Study Of Mechano-sensitive Piezo1 Ca²⁺ Channel-PI3K/Akt-Cx43 HCs Pathway And Regulation Factors

Objectives:1.To determine the upstream/downstream relationship of mechano-sensitive Piezo1 Ca²⁺channel and Cx43 hemichannels（Cx43 HCs）both in vitro and in vivo;2.To study the mechano-sensitive Piezo1 Ca²⁺channel-PI3K/Akt-Cx43 HCs activation pathway and evaluate the regulation factors involved in Piezo1 induced intracellular Ca²⁺level;3.To establish the structural relationship model of Piezo1 Ca²⁺channel and Cx43 HCs on the plasma membrane.Methods:In Vitro experiments:1.Osteocytic MLO-Y4 cells derived from murine long bones were used in all in vitro experiments,including normal MLO-Y4 cells,Cx43 specific knockdown（Cx43 KD）MLO-Y4 cells and its specific control Rosa26MLO-Y4 cells.Et Br dye uptake assay were performed on these three types of cells with specific inhibition of Cx43 HCs or Piezo1 channel,unspecific inhibition of membrane HCs,low extracellular/intracellular Ca²⁺level or specific inhibition on Panx1 channel pretreatments and followed with Yoda1or FFSS stimulus to evaluate the upstream/downstream relationship between Piezo1 Ca²⁺channel and Cx43 HCs;2.Live cell calcium imaging were performed with specific inhibition of Cx43 HCs or Piezo1 channel,low extracellular/intracellular Ca²⁺level or specific inhibition on Panx1 channel or ATP-P2X7 receptor pathway pretreatments and followed with Yoda1 or FFSS stimulus to evaluate the regulation factor on intracellualr Ca²⁺of Piezo1 Ca²⁺channel-Cx43 HCs activation pathway;3.Cell surface staining experiments were performed on normal MLO-Y4 cells with Yoda1 or FFSS stimulation,western blot of Cx43 KD and Rosa26 MLO-Y4 membrane protein samples as well as Piezo1 antibody immunoprecipitation of MLO-Y4 protein were performed to study the structural relationship of Piezo1 Ca²⁺channel and Cx43 HCs on osteocytic membrane;4.Western blot of different duration of Yoda1 treated MLO-Y4 whole protein samples and Et Br dye uptake assay with specific inhibition of PI3K/Akt were performed to study the Piezo1 Ca²⁺channel-PI3K/Akt-Cx43HCs activation pathway;In Vivo experiments:5.15-week-old male C57BL/6J mice were used in in vivo experiments and were randomly grouped with 5 mice per group.Evans Blue dye uptake assay were performed with specific inhibition of Cx43 HCs plus Yoda1stimulation or specific inhibition of Piezo1 plus tibia compression experiment to evaluate the functional relationship of Piezo1 channel and Cx43 HCs in vivo.Statistic methods:6.NIH Image J software was used for image processing and quantification.Graph Pad Prism8 statistics software was used for statistical analysis and graph manipulation.Student-Newman Keul’s test was used for comparison between two groups for single factor and One-way ANOVA was used for multiple comparison analysis.Two-way ANOVA with post hoc Tukey test was used to assess the difference between multiple groups with two variables.Data were shown as mean±SD,*P<0.05,**P<0.01,***P<0.001,****P<0.0001.Results:1.In vitro experiments indicated that Piezo1 specific agonist Yoda1could significantly increase Et Br dye uptake of MLO-Y4 cells in a dose-dependent and time-dependent manner.Specific inhibition of Cx43 HCs or Cx43 KD could significantly inhibit Yoda1-induced Et Br increase and unspecific inhibition of membrane HCs could reduce the Et Br to basal level suggested Cx43 HCs are activated by Piezo1 channel and MLO-Y4 cells uptake Et Br mainly through Cx43 HCs;3.Dooku1 could significantly inhibit FFSS induced Ca²⁺influx of MLO-Y4 cells as well Yoda1,FFSS induced Et Br dye uptake but had no effect on low extracellular Ca²⁺induced Et Br level,suggesting Dooku1serves as Piezo1 channel specific inhibitor and Piezo1 is the upstream of Cx43 HCs.3.Inhibition on intracellular Ca²⁺level could significantly reduce Yoda1,FFSS induced Et Br dye uptake in MLO-Y4 cells.Ionomycin could induce Et Br dye uptake.Yoda1 could increase p-Akt/Akt ratio significantly and specific inhibition of PI3K/Akt pathway reduced Yoda1 induced Et Br dye uptake as well.Together,activated-Piezo1 induced Ca²⁺influx and PI3K/Akt pathway play important role in inducing Cx43 HCs opening;4.Specific inhibition of Cx43 HCs,low extracellular/intracellular Ca²⁺level,specific inhibition on Panx1 channel or ATP-P2X7 receptor pathway reduced Yoda1 induced Ca²⁺influx on MLO-Y4 cells,indicating the major source of Ca²⁺influx is from extracellular Ca²⁺and Piezo1 calcium channel-PI3K/Akt-Cx43 HCs activation pathway exist,;5.Cell surface staining showed Cx43 and Piezo1 protein co-localization signals on MLO-Y4 cell membrane which was enhanced by FFSS.The Piezo1 protein level decreased in Cx43 KD cells;6.In C57BL/6J mice,specific inhibition of Cx43 HCs could significantly reduce the Yoda1 induced EB dye uptake of osteocytes in cortical bone of mice tibia.Specific inhibition of Piezo1 could significantly reduce the tibia compression induced EB dye uptake of osteocytes.Together,mechano-sensitive Piezo1 channel activation could induce the opening of Cx43 HCs in vivo;Conclusion:1.Mechanical-activated Piezo1 induces Ca²⁺influx and P3IK/Akt pathway activation to open Cx43 HCs;2.Cx43 HCs,Panx1 channels and ATP-P2X7 receptor pathway participate in the regulation intracellular Ca²⁺;3.Cx43 HCs and Piezo1 co-localized on the osteocytic cell membrane.