TGFB1 disrupts the angiogenic potential of microvascular endothelial cells of the corpus luteum.

Cyclical formation and regression of the ovarian corpus luteum is required for reproduction. During luteal regression, the microvasculature of the corpus luteum is extensively disrupted. Prostaglandin F2α, a primary signal for luteal regression, induces the expression of transforming growth factor β1 (TGFB1) in the corpus luteum. This study determined the actions of TGFB1 on microvascular endothelial cells isolated from the bovine corpus luteum (CLENDO cells). We hypothesized that TGFB1 participates in the disruption of the microvasculature during luteal regression. TGFB1 activated the canonical SMAD signaling pathway in CLENDO cells. TGFB1 (1 ng/ml) significantly reduced both basal and fetal-calf-serum-stimulated DNA synthesis, without reducing cell viability. TGFB1 also significantly reduced CLENDO cell transwell migration and disrupted the formation of capillary-like structures when CLENDO cells were plated on Matrigel. By contrast, CLENDO cells plated on fibrillar collagen I gels did not form capillary-like structures and TGFB1 induced cell death. Additionally, TGFB1 caused loss of VE-cadherin from cellular junctions and loss of cell-cell contacts, and increased the permeability of confluent CLENDO cell monolayers. These studies demonstrate that TGFB1 acts directly on CLENDO cells to limit endothelial cell function and suggest that TGFB1 might act in the disassembly of capillaries observed during luteal regression

Distinct effects of EGFR ligands on human mammary epithelial cell differentiation.

Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs). Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR) and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT)-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+)K19(-) hMECs). As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+)K19(-) hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF), amphiregulin (AREG) and transforming growth factor alpha (TGFα) in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, interfered with K5(+)K19(-) hMEC differentiation. The present data validate the utility of the K5(+)K19(-) hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation

MEK inhibitor blocks differentiation of K5⁺K19^- hMECs.

<p>(<b>A</b>) Undifferentiated K5⁺K19^- hMECs were starved of serum/growth factors for 48 hours in D3 medium, treated with 1 µM U0126 or vehicle (DMSO) alone for 4 hours before stimulation with 5 nM EGF for the indicated period. Cell lysate was analyzed by immunoblotting. (<b>B</b>) K5⁺K19^- hMECs were propagated in MEGM medium (containing EGF) with or without 1 µM U0126 for three weeks. Medium was replaced every two days. Overall cell morphology was assessed by Wright-Giemsa staining (top panels) and K5 (green) and MUC1 (red) expression was assessed by immunofluorescence microscopy (middle panels). Nuclei were visualized with DAPI (blue). Red bars indicate 50 µM. Expression of CD49f and EpCAM was analyzed by flow cytometry (bottom panels). Gates and percentages for CD49f^loEpCAM^hi (luminal, green box) and EpCAM^lo (myoepithelial, red box) populations are indicated. Shown are representative results from 3 independent experiments. (<b>C</b>) Undifferentiated K5⁺K19^- hMECs were starved of serum/growth factors for 48 hours in D3 medium, treated with 5 µM wortmannin or vehicle (DMSO) alone for 4 hours before stimulation with 5 nM EGF for the indicated period. Cell lysate was analyzed by immunoblotting. (<b>D</b>) K5⁺K19^- hMECs were propagated in MEGM medium (containing EGF) with or without 5 µM wortmannin for ten days. At this time point, control culture has not differentiated yet. Medium was replaced every two days. Overall cell morphology was assessed by Wright-Giemsa staining (top panels) and K5 (green) and MUC1 (red) expression was assessed by immunofluorescence microscopy (middle panels). Nuclei were visualized with DAPI (blue). Red bars indicate 50 µM. Expression of CD49f and EpCAM was analyzed by flow cytometry (bottom panels). Gates and percentages for CD49f^loEpCAM^hi (luminal, green box) and EpCAM^lo (myoepithelial, red box) populations are indicated. Shown are representative results from 2 independent experiments.</p

All EGFR ligands support growth of K5⁺K19^- hMECs before and after differentiation.

<p>(<b>A</b>) Undifferentiated K5⁺K19^- hMECs were starved of serum/growth factors for 24 hours in D3 medium before being left unstimulated (Control) or stimulated with AREG, EGF or TGFα (all at 5 nM) for 24 hours. Cell growth was assessed by [³H] thymidine incorporation for the last 6 hours of incubation. A representative result from 2 independent experiments run in triplicates is shown. Error bars indicate standard errors. (<b>B</b>) K5⁺K19^- hMECs were propagated in MEGM medium (with EGF) to induce differentiation. Differentiated luminal (CD49f^loEpCAM^hi) and myoepithelial (EpCAM^lo) cells were separated by FACS and plated in modified MEGM medium containing either AREG, EGF or TGFα (all at 5 nM). The percentage of proliferating cells was assessed by the expression of Ki67. Each condition was run in 5 replicates. Error bars represent standard errors. The difference between EGF and TGFα, as well as that between AREG and TGFα in CD49f^loEpCAM^hi cells was statistically significant at p<0.05 when analyzed by one-way ANOVA with Bonferroni multiple comparisons.</p

Biochemical consequences of EGFR engagement with various ligands.

<p>Undifferentiated K5⁺K19^- hMECs were starved of serum/growth factors for 48 hours in D3 medium before being stimulated with 5 nM EGF, AREG or TGFα for indicated period. Cell lysate was analyzed by immunoblotting. (<b>A</b>) Immunoblot results of phosphotyrosine (p-Tyr), total EGFR, phospho-p44/42 MAPK (p-Erk1/2), total p44/42 MAPK (Erk1/2), phospho-Akt (p-Akt) and total Akt. HSC70 was used as loading control. A representative of 2 independent experiments is shown. (<b>B</b>) Results from 2 independent experiments were quantitated by densitometry and ratios of p-Erk/total Erk and p-Akt/total Akt were plotted. Shown are averages of 2 experiments; error bars indicate standard errors. Y axis is in arbitrary unit.</p

In vitro differentiation of K5⁺K19^- h<i>TERT</i>-immortalized mammary epithelial cells.

<p>Cells were either maintained under non-differentiating condition (DFCI-1 medium) or propagated under differentiation-promoting condition (MEGM medium containing 5 nM EGF) for three weeks and cell morphology and marker expressions were evaluated. Shown are representative results from more than 10 independent experiments with similar outcome. (<b>A</b>) Overall cell morphology was assessed by Wright-Giemsa staining (top panels) and K5 (green) and MUC1 (purple) expression was assessed by immunofluorescence microscopy (bottom panels). Nuclei were visualized with DAPI (blue). Red bars indicate 50 µM. (<b>B</b>) Expression of CD49f, EpCAM, MUC1 and CD10 was assessed by flow cytometry. Gates for CD49f^loEpCAM^hi (luminal, green box), EpCAM^lo (myoepithelial, red box) and CD49f^hiEpCAM^hi (undifferentiated, black box) cells are indicated. Histograms on the right indicate levels of MUC1 (luminal marker, top) and CD10 (myoepithelial marker, bottom) in cells propagated in MEGM medium. Green lines represent the levels of MUC1 (top) or CD10 (bottom) in the CD49f^loEpCAM^hi (luminal) population, red lines are for the EpCAM^lo (myoepithelial) population and black lines for the CD49f^hiEpCAM^hi (undifferentiated) population. (<b>C</b>) Expression of α-smooth muscle actin (αSMA) and K5 was assessed by immunoblotting. NIH3T3 cells (lane 1) were included as a positive control for αSMA. Lane 2: K5⁺K19^- hMECs maintained in DFCI-1 medium; Lanes 3-5: K5⁺K19^- hMECs were differentiated in MEGM medium and sorted into CD49f^loEpCAM^hi (luminal), EpCAM^lo (myoepithelial) and CD49f^hiEpCAM^hi (undifferentiated) populations. Membrane was probed for HSC70 to ensure equal loading.</p

Effects of various EGFR ligands on K5⁺K19^- hMEC differentiation in MEGM medium.

<p>Cells were propagated in modified MEGM media where EGF was substituted with either AREG or TGFα and morphology and marker expressions were analyzed after three weeks. (<b>A</b>) Cell growth during differentiation. Two hundred thousand (2 x 10⁵) K5⁺K19^- hMECs were seeded in 60 mm dishes in modified MEGM media with indicated EGFR ligands. Cell numbers were determined every week. Shown are averages from 4 independent experiments. Error bars indicate standard errors. There was no statistically significant difference between groups in cell number at each time point by one-way ANOVA with Bonferroni multiple comparisons. (<b>B</b>) Overall cell morphology was assessed by Wright-Giemsa staining (left panels) and K5 (green) and MUC1 (purple) expression was assessed by immunofluorescence microscopy (right panels). Nuclei were visualized with DAPI (blue). Red bars indicate 50 µM. (<b>C</b>) Expression of CD49f, EpCAM, MUC1 and CD10 was analyzed by flow cytometry. Gates and percentages for CD49f^loEpCAM^hi (luminal, green box), EpCAM^lo (myoepithelial, red box) and CD49f^hiEpCAM^hi (undifferentiated, black box) populations are indicated in the top panels. Middle and bottom panels are histograms for MUC1 (middle) and CD10 (bottom). Green lines represent the levels of MUC1 (middle) or CD10 (bottom) in the CD49f^loEpCAM^hi (luminal) population, red lines are for the EpCAM^lo (myoepithelial) population and black lines for the CD49f^hiEpCAM^hi (undifferentiated) population. Histograms for EpCAM^lo (myoepithelial) populations in AREG-treated cells and CD49f^loEpCAM^hi (luminal) and CD49f^hiEpCAM^hi (undifferentiated) populations in TGFα-treated are not shown in the overlays due to extremely small cell numbers. Though the difference in MUC1 expression between EGF-treated cell populations was not as robust as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075907#pone-0075907-g001" target="_blank">Figure 1B</a> in this particular experiment, MUC1 mean fluorescence intensity for CD49f^loEpCAM^hi (luminal) cells (642) was higher than that of EpCAM^lo (myoepithelial) or CD49f^hiEpCAM^hi (undifferentiated) cells (437 and 337, respectively). (<b>B</b>) and (<b>C</b>) are representative results from 6 independent experiments with similar outcome.</p