228 Chapter 7 RESULTS To investigate the cytotoxic effects of dexamethasone, prednisolone and hydrocortisone via the GR or the MR in leukemic cells, we generated bulk-transduced Reh cells with a doxycyclineinducible DDK-tagged NR3C1 or NR3C2 construct, respectively. Doxycycline exposure induced the expression of DDK-tagged NR3C1 (referred to as RehNR3C1) or DDK-tagged NR3C2 (referred to as RehNR3C2) in Reh parental cells that lack a functional GR and MR (Figure 1A, Supplemental Figures 1A-B).20 In both cell lines, the DDK signal became apparent after induction with doxycycline, and steroid treatment further enhanced this signal and correspondingly showed strong induction of BIM. Next, we studied the expression of NR3C1 and NR3C2 in both cell line models upon treatment with dexamethasone (known to specifically bind the GR) or hydrocortisone (known to specifically bind the MR). As expected, exposure to doxycycline induced the expression of NR3C1 in the RehNR3C1 cell line (Figure 1B, left panels). Treatment with dexamethasone and hydrocortisone significantly enhanced the expression of the NR3C1 gene in these cells. Interestingly, both dexamethasone and hydrocortisone treatment also induced the expression of the endogenous NR3C2 gene, signifying NR3C2 as a target gene of NR3C1. Of note, this induced expression of NR3C2 was significantly lower than the induced expression of NR3C1 in RehNR3C1 cells. In the RehNR3C2 cell line, doxycycline exposure induced the expression of NR3C2, which was enhanced by hydrocortisone and to a lesser extent by dexamethasone treatment (Figure 1B, right panels). Moreover, and in contrast to RehNR3C1 cells, only hydrocortisone treatment was able to induce the expression of NR3C1 in RehNR3C2 cells. To further study the differences in GR and MR activation by hydrocortisone and dexamethasone, we further explored steroidinduced expression of known NR3C1 transcriptional target genes. In line with the induced expression of NR3C1 or NR3C2, the expression of BIM, GILZ and FKBP5 was strongly induced by hydrocortisone and dexamethasone in RehNR3C1 cells (Figure 1C, upper panels). In contrast, strong transcriptional upregulation of these genes was only achieved by hydrocortisone treatment in RehNR3C2 cells (Figure 1C, lower panels), confirming the low potency of dexamethasone to activate the MR. Combined, these data show that hydrocortisone can induce the expression of steroid-regulated genes via both the GR and the MR, while dexamethasone predominantly induces transcription via the GR. The strong induction of BIM by hydrocortisone treatment in RehNR3C2 cells (Figure 1A, C) is of specific interest since BIM mediates steroid-induced apoptosis of lymphoid cells.21 The induction of BIM by dexamethasone, prednisolone and hydrocortisone in RehNR3C2 cells indicates that the MR may play a role in steroid-induced cytotoxicity
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