Effect of anabolic-androgenic steroids and glucocorticoids on the kinetics of hAR and hGR nucleocytoplasmic translocation

Abstract

Although the qualitative nucleocytoplasmic transport of nuclear hormone receptors (NHRs) has been studied, there is little documentation of the cellular kinetics of this transport. Here, translocation studies using the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) were performed to aid in identifying the mechanism by which anabolic-androgenic steroids (AAS) were activating hAR and potentially interacting with hGR and how glucocorticoid ligands were interacting with the hGR and hAR. The real-time analysis of EGFP-labeled hAR and hGR ligand-induced cytoplasm-to-nucleus translocation was performed using fluorescence microscopy to better understand the action of these NHRs in a physiologically relevant cell-based model. After transient transfection, the hAR and hGR individually translocate as expected (i.e., transport is ligand-induced and dose-dependent) in this model biological system. Testosterone (TEST) had the fastest translocation rate for the hAR of 0.0525 min(-1). The other endogenous steroids, androstenedione (ANE) and dihydrotestosterone (DHT), had considerably lower hAR transport rates. The rates of hAR transport for the exogenous steroids methyltrienelone (MET), nandrolone (NAN), and oxandrolone (OXA) are lower than that of testosterone and similar to those of the endogenous steroids ANE and DHT. The hGR transport rates for cortisol (COR) and dexamethasone (DEX) are also presented. The synthetic GC, DEX, had a more rapid translocation rate (0.1599 min(-1)) at the highest dose of 100 nM compared to the endogenous GC COR (0.0431 min(-1)). The data obtained agrees with the existing qualitative data and adds an important ligand-dependent kinetic component to hAR and hGR transport. These kinetic data can aid our understanding of NHR action and interaction with other regulatory proteins, and can be useful in the development of new therapies.

Figure 1

Translocation of EGFP-hAR in COS-7 cells after vehicle treatment. Representative time points of 0 minutes, 45 minutes, and 240 minutes are shown. At all time points, the majority of EGFP-hAR is cytosolic.

Figure 2

Translocation of EGFP-hAR in COS-7 cells after TEST treatment. Representative time points of 0 minutes, 45 minutes, and 240 minutes are shown for 4 treatment concentrations of TEST (1 nM, 10 nM, 50 nM, and 100 nM). At 0 minutes, the majority of EGFP-hAR is cytosolic. At 45 minutes, EGFP-hAR has translocated to the nucleus. At 240 minutes, transport of EGFP-hAR to the nucleus has plateaued in a dose-dependent manner; more EGFP-hAR is nuclear in the cells treated with the highest dose of TEST. Translocation of other AAS was similar.

Figure 3

Translocation data for hAR after treatment with hAR ligands. The graphs depict the change in percent intensity of EGFP-hAR in the nucleus over time after induction with 1 nM, 10 nM, 50 nM, or 100 nM of the ligand. Translocation was observed for 240 minutes. Data reported is mean ± SEM, n = 3 experiments with 10 cells analyzed per experiment. The varying steepness of the curves represents the varying rate of hAR transport depending on the dose of ligand. See Table 2.1 for rates. Significant difference from corresponding vehicle time point indicated with *, p < 0.05. A, testosterone (TEST); B, dihydrotestosterone (DHT); C, androstenedione (ANE); D, epitestosterone (EPI); E, methyltrienelone (MET); F, nandrolone (NAN); G, oxandrolone (OXA); H, flutamide (FLU).

Figure 4

Translocation of EGFP-hAR in COS-7 cells after 100 nM COR treatment. A. Cells at representative time points of 0 minutes, 45 minutes, and 240 minutes are shown. At all time points, the majority of EGFP-hAR is cytosolic. Transport of DEX and E2 were similar. B. Translocation data for COR and hAR. The graph depicts the change in percent intensity of EGFP-hAR in the nucleus over time after induction with 100 nM COR. Translocation was observed for 240 minutes. Data reported is mean ± SEM, n = 3 experiments with 10 cells analyzed per experiment. There was no change in the rate of hAR transport with the highest dose of COR used. See Table 2.3 for rate. No significant difference from corresponding vehicle time point was seen.

Figure 5

Translocation of EGFP-hGR in COS-7 cells after vehicle treatment. Representative time points of 0 minutes, 45 minutes, and 240 minutes are shown. At all time points, the majority of EGFP-hGR is cytosolic.

Figure 6

Translocation of EGFP-hGR in COS-7 cells after COR treatment. Representative time points of 0 minutes, 45 minutes, and 240 minutes are shown for 4 treatment concentrations of COR (1 nM, 10 nM, 50 nM, and 100 nM). At 0 minutes, the majority of EGFP-hGR is cytosolic. At 45 minutes, EGFP-hGR has translocated to the nucleus. At 240 minutes, transport of EGFP-hGR to the nucleus has plateaued in a dose-dependent manner; more EGFP-hGR is nuclear in the cells treated with the highest dose of TEST. Translocation of DEX was similar.

Figure 7

Translocation data for hGR after COR and DEX treatment. The graph depicts the change in percent intensity of EGFP-hGR in the nucleus over time after induction with 1 nM, 10 nM, 50 nM, or 100 nM COR or DEX. Translocation was observed for 240 minutes. Data reported is mean ± SEM, n = 3 experiments with 10 cells analyzed per experiment. The varying steepness of the curves represents the varying rate of hGR transport depending on the dose of COR or DEX. See Table 2.4 for rates. Significant difference from corresponding vehicle time point indicated with *, p < 0.05. A, cortisol (COR); B, dexamethasone (DEX).

Figure 8

Translocation of EGFP-hGR in COS-7 cells after 100 nM TEST treatment. A. Cells at representative time points of 0 minutes, 45 minutes, and 240 minutes are shown. At all time points, the majority of EGFP-hGR is cytosolic. Transport of other AAS, FLU, and E2 was similar. B. Translocation data for TEST and hGR. The graph depicts the change in percent intensity of EGFP-hGR in the nucleus over time after induction with 100 nM TEST. Translocation was observed for 240 minutes. Data reported is mean ± SEM, n = 3 experiments with 10 cells analyzed per experiment. There was no change in the rate of hGR transport with the highest dose of TEST used. See Table 2.5 for rate. No significant difference from corresponding vehicle time point was seen.