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. 2014:2014:382653.
doi: 10.1155/2014/382653. Epub 2014 May 7.

Functional interactions between 17 β -estradiol and progesterone regulate autophagy during acini formation by bovine mammary epithelial cells in 3D cultures

Katarzyna Zielniok  1 , Tomasz Motyl  1 , Malgorzata Gajewska  1
Affiliations

Functional interactions between 17 β -estradiol and progesterone regulate autophagy during acini formation by bovine mammary epithelial cells in 3D cultures

Katarzyna Zielniok et al. Biomed Res Int. 2014.

Abstract

Mammary gland epithelium forms a network of ducts and alveolar units under control of ovarian hormones: 17-beta-estradiol (E2) and progesterone (P4). Mammary epithelial cells (MECs) cultured on reconstituted basement membrane (rBM) form three-dimensional (3D) acini composed of polarized monolayers surrounding a lumen. Using the 3D culture of BME-UV1 bovine MECs we previously demonstrated that autophagy was induced in the centrally located cells of developing spheroids, and sex steroids increased this process. In the present study we showed that E2 and P4 enhanced the expression of ATG3, ATG5, and BECN1 genes during acini formation, and this effect was accelerated in the presence of both hormones together. The stimulatory action of E2 and P4 was also reflected by increased levels of Atg5, Atg3, and LC3-II proteins. Additionally, the activity of kinases involved in autophagy regulation, Akt, ERK, AMPK, and mTOR, was examined. E2 + P4 slightly increased the level of phosphorylated AMPK but diminished phosphorylated Akt and mTOR on day 9 of 3D culture. Thus, the synergistic actions of E2 and P4 accelerate the development of bovine mammary acini, which may be connected with stimulation of ATGs expression, as well as regulation of signaling pathways (PI3K/Akt/mTOR; AMPK/mTOR) involved in autophagy induction.

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Figures

Figure 1
Figure 1
E-cadherin localization in acinar structures formed by BME-UV1 cells cultured on Matrigel for 12 days in differentiation medium (control), enriched with 17β-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E2 + P4); E-cadherin (epithelial cell marker) was labeled with antibodies conjugated with Alexa Fluor 488 (green fluorescence) and DNA was counterstained with 7AAD (red fluorescence).
Figure 2
Figure 2
Cleaved caspase-3 expression in BME-UV1 cells cultured on Matrigel in differentiation medium (control), enriched with 17β-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E2 + P4) for 3, 6, 9, 12, or 14 days; (a) images present immunofluorescence staining of cleaved caspase-3 (green fluorescence) and DNA counterstained with 7AAD (red fluorescence) and graph beside represents quantitative analysis of the intensity of green fluorescence of cleaved caspase-3 immunostaining, presented as the ratio of integrated optical density (IOD) of caspase-3 to IOD of nuclei in each acinus analysed; (b) Western blot analysis of the levels of cleaved caspase-3 in cell lysates: expression of β-actin was used as a loading control; graph beside the image represents the obtained results of densitometric analysis, in which IOD of each band was measured, and the values were normalized to IOD of β-actin; the IOD results are presented as means ± SEM from at least three separate experiments; statistically significant differences (P < 0.05) in comparison with control conditions were marked with *.
Figure 3
Figure 3
Confocal images of acinar structures formed by BME-UV1 cells cultured on Matrigel for 6, 9, or 12 days in differentiation medium (control), enriched with 17β-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E2 + P4); (a) panel of images presents immunofluorescence staining of p62 (green fluorescence): DNA was counterstained with 7AAD (red fluorescence); (b) graph representing quantitative analysis of the intensity of green fluorescence of p62 immunostaining, presented as the ratio of integrated optical density (IOD) of p62 to IOD of nuclei in each acinar structure analysed; *statistically significant difference (P < 0.05) in comparison with control conditions.
Figure 4
Figure 4
Expression of autophagic proteins (Atg5, Atg3, LC3, and beclin-1) and phosphorylated Bcl-2 (Ser70) in BME-UV1 cells forming acinar structures on Matrigel. Representative images of Western blot analysis of Atg5 (a), Atg3 (b), LC3 (c), beclin-1 (d), and phosphorylated Bcl-2 (Ser70) (f) in bovine MECs grown in 3D culture for 3, 6, 9, and 14 days in differentiation medium (control), enriched with 17β-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E + P); expression of β-actin was used as a loading control; graphs below the images show the results of densitometric analysis, in which IOD of each band was measured, and the values were normalized to IOD of β-actin; the IOD results are presented as means ± SEM from at least three separate experiments. (e) Confocal images of immunofluorescence staining of beclin-1 (green fluorescence) in cells grown in 3D culture for 3 or 9 days in control conditions: DNA was counterstained with 7AAD (red fluorescence) and white arrows indicate the nuclear localization of beclin-1.
Figure 5
Figure 5
Real-time PCR analysis showing the effects of (a) 17β-estradiol (1 nM), (b) progesterone (5 ng/mL), or (c) both steroid hormones administered simultaneously on the expression of chosen autophagy-related genes: ATG5, BECN1, ATG3, and LC3B in BME-UV1 cells cultured on Matrigel for 3, 6, 9, and 14 days; the levels of analyzed mRNA were normalized according to the relative GAPDH mRNA expression of each sample; the expression of each ATG under control conditions was appointed as 1 at each time point; a change in expression was considered significant (*) when at least 1.5-fold increase was obtained.
Figure 6
Figure 6
(a) Western blot analysis of the levels of chosen kinases (Akt, ERK, AMPK, and mTOR) involved in autophagy regulation, detected in BME-UV1 cells cultured on Matrigel for 3, 6, 9, and 14 days in differentiation medium (control), enriched with 17β-estradiol (E2, 1 nM), progesterone (P4, 5 ng/mL), or both (E + P): expression of β-actin was used as a loading control; (b) graphs represent the obtained results of densitometric analysis, in which IOD of each band was measured, and the IOD values for phosphorylated forms of kinases were normalized to the respective IOD of the total forms, with an exception of phos-mTOR, which was normalized to IOD of β-actin; results are presented as means ± SEM from at least three separate experiments; *statistically significant difference (P < 0.05) in comparison with control conditions.
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