Regulation of osteogenic differentiation under hypoxia by EphrinB2/EphB4 in MC3T3-E1 cells
10.12016/j.issn.2096-1456.2023.09.002
- Author:
LIU Zishan
1
,
2
;
WANG Yixin
1
,
2
;
LI Yongming
1
,
2
Author Information
1. Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Department of Orthodontic, School &
2. Hospital of Stomatology, Tongji University
- Publication Type:Journal Article
- Keywords:
hypoxia inducible factor-1α / erythropoietin producing hepatocyte kinase receptor ligand B2 / erythropoietin producing hepatocyte kinase receptor B4 / osteogenic differentiation / hypoxia / bone metabolism / alkaline phosphatase / osteocalcin
- From:
Journal of Prevention and Treatment for Stomatological Diseases
2023;31(9):618-624
- CountryChina
- Language:Chinese
-
Abstract:
Objective : To investigate the effect of erythropoietin producing hepatocyte kinase receptor ligand B2-erythropoietin producing hepatocyte kinase receptor B4 (EphrinB2/EphB4) on the osteogenic differentiation of MC3T3-E1 cells in a hypoxic environment to provide experimental evidence for hypoxia regulation of osteoblast differentiation.
Methods : Control groups and cobalt chloride (CoCl2)-induced hypoxia groups were set up first. qRT-PCR was used to detect the mRNA expression of the osteogenic markers alkaline phosphatase (ALP), collogen1 (COL I), runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN). ALP staining was used to detect the activity of cell alkaline phosphatase after osteogenic induction. The mRNA and protein expression levels of hypoxia inducible factor-1α (HIF-1α), EphrinB2 and EphB4 in the two groups were detected via qRT-PCR and Western blot. Then, the CoCl2 + inhibitor group was established. NVP-BHG712, an EphB4 phosphorylation inhibitor, was added to this group to prevent EphrinB2 from binding to EphB4 and producing signals. qRT-PCR and Western blot were used to detect the mRNA and protein expression of osteogenic markers, including ALP, RUNX2, COL I, and OCN. ALP staining and Alizarin red S staining were used to measure osteoblast differentiation and mineralization.
Results : Compared with the control group, the mRNA expression of the osteogenic differentiation markers ALP, RUNX2, COL-1, and OCN in MC3T3-E1 cells increased, and ALP activity and mineralization were enhanced under CoCl2-induced hypoxia in vitro (P<0.05). Additionally, the expression of HIF-1α, EphrinB2 and EphB4 was upregulated at the mRNA and protein levels under hypoxia (P<0.05). When NVP-BHG712 was used to block the connection between EphrinB2 and EphB4, the expression of osteogenic markers and ALP activity and mineralization were decreased (P<0.05).
Conclusion:EphrinB2/EphB4 can promote osteogenic differentiation of MC3T3-E1 cells and increase the expression of osteogenic markers and tissue mineralization in a hypoxic environment.
