Effect Of Piezo1 On Electrotaxis Of HaCaT Cells

The endogenous physiological electric field around the wound is an important factor to guide the directional migration of cells and promote the wound healing.The phenomenon that cells may sense small directed electric field and migrate directionally is called electrotaxis.Experinmental data showed that electrotaxis play an important role in p embryonic development,tissue regeneration and tumorigenesis.Piezo1 is an ion channel which is mainly distributed in non-nerve cells in mechanical sensing to participates in fluid pressure perception,touch sensation,maintaining epithelial cell homeostasis and other biological processes.Studies have found that Piezo1 can affect the chemotactic migration of cells,participate in cell movement,and play an important role in the wound healing.Piezo1 is regulated by voltage,and can even switch to pure voltage-gated mode.However,it is not clear whether Piezo1 participates in electric field guided cell migration.In this study,HaCaT cells were used as a model to study the role of Piezo1 in electrotaxis to the signal transduction pathway involved in Piezo1 were explored the mechanism of electrotaxis underpin were discussed.In this study,live cell Imaging System was used to track the migration of human keratinocyte HaCaT cells in physiological strength of directed electric field.Inhibitors,activators and RNAi techniques were applied to study the role of Piezo1 in electrotaxis.Western blotting was used to explore the mechanism of electrotaxis involved in Piezo1,and the molecuars involved insignaling transduction pathway mediated by Piezo1 were discussed.The results showed that 1)With the action of electric field,HaCaT cells migrated to anode electric field.Western blot results showed that electric field promoted phosphorylation level of FAK and the expression of Integrin β1.2)ruthenium red,a broad-spectrum inhibitor of Piezo1,significantly inhibited the directional migration of HaCaT cells to the anode electric field(P < 0.001);GsMTx4 of Piezo1 inhibitor significantly decreased the trajectory velocity(P < 0.05),displacement velocity and directional persistence of HaCaT cells to the anode in electric field(P < 0.001),but the direction were not significantly affect.Western blotting showed that the GsMTx4 prevented the electric field from further increasing the phosphorylation level of FAK and the expression of integrin β1.3)After interfering with Piezo1 expression by si RNA,the direction of electrotaxis migration of HaCaT cells decreased significantly(P <0.001),si RNA interfereence significantly hinderedthe expression level of integrin β1promoted by electric field.The down-regulation of Piezo1 also significantly decreased the phosphorylation level of FAK.4)Chemical activation of Piezo1 by Yoda1,the electrotaxis of HaCaT cells was also significantly decreased(P < 0.001).Both electric field and Yoda1 treatment may enhance the expression level of integrin β1.and the synergistic effect can increase the expression of integrin β1;Yoda1 treatment alone may promotes the phosphorylation of FAK,the electric field may enhance the phosphorylation level of FAK.5)When integrin β1 inhibitor P5D2 and FAK inhibitor PF562271 were applied,the electrotaxis of HaCaT cells was significantly decreased.Western blotting showed that the inhibition of integrin β1 function by P5D2 hindered the activation of Akt and other molecules by electric field.Based on the experimental results,a)It is concluded that the Piezo1 participates in the electrotaxis migration of HaCaT cells and is one of an important molecules for HaCaT cells to perceive electric field signals;b)Inhibition of integrin β1 and FAK also affects the directional migration process of cells responding to electric field,and electric field signals may participate in the regulation of integrin β1 through Piezo1,and FAK.