Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland

Abstract

The mouse mammary gland develops postnatally under the control of female reproductive hormones. Estrogens and progesterone trigger morphogenesis by poorly understood mechanisms acting on a subset of mammary epithelial cells (MECs) that express their cognate receptors, estrogen receptor alpha (ERalpha) and progesterone receptor (PR). Here, we show that in the adult female, progesterone drives proliferation of MECs in two waves. The first, small wave, encompasses PR(+) cells and requires cyclin D1, the second, large wave, comprises mostly PR(-) cells and relies on the tumor necrosis factor (TNF) family member, receptor activator of NF-kappaB-ligand (RANKL). RANKL elicits proliferation by a paracrine mechanism. Ablation of RANKL in the mammary epithelium blocks progesterone-induced morphogenesis, and ectopic expression of RANKL in MECs completely rescues the PR(-/-) phenotype. Systemic administration of RANKL triggers proliferation in the absence of PR signaling, and injection of a RANK signaling inhibitor interferes with progesterone-induced proliferation. Thus, progesterone elicits proliferation by a cell-intrinsic and a, more important, paracrine mechanism.

Fig. 1.

Progesterone induces two waves of proliferation. (A–E) Ten-week-old female mice were ovariectomized, pretreated with 17-β-estradiol 10 days later, and then injected with vehicle, 17-β-estradiol, or 17-β-estradiol and progesterone. BrdU was administered repeatedly for analysis at 24 h (A and C) or as a single bolus at 46 h (B and D). Bars show BrdU incorporating MECs ± SEM in different treatment groups 24 h (A) and 48 h (B) after injection (n = 3). Double immunofluorescence after 24 h (C) and 48 h (D) of stimulation. Green, PR; red, BrdU; blue, DAPI. (Scale bar: 40 μm.) (E) Percentage of PR(+) and PR(−) cells ± SEM among BrdU incorporating MECs 24 h and 48 h after progesterone stimulation. (F and G) Ten-week-old female mice were ovariectomized and treated every 24 h with 17-β-estradiol and progesterone. Percentage of PR(+) and PR(−) BrdU incorporating MECs determined by double immunofluorescence; 40–400 BrdU(+) MECs counted per mouse (n = 3–4) (F). BrdU incorporation indices were determined by counting 3,000 cells per mouse (n = 3–4), and plotted over time. Percentage of PR(+) (red) and PR(−) cells (blue) incorporating BrdU was calculated based on F (G).

Fig. 2.

Cyclin D1- and progesterone-induced proliferation. (A and D) Mice engrafted with cyclin D1^−/− and WT epithelia were stimulated with progesterone for 24 h (A, B, and E) or 48 h (C, D, and F). Histological sections of contralateral mammary glands engrafted with cyclin D1^−/− (A and C) or WT (B and D) epithelia and stained with an anti-BrdU antibody. (Scale bar: 40 μm.) (E and F) Bar graphs showing BrdU incorporation in cyclin D1^−/− and WT MECs ± SEM (n = 3), 1,000 cells counted per mouse.

Fig. 3.

Response to progesterone in RANKL^−/− MECs. (A–F) Mice engrafted with RANKL^−/− or WT MECs stimulated with progesterone for 24 h (A, B, and C) or 48 h (D, E, and F). The percentage of BrdU-incorporating cells ± SEM in contralateral mammary epithelia of three mice after 24 h (A) and 48 h (D) of stimulation. Open bars, RANKL^−/−; black bars, WT MECS; total of 1,000 cells counted in three different sections from each mouse. (B, C, E, and F) Double-immunofluorescence at 24 h (B and C) and 48 h (E and F) of stimulation. Green, PR; red, BrdU; blue, DAPI. (Scale bar: 40 μm.) Note: at 24 h, RANKL^−/− (B) and WT (C) MECs incorporate BrdU similarly and are PR(+). At 48 h, fewer mutant MECs incorporate BrdU (E) vs. WT (F), and most BrdU-incorporating cells are hormone receptor negative.

Fig. 4.

RANKL is sufficient to mediate PR function in the mammary epithelium. (A–D) Whole-mount micrographs of mammary glands from WT hosts postpartum. (A) Endogenous gland showing highly branched ductal system with dilated alveoli. (B) Gland engrafted with PR^−/− epithelium infected with control virus. Note the simple ductal system. (Scale bar: 300 μm.) (C and D) Two representative mammary glands engrafted with PR^−/− MECs infected with a retrovirus expressing RANKL showing extensive alveologenesis (C) or PR^−/− phenotype in one area (arrow) next to a fully developed sector (arrowhead) (D). (E and F) Histological sections of postpartum mammary glands stained with an anti-β-casein antibody, gland engrafted with PR^−/− MECs infected with a retrovirus expressing RANKL (E), and endogenous control gland (F). Note the presence of fat droplets and β-casein expression in both WT epithelium and PR^−/− MECs expressing ectopic RANKL. (Scale bar: 40 μm.) (G–I) Stereomicrographs of a mammary gland reconstituted with PR^−/− MECs expressing ectopic RANKL and GFP. The sector highlighted in G is magnified in H; same sector shown with a fluorescent image taken before tissue processing (I). Note: area of rescue coincides with GFP expression. Areas showing the PR^−/− phenotype (arrows; G) do not express detectable levels of GFP (G). (Scale bar: 1 mm.) (H and I) 400 μm. (K) Double immunofluorescence for GFP (green) and BrdU (red) on mammary gland reconstituted with PR^−/− MECs expressing ectopic RANKL and GFP. Blue, DAPI. Note: BrdU incorporation in MECs adjacent to cells expressing viral GFP. (L) Anti-RANKL (green) and BrdU (red) immunofluorescence of histological sections of mammary glands from WT female at day 12.5 of pregnancy. Note: BrdU incorporation in MECs adjacent to cells expressing RANKL. (K and L Right) Zoom of marked area. (Scale bar: 40 μm.)

Fig. 5.

Systemic manipulation of RANKL signaling affects the mammary epithelium. (A) Twelve-week-old PR^−/− females were injected i.v. with either 8 μg of Fc-RANKL or PBS. BrdU was administered continuously for 72 h. BrdU-incorporating cells ± SEM; 1,200 cells were counted per mouse (n = 6), representing two independent experiments. (B and C) WT females were stimulated with progesterone and treated either with PBS or OPG. (B) BrdU incorporation in MECs is plotted ± SEM. Open bars, PBS treated (n = 9); filled bars, OPG treated (n = 12); three independent experiments were performed. (C) Double immunofluorescence of histological sections from mammary glands stimulated with progesterone and treated with PBS (Left) or OPG (Right). Green, PR; red, BrdU; blue, DAPI; bottom, overlay. Note: most BrdU(+) MECs in OPG-treated animals are PR(+).