The Mechanism Of CaSR-mediated Irregular Calcium Oscillations Regulate The Transcription Of NFκB In A549 Cells

Objective:Calcium sensitive receptors（CaSR）are expressed in most tumor cells,including lung adenocarcinoma cells,especially in highly differentiated lung cancer.Calcium ions act as important second messengers and participate in many cytological behaviors.Irregular calcium oscillations participate in the transduction of signaling pathways as one of its manifestations,while CaSR can mediate irregular calcium oscillations.This experiment initially explored the possible mechanism by which CaSR-mediated irregular calcium oscillations regulate NFκB transcription in A549 cells.Methods:1.A549 cells were incubated with calcium ion probe Fura-2/AM,and treated with or without histamine（His）under normoxia,hypoxia for 24 h and hypoxia for 48 h,respectively.At the same time,calcium ion imaging system was used to detect cytosolic free calcium ions.After transfection with CaSR-overexpression plasmid,the changes of calcium concentration were detected under the above conditions.The kinetic parameters such as frequency,cumulative spike duration（Cumulative Spike Duration-NFκB and Cumulative Spike Duration-STAT3）and amplitude were recorded and compared.2.After transfection with GFP-RelA plasmid,A549 cells were stimulated with 10μM His after 48h of hypoxia,and after incubated the calcium ion probe Fura-red/AM,simultaneously monitored the cytosolic calcium concentration and transcription factor NFκB nuclear translocation fluorescence by confocal microscopy.The relationships between the parameters of calcium oscillation and the change of nuclear translocation fluorescence intensity were recorded and analyzed.Results:1.Analysis of kinetic parameters of irregular calcium oscillation in wild type A549cells CSD（S）-NFκB（180 nM）:There were statistical differences between the hypoxia 24h untreated group,the hypoxia 48h untreated group,the hypoxia 24h histamine treated group,and the hypoxia 48h histamine treated group（*p<0.05）.CSD（S）-STAT3（395 nM）:There was significant difference between the hyoxia 24h untreated group and hyoxia 48h untreated group,hyoxia 24h untreated group and hyoxia 24h histamine treated group,hyoxia 48h untreated group and hypoxia 48h histamine treated group（*p<0.05）.There was no significant difference between the hypoxia 24h histamine treated group and the hypoxia48h histamine treated group（*p>0.05）.The frequency(min^-1):the hyoxia 24h untreated group and the hypoxia 24h histamine treated group,the hyoxia 48h untreated group and the hypoxia 48h histamine treated group were statistically different（*p<0.05）.There was no significant difference between the hyoxia 24h untreated group and the hypoxia 48h untreated group,the hypoxia 24h histamine treated group and the hypoxia 48h histamine treated group（*p>0.05）.Amplitude（nM）:There was no significant difference between the hypoxia 24h untreated group and the hypoxia 48h untreated group（*p>0.05）;there were significant differences between the hypoxia 24h untreated group and the hypoxia 24h histamine treated group,hypoxia 48h untreated group and the hypoxia 48h histamine treated group,the hypoxia 24h histamine treated group and the hypoxia 48h histamine treated group（*p<0.001）.2.Analysis of kinetic parameters of irregular calcium oscillation in CaSR-overexpression A549 cells CSD（S）-NFκB（180 nM）:CaSR-overexpression A549 cells and CaSR-vector A549 cells were statistically different under various conditions（*p<0.001）;there were significant differences between CaSR-overexpression A549 cells with histamine treatment in normoxic group,hypoxia 24h group and hypoxia 48h group（*p<0.05）.CSD（S）-STAT3（395 nM）:CaSR-overexpression A549 cells and CaSR-vector A549 cells were statistically different under various conditions（*p<0.001）;there were significant differences between CaSR-overexpression A549 cells with histamine treatment in normoxic group,hypoxia 24h group and hypoxia 48h group（*p<0.05）.The frequency(min^-1):CaSR-overexpression A549 cells and CaSR-vector A549 cells were statistically different under various conditions（*p<0.001）;there was no significant difference between CaSR-overexpressing A549 cells with histamine treatment in normoxic group,hypoxia 24h group and hypoxia 48h group（*p>0.05）.Amplitude（nM）:CaSR-overexpression A549 cells and CaSR-vector A549 cells were statistically different under various conditions（*p<0.001）;there were significant differences between CaSR-overexpression A549 cells with histamine treatment in normoxic group and hypoxia 24h group,normoxic group and hypoxia 48h group（*p<0.001）;there was no significant difference between CaSR-overexpression A549 cells with histamine treatment in hypoxia 24h hroup and hypoxia 48h group（*p>0.05）.3.Analysis of the relationship between NFκB nuclear translocation and irregular calcium oscillation kinetic parameters The analysis by four parameter logistic regression method indicated that the fluorescence intensity of nuclear factor translocation NFκB nuclear transcription was well fitted to amplitude and cumulative spike duration regression equation,and there was statistical difference（*p<0.001）,n=79;however the fluorescence intensity of the transcription factor NFκB nuclear translocation was not well fitted to the frequency regression equation,and there was no statistical difference（*p>0.05）,n=79.Conclusions:At the single-cell level,wild-type/CaSR overexpressing A549 cells can undergo spontaneous calcium oscillation under hypoxic conditions,a process that is dependent on CaSR.With the prolongation of hypoxia and the stimulation of inflammatory factors,the incidence of irregular calcium oscillations and kinetic parameters of A549 cells increased.However,the length of hypoxia had no effect on frequency,probably due to the sensitivity of the histamine is greater than the sensitivity to the duration of hypoxia.A549 cells regulate the downstream transcription factor NFκB activity with cumulative spike duration and amplitude dependence.