated with TNF-α at doses of 0, 0.1 or 1 ng/mL. The ephrinB2-EphB4 signalling pathway was activated using ephrinB2-fc, or inhibited using lentiviruses encoding siRNAs specifically targeting EphB4. Cell proliferation/survival was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and expression levels of Runx2, BSP, ephrinB2 and EphB4 were determined using RT-PCR and Western blotting. ALP activity in these cells was also determined, and mineral nodule formation was evaluated with alizarin red S staining. RESULTS: Low concentrations of TNF-α had no influence on cell proliferation/survival. However, expression levels of Runx2, BSP, ephrinB2 and EphB4, as well as ALP activity and mineral nodule formation, were significantly enhanced in MC3T3-E1 cells treated with low concentrations of TNF-α. Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-fc enhanced TNF-α-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-α. CONCLUSIONS: Low concentrations of TNF-α promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.
OBJECTIVES: Low concentrations of tumour necrosis factor-alpha (TNF-α) have been reported to promote osteogenic differentiation. In this study, a series of in vitro experiments was performed to investigate underlying molecular mechanisms involved. MATERIALS AND METHODS: MC3T3-E1 murine preosteoblasts were treated with TNF-α at doses of 0, 0.1 or 1 ng/mL. The ephrinB2-EphB4 signalling pathway was activated using ephrinB2-fc, or inhibited using lentiviruses encoding siRNAs specifically targeting EphB4. Cell proliferation/survival was evaluated using the Cell Counting Kit-8 (CCK-8) assay, and expression levels of Runx2, BSP, ephrinB2 and EphB4 were determined using RT-PCR and Western blotting. ALP activity in these cells was also determined, and mineral nodule formation was evaluated with alizarin red S staining. RESULTS: Low concentrations of TNF-α had no influence on cell proliferation/survival. However, expression levels of Runx2, BSP, ephrinB2 and EphB4, as well as ALP activity and mineral nodule formation, were significantly enhanced in MC3T3-E1 cells treated with low concentrations of TNF-α. Moreover, activation of the ephrinB2-EphB4 signalling pathway by ephrinB2-fc enhanced TNF-α-induced osteogenic differentiation, while down-regulation of EphB4 level reversed the positive effect of TNF-α. CONCLUSIONS: Low concentrations of TNF-α promoted osteogenic differentiation via activation of the ephrinB2-EphB4 signalling pathway.
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