Role and regulation of autophagy in the development of acinar structures formed by bovine BME-UV1 mammary epithelial cells

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Abstract

Autophagy is a catabolic process providing an alternative energy source for cells under stressful conditions such as starvation, growth factor deprivation or hypoxia. During involution of the bovine mammary gland autophagy is induced in mammary epithelial cells (MECs) as a survival mechanism, and is tightly regulated by hormones and growth factors necessary for gland development. In the present study we adapted the three-dimensional culture model to investigate the role of autophagy during formation of alveoli-like structures by bovine BME-UV1 MECs grown on extracellular matrix (ECM) components. Using confocal microscopy and Western-blot analyses of autophagic and apoptotic markers: LC3, and cleaved caspase-3, we showed that autophagy was induced in centrally localized cells within the developing acini. These cells lacked a direct contact with ECM, and formed a distinct population from the outer layer of cells. Induction of autophagy preceded apoptosis, but did not inhibit the formation of a hollow lumen. In the presence of steroid hormones: 17β-estradiol and progesterone, although autophagy was augmented, acini formation proceeded normally. In contrast, the major lactogenic hormone: prolactin, which supports functional differentiation of alveoli, did not alter induction of autophagy within the spheroids. BME-UV1 cells cultured on Matrigel in the presence of growth factors IGF-I and EGF formed larger, underdeveloped acini without lumens due to caspase-3 inhibition, and sustained autophagy in the centre of the spheroids, while TGF-β1 accelerated apoptosis, and increased autophagy significantly. Our observations suggest that sex steroids 17β-estradiol and progesterone, as well as growth factor TGF-β1 may regulate the development of the bovine mammary gland by inducing autophagy in addition to regulating proliferation and apoptosis of MECs. These data indicate that autophagy may play an important role during alveolargenesis.

Introduction

Mammary gland development occurs mainly in the post-natal life of mammals, especially during puberty and gestation. The prepubertal period in ruminants is characterized by the development of a highly arborescent ductal network within loose connective tissue. During puberty ductal elongation is accomplished through the coordinated growth, branching and extension of terminal ductal units (TDU) (Capuco and Ellis, 2005). A fully developed organ is formed during gestation, when pregnancy hormones control the extensive growth of epithelium. This forms a network of ductules terminated by alveoli, which constitute the basic secretory units of the gland. In dairy cattle most of the mammary growth occurs prior to parturition and the initial peak of milk production is due to increased milk secretory activity per epithelial cell. A gradual decline in milk production is caused by the loss of secretory epithelial cells via programmed cell death (Capuco and Ellis, 2005). The most pronounced induction of apoptosis in mammary epithelial cells (MECs) is associated with cessation of milking at the beginning of the dry period (Wilde et al., 1997, Wilde et al., 1999). The cycle of proliferation, differentiation, and regression is repeated many times throughout the animal's life, and depends on the dynamic equilibrium between mitosis and apoptosis of MECs.

We have previously reported enhanced autophagy in bovine mammary glands at the end of lactation and during the dry period (Zarzyńska et al., 2007, Motyl et al., 2007). This was shown by both increased expression of beclin1 and morphological features of autophagy (autophagosomes and autophagolysosomes) in the majority of cells.

Autophagy is an evolutionarily conserved catabolic process whereby cytoplasmic proteins and organelles are targeted to lysosomes for degradation (Yorimitsu and Klionsky, 2005). Autophagy is a survival mechanism during starvation or growth factors deprivation as self-digestion provides an alternative energy source (Boya et al., 2005, Degenhardt et al., 2006, Lum et al., 2005). Our recent studies using the bovine mammary epithelial cell line BME-UV1 have shown that reduction of FBS concentration in culture medium (from 10% to 0.5%) mimics the naturally occurring decline in bioactive compounds and nutrients at the end of lactation and the dry period, causing induction of autophagy. This process is tightly regulated by hormones and growth factors responsible for the development of the mammary gland. IGF-I and EGF were shown to inhibit autophagy via the mTOR kinase pathway while steroid hormones: 17β-estradiol and progesterone stimulated autophagy in BME-UV1 cells (Sobolewska et al., 2009).

Autophagy also seems to play an important role during alveolar morphogenesis demonstrated in studies using three-dimensional (3D) culture models. In the 3D culture model mammary epithelial cells are cultured on extracellular matrix (ECM) components allowing them to organize into spherical structures that resemble alveoli formed in the gland during pregnancy (Debnath and Brugge, 2005). Upon seeding within a reconstituted basement membrane normal MECs first proliferate to form small cell masses, which then organize into acinar structures notable for the presence of a centrally localized, hollow lumen, and a single layer of polarized epithelial cells surrounding this lumen (Debnath and Brugge, 2005, Shaw et al., 2004). Formation of the lumen involves apoptosis of centrally localized cells lacking direct contact with the ECM, this process is known as anoikis (Debnath et al., 2002, Reginato and Muthuswamy, 2006). However, not only apoptosis is observed during lumen formation. Electron microscopic analysis of acini formed by human MEC line MCF-10A cultured in the 3D model revealed numerous autophagic vacuoles present in the centre of spheroids (Debnath et al., 2002). Further studies by Fung et al. (2008) have shown that depletion of autophagy-related genes (ATGs) inhibited autophagy induced during formation of acinar structures by MCF-10A epithelial cells. Loss of autophagy led to apoptosis and reduced clonogenic recovery after anoikis. Moreover, a stable reduction of ATG5 or ATG7 in MCF-10A cells enhanced luminal apoptosis during acini maturation, and failed to elicit long-term luminal filling even when the MECs overexpressed anti-apoptotic gene – Bcl-2. These finding suggest that induction of autophagy contributes to survival of epithelial cells during anoikis, and to lumen formation (Debnath, 2008, Fung et al., 2008).

Studies on mechanisms controlling alveolar morphogenesis in cattle are scarce. There are only a few reports on differentiation of bovine MECs grown in the 3D culture model (Delabarre et al., 1997, Kozlowski et al., 2009, Riley et al., 2010) and none refer to the role of autophagy in this process. Since our previous studies have shown the importance of autophagy in bovine BME-UV1 mammary epithelial cells survival, when exposed to stressful conditions, we decided to elucidate the role of this process during in vitro alveolar morphogenesis of the bovine mammary gland. We adapted the 3D model to culture bovine BME-UV1 cells on Matrigel – a commercially available reconstituted basement membrane (rBM). The aim of the present study was to determine the participation of autophagy in the process of acinar structures formation. Additionally, we investigated the influence of hormones (prolactin, 17β-estradiol and progesterone), and growth factors (IGF-I, EGF, TGF-β1), known to participate in the morphogenesis and remodelling of the mammary gland, on the induction of autophagy in 3D structures formed by bovine MECs.

Section snippets

Media and reagents

DME/F-12, RPMI-1640, NCTC 135, prolactin, α-lactose, lactalbumin hydrolysate, glutathione, bovine insulin, bovine holo-transferin, hydrocortisone, l-ascorbic acid, insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), prolactin (PRL), progesterone (P4), 17β-estradiol (E2), rapamycin, 3-methyladenine (3-MA) and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Antibodies against E-cadherin, and gapdh, as well as HRP-conjugated secondary antibodies were

Role of autophagy in the development of acinar structures by BME-UV1 cells cultured on Matrigel

BME-UV1 cells seeded on Matrigel formed spherical cell organoids, which developed a hollow lumen within 12 to 16 days of 3D culture. Immunostaining and confocal imaging for chosen markers of proliferation (Ki67), polarization (E-cadherin), and apoptosis (cleaved caspase-3) allowed us to determine the specific time points at which bovine MECs undergo changes, and form fully developed alveoli-like structures in vitro. Cells proliferated until the 9th day of 3D cell culture, as judged by the

Discussion

Three-dimensional basement membrane cultures of mouse and human mammary epithelial cells (primary and immortalized) have led to understanding of the molecular mechanisms responsible for development and function of mammary alveoli (Blatchford et al., 1999, Debnath et al., 2003, Murtagh et al., 2004, Petersen et al., 1992, Xian et al., 2005). There are very few studies using bovine mammary epithelial cells cultured on rBM matrix. We adapted the 3D model to culture the immortalized bovine MEC line

Acknowledgements

This work was supported by the Ministry of Science and Higher Education (grant no.: N N308 3067 33). The authors would like to thank Dr. Madeline Murphy from Conway Institute, University College Dublin for language corrections, and all valuable remarks.

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