Overlapping but Distinct Profiles of Gene Expression Elicited by Glucocorticoids and Progestins

Abstract

Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, we have employed two systematic approaches to identify genes that are differentially regulated by the two hormones. The first strategy is to utilize Affymetrix oligonucleotide arrays to compare glucocorticoid-and progestinregulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses. Treatment with cycloheximide or mifepristone (RU486) indicates that the regulation is a primary, receptor-mediated event. The second strategy is to employ a retroviral promoter trap and Cre/loxP-mediated, sitespecific recombination to identify genes that are differentially regulated by glucocorticoids and progestins. A mouse fibroblast cell line (4F) stably expressing both glucocorticoid receptor (GR) and progesterone receptor (PR) and containing a single copy of a multifunctional selection plasmid was generated. This line was transduced with a self-inactivating retroviral promoter trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Integration of the provirus places Cre expression under the control of genomic flanking sequence. Activation of Cre expression from integration into active genes results in a permanent switch between the selectable marker genes that convert the cells from neomycin resistant to hygromycin resistant. Selection for hygromycin resistance after hormone treatment yields recombinants in which Cre sequences in the U3 region are expressed from hormone-inducible, upstream cellular promoters. Because Cre-mediated recombination is a permanent event, the expression of the selectable marker genes is independent of ongoing Cre expression. Thus, this system permits the identification of genes that are transiently or weakly induced by hormone. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.

Microarray Analysis Reveals Glucocorticoid-Regulated Survival Genes That Are Associated With Inhibition of Apoptosis in Breast Epithelial Cells

“…To explore the underlying molecular mechanisms of GC-induced breast epithelial cell survival, we performed a genome-wide screen using high-density oligonucleotide microarrays to identify GC-regulated genes. Interestingly, the gene expression changes we observed in MCF10A-Myc cells differ significantly from those previously identified both in lymphocytes (32) and in the breast cancer tumor cell line T47D-GR (33). A possible explanation for these results is the differential expression of GR coactivator and corepressors in different cell types, a mechanism that has been proposed to explain the opposite effects of tamoxifen on mammary versus endometrial tissue (34).…”

Progesterone Attenuates Corticotropin-Releasing Factor-Enhanced But Not Fear-Potentiated Startle via the Activity of Its Neuroactive Metabolite, Allopregnanolone

“…However, P and MPA may not affect GR in the same manner as glucocorticoids do. Indeed, activation of GR via progestins versus glucocorticoids results in differential gene transcription (Wan and Nordeen, 2003).…”

Nicotinamide Uncouples Hormone-Dependent Chromatin Remodeling from Transcription Complex Assembly

“…NAM is a reaction intermediate of SIRT1 deacetylation activity that feeds back to inhibit the deacetylation activity of SIRT1 (4,17). The effect of NAM on PR-mediated transcription was determined by using T47D/2963.1 (2963.1) human breast cancer cells that endogenously express PR (45) that were treated with or without NAM for 30 min, followed by treatment with synthetic progesterone R5020 or vehicle (EtOH) for 4 h. RT-PCRs were performed from isolated RNA, and the expression levels of known PR-regulated genes CEBP/␤, EZF and SGK (53,62) were analyzed by real-time PCR. The RT-PCR analysis revealed that NAM inhibits hormone-dependent activation of PR-regulated genes in a dosedependent manner (Fig.…”