Dvl Regulates The Migration Of Mesenchymal Stem Cells Toward HGF

Mesenchymal stromal cells (MSCs) are stromal cells that are found in a wide range of adult post-natal organs and tissues, including bone marrow, blood, and adipose tissues. Because these cells can be obtained from various tissues, can be expanded easily ex vivo, and can exert their beneficial effects by cell replacement and also by neuroprotection, trophic support, and angiogenesis, MSCs are considered promising therapeutic stem cell candidates with wide clinical trial performance. However, to maximize the effect of transplanted MSCs, enhancing their migration to sites of pathological insult is critical. Therefore,several research have explored the mechanisms of directional MSCs migration. Studies showed that activation of P-catenin by HGF/c-Met signaling promotes cell migration, and that Dvl2can regulate the invasive capacity of human cancers induced by HGF. Regardless, the precise mechanisms underlying MSC migration have yet to be elucidated. The aim of our experiments was to investigate the key protein Dvl which regulate the directed migration of MSCs toward Wnt3a and HGF.MSCs were isolated from bone marrow of rats and expanded. In our experiments, Dvl and variants individually missing each of the two conserved regions (△DIX,△DEP) were cloned by RT-PCR. Then cDNA of Dvl,△DIX and△DEP were cloned into a pAdTrack-CMV transfer vector by direct cloning after Kpn I and Xba I endonucleases digestion. pAdTrack-CMV-Dvl, pAdTrack-CMV-△DIX, pAd-Track-CMV-△DEP transfer vector linearized with Pme I digestion and pAdEasy-1backbone vector were further cotransformed into the bacteria BJ5183competent cells for homologous recombination adenoviruses Ad-Dvl, Ad-△DIX and Ad-ADEP. Then they were linearized with PacI digestion and transfected into the human embryonic kidney293(QBI-293A) cells by LipofectamineTM2000, About107pfu/ml of high titer recombinant virus was successfully obtained, the optimal virus concentration for MSCs was150MOI. At the same time, FAM fluorescently labeled siRNA used for interfere Dvl gene.First, we studied the effect of Wnt/β-catenin signaling on MSCs migration. To activate or inhibit Wnt/β-catenin signaling, LiCl and Wnt3a, activitors of Wnt/β-catenin signaling, and Dkkl, a specific antagonist of Wnt/β-catenin signaling that binds directly to LRP5/LRP6and prevents formation of the Wnt/Fzd/LRP complex, and FH535, a synthetic inhibitor of the canonical Wnt/β-catenin signaling were utilized. We performed Boyden chamber and Dunn chamber assays to test the migration of MSCs and observed that LiCl and Wnt3a promoted the migration of MSCs, and that FH535not only significantly inhibited MSCs migration but also blocked the effects of Wnt3a and LiCl on MSCs migration. Western blot showed that LiCl and Wnt3a upregulated the expression of β-catenin. Surprisingly, Dkk-1increased cell migration; furthermore, Dkk-1did not inhibit Wnt3a-associated chemotaxis when cells were exposed to Wnt-3a. Moreover, Dkkl dramatically reduced the accumulation of β-catenin, confirming that Dkkl inhibited the β-catenin pathway. However, we observed that Dkkl also could promote JNK phosphorylation, demonstrating that DKK1maybe promote migration of MSCs through non-canonical Wnt signaling.Dvl proteins are critical effector molecules in multiple Wnt signaling pathways and have been implicated in Wnt-dependent cell migration. To test whether Dvl is required for Wnt-3a-dependent and HGF-dependent MSCs migration, siRNA was used to knockdown the expression of Dvl. Specific knockdown of Dvl led to a decreased cell migration. Furthermore, MSCs were infected with Ad-Dvl, Ad-△DIX and Ad-ADEP which could affect the expression of Dvl. These results suggested that over-expression of Dvl or△DIX,△DEP significantly increased cell migration, indicating that Dvl proteins contribute to Wnt3a-dependent and HGF-dependent cell migration.In conclusion, our experiment showed that Dvl promotes the directional migration of MSCs by up-regulating β-catenin, which induced by Wnt3a as well as HGF. siRNA suppressed the directional migration of MSCs toward Wnt3a and HGF, and high expression of Dvl enhances Wnt3a-dependent and HGF-dependent directional migration of MSCs by activing the Wnt/β-catenin signaling. During this process the DIX domain plays an important role. These data reveal the mechanism of MSCs migration and help design strategies for the clinical application of MSCs.