Abstract
Although glucocorticoids suppress proliferation of many cell types and are used in the treatment of certain cancers, trials of glucocorticoid therapy in breast cancer have been a disappointment. Another suggestion that estrogens may affect glucocorticoid action is that the course of some inflammatory diseases tends to be more severe and less responsive to corticosteroid treatment in females. To date, the molecular mechanism of cross-talk between estrogens and glucocorticoids is poorly understood. Here we show that, in both MCF-7 and T47D breast cancer cells, estrogen inhibits glucocorticoid induction of the MKP-1 (mitogen-activated protein kinase phosphatase-1) and serum/glucocorticoid-regulated kinase genes. Estrogen did not affect glucocorticoid-induced glucocorticoid receptor (GR) nuclear translocation but reduced ligand-induced GR phosphorylation at Ser-211, which is associated with the active form of GR. We show that estrogen increases expression of protein phosphatase 5 (PP5), which mediates the dephosphorylation of GR at Ser-211. Gene knockdown of PP5 abolished the estrogen-mediated suppression of GR phosphorylation and induction of MKP-1 and serum/glucocorticoid-regulated kinase. More importantly, after PP5 knockdown estrogen-promoted cell proliferation was significantly suppressed by glucocorticoids. This study demonstrates cross-talk between estrogen-induced PP5 and GR action. It also reveals that PP5 inhibition may antagonize estrogen-promoted events in response to corticosteroid therapy.
Highlights
Three pathways have been reported to affect glucocorticoid receptor (GR) phosphorylation and activity
We investigated the three GR-regulating pathways discussed above, and we identified PP5 to be involved in the inhibition of GR activity by estrogen providing a novel mechanism of cross-talk between estrogen and glucocorticoids
The purpose of pretreating the cells with estrogen was to mimic the in vivo state, because the breast cancer cells are under estrogen influence before glucocorticoid treatment
Summary
Materials—17-Estradiol (E2), dexamethasone (DEX), ICI 182,780, nonimmune rabbit serum, and monoclonal anti--actin antibody were purchased from Sigma. The ⌬⌬Ct method was utilized to calculate the relative change in target gene expression as an approximation of transcription based on the change in threshold values for control versus treated cells (the cycle number at which the fluorescent signaling crosses the “threshold” or logarithmic increases in cDNA concentration). The cells were washed in PBS containing 0.1% Tween 20 for 20 min, followed by incubation with Cy3-conjugated secondary antibody (donkey anti-mouse or donkey antirabbit, diluted 1:500 in Permeabilization Buffer containing 300 nM 4Ј,6-diamidino-2-phenylindole) at room temperature for 1 h. Membranes were washed in PBS, 0.05% Tween 20 and incubated for 1 h at room temperature with anti-mouse or anti-rabbit horseradish peroxidase-labeled IgG (1:10,000), washed, incubated with chemiluminescent reagent, and processed for autoradiography. A minimum of three independent experiments were conducted to allow for statistical comparisons
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