Transcriptional response of the murine mammary gland to acute progesterone exposure - PubMed (original) (raw)

Transcriptional response of the murine mammary gland to acute progesterone exposure

Rodrigo Fernandez-Valdivia et al. Endocrinology. 2008 Dec.

Abstract

Our mechanistic understanding of progesterone's involvement in murine mammary morphogenesis and tumorigenesis is dependent on defining effector pathways responsible for transducing the progesterone signal into a morphogenetic response. Toward this goal, microarray methods were applied to the murine mammary gland to identify novel downstream gene targets of progesterone. Consistent with a tissue undergoing epithelial expansion, mining of the progesterone-responsive transcriptome revealed the up-regulation of functional gene classes involved in epithelial proliferation and survival. Reassuringly, signaling pathways previously reported to be responsive to progesterone were also identified. Mining this informational resource for rapidly induced genes, we identified "inhibitor of differentiation 4" (Id4) as a new molecular target acutely induced by progesterone exposure. Mammary Id4 is transiently induced during early pregnancy and colocalizes with progesterone receptor (PR) expression, suggesting that Id4 mediates the early events of PR-dependent mammary morphogenesis. Chromatin immunoprecipitation assay detecting direct recruitment of ligand occupied PR to the Id4 promoter supports this proposal. Given that Id4 is a member of the Id family of transcriptional regulators that have been linked to the maintenance of proliferative status and tumorigenesis, the establishment of a mechanistic link between PR signaling and Id4 promises to furnish a wider conceptual framework with which to advance our understanding of normal and abnormal mammary epithelial responses to progestins. In sum, the progesterone-responsive transcriptome described herein not only reinforces the importance of progesterone in mammary epithelial expansion but also represents an invaluable information resource with which to identify novel signaling paradigms for mammary PR action.

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Figures

Figure 1

Figure 1

Retention of mammary PR expression in the ovariectomized mouse. A, Immunohistochemical detection of PR is clearly evident in a transverse section of a mammary duct from an intact 8-wk-old wild-type virgin mouse (arrowhead). Inset shows robust β-gal activity (arrow) in a lacZ stained mammary whole mount from a similar aged intact PRlacZ knock-in mouse. B, Two weeks after ovariectomy, an 8-wk-old wild-type mouse exhibits a lower but detectable level of PR immunoreactivity in the mammary gland (arrowhead). Upper-right corner inset shows detectable β-gal activity (arrow) in a lacZ stained mammary gland whole mount from an ovariectomized 8-wk-old PRlacZ knock-in mouse. The histogram in the lower-left corner shows the percentage of mammary luminal epithelial cells positive for PR immunoreactivity in the intact and ovariectomized mouse (bars 1 and 2, respectively). The values represent the mean ±

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from five animals per group. C and D, Higher magnifications of regions shown in A and B, respectively. C, Note that robust PR immunoreactivity is restricted to the luminal epithelial compartment of the mammary gland (black arrowhead); cells scoring negative for PR expression are indicated by the white arrowhead. D, PR immunoreactivity is detected in the luminal epithelial compartment (black arrowhead); a subset of cells is negative for PR expression (white arrowhead). Insets in C and D show lacZ stained sections of mammary glands from the intact and ovariectomized PRlacZ knock-in mouse, respectively. Note the clear β-gal activity in both panels (black arrowhead); a cell negative for β-gal activity is indicated by a white arrowhead. E, Robust PR immunoreactivity is clearly detectable in the luminal and glandular epithelial (LE and GE, respectively) compartments of the uterus from an 8-wk-old ovariectomized mouse. Inset shows strong β-gal activity (black arrowhead) in the luminal epithelial compartment of a lacZ stained uterine transverse section from an 8-wk-old ovariectomized PRlacZ knock-in mouse. F, A higher magnification of a region shown in E; note the strong PR immunoreactivity in most cells of the luminal and glandular epithelial compartments (black arrowheads) and in a subset of stromal (S) cells. The luminal epithelial, glandular epithelial, stromal, and myometrial compartments are denoted by LE, GE, S, and M, respectively. Scale bars in A and C, apply to B and D, respectively; scale bars in insets in A and C apply to insets shown in B and D, respectively.

Figure 2

Figure 2

Acute progesterone exposure elicits branching morphogenesis in the mammary gland of the ovariectomized mouse. A–C are high-magnification images of mammary gland whole mounts from untreated, progesterone-treated (for 76 h), or sesame oil-treated (for 76 h) ovariectomized wild-type mice, respectively. Note the overt dilation of the ductal network and the emergence of numerous ductal side branches (arrows) in response to 76 h progesterone exposure (compare B with A). Sesame oil treatment for 76 h fails to induce these morphological responses (compare C with B). D–F show representative H&E stained sections of the whole mounts shown in A–C, respectively. Note the significant expansion of the epithelial compartment (arrow) after progesterone treatment for 76 h (compare E with D and F). G and H show whole mounts of mammary glands from ovariectomized mice continuously treated with progesterone for 1 and 2 wk, respectively. In both panels, note the clear increase in ductal side branching and limited alveolar budding with progesterone exposure (arrows). I, Absence of these mammary morphological changes in the ovariectomized wild-type mouse after 2 wk sesame oil treatment. J–L, H&E stained sections of the whole mounts shown in G–I, respectively; a marked increase in epithelial content is observed with progesterone exposure for 1 and 2 wk (compare J and K with L). Scale bar in A applies to B, C, and G–I, respectively; scale bar in D applies to E, F, and J–L, respectively. Also see supplemental Figs. 1 and 2.

Figure 3

Figure 3

Short progesterone exposure induces mammary epithelial proliferation in the ovariectomized mouse. High-magnification images of mammary gland sections from different hormone treatment groups [i.e. 8-wk-old intact or ovariectomized (OVX) wild-type mice treated with sesame oil (SO) or progesterone (P)] stained for BrdU incorporation, PR, and ER-α expression are shown as three sets of seven panels. Although the mammary epithelium of sesame oil-treated intact and ovariectomized mice does not exhibit BrdU incorporation, a significant increase in the number of luminal epithelial cells scoring positive for BrdU incorporation (arrow) in the mammary gland of the ovariectomized mouse is clearly evident by 16 h progesterone exposure, and this number significantly increases by 76 h progesterone exposure (arrows). Although ovariectomy significantly decreases the levels of mammary PR expression in the ovariectomized mouse [compare ovariectomized (sesame oil 4 h) with INTACT (sesame oil 4 h), and also see Fig. 1], mammary PR expression is further attenuated in the ovariectomized mouse when treated with progesterone for 16 h (arrow) and is absent by 76 h progesterone exposure (arrow). Unlike progesterone treatment, PR expression levels are not changed in the ovariectomized mouse with continuous sesame oil treatment for 76 h [arrow in ovariectomized (sesame oil 76 h)]. In contrast to PR expression, ER-α expression levels are not changed with progesterone exposure. ER-α expression levels in the luminal (vertical arrow) and stromal (horizontal arrow) compartments do not change in any hormone treatment group. Scale bar applies to all panels.

Figure 4

Figure 4

Areg, calcitonin, RANKL, and Wnt-4 transcription is induced in the mammary gland of the ovariectomized mouse after acute progesterone exposure. A–D show RPA for Areg, calcitonin, RANKL, and Wnt-4 in the mammary gland of ovariectomized 8-wk-old mice that are untreated (UT) or progesterone (P) treated for 76 h, respectively. Lanes 1–3 in the untreated control and progesterone test group denote total mammary RNA isolated from three separate sets of mice, each set containing total RNA pooled from five mice for a total of 15 mice tested in the untreated and progesterone group for each RPA experiment. The RPA experiment was performed three times using different mice in each experiment; therefore, 45 mice in total were examined per test and control group in this study. A–D show that Areg (black arrowhead; protected fragment: 330 bp), calcitonin (black arrowhead; protected fragment: 350 bp), RANKL (black arrowhead; protected fragment: 205 bp), and Wnt-4 (black arrowhead; protected fragment: 400 bp) are induced by 76 h progesterone exposure, respectively. For each RPA experiment, cyclophilin served as a loading control (white arrowhead; protected fragment: 100 bp). Located on the right side of each RPA gel are probe controls in the presence of yeast RNA consisting of target gene and cyclophilin antisense riboprobes in the presence (+) or absence (−) of RNase; M, marker lanes on either side of each gel. The histogram in each panel quantitatively displays target gene transcript induction as a percentage of cyclophilin mRNA from triplicate experiments (error bar denotes ±

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).

Figure 5

Figure 5

Significant transcriptional activity is induced in the mammary gland of the ovariectomized 8-wk-old mouse in response to acute progesterone exposure. A, Overall hormone treatment strategy to stimulate a progesterone-induced transcriptional response in the 8-wk-old wild-type ovariectomized mouse at the times indicated. Mice were ovariectomized at 6 wk of age and rested for 2 wk to purge endogenous ovarian hormones. At 8 wk of age, ovariectomized mice were treated with 1 mg progesterone daily for 4, 16, 28, or 76 h. Ovariectomized mice treated with sesame oil (hormone vehicle) for the same time periods served as controls. B, Expression data matrix of 936 RNA transcripts (representing 711 unique genes) showing either induction or repression in the mammary gland of ovariectomized mice after progesterone (P) exposure for 4, 16, 28, or 76 h (fold change >1.8; P < 0.01). Each _row_ represents a gene, whereas each _column_ denotes sesame oil (SO) or progesterone treatment to a given time point in hours. The level of expression of each gene in each treatment group is represented using a _red-green_ color scale (_black_: no change; and _green_: low expression, with _bright red_ >2-fold change from control). To the right of the data matrix, color bars denote the corresponding annotation of genes in the matrix using selected GO terms. C, Table of the number of transcripts (with the number of unique genes in parentheses) induced or repressed by progesterone at each time point with the “Total” column listing the percentages of genes represented on the expression array. The complete list of mammary genes induced or repressed by progesterone during each of these time points is recorded in supplemental Table 2. D, Venn diagram of 512 unique progesterone-induced genes at 76 h (from panel B) and 214 PRL-induced genes as described by Harris et al. (31). The P value for the overlap between the two gene sets using one-sided Fisher’s exact test is indicated (based on the 20,964 unique named genes represented on the 430 2.0 array). The identity of 56 genes in the overlap between progesterone and PRL stimulated genes is listed in supplemental Table 3.

Figure 6

Figure 6

Id4 expression is markedly induced in the mammary epithelium of the ovariectomized mouse in response to acute progesterone exposure. A, Heat map analysis (from data shown in Fig. 5) for the transcriptional expression of Areg, calcitonin (Calca), RANKL (Tnfsf11), Wnt4, Mt2, Id4, PR (Pgr), and ER-α (Esr1) in mammary tissue from ovariectomized mice treated with sesame oil (SO) or progesterone (P) for the times indicated in hours. Note that Id4 transcription displays similar induction kinetics as Areg, calcitonin, RANKL, Wnt-4, and Mt2. In agreement with Fig. 3, PR transcription is predominantly repressed, and ER-α transcription remains essentially unchanged with progesterone exposure. B, Real-time PCR results for the expression of Areg, calcitonin, RANKL, Wnt-4, Mt2, and Id4 transcripts in mammary tissue from ovariectomized mice treated with sesame oil or progesterone for 76 h. Note that Id4 exhibits robust transcriptional induction in response to 76 h progesterone exposure. The results represent the mean ±

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of three independent RNA sets (*, P < 0.01). Id4 immunohistochemically stained mammary sections from an untreated intact mouse (C), from an ovariectomized mouse treated with progesterone for 4 h (D), 16 h (E), 28 h (F), and 76 h (G), as well as sesame oil for 76 h (H) are shown. Note negligible Id4 nuclear staining in the luminal epithelium of the untreated intact mouse [C (blue arrowhead)]. With prolonged progesterone exposure overtime, a clear induction of nuclear staining for Id4 is observed in D–G (black arrowhead); cells scoring negative for Id4 expression are indicated by the blue arrowhead. Approximately 30% of luminal epithelial cells scored positive for Id4 expression after progesterone exposure. H shows that sesame oil treatment for 76 h fails to induce Id4 nuclear staining (blue arrowhead) in the mammary epithelium of the ovariectomized wild-type mouse.

Figure 7

Figure 7

Id4 expression is induced in the epithelial compartment of the murine mammary gland during early pregnancy and colocalizes with PR expression. A–D, Mammary gland sections immunohistochemically stained for Id4 expression from adult virgin (12 wk old), early pregnant (5.5 d post coitum), late pregnant (18 dpc), and lactating (d 7) mice, respectively. Although Id4 is not detected in the epithelial compartment of the mammary gland of the virgin, late pregnant or lactating mouse (A, C, and D, respectively), note the striking increase in Id4 expression in a subset of cells in the luminal epithelial compartment of the mammary gland of the early pregnant mouse [B (arrow)]. E–H represent higher magnification images of regions shown in A–D, respectively. Note the strong nuclear expression for Id4 in a subset of luminal epithelial cells of mammary gland at early pregnancy [F (arrows)]; approximately 30% of luminal epithelial cells score positive for Id4 expression. Dual immunofluorescence clearly demonstrates that Id4 and PR expression colocalize in the mammary epithelium of the early pregnant mouse. I, A 4′, 6′-diamidino-2-phenylindole-stained section of the mammary epithelium from an early pregnant (5.5 dpc) mouse. J, The same section as in I immunofluorescently stained for Id4. Note the punctate pattern for Id4 expression (green arrows), which agrees with the immunohistochemical data shown in B and F. K shows the same section immunofluorescently stained for PR expression; again note a similar punctate pattern for PR expression in this section (red arrows). Superimposing J and K confirms that Id4 and PR expression localize to identical cells (yellow arrows in L). Scale bar in A, E, and I apply to B–D, F–H, and J–L, respectively.

Figure 8

Figure 8

Progesterone induction of Id4 gene expression is mediated by a direct interaction of PR and functionally synergizes with PRL/Stat5a signaling. A, Schematic of the predicted S5RE and PRE half-sites in the upstream promoter region of Id4, and PCR primers used for ChIP assay. The numbering represents the number of base pairs upstream of the transcription start site of the murine Id4 gene. B, HC-11 cells grown and differentiated, as described in Materials and Methods, were transduced at an multiplicity of infection of 50 pfu per cell with an adenoviral vector that encodes mouse PR-B. Cells were treated for 24 h with vehicle (−), R5020 (100 n

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), PRL (3 μg/ml), or R5020 (100 n

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) plus PRL (3 μg/ml), total RNA was prepared, and Id4 mRNA was measured by real-time quantitative PCR as described in Experimental procedures. The Id4 mRNA was normalized to 18S RNA, and data represent the mean ±

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of triplicate biological RNA samples measured by duplicate PCRs. The fold increase by hormone treatment relative to the vehicle control is indicated over each bar. This is a single experiment that is representative of three independent experiments. C–E, Differentiated HC-11 cells transduced with an adenovirus vector encoding hPR-B were treated for 1 h with vehicle (−), R5020 (100 n

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), PRL (3 μg/ml), or R5020 plus PRL (3 μg/ml), and ChIP assays were performed. Lower panels, Agarose gels of PCR products from input DNA and DNA immunoprecipitated by a control antibody, antibody specific for PR (1294), or antibody specific for Stat5a (L-20). Upper panels, Quantification of ChIP results using Syngene gel imager and Genetools software calculated as the average density of the immunoprecipitated DNA as a percentage (%) of input DNA after subtraction of signal obtained with the control antibody. Shown is a representative ChIP result of these independent experiments.

Figure 9

Figure 9

Id4 is overexpressed in MMTV-Wnt-1 mammary tumors. A, Comparative Western blot analysis of adult wild-type virgin mammary gland (MG) and MMTV-Wnt1 tumors (TUMOR) for Id4 and cyclin D1 expression (β-actin is included as a loading control). Mammary gland protein lysates from three individual wild-type mice were analyzed (lanes 1–3), whereas protein lysate from six tumors (lanes 1–6) from six individual MMTV-Wnt1 transgenic mice was analyzed by Western blot. Although not observed in the wild-type mammary gland, Id4 protein is clearly detected in all six MMTV-Wnt1 mammary tumors (TUMOR). The immunohistochemical detection of Id4 nuclear expression (black arrowhead) in MMTV-Wnt1 mammary tumors is clearly evident in B and C, which confirms the Western blot data shown in A. C is a higher magnification of a region shown in B, which shows tumor cells positive and negative for Id4 immunoreactivity (black and gray arrowheads, respectively).

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