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. 2012 Sep;16(9):2035-48.
doi: 10.1111/j.1582-4934.2011.01499.x.

Functional consequences of prolactin signalling in endothelial cells: a potential link with angiogenesis in pathophysiology?

Anne Q Reuwer  1 , Patrycja Nowak-Sliwinska, Laurie A Mans, Chris M van der Loos, Jan H von der Thüsen, Marcel Th B Twickler, C Arnold Spek, Vincent Goffin, Arjan W Griffioen, Keren S Borensztajn
Affiliations

Functional consequences of prolactin signalling in endothelial cells: a potential link with angiogenesis in pathophysiology?

Anne Q Reuwer et al. J Cell Mol Med. 2012 Sep.

Abstract

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.

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Figures

Fig 1
Fig 1
Prolactin exerts a direct effect on endothelial cells. 2H11 endothelial cells, serum starved for 8 hrs, were stimulated for 30 min. with prolactin (10, 100, 500 or 1000 μg/l; lane 5–9) or with prolactin (100 or 500 μg/l) in combination with the prolactin receptor antagonist del1-9-G129R-hPRL (‘Anta’; 2000 or 10,000 μg/l; lane 1 and 2) or with del1-9-G129R-hPRL alone (2000 or 10,000 μg/l; lane 3 and 4). Cell lysates were analysed using Western blot for phosphorylated STAT5 (‘Phospho-STAT5’; top panel), phosphorylated ERK1/2 (‘Phospho-ERK1/2’; middle panel) and tubulin as loading control (bottom panel). A representative picture of an experiment is shown, which was performed three times with similar results.
Fig 2
Fig 2
Effect of prolactin on in vivo angiogenesis in the CAM. Images show the CAM (EDD9) with or without treatment with prolactin (PRL) and prolactin with its receptor antagonist (PRL+Anta). The vasculature is visualized by FITC-dextran fluorescence angiography (25 mg/kg, 20 kD, λex= 470 nm). (A) Untreated CAM with small vessels and the capillary network. (B) Angiography of a CAM treated with prolactin (1 μg/embryo/day). (C) Angiography after treatment with prolactin (1 μg/embryo/day) mixed with its receptor antagonist (10 μg/embryo/day). Scale bar in (A) is valid for all three images. Arrows indicate the induced pillar formation and intussusceptive angiogenesis in the larger blood vessels. (D, E) Quantification of digital analysis of the angiography images. (D) Branching points/mm2 for CAM, stimulated with 0.9% NaCl alone (control, white bar) or 0.9% NaCl supplemented with the indicated amounts of prolactin (PRL, black bars) or prolactin with antagonist (PRL + Anta, grey bar). (E) Number of segments/mm2 as a marker of vessel density for CAM, stimulated with 0.9% NaCl alone (control, white bar) or 0.9% NaCl supplemented with the indicated amounts of prolactin (PRL, black bars) or prolactin with antagonist (PRL + Anta, grey bar). Mean values (± standard error of the mean) are shown for two different experiments, in which each condition was included four times (thus eight individual eggs). **P < 0.01.
Fig 3
Fig 3
Prolactin slightly stimulates proliferation of endothelial cell cultures. (A) MTT assay on a cell culture of the 2H11 endothelial cell line in serum-free medium (control, white bar) or in serum-free medium supplemented with the indicated concentrations of prolactin (PRL, black bars). The mean (± standard error of the mean) of six different experiments performed in octuplicate is shown. (B) CellTiterGlo assay using primary human endothelial cells HUVECs in serum-free medium (control, white bar) or serum-free medium supplemented with the indicated concentrations of prolactin (PRL, black bars). The mean (± standard error of the mean) of two different experiments is shown, in which each condition was included either two or five times. Results shown in (A) and (B) are expressed as relative induction of proliferation (% of control). ***P < 0.001, **P < 0.01, *P < 0.05.
Fig 4
Fig 4
Prolactin stimulates endothelial cell migration. (A) Trans-well migration towards serum-free medium of 2H11 cells. Cells were stimulated for 18 hrs either with 1000 μg/l prolactin (PRL), serum-free medium (SF), the prolactin receptor antagonist del1-9-G129R-hPRL (Anta) alone (20,000 μg/l) or prolactin 1000 μg/l in combination with del1-9-G129R-hPRL 20,000 μg/l (PRL+Anta). Fluorescence was measured every 2 min. at the bottom side of the well. The level of fluorescence is shown and is representative for the number of migrated cells in time [relative fluorescence units (RFU)]. A representative example of an experiment performed three times is shown, in which each condition was included three- to sixfold. (B–E) Migration of HUVECs as assessed in the wound scratch assay. After the wound scratch was performed, serum-free medium (control) or serum-free medium supplemented with prolactin (PRL, 1000 μg/l) was added and wound closure was monitored for 8 hrs. (B) and (D) are representative pictures of the wound at t = 0 hr. (C) Representative picture of wound closure after 8-hr incubation in the control condition. (E) Representative picture of wound closure after 8-hr incubation with prolactin (1000 μg/l) over this period. Blue colour represents the digital calculated wound area, used for the analyses. (F) Quantification of wound closure with prolactin (PRL, black bar) expressed as percentage of the control (white bar). Mean values (± standard error of the mean) for three different experiments are shown. Each condition was included at least fivefold. ***P < 0.001, *P < 0.05.
Fig 5
Fig 5
Prolactin promotes endothelial tube formation. The 2H11 endothelial cells (5*104/well) were resuspended in (A) serum-free medium (control), (B) prolactin (PRL, 1000 μg/l) and (C) prolactin (1000 μg/l) in combination with antagonist del1-9-G129R-hPRL (PRL+Anta 20,000 μg/l) and tube formation was assessed after 18 hrs. The representative pictures of an experiment performed three times are shown. (D) Quantification of tube formation in the control condition (white bar) or 10–10,000 μg/l prolactin (PRL, black bars). (E) Quantification of tube formation in the control condition (white bar) or del1-9-G129R-hPRL alone (striped bar) or 1000 μg/l prolactin (PRL, black bar) or prolactin in combination with del1-9-G129R-hPRL (grey bar). (D–E) are shown as mean values (± standard error of the mean) for three different experiments. *P < 0.05 compared to the control, ∧P < 0.05 in comparison to prolactin 1000 μg/l.
Fig 6
Fig 6
Immunohistochemical staining of the prolactin receptor and CD34 in mammary carcinoma. (A) Staining of the prolactin receptor (turquoise) and CD34 (endothelial cells, purple), counterstaining with haematoxylin (blue). The pictures were digitally processed to unmix the different colours. (B) Fluorescence-like image of both prolactin receptor (green) and CD34 (red) in pseudo-colours. (C) Areas where prolactin receptor and CD34 co-localize, and endothelial expression of prolactin receptor. (D) and (E), respectively, show pseudo-fluorescent staining for the prolactin receptor (green), endothelial cells (red) and nuclei (blue). Scale bar = 0.075 μm in (A–E).
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