Membrane expression of the estrogen receptor ERα is required for intercellular communications in the mammary epithelium

ABSTRACT 17β-Estradiol induces the postnatal development of mammary gland and influences breast carcinogenesis by binding to the estrogen receptor ERα. ERα acts as a transcription factor but also elicits rapid signaling through a fraction of ERα expressed at the membrane. Here, we have used the C451A-ERα mouse model mutated for the palmitoylation site to understand how ERα membrane signaling affects mammary gland development. Although the overall structure of physiological mammary gland development is slightly affected, both epithelial fragments and basal cells isolated from C451A-ERα mammary glands failed to grow when engrafted into cleared wild-type fat pads, even in pregnant hosts. Similarly, basal cells purified from hormone-stimulated ovariectomized C451A-ERα mice did not produce normal outgrowths. Ex vivo, C451A-ERα basal cells displayed reduced matrix degradation capacities, suggesting altered migration properties. More importantly, C451A-ERα basal cells recovered in vivo repopulating ability when co-transplanted with wild-type luminal cells and specifically with ERα-positive luminal cells. Transcriptional profiling identified crucial paracrine luminal-to-basal signals. Altogether, our findings uncover an important role for membrane ERα expression in promoting intercellular communications that are essential for mammary gland development.

: A-B) Representative images of Ki67, ERα, PR and active-caspase 3 immunostaining of mammary glands from 5-week-old (A) and 3 to 6-month-old (B) virgin WT and C451A-ERα mice (t-test, *P <0,05; scale bar=100 µm). The proportion of epithelial cells expressing Ki67 (both on distal ducts and terminal end buds (TEB), ERα, PR and active caspase 3 was quantified as percentage of total epithelial cells. On B) Confocal images of mammary gland epithelium after immunostaining with anti-K5(Green) and K8 (magenta) primary antibodies (scale bar=20μm or 5µm). C) Levels of the ERα protein normalized to GAPDH in mammary glands from 5-week-old or 3-6-month-old C451A-ERα and WT mice were assessed using Western blotting. Uteri were used as positive controls (C+) (two-way ANOVA, ns: not significant).

Figure S4: The C451A-ERα mutation does not modify the clonogenic capacity of luminal and basal cells in vitro, but impacts matrix degradation A)
Hematoxylin-eosin staining was performed to evaluate the clonogenic capacity of luminal CD24 + CD29 lo cells seeded on irradiated fibroblast feeder cells after 7 days in culture. Box plots show the number of luminal colonies generated when 1000 cells were plated, and area of the different obtained clones (n= 3 independent experiments from luminal WT or ERα-C451A cells pooled from 2 to 4 mice (t-test, the P value was not significant)). B) Representative images of mammosphere colony forming assays from luminal and basal cells grown on matrigel. After one generation, cells from the colonies were collected and plated again for the colony forming assay at generations II and III. The data indicate the number of mammospheres obtained from 5000 cells (n=4 independent experiments from luminal WT or C451A-ERα cells pooled from 2 to 4 mice each (t-test, the P value was not significant).

Figure S5: Images of immunofluorescence staining of green-fluorescent gelatin degradation by double-sorted CD29 hi CD24 + basal cells in vitro
A) Representative images of degraded A488-labeled gelatin appearing in black. The areas of degraded gelatin were measured in at least 18 fields per genotype in each experiment and an average of 281 cells was counted from each genotype in each experiment (scale bar=50µm). B) The area of gelatin degraded by gate-sorted C451A-ERα basal cells was compared to the area of gelatin degraded by cells purified from their WT littermates within the same experiment. The graph shows the means of four independent experiments using WT or C451A-ERα cells pooled from 2 to 4 mice each (t-test, **P value<0.01). C) Inhibition of gelatin degradation was observed when the non selective MMP inhibitor (marimastat) was added on the culture medium. The proportion of ERα positive cells following immunohistochemistry using anti-ERa antibody was quantified in the regenerated mammary gland obtained 8 weeks after co-transplantations of GFP-positive C451A-ERα or WT basal cells with GFP-negative ERα-WT luminal cells.

Figure S7: A)
Predicted gene-gene interaction network (Search tool for the Retrievel of interacting Genes/StringV10) among genes significantly down-regulated in C451A versus WT mice by E2 (FC>1.5, P>0.05). Genes coloured in red belong to the GO category of «extracellular region», in blue to the «Plasma membrane» and in green to the « basal membrane protein». B) Gene network among the 9 genes found in the GO category of genes belonging to the positive regulation of cell migration, including Stat5a, a related gene with C) the corresponding Log fold change indicated in the right. D) Model of impact of membrane ERα on stem cells interfering with pathways induced by Growth-Hormone Receptors (GH-R) and FGF-R receptors impacting the Jak2/Stat5 signalling pathways, modified from Furth et al., 2011.

Figure S8: A)
Predicted gene-gene interaction network (Search tool for the Retrievel of interacting Genes/StringV10) among genes significantly down-regulated in C451A versus WT mice by E2+Progesterone (Pg) (FC>1.5, P>0.05). Genes coloured in red belong to the GO category of «extracellular matrix protein», in blue to the «membrane protein». In B) the Log fold change of genes found in the network are indicated. Table S1: Summary of co-transplantation experiments performed mixing C451A-ERα MaSCs with either Sca1 + CD133+ or Sca1 -CD133 -WT luminal cells at a ratio of 5 basal/1 luminal cells.

Movie 1:
Movie showing different angles of the confocal images of the 3D-reconstructed outgrowths obtained 8 weeks after transplantation of a mixture of GFP positive C451A MaSCs with WT luminal cells, after labeling with anti-ERα (red) and anti-GFP (green) antibodies.