ABSTRACT
Objective: To investigate the effects of FTY720-P on EphA2-EphrinA2 bidirectional signaling in osteoclasts. Methods: Murine RAW264.7 macrophages were induced into osteoclasts by dexamethasone and 1α, 25-dihydroxyvitamin D 3, and identified by tartrate resistant acid phosphatase (TRAP) staining. Then, the osteoclasts were divided into 2 groups. The osteoclasts were treated with 400 ng/mL FTY720-P in experimental group and without FTY720-P in control group, respectively. After 48 hours of culture, the cells in 2 groups were detected by real-time fluorescent quantitative PCR, Western blot, and immunofluorescence staining. The expressions of EphA2, EphrinA2, RhoA, and the bone reconstruction associated proteins［bone morphogenetic protein 2 (BMP-2) and transform growth factor β 1 (TGF-β 1)］were analyzed and compared. Results: RAW264.7 cells were successfully induced into osteoclasts identified by TRAP staining. Compared with control group, the relative expressions of EphA2 and EphrinA2 mRNAs and proteins in experimental group significantly decreased after 48 hours ( P<0.05), and the relative expression of RhoA protein also significantly decreased ( P<0.05). The relative expressions of BMP-2 and TGF-β 1 mRNAs were significantly increased ( P<0.05), and those protein expressions were enhanced. Conclusion: FTY720-P can down-regulate the expression of RhoA and promote the expressions of TGF- β 1 and BMP-2 by affecting the transduction of EphA2-EphrinA2 bidirectional signaling in osteoclasts.
ABSTRACT
The Eph family is thought to exert its function through the complementary expression of receptors and ligands. The dorsal mesencephalon appears to be segmented into two broad regions demarcated by the mutually exclusive expression of EphA receptors and ephrinA ligands. In this study, we analyzed transgenic embryos expressing ephrinA2 in the anterior region of the developing midbrain where the EphA8 receptor is expressed. First, 1% of transgenic embryos showed cephalic neural tube closure defects. Second, it was confirmed that mis-expression of ephrin-A2 in the anterior mesencephalon induced an increase in the EphA8 tyrosine kinase activity. Accordingly, an increased MAPK activity was also detected in the anterior mesencephalon of E14.5 transgenic embryo. Third, cell adhesion assay revealed that mis-expression of ephrinA2 promoted cell attachment to fibronectin. Taken together, these findings suggest that co-expression of EphA receptors and ephrinA ligands significantly alter cell behaviors including cell adhesion.
Subject(s)
Animals , Humans , Mice , Cell Adhesion , Diencephalon , Embryonic Structures , Ephrin-A2 , Fibronectins , Ligands , Mesencephalon , Mice, Transgenic , Neural Tube , Protein-Tyrosine Kinases , Receptor, EphA8 , Receptors, Eph FamilyABSTRACT
Objective To investigate the role of Ephrin A2 in the regeneration and reinnervation of hair cells in the chick cochlea following kanamycin ototoxicity.Methods 66 newly hatched Roman chickens (3 days old) were randomly divided into experimental group and control groups. Experimental chickens (n=48) received intramuscular kanamycin (200 mg/kg:Sigma,St Louis,MO) for 10 consecutive days and were subsequently sacrificed 2 days before the last injection,and 1,3,7,10,15,21,30,and 60 days after the last injection (n=6 per subgroup). Control chickens (n=18) were untreated and sacrificed 3,13 and 43 days after hatching (n=6 per subgroup). Ephrin A2 protein expression in acoustic ganglia was determined by western blot analysis in all chickens after sacrifice. Results Ephrin A2 protein expression was found and the protein level was almost same in acoustical ganglia of all normal chickens. After kanamycin exposure,the Ephrin A2 protein expression level in the cochlea of the experiment chickens from 2 to 7 days after last kanamycin injection was lower than that in control chickens,respectively. Ephrin A2 expression increased obviously at 15 days after kanamycin last injection. By 30 days after the cessation of kanamycin treatment,the level of Ephrin A2 protein approximated to that in normal control group.Conclusion The expression of Ephrin A2 protein in the acoustical ganglia basically synchronizes with the regeneration and the reinnervation of the hair cells in the chicken cochlea following kanamycin damage,indicating that Ephrin A2 may play an important role in this process.