FP41-2 Progesterone Receptor Phosphorylation Regulates Transcription in a Gene-Specific Manner in vitro and in vivo

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: FP41-Sex Hormone Receptors in Development, Aging and Cancer: Omics Approaches
Monday, June 17, 2013: 10:45 AM-11:15 AM
Presentation Start Time: 10:50 AM
Room 121 (Moscone Center)

Poster Board MON-356
Lindsey S Trevino*, Sandra L Grimm, Alison E Obr, Robert D Ward, Francesco J DeMayo, John P Lydon, Dean Paul Edwards* and Nancy L Weigel
Baylor College of Medicine, Houston, TX
Progestin action is conveyed through two isoforms of the progesterone receptor (PR), PR-B and PR-A, whose functions are regulated by phosphorylation. Multiple PR phosphorylation sites have been identified, but the mechanisms by which site-specific phosphorylation regulates PR function are largely undetermined. Mutation of phosphorylation sites has minimal effect on PR-responsive reporter activity. We are using two approaches to examine the role of PR phosphorylation in the regulation of endogenous target genes. First, we have generated breast cancer cell lines that stably express inducible forms of PR-B (PR-B), a single-site phosphorylation mutant form of PR-B (S162A), or a multi-site phosphorylation mutant form of PR-B (6-phospho mutant; 6pm). These cell lines allow us to control the expression levels of PR-B and to compare the effects of site-specific phosphorylation on PR-mediated gene expression. Second, we have generated a mouse with a single alanine substitution for Ser191 (Ala191), a phosphorylation site common to the PR-B and PR-A isoforms. In the human PR, loss of phosphorylation has little effect on transcription of SGK1, genes such as FKBP5 and S100P exhibit diminished activation in both mutant lines, but induction of HSD11β2 is only reduced in the 6pm line. Chromatin immunoprecipitation (ChIP) assays show that there are site-specific differences in recruitment of PR mutants to regions of the FKBP5 gene. To examine the role of S191 phosphorylation in mouse mammary epithelial cells (MECs), we have established three-dimensional cultures in Matrigel that develop acini with characteristics of mammary glands in vivo including heterogeneity of PR expression and paracrine-mediated proliferation. Gene expression studies revealed that whereas the induction of RANKL and calcitonin, two genes important for paracrine-mediated actions of progesterone, was reduced in the Ala191 MECs, regulation of other genes was unaffected. A similar gene regulation pattern was observed in MECs isolated from glands of mice treated with progesterone in vivo. To examine the molecular basis for reduced induction, isolated MECs were treated with progesterone, and ChIP was performed. Similar to human PR, we observed site-specific differences between wild-type and Ala191 PR binding to distal enhancers of the RANKL gene. These data suggest a role for phosphorylation in the regulation of protein-protein interactions facilitating binding to specific sites in target genes.

Nothing to Disclose: LST, SLG, AEO, RDW, FJD, JPL, DPE, NLW

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm