The Mechanism Of Activating TRPV4 Induces Exotycosis In Human Melanoma Cells

Objective:Endocytosis and exocytosis are common physiological phenomena in eukaryotic cells,which promote the transport of biomacromolecules and particulate matter inside and outside cells through a series of processes such as membrane vesicle formation,displacement and fusion,so as to maintain the renewal of cell membrane and the normal growth and survival of cells.TRPV4（Transient receptor potential vanilloid 4）is a member of the transient vanilloid receptor potential TRPV channel family.TRPV4 is a calcium permeable TRP ion channel,which can be activated by a variety of stimuli,including cell swelling,temperature and chemical stimulation.TRPV4 is widely expressed in a variety of cells and tissues,and is involved in various physiological processes such as homeostasis,temperature sensitivity,inflammation and metabolism.Previous studies have shown that TRPV4 plays a key role in cellular entosis,but whether it helps to participate in the regulation of cellular exocytosis remains unclear.Our previous studies have found that TRPV4 is highly expressed in melanoma cells,and the addition of TRPV4 channel agonists can induce cell death.Therefore,the purpose of this study is to clarify the relationship between TRPV4 channel and cellular exocytosis,as well as to determine the molecular mechanism of activating TRPV4 channel to induce and regulate the exocytosis of melanoma A375 cells,so as to lay the foundation for subsequent research on the influence of TRPV4 channel and mediated cellular exocytosis on the fate of melanoma cells.Methods:（1）The association between functional TRPV4-activated ion channels and cellular exocytosis was examined in cell Ca²⁺imaging experiments and cell morphology observation experiments using chemical agonists and inhibitors of TRPV4 and TRPV4knockdown in human melanoma A375 cell with high expression of TRPV4.（2）Transient transfect TRPV4 in 3T3 cell that did not express TRPV4,further detected the relationship between TRPV4 activation and exocytosis in this expression system with activators GSK1016790A.（3）Under the conditions of chelating extracellular calcium ions,inhibiting intracellular calcium ions and using ionomycin,Fluo-4 AM was used to label intracellular calcium ion concentration,and to explore the relationship between exocytosis and changes in intracellular calcium ion concentration.（4）By inhibiting intracellular calmodulin and calmodulin kinaseⅡsignal and by using potassium channel inhibitors,we explored the intracellular signaling pathway after activation of TRPV4 to induce calcium influx.（5）In wild-type and TRPV4-knockdown A375 cells,the expression changes of lysosome related proteins after TRPV4 activation were detected by western blotting assay and immunofluorescence assay,and analyze whether lysosome related proteins were involved in exocytosis after TRPV4 activation.（6）The changes of A375 cell mitochondria in exocytosis after TRPV4 activation were observed by electron microscopy and mitochondrial metabolism test.Results:（1）In human melanoma A375 cells,activation of TRPV4 induces exocytosis and calcium influx was observed in cell morphology observation experiments and cell Ca²⁺imaging experiments,whereas in the presence of TRPV4 inhibitors or TRPV4knockdown,TRPV4 activators did not induce exocytosis and calcium influx.（2）In 3T3cell that did not express TRPV4,after transient transfer of TRPV4,the cells were stimulated with an activator and found to have significant exocytosis and calcium influx.（3）Activation of TRPV4 in the presence of extracellular calcium chelators did not result in calcium influx and exocytosis,whereas intracellular calcium chelators did not.Exocytosis was not induced by calcium influx caused by ionomycin use.（4）Inhibition of intracellular calmodulin or calmodulin kinaseⅡ,and then activated TRPV4,exocytosis did not occur.However,when the intracellular potassium channel was inhibited and TRPV4 was activated,exocytosis still occurred.（5）Western blotting and Immunofluorescence experiment showed that the expression of lysosome associated protein 2（LAMP2）increased during the exocytosis induced by activation of TRPV4,but LAMP2 unchanged after activation of TRPV4 in TRPV4 knockdown cells.（6）The results of electron microscopy and mitochondrial metabolism experiments showed that the mitochondrial structure of A375 cell was damaged and mitochondrial metabolism decreased after activation of TRPV4 to induce exocytosis.Conclusion:Activation of TRPV4 induces massive exocytosis in melanoma A375 cell and in allogeneic expression systems.This exocytosis is triggered by the intracellular flow of calcium ions mediated by activation of TRPV4 through activated TRPV4,and induced by activation of related downstream Ca²⁺/Ca M/Ca MKⅡsignaling pathways.In the process,lysosome associated proteins participate in the exocytosis process.Mitochondria are damaged in this exocytosis process.