Identification of Murine Uterine Genes Regulated in a Ligand-Dependent Manner by the Progesterone Receptor

0013-7227/05/$15.00/0 Printed in U.S.A. Endocrinology 146(8):3490 –3505 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2005-0016 Identification of Murine Uterine Genes Regulated in a Ligand-Dependent Manner by the Progesterone Receptor Jae-Wook Jeong, Kevin Y. Lee, Inseok Kwak, Lisa D. White, Susan G. Hilsenbeck, John P. Lydon, and Francesco J. DeMayo Department of Molecular and Cellular Biology (J.-W.J., K.Y.L., I.K., J.P.L., F.J.D.); Microarray Core Facility, Department of Molecular and Human Genetics (L.D.W.); and Breast Center (S.G.H.), Baylor College of Medicine, Houston, Texas 77030 Progesterone (P4) acting through its cognate receptor, the progesterone receptor (PR), plays an important role in uterine physiology. The PR knockout (PRKO) mouse has demonstrated the importance of the P4-PR axis in the regulation of uterine function. To define the molecular pathways regulated by P4-PR in the mouse uterus, Affymetrix MG U74Av2 oligonucleotide arrays were used to identify alterations in gene expression after acute and chronic P4 treatments. PRKO and wild-type mice were ovariectomized and then treated with vehicle or 1 mg P4 every 12 h. Mice were killed either 4 h after the first injection (acute P4 treatment) or after the fourth injection of P4 (chronic P4 treatment). At the genomic level, the major change in gene expression after acute P4 treatment T HE OVARIAN STEROID hormone progesterone (P4) is an essential regulator of reproductive events associated with all aspects of the establishment and maintenance of pregnancy (1, 2). Most of the physiological affects of P4 are mediated through its receptor, the progesterone receptor (PR). This signaling axis, the P4-PR axis, has been investigated by dissecting the role of PR (3). PR is a transcription factor that belongs to the nuclear receptor superfamily (4 – 6). PR is encoded in one gene and exists as one of two isoforms, PR-A and PR-B. These isoforms arise from the alternate translation start sites in the PR gene (7). Genetic ablation of the PR gene in mice (PRKO) leads to pleiotropic reproductive abnormalities, including defects in female reproductive behavior (8), failure to ovulate, failure of the uterus to support embryo implantation, and defects in branching and glandular development in the mammary glands (2, 9). Site-directed mutagenesis of the PR gene in vivo has demonstrated that the PR-A isoform is the major mediator of P4 signaling in the mouse uterus regulating uterine function, whereas the PR-B isoform regulates uterine epithelial cell proliferation (10, 11). Although the physiological processes governed by the P4-PR signaling axis in the uterus have been identified, the molecFirst Published Online April 21, 2005 Abbreviations: ADH5, Alcohol dehydrogenase 5; Aldh1a1, aldehyde dehydrogenase 1a1; Cited2, cAMP-corticosterone-binding protein/ p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail; Cyp26a1, cytochrome P 450 26a1; Klk, kallikrein; P4, progesterone; PR, progesterone receptor; PRKO, progesterone receptor knockout; RA, retinoic acid; RAR, retinoic acid receptor; Rbp, retinolbinding protein; RXR, retinoid X receptor. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. was an increase in the expression of 55 genes. Conversely, the major change in gene expression after chronic P4 treatment was an overall reduction in the expression of 102 genes. In the analysis, retinoic acid metabolic genes, cytochrome P 450 26a1 (Cyp26a1), alcohol dehydrogenase 5, and aldehyde dehydrogenase 1a1 (Aldh1a1); kallikrein genes, Klk5 and Klk6; and specific transcription factors, GATA-2 and Cited2 [cAMP-corticosterone-binding protein/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail], were validated as regulated by the P4-PR axis. Identification and analysis of these responsive genes will help define the role of PR in regulating uterine biology. (Endocrinology 146: 3490 –3505, 2005) ular pathways governed by PR are not fully understood. Therefore, elucidation of the molecular pathways regulated by PR in the uterus by identification of the target genes whose transcription is regulated by PR is of great importance. To date, only a few P4-PR-regulated genes have been identified. These include the genes encoding amphiregulin (Areg) (12), histidine decarboxylase (Hdc) (13), Hoxa-10 and -11 (14), calcitonin (15, 16), calbindin-D9K (17), Indian hedgehog (Ihh) (18), hypoxia-inducible factor 1 (HIF1A) (19), and immune-responsive gene 1 (20). These target genes have been identified by testing candidate genes (12), differential library screening (15), and DNA microarray approaches (18, 20). High-density DNA microarray technology has immensely improved the ability to identify PR-regulated genes in the uterus. Cheon and co-workers (3) used high-density DNA microarray technology to identify PR-responsive genes in the mouse uterus by treating female mice on d 3 of pregnancy with the antiprogestin RU486 and assaying the impact on uterine target genes 24 h later. This approach identified PRregulated genes by inhibiting PR action at a time when PR levels were elevated in all compartments. In a sense, this approach identified the impact of withdrawal of P4-PR signaling on uterine gene expression and successfully identified 148 genes that were regulated by this axis (3). In this report, high-density DNA microarray technology in combination with the PRKO mouse were used to identify genes affected by acute and chronic stimulation of the P4-PR axis. In this study, we have taken a pharmacological approach to identify the impact of activation of the P4-PR axis on the mouse uterus. This analysis shows the impact of acute and chronic pharmacological stimulation of the P4-PR axis, al- 3490 Jeong et al. • Ligand-Dependent, PR-Regulated Murine Uterine Genes Endocrinology, August 2005, 146(8):3490 –3505 FIG. 1. The five physiologically relevant comparisons used to identify significantly P4- and/or PR-regulated uterine genes: comparison 1, vehicle (veh)-treated wild-type (WT) vs. P4-treated wild-type mice; comparison 2, P4-treated PRKO vs. P4-treated wild-type mice; comparison 3, vehicle-treated PRKO vs. P4-treated wild-type mice; comparison 4, vehicle-treated wild-type vs. vehicle-treated PRKO mice; and comparison 5, vehicle-treated PRKO vs. P4-treated PRKO mice. Differentially expressed genes were selected using two-sample comparison according to the lower boundary of a 90% confidence interval of fold change greater than 1.2 and an absolute value of difference between group means greater than 80. lowing additional identification of how these target genes function to regulate uterine biology. The groups of genes that were validated were those involved in retinoic acid (RA) metabolism, the kallikrein (Klk) family of genes, and specific developmentally importan (...truncated)