Cyclic AMP regulates formation of mammary epithelial acini in vitro

Abstract

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

FIGURE 1:

cAMP is required for MCF10A acinus formation. (A) MCF10A cells were grown in three-dimensional culture in the absence or the presence of 500 μM CPT-cAMP for different periods of time, and fixed and stained for β-catenin (red), GM130 (green), and nuclei (blue). Scale bars: 20 μm. (B) Top, representative images of acini with filled, mostly filled, mostly clear, and clear lumens. Nuclei were detected by DAPI (blue) and apoptotic cells by cleaved caspase 3 staining (red). Bottom, quantification of acini with varying degrees of luminal filling (as depicted above) when grown in the absence (DMSO, left) or presence of 500 μM CPT-cAMP (right). Data are means of three independent experiments with at least 100 acini counted for each time point. (C) MCF10A cells were grown in three-dimensional culture for 17 d in the presence of 100 ng/ml cholera toxin (ChTx), 10 μM forskolin (FSK), or 500 μM CPT-2′-O-Me-cAMP (2′-Me-cAMP), and fixed and stained as in (A). Scale bars: 20 μm. (D) MCF10A cells were grown in the absence (DMSO) and presence (cAMP) of 500 μM CPT-cAMP, and the areas of equatorial cross-sections were measured. Data are mean ± SEM for three independent experiments with more than 100 acini counted for each condition. ***, p < 0.01. (E) HPV E7-expressing MCF10A cells were grown in three-dimensional culture for 17 d in the presence or absence of 500 μM CPT-cAMP (cAMP), and fixed and stained to detect nuclei (blue) and F-actin (red). Scale bars: 50 μm.

FIGURE 2:

cAMP induces luminal apoptosis and increases BIM in MCF10A acini. (A and B) MCF10A cells were grown in three-dimensional culture in the presence or absence of 500 μM CPT-cAMP for indicated times. Dead cells were visualized by EtBr staining (A, left); apoptotic cells were visualized by TUNEL-staining (A, right). Note: positive acini were considered to have at least one EtBr- or TUNEL-positive cell. Quantitative data are means ± SEM of three independent experiments with more than 100 total structures analyzed for each condition. (B) Representative images of 10-d-old TUNEL-stained (red) acini are shown. The numbers on the images are means ± SEM of TUNEL-positive cells per acinus (three independent experiments with 36 and 30 randomly selected acini analyzed for DMSO and cAMP treatment, respectively). Statistically significant differences vs. DMSO-treated cells are indicated: ***, p < 0.001. Scale bar: 20 μm. (C) MCF10A cells were grown as in (A). BIM, pERK, and GAPDH were detected by Western blotting. Representative blots are shown. BIM_EL (*), BIM_L (º), and BIM_S (†) splice variants are marked. (D) MCF10A cells were grown in three-dimensional culture for 7 d and treated thereafter with 500 μM CPT-cAMP or 0.5% DMSO for another 24 h. BIM, pERK, ERK, and GAPDH were detected by Western blotting.

FIGURE 3:

cAMP and ECM detachment have distinct and separable effects on BIM and pERK levels in mammary epithelial cells. (A) MCF10A cells were grown in suspension culture in the presence or absence of ECM (2% Matrigel) and/or 500 μM CPT-cAMP. At 24 h, mRNA expression levels of BIM and Bmf were quantified by qPCR and normalized to those for DMSO controls grown in the presence of ECM. Shown are means ± SEM of five independent experiments, each performed in duplicate. Statistically significant differences compared with control (presence of ECM, absence of cAMP; open black bars) are indicated: **, p < 0.001; ***, p < 0.001. (B) MCF10A cells were grown in suspension culture in the presence or absence of ECM (2% Matrigel) or combination of ECM and inhibiting anti–β1-integrin antibody (AIIB2; 1/200), and treated with 0.5% DMSO or 500 μM CPT-cAMP for 24 h. (C) MCF10A cells or NMuMG cells were treated with 0.5% DMSO, 500 μM CPT-cAMP (CPT-), 500 μM CPT-2′-Me-cAMP (2′-Me-), or 500 μM Bnz-cAMP (Bnz-) for 24 h. (D) Primary HMEC were grown in the presence of ECM (2% Matrigel) or a combination of ECM and inhibiting anti–β1-integrin antibody (AIIB2; 1/200), and treated with 0.5% DMSO or 500 μM CPT-cAMP for 24 h. (E) Control or EGFR-expressing MCF10A cells were grown in suspension culture in the presence or absence of 500 μM CPT-cAMP for 24 h. Indicated proteins were detected by Western blotting.

FIGURE 4:

cAMP accelerates polarization of MCF10A acini. (A and B) MCF10A acini were treated with 0.5% DMSO or 500 μM CPT-cAMP starting at day 3, fixed at time points indicated, and stained for α6-integrin. Shown are representative images (A) and the percentage of α6-integrin at the peripheral rim of the acinus (B; for quantification details see Materials and Methods). Data are means ± SEM of three independent experiments. Between 50 and 90 acini were analyzed for each condition. Statistically significant differences vs. DMSO-treated cells are indicated: ***, p < 0.001. (C) MCF10A acini were treated with 10 μM gefitinib or 10 μM U0126 starting at day 3 and were fixed 24 h later. Top, β-catenin (red), GM130 (green), and nuclei (blue); bottom, α6-integrin staining. (D) Day 6 MCF10A acini treated with 0.5% DMSO, 500 μM CPT-cAMP, anti–β1-integrin (AIIB2), or anti–α6-integrin (GoH3 1/50) antibodies and stained for β-catenin (red), GM130 (green), and nuclei (blue). (E) Quantification of percentage of polarized acini at day 6 when treated as indicated in (D). Data are means ± SEM of three independent experiments; more than 100 acini were scored for each condition. Statistically significant differences vs. DMSO-treated cells are indicated: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bars: 20 μm.

FIGURE 5:

β-Adrenergic agonist, isoproterenol, increases cAMP and induces acinus formation in MCF10A cells. (A) MCF10A cells were treated with 10 μM forskolin (FSK) or 100 nM or 1 μM isoproterenol (ISO); cAMP levels were measured and expressed as fold increase of untreated cells. Shown are means ± SEM of three independent experiments. Statistically significant differences vs. control cells are indicated: *, p < 0.05; ***, p < 0.001. (B) MCF10A cells were grown in three-dimensional culture in the presence or absence of 100 nM isoproterenol (ISO) for 15 d, fixed, and stained for β-catenin (red), GM130 (green), and nuclei (blue). Bottom, quantification of acini with varying degrees of luminal filling (as depicted in Figure 1B) when grown in the absence (Control) or presence (ISO) of 100 nM isoproterenol for 14 d. Data are means of three independent experiments with at least 100 acini counted for each time point.