EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

Abstract

Background: Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α upregulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. Results: The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, pERK and p-p38. However, only the pERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 μM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. Conclusions: The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

Alterations in bone fracture healing associated with TNFRSF signaling pathways

“…As mentioned in 2.2, the Runx2 level is potentially associated with TNFR2 signaling. ( Zhang et al, 2020 ) MSCs: mesenchymal stem cells; CFU-M: Colony forming unit M, namely monoblast. M-CSF: macrophage colony-stimulating factor.…”

“…In 2020, Y Zhang et al led a study on EphB4/TNFR2/ERK/MAPK signaling. ( Zhang et al, 2020 ). Previous studies showed that low concentrations of TNFα and TNFR2 could upregulate EphB4 expression and promote osteogenic differentiation of osteoblast precursor cells.…”

“…( Wang et al, 2017 ). Zhang et al, (2020) used lentivirus-mediated shRNA to knockdown TNFR2 expression level in MC3T3-E1 osteoblasts, which showed an approximately 70% reduction in TNFR2 expression compared with the control group. RUNX 2 and bone sialoprotein (BSP) were selected as indicators downstream of TNFR2 in osteoblast differentiation.…”

“…FIGURE 5General overview of the four-stage fracture healing process and the respective signaling pathways and cytokines involved in initial inflammation, osteoblastogenesis, and osteoclastogenesis. As mentioned in 2.2, the Runx2 level is potentially associated with TNFR2 signaling (Zhang et al, 2020). MSCs: mesenchymal stem cells; CFU-M: Colony forming unit M, namely monoblast.…”

Strategies of Macrophages to Maintain Bone Homeostasis and Promote Bone Repair: A Narrative Review

“…These opposing experimental results may be due to the dose-dependent effect of TNF-α on osteoblasts. It has been shown that low concentrations (0.5 ng/mL) of TNF-α significantly promoted osteoblastic differentiation of osteoblasts in vitro [ 163 ]. It increased the expression of osteoblast Runx2 and BSP activity, and the expression of TNFR2, the receptor of TNF-α, was elevated.…”