| Mesenchymal stem cells are multipotential stem cells with low immunogenicity and are widely existed in umbilical cord,bone marrow and adipose tissue.The convenience of isolation,abundance in origin and easy to autoallergic transplantation make MSCs as one of the ideal candidates of cell therapy.They have the ability to expand many folds in culture while retaining their growth and multilineage potential such as to neural cells.However,when they are used in treatment for neurodegenerative disorders,only a few of MSCs successfully reached the damaged areas,resulting in the low rate of cellsreplacement.Thus,improving the migration ability of MSCs is the key point of in effective treatment.Our previous study demonstrated that activation of Wnt/?-catenin signaling promoted the migratory ability of MSCs and could also activate MAPKs signaling correspondly.As known to us,the key event of Wnt/?-catenin signaling is that ?-catenin enters into the nucleus,and Wnt target genes are following activated by the DNA-bound T cell factor/lymphoid enhancer factor(TCF/LEF)family of proteins.However,the mechanism of how Wnt/?-catenin signaling regulates the target genes involved in cell migration is not well known.Our research aims to study the role of Wnt/?-catenin signaling target genes Nedd9 in mediating cell migration.MSCs were isolated from rat femur and tibia and used as an object to study the influence of Wnt/?-catenin signaling on MSC migration.By Boyden chamber assays,we found that Compared to control,the migrating ability of MSCs was improved to 1.6 folds by Wnt3 a 50 ng/ml,while it was decreased to 0.6 fold by FH535 5 ?M,the inhibitor of Wnt/?-catenin signaling.We next found thatthe RNA level of Nedd9 was increased by treating with Wnt3 a from 1h and reached a peak at 4 h by q PCR.As reported,Nedd9 can promote the migration and invasion of tumor cells.So we speculated whether the promotion of MSC migration by Wnt/?-catenin signaling is through Nedd9.So we cloned full-length rat Nedd9 into the Ad Easy Adenoviral Vector System.The recombinant adenoviruses were generated,identified,and designated Ad-Nedd9.The migratory capacity of MSCs infected with Ad-Nedd9 was determined Boyden chamber assay and the results demonstrate that up-regulating of Nedd9 can improve the migratory capacity of MSCs.Surprisingly,compared with control,overexpressing of Nedd9 in MSCs didn't alter its chemotactic migration toward HGF.Furthermore,we utilized a Dunn chamber in conjunction with time-lapse video analysis to directly observe and follow the HGF-induced migration of Nedd9-overexpressed MSCs and the data showed a significant increase in migration speed but not migration efficiency.Then we examined the molecular mechanism of Nedd9-promoted migration.Our previous study showed that Wnt/?-catenin signaling improved migration of MSCs was partly through MAPKs signaling.So we doubted whether overexpression of Nedd9 can activate MAPKs signaling.Western bloting showed that the total proteins of Akt,ERK1/2,and p38 MAPK in MSCs infected with Ad-Nedd9 remained unchanged while the phosphorylation of SAPK/JNK elevated but not the phosphorylation of ERK1/2 and p38 MAPK.These results showed that Nedd9-promoted migration of MSCs might be partly through SAPK/JNK signaling.Nedd9 is a scaffolding protein in focal adhesions.For further study,we detected the expression and phosphorylation of focal adhesion(FA)associated protein including FAK and paxillin and we found that overexpressing Nedd9 did not change the total proteins of them but promoted the phosphorylation of paxillin at tyrosine 118.To our surprise,MSCs infected with Ad-Nedd9 didn't have an elevation in phosphorylation of FAK at tyrosine 397.Cell migration is closely related to the turnover of FAs and reorganization of F-actin.Immunocytochemical analysis showed that up-regulation of Nedd9 can increase the number of FAs and ratio of the peripheral/intermediate FAs.In vitro study showed that overexpression of Nedd9 can improve migratory ability of MSCs,so we conducted in vivo study to further verify this hypothesis.We conducted rat transected spinal cord injury(SCI)model and the rat's spinal cords were completely transected in T10.MSCs infected with Ad or Ad-Nedd9 were intrathecal injected after injury for 7 days.7 days later,frozen section of the spinal cord were performed to tracing the distribution of MSCs around the injured area and we found that overexpression of Nedd9 significantly promoted the migration of MSCs toward injured area.In conclusion,Wnt/?-catenin signaling improves the migration of MSCs by activating its target gene Nedd9,revealing the molecular mechanism of MSCs migration and shedding a new light on clinical transplantation therapy by using MSCs. |
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