| Mesenchymal stem cells (MSCs) are a group of adult stem cells that is widely usedfor clinical therapies in neuronal degenerative disease and tissue injury due to itsextensive sources, high ex vivo proliferating capacity, and less immune rejection.However, the new therapeutic strategy of stem cell transplantation in clinic is limitedbecause of the low effective number of targeted migrated cells to injury site. Therefore,it is particularly important to promote cell migration, and improve numbers of cells thatdirectly migrated to the injury and inflammation sites. Previously, we demonstrated thatHGF could promote Wnt-independent-β-catenin signaling pathway, while MSCsmigration was attenuated after the inhibition of Wnt/β-catenin signaling, whichillustrates that enhanced migration of MSCs by HGF is related to β-catenin signaling.Meanwhile, blocking the signal pathway PI3K/Akt and MAPKs also influence theMSCs migration to HGF differently. The change of cell migration behavior is reflectedby the change of the formation of focal adhesions and reorganization of F-actin. In thisstudy, we focus on the relationship between β-catenin signaling and PI3K/Akt orMAPKs pathways during MSCs migration or HGF-induced cell migration, and theinfluence on the change of focal adhesions by these signaling pathways.Relationship between β-catenin signaling, PI3K/Akt signaling, and MAPKssignaling in MSCs. The effect of Wnt/β-catenin signaling pathway activation is theexpression of downstream target genes, while Nedd9, Runx2and Tiam1are predicted asdownstream target genes of Wnt/β-catenin signaling pathway, and research has provedthat these three genes are related with migration in other cells. Dkk1or FH535, thespecific inhibitor of Wnt/β-catenin signaling pathway, markedly suppressed the basal orWnt3a-induced transcription of signaling downstream target genes, which confirm thatNedd9, Runx2, and Tiam1are downstream target genes of Wnt/β-catenin signaling.Runx2, an essential transcription factor for skeletal morphogenesis, enhanced the migration of osteoblast and chondrocyte by coupling with PI3K/Akt signaling. Presentstudy has confirmed that Runx2is downstream target gene of Wnt/β-catenin signalingpathway, and what relationship between the PI3K/Akt and Wnt/β-catenin signalingpathway? Therefore, we examined the phosphorylation of Akt and MAPKs (ERK1/2,p38MAPK or JNK/SAPK) by blocking Wnt/β-catenin signaling, and we found thatinterruption of Wnt/β-catenin signaling, the transcription of Runx2and thephosphorylation of Akt and ERK1/2were decreased, while activation of the signalingpathway did not increase the level of Akt and ERK1/2phosphorylation, implying thatthe activation of PI3K/Akt and ERK1/2signaling is regulated by the basalWnt/β-catenin pathway. As PI3K/Akt and MAPKs signaling pathways are involved inthe regulation of cell migration, we blocking PI3K/Akt, ERK1/2, p38MAPK orJNK/SAPK signaling by using their specific chemical inhibitors, LY294002, PD98059,SB203580or SP600125, results showed that Wnt/β-catenin signaling pathway wassuppressed after inhibiting PI3K/Akt signaling, while it was activated by theinterruption of p38MAPK or JNK/SAPK, but not ERK1/2pathway. Both Nedd9andTiam1, which are regulated by canonical Wnt signaling participate inintegrin-dependent focal adhesion signaling. Expression of Nedd9and Tiam1alsochanged with the classic Wnt signaling by regulating integrin in focal adhesionsignaling and further influence cell migration differently. Abolishment of Wnt/β-cateninsignaling pathway effectively suppressed the level of Y397-FAK and Y118-paxillinphosphorylation and paxillin, while enhancement of the signaling produced the oppositeeffect. Interference with PI3K/Akt or MAPKs pathways influenced the distribution,morphous, and number of focal adhesions. Inactivation of PI3K/Akt signaling byLY294002, focal adhesions became bigger, less and most of them located around cellperiphery. Inhibition of p38MAPK or JNK/SAPK signaling, the phosphorylation ofY397-FAK and Y118-paxillin was increased, MSCs showed a characteristic polarizedmorphology, exhibiting a leading edge with extension of lamellipodia, and focaladhesion became tinier, more and most of them distributed around the cell peripherywhich are features of slower migration. Suppression of ERK1/2signling led to thedecrease of Y397-FAK and Y118-paxillin phosphorylation and the accumulation oflarge numbers of focal adhesions around the cell periphery.Relationship between β-catenin signaling, PI3K/Akt signaling, and MAPKssignaling in HGF-induced MSCs. After treating with HGF, the mRNA levels of Nedd9, Runx2and Tiam1increased, while the effect was abolished by addition of FH535. Theresult indicates that HGF can activate β-catenin signaling, then, if there is anyrelationship between β-catenin signaling and PI3K/Akt and MAPKs signaling, whichcan also influence cell migration and be activated by HGF? So, we detected the proteinlevel and the location of β-catenin and the mRNA level of downstream target genes afterblocking PI3K/Akt and MAPKs signaling pathways with their specific inhibitors. Theresults showed that inhibition of PI3K/Akt and JNK/SAPK signaling enhanced theHGF-induced activation of β-catenin signaling, while inhibition of P38MAPK signalingreduced the HGF-induced activation of β-catenin signaling, and no effect was observedafter inhibition of ERK signaling. Analysis of FAK and paxillin activity and focaladhesions location, we found that treatment with HGF led to an increase of Y397-FAKand Y118-paxillin phosphorylation, polarized cell shape, and formation of largenumbers of focal adhesions which are features of bona fide cell migration, while theeffect was eliminated after interrupting Wnt/β-catenin signaling pathway. BlockingPI3K/Akt and SAPK/JNK did not affect HGF-induced paxillin Y118phosphorylationand cell polarity, while the level of FAK Y397phosphorylation further improved afterinactivation of ERK1/2pathway. However, interference with p38MAPK inhibitedHGF-promoted cell polarity.In conclusion, we focus on the relationship between β-catenin, PI3K/Akt, andMAPKs signalings in HGF-induced MSCs migration, and the influence of thesesignaling pathways on the distribution, morphous and numbers of focal adhesions. Thisstudy provide a directly theory on the regulation of focal adhesion turnover and cellmigration, and is meaningful for the clinical therapies in surgical injury and woundhealing. |
PDF Full Text Request