title: An evaluation of Glucocorticoid Receptors in A549 cells and cardiac fibroblasts: Potential in vitro models of COPD and Heart Failure
Creators: name: Lang-Lemckert, Julian Charles
Publisher: name: Griffith University
Year: 2018
Subjects: subject: Glucocorticoid receptors
subject: A549 cells
subject: Cardiac fibroblasts
subject: in vitro models
subject: COPD
subject: Heart failure

description: Introduction Chronic Obstructive Pulmonary Disease (COPD) and Heart Failure (HF) are two diseases that contribute significantly to disease morbidity and mortality within Australia. Both conditions exhibit excessive inflammation of the pulmonary tissue and cardiac tissue respectively which plays a significant role in disease onset and progression. Recent clinical and in vitro studies have begun to focus upon glucocorticoid receptor (GR) function associated with these conditions, as the GRs produce anti-inflammatory responses when activated. Studies using pulmonary and cardiac cells, in vitro, have observed that decreases within GR signalling (i.e. GR resistance) may accelerate the progression of the disease and worsen prognosis. Additionally, recent studies have also shown that the administration of mineralocorticoid receptor (MR) antagonists to cardiac and pulmonary cells are capable of reducing inflammation. Therefore understanding the changes occurring in GR and MR populations associated with COPD and HF may provide further insight into the inflammatory pathways underlying these diseases; therefore allowing for the development of more effective treatments for both conditions. Experimental Aims The aim of this study was to investigate the effect of 24 hours incubation with the GR agonist dexamethasone in both adenocarcinoma cell lines (A549 cells) and primary Human Cardiac Fibroblasts (HCF), in order to identify whether GR resistance could be induced in these cells lines (simulating the decreased GR signalling noted within COPD and HF patients). Following treatment, Western blotting analysis of GR and MR populations, as well as fluorescent proliferation assays were utilized to determine if GR resistance could be accurately measured within these cells. Methods The cellular viability (as measured via MTT and LDH assays) of A549 and HCF cells in response to 24 hours treatment with concentrations of the GR agonist dexamethasone in the presence or absence of the GR inhibitor mifepristone (and vehicle controls) was measured in order to determine optimum concentrations of these drugs for the 24 hour study period. Following viability studies, cells were then exposed to dexamethasone (100nM) and/or mifepristone (10nM) for 24 hours. The A549 and HCF cell proliferation rates were then measured using the fluorescence-based 2-deoxybromouridine (BRDU) proliferation assay to determine if any changes in dexamethasone responsiveness (as measured via a ratio between dexamethasone : vehicle control absorbance) could be detected. Further, A549 and HCF cells were then treated for 24 hours with dexamethasone (100nM) in the presence or absence of mifepristone (10nM) then lysed with 2X RIPA lysis buffer supplemented with protease and phosphatase inhibitors in order to form protein lysates for Western blotting. Western blotting analysis was then conducted in order to determine the expression of the three GR gene variants GRα-A, GRβ and GRγ, as well as MR. All Western blots were normalized to both an internal standard and housekeeping protein (cofilin). All data was presented as mean ± SEM and one-way ANOVA performed using GraphPad Prism, with a p value of 0.05 utilized to register statistical significance. Results MTT and LDH cellular viability assays confirmed that dexamethasone (100nM) and mifepristone (10nM) did not have any significant impact upon the cellular viability of A549 and HCF cells over a 24 hour period (p&gt;0.05). Subsequent BRDU proliferation assays on A549 cells revealed only a significant difference in cell proliferation rates between the dexamethasone (100nM) only treated and the combined dexamethasone (100nM) and mifepristone (10nM) treatment groups (p&lt;0.05). These results were not repeated in the HCF cell experiments, which showed no significant difference in proliferation rates between any of the treatment groups (p&gt;0.05). Subsequent protein analysis via Western blotting revealed that all treatments significantly reduced the protein expression of GRα-A within A549 cells relative to control (p&lt;0.05). In contrast, protein analysis of HCF cells revealed only a significant decrease in GRα-A protein expression in HCF cells treated with dexamethasone (100nM, p&lt;0.01). GRβ and GRγ protein expression was found to be significantly decreased in A549 cells treated with the combined treatment of dexamethasone (100nM) and mifepristone (10nM) only when compared to protein levels in untreated cells (p&lt;0.05). Alternatively, GRγ protein expression did not differ significantly differ between HCF treatment groups (p&gt;0.05) and GRβ protein was not expressed within measurable concentrations within any of the HCF treatment groups. Finally, both A549 and HCF cells did not express MR within measurable quantities. Conclusion This research is the first project to investigate the effects of dexamethasone treatment on both the development of GR resistance (as measured via BRDU fluorescence assays) and GR and MR protein expression in both A549 and HCF cells. Novel findings include the BRDU analysis within A549 cells, which indicated the method was capable of detecting some changes in proliferation rates which correlated well with changes within GRβ and GRγ protein expression changes measured using Western blot procedures. This method was also found to be ineffective in measuring GR resistance within HCF cells, as the decrease in GRα-A protein expression observed in the Western blots produced no change in BRDU cell proliferation results. Overall, further studies should be conducted into developing the BRDU method as a measurement of GR resistance for use in primary cells, in vitro. Due to the inconsistencies noted between the findings this study and those presented in literature, further investigations on the impact of dexamethasone and mifepristone on GR regulation in A549 and HCF cells should be conducted.