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

Abstract Low concentrations of tumor necrosis factor-alpha and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate 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. This study showed that with increase in ALP activity, mRNA and protein expression of Runx2, BSP and EphB4, TNFR2 expression was significantly promoted under 0.5 ng/ml TNF-α stimulation in osteoblast precursor cells MC3T3-E1. 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, but up-regulation of EphB4 by TNF-α kept 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 augmented expression of p-JNK, p-Erk and p-p38, however, only p-Erk expression was significantly inhibited in TNFR2-knockdown cells. In addition, the Erk pathway inhibitor, U0126 (10μM), significantly inhibited the protein levels of Runx2 and BSP when compared with cells treated with TNF-α only. In conclusion, the EphB4, TNFR2 and Erk/MAPK signaling pathway comprise a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

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

10.21203/rs.2.18101/v2 ◽

2020 ◽

Author(s):

Yu Zhang ◽

Chengzhe Yang ◽

Shaohua Ge ◽

Limei Wang ◽

Jin Zhang ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Mapk Signaling ◽

Precursor Cells ◽

Mapk Signaling Pathway ◽

Erk Mapk ◽

Low Concentrations ◽

Tnf Α ◽

Signaling Axis ◽

Positive Effect

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 up-regulate 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 : This study showed that with increase in ALP activity, mRNA and protein expression of RUNX2, BSP and EphB4, TNFR2 expression was significantly promoted under 0.5 ng/ml TNF-α stimulation in osteoblast precursor cells MC3T3-E1. 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, but up-regulation of EphB4 by TNF-α kept 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 augmented expression of p -JNK, p -ERK and p -p38, however, only p -ERK expression was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10μM), significantly inhibited the protein levels of RUNX2 and BSP when compared with cells treated with TNF-α only. Conclusions : The EphB4, TNFR2 and ERK/MAPK signaling pathway comprise a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

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

10.21203/rs.2.18101/v3 ◽

2020 ◽

Author(s):

Yu Zhang ◽

Chengzhe Yang ◽

Shaohua Ge ◽

Limei Wang ◽

Jin Zhang ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Mapk Signaling ◽

Precursor Cells ◽

Mapk Signaling Pathway ◽

Protein Levels ◽

Low Concentrations ◽

Tnf Α ◽

Signaling Axis ◽

Positive Effect

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 up-regulate 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, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10μM), 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.

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Inhibition of miR-200c Expression Promotes Activation of Extracellular Signal-Regulated Kinase (ERK)/ Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway and Inhibits Osteogenic Differentiation in Osteoporosis Mice

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2242 ◽

2020 ◽

Vol 10 (2) ◽

pp. 163-168

Author(s):

Sheng Wang ◽

Zhonghan Min ◽

Run Gu ◽

Zhongwei Yu ◽

Pingquan Chen ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Mapk Signaling ◽

Bone Diseases ◽

Mapk Signaling Pathway ◽

Mitogen Activated Protein Kinase ◽

Erk Mapk ◽

Mitogen Activated Protein ◽

Marrow Mesenchymal Stem Cells ◽

Sirna Group

During OP bone metabolism, activated MAPK signaling can promote the proliferation and differentiation of osteoclasts. miRNAs involve in bone diseases. Our study aimed to evaluate miR-200c’s effect on ERK/MAPK signaling pathway in OP. miR-200c expression in OP mice and normal mice was detected by qPCR. BMSCs were cultured and transfected with siRNA to establish a miR-200c knockout model. Flow cytometry was used to detect cell apoptosis and ERK/MAPK signaling protein was detected by Western blot. miR-200c expression in OP mice was significantly lower than that in normal mice. Bone marrow mesenchymal stem cells (BMSCs) contain a large amount of siRNA particles under a fluorescence microscope. siRNA transfection can effectively inhibit miR-200c expression without difference of BMSCs apoptosis between miR-200c siRNA group and NC group. However, ERK1/2 and P38 expression in experimental group were significantly higher than those in NC siRNA group with reduced ALP activity. In addition, BMSCs osteogenic differentiation was further diminished when miR-200c expression was inhibited. miR-200c expression is lower in OP mice. miR-200c siRNA inhibits BMSCs osteogenic differentiation via ERK/MAPK signaling, thereby promoting OP progression.

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