Regulation Of Cell Migration By HSP27 Under Shear Stress

Introduction:Heat shock protein 27(HSP27)is a multifunctional protein that undergoes significant changes in expression and phosphorylation in response to shear stress stimuli,suggesting that HSP27 may be involved in the mechanotransduction.However,the mechanism of HSP27 affecting tumor cell migration under shear stress is still not clear.Methods:HSP27-ECFP and HSP27-Ypet are constructed to visualize the self-polymerization of HSP27 in living cells based on Fluorescence Resonance Energy Transfer(FRET)technology.Ser15,Ser78,and Ser82 sites in HSP27 are mutated to Ala throughsite-directedmutagenesistoconstructnon-phosphorylatedvariants(HSP27-3A-ECFP and HSP27-3A-Ypet).In order to observe the distribution and polymerization of HSP27 in HeLa cells under flow,HeLa cells transfected with various biosensors are exposed to different magnitude of shear stress by a parallel-plate flow chamber.In addition,mutants or inhibitors are used for the inhibition of HSP27 phosphorylation to explore the effect of phosphorylation in shear stress-induced HSP27 distribution and polymerization changes.Actin-mcherry and Cyto-FAK FRET biosensor are used to detect the distribution of actin and the activity of FAK in response to shear stress.In addition,the migration ability of HeLa cells is measured using Transwell chamber.Results:(1)The fluorescence intensity of HSP27-Ypet at the downstream edge of the cell is significantly higher than upstream upon 30 min of 20 and 40 dyn/cm~2 shear stress application,and this polar distribution is also observed in the non-phosphprylated group.However,the distribution of HSP27 is uniform in 5 dyn/cm~2 group of Hela cell.(2)The FRET ratio between HSP27-ECFP and HSP27-Ypet in HeLa cells decreases significantly after 30 min of different shear stress application,which is depressed by KRIBB3,inhibitor of HSP27 phosphorylation.Consistently,the decrease of FRET ratio in non-phosphorylated group is also lower than control group.(3)The FRET ratio of Cyto-FAK increases after 30min of shear stress stimulation,and the ratio in 20 dyn/cm~2 group is higher than that in 5 and40 dyn/cm~2 groups.Overexpression of HSP27 or inhibition of HSP27 phosphorylation can both inhibit shear stress-induced increase of Cyto-FAK FRET ratio.(4)The fluorescence intensity of actin-Mcherry exhibits a polar distribution after 30 min of shear stress stimulation,which is blocked by overexpression HSP27-3A.(5)KRIBB3 reduces the number of shear stress-induced migrating HeLa cells.Conclusions:The probes for detecting HSP27 self-polymerization in living cells are constructed successfully.Shear stress induces polar distribution of HSP27 and then regulates the dynamic structure at the cell leading edge.Shear stress also promotes HSP27depolymerizing to small molecule,which regulates polar actin accumulation and FAK activity,and further promotes tumor cell migration.This study suggests that HSP27 plays an important role in the regulation of shear stress-induced He La cell migration,which provides a theoretical basis for HSP27 as a potential drug target for tumor metastasis.