Progesterone receptor A-regulated gene expression in mammary organoid cultures - PubMed (original) (raw)

Progesterone receptor A-regulated gene expression in mammary organoid cultures

Sarah J Santos et al. J Steroid Biochem Mol Biol. 2009 Jul.

Abstract

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

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Figures

Fig. 1

Fig. 1. Effect of R5020 and P on epithelial cell proliferation and organoid morphology

A. Mammary epithelial cells suspended in collagen I gels were cultured in BM, R5020 (20 nM), or P (20 nM). [3H] Thymidine incorporation into DNA was assayed after 3 d of culture. Each bar represents the mean ± SEM of triplicate values from a representative experiment. The data are expressed as the fold increase over the BM control. B. Organoid morphology was visualized in situ in collagen gels with the aid of an inverted microscope. Note the solid appearance of organoids from BM, whereas lumens are visible in R5020- and P-treated organoids. Scale bar = 100 μm.

Fig. 2

Fig. 2. R5020 treatment modulates levels of RNA expression in mouse mammary organoids

A. Table of the number of genes induced or repressed by R5020 in primary cultures from either adult or pubertal mice (fold change ≥ 2, P ≤ 0.05). B. Venn diagram of R5020-regulated genes in the adult and pubertal organoids.

Fig. 3

Fig. 3. Fold inductions of twelve R5020-regulated genes as determined by quantitative RT-PCR

Three to four pubertal (●) and adult (○) primary cultures were analyzed for expression of each gene, and the average inductions are shown as solid black lines. * = P ≤ 0.05, Agtr1, Calca, Defb1 and Tnfsf11 were more highly induced by R5020 in adult organoids than pubertal organoids.

Fig. 4

Fig. 4. Progestins induce SAA1 protein expression, and recruit leukocytes to the peri-epithelial stroma of small ducts, duct ends, and alveoli

A. Immunofluorescent detection of SAA1 (green) in adult organoids treated with basal media (BM) or R5020 for 24 hours. Nuclei (blue) were counterstained with DAPI. B. Immunofluorescent detection of SAA1 (green) in tissue sections from adult OVX mice treated with vehicle control (C) or progesterone (P) for 1 day. Nuclei (blue) were counterstained with DAPI. Scale bars (A,B) = 25 μm. C. Immunohistochemical detection of CD45-expressing leukocytes in mammary glands of OVX adult mice after 5 d treatment with vehicle control (i, ii) or progesterone (iii, iv, v). In control-treated sections, CD45+ leukocytes (brown cells marked by arrows) were sparsely localized in both ductal epithelium and peri-epithelial stroma (i, ii). In P-treated sections, CD45+ leukocytes were localized mainly in peri-epithelial stroma of ducts (iii), duct ends (iv), and alveoli (v). Scale bar = 50 μm. D. Quantitation of CD45+ leukocytes per unit area in the peri-epithelial stroma and epithelium of large ducts, small ducts, duct ends and alveoli after 5d C or P treatment. All structures were compared to control large ducts, and normalized to a value of 1 for CD45+ cells in control-treated large ducts. * = P ≤ 0.05.

Fig. 5

Fig. 5. Angiotensin II induces proliferation of organoids by itself and in synergy with R5020

Proliferation was measured by 3H-thymidine incorporation after 48-hour treatment with basal medium (BM), R5020 (20 nM), basal medium plus ANG II (BM + ANG II, 100nM), and R5020 plus ANG II (R5020 20 nM + ANG II 100 nM). Each bar represents the mean ± SEM of triplicate values from a representative experiment. * P≤0.05, BM + ANG II increased proliferation over BM alone. ** P≤0.05, R5020 + ANG II increased proliferation over any other treatment.

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