Tumor necrosis factor-alpha inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated nuclear factor-kappaB signaling pathway.

BACKGROUND AND OBJECTIVE: The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. MATERIAL AND METHODS: Murine MC3T3-E1 pre-osteoblasts were treated with 10 ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. RESULTS: TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. CONCLUSION: TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

Porphyromonas gingivalis lipopolysaccharide regulates ephrin/Eph signalling in human periodontal ligament fibroblasts.

OBJECTIVE: EphrinA2-EphA2 and ephrinB2-EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs). MATERIAL AND METHODS: The primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalisLPS at 0.001-10 µg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalisLPS at the optimal concentration (0.1 µg/mL) for different periods (6-48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse-transcription real-time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt-related transcription factor 2 (Runx2), and the osteoclastogenesis-related factors receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin were also evaluated. RESULTS: The ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalisLPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased. CONCLUSION: The present study suggest that P. gingivalisLPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.

[Moderate Angiogenic Effect of Xuefu Zhuyu Capsule by Regulating EphB4/EphrinB2].

Objective To observe moderate angiogenic effect of Xuefu Zhuyu Capsule (XFZYC) on human microvascular endothelial cell line 1 ( HMEC-1) , and its regulation effect on expression of EphB4/EphrinB2. Methods The moderate angiogenic effect of XFZYC was clarified by detecting XFZYC containing serum on cell viability, cell cycle, migration, adhesion and in vitro angiogenesis. Its effects on expressions of EphB4/EphrinB2 were detected by Real-time quantitative PCR and Western blot. Results XFZYC containing serum (XFZYC-CS) had no effect on the cell viability or cell ratio in phase S endothelial cells. Cell migration was significantly improved by 1.25% XFZYC-CS after 24, 48, and 72 h of action 2. 50% XFZYC-CS inhibited cell migration at the primary 24 h, but it significantly promoted cell migration at 48 and 72 h afterwards. It showed just an opposite tendency to 5. 00% XFZYC-CS. Cellular adhesion number was significantly reduced by 1. 25% XFZYC-CS at 72 h. Cellular adhesion number was significant- ly increased by 2. 50% XFZYC-CS at 24 and 48 h, but inhibited at 72 h 5. 00% XFZYC-CS showed inhibition at 24 h, but turned to promotion, and disappeared afterwards. In vessel formation aspect, only 2.50% XFZYC-CS showed vessel formation promotion 5. 00% XFZYC-CS showed inhibition on vessel formation at 48 and 72 h. Results of Real-time quantitative PCR and Western blot in 2. 50% XFZYC-CS showed EphB4 expression was up-regulated at 12 h; EphB4 expression was down-regulated while EphrinB2 expression was up-regulated at 24 h. Conclusions Only 2. 50% XFZYC-CS at 48 h had promotion of migration, adhe- sion, and in vitro angiogenesis of HMEC-1 , which was the optimal condition for vessel growth. These re- sults suggested XFZYC promoted angiogenesis in certain conditional limitations. But it regulated the ex- pression of EphB4/EphrinB2, which might be one of important factors.

Erythropoietin promotes bone formation through EphrinB2/EphB4 signaling.

Recent studies have demonstrated that erythropoietin (EPO) has extensive nonhematopoietic biological functions. However, little is known about how EPO regulates bone formation, although several studies suggested that EPO can affect bone homeostasis. In this study, we investigated the effects of EPO on the communication between osteoclasts and osteoblasts through the ephrinB2/EphB4 signaling pathway. We found that EPO slightly promotes osteoblastic differentiation with the increased expression of EphB4 in ST2 cells. However, EPO increased the expression of Nfatc1 and ephrinB2 but decreased the expression of Mmp9 in RAW264.7 cells, resulting in an increase of ephrinB2-expressing osteoclasts and a decrease in resorption activity. The stimulation of ephrinB2/EphB4 signaling via ephrinB2-Fc significantly promoted EPO-mediated osteoblastic differentiation in ST2 cells. EphB4 knockdown through EphB4 shRNA inhibited EPO-mediated osteoblastic phenotypes. Furthermore, in vivo assays clearly demonstrated that EPO efficiently induces new bone formation in the alveolar bone regeneration model. Taken together, these results suggest that ephrinB2/EphB4 signaling may play an important role in EPO-mediated bone formation.