Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo

JUDITH AGGELER 0 1 JEROME WARD 0 1 LESLIE MACKENZIE BLACKIE 0 0 San Francisco , CA 94121 , USA 1 Department of Cell Biology and Human Anatomy, School of Medicine, University of California , Davis, CA 95616 , USA 2 Division of Cell and Molecular Biology, Lawrence Berkeley Laboratory , Berkeley, CA 94720 , USA Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to - In the present study we provide evidence that the cytodifferentiation of primary mouse mammary epithelial cells within the alveolar-like structures formed after culture on a reconstituted basement membrane resembles development in vivo during late pregnancy and early lactation. During the first two days in culture on a basement membrane gel in the presence of lactogenic hormones, epithelial cells isolated from mid-pregnant mice are disorganized and central lumina are largely absent. Levels of mRNA for the milk proteins, /J-casein and transferrin, are dramatically reduced. By the second or third day in culture, cytoplasmic polarization becomes evident and prominent apical junctional complexes are formed. Synthesis of both mRNA and milk protein is reinitiated at this time. By day 4, welldefined lumina appear, and abundant synthesis and secretion of casein and lipid is observed. A striking feature of this differentiation in culture is the specific The development and function of the mammary gland have been subjects of intense interest for many years. Pioneering studies of its morphological differentiation during pregnancy and lactation were among the earliest electron-microscopic investigations to trace the subcellular route of newly synthesized secretory proteins from the rough endoplasmic reticulum (RER) to the trans Golgi face, where secretory vesicles are formed (Wellings et al. 1960; Bargmann, 1962; Kurosumi et al. 1968; Wellings, 1969). During the past decade, many studies have been focussed on both molecular and cellular aspects of milk protein gene expression. A variety of milk protein genes have now been cloned, and their regulatory sequences are beginning to be dissected (Bisbee and Rosen, 1987). At the same time the cellular mechanisms underlying the expression of milk proteins have been studied in some detail, especially through the development of mammary epithelial cell culture systems (Bissell and Hall, 1987). These cell culture models confer many advantages for localization of milk protein gene expression (/f-casein mRNA) to lumina] epithelial cells in the alveolar-like structures. At the ultrastructural level, increased milk protein synthesis and secretion are paralleled by a fourfold increase in rough ER that resembles the dramatic increase in the ER observed in vivo following parturition. One indication of tissuespecific differentiation observed in later cultures (days 4-11) is the synthesis and secretion of abundant casein micelles. A second characteristic of lactating mammary epithelial cells in vivo that has not previously been observed in culture is the secretion of milk fat globules. Taken together, these observations indicate that mammary epithelial cells plated onto a reconstituted basement membrane differentiate to the lactating phenotype in culture. investigating the mechanisms underlying differentiation and tissue-specific gene expression, including the ability to isolate a single cell type, to manipulate hormonal stimuli and other growth conditions, and to control the microenvironment, including cell-cell and cell-matrix interactions. In the case of the mammary epithelium, such studies have made it clear that the underlying basal lamina and its components exert important control over milk protein expression (Li et al. 1987; Aggeler et al. 1988; Streuli and Bissell, 1990). Our previous work has indicated that mammary epithelial cells derived from mid-pregnant animals and cultured on a reconstituted basement membrane re-form alveolar-like structures and synthesize and secrete milk proteins into sealed lumina (Barcellos-Hoff et al. 1989). We now report that alveolar re-formation is accompanied by differentiation of these cultured mammary epithelial cells from a mid-pregnant phenotype to one closely resembling lactation. In addition, expression of the tissue-specific milk protein, /3-casein, appears to be localized to highly differentiated luminal epithelial cells. Materials and methods Cell culture Primary mouse mammary epithelial cells were isolated from midpregnant (13- to 15-day) CD-I mice (Charles Rivers Laboratories, Wilmington, MA), as previously described (Lee et al. 1984; Bissell etal. 1987; Barcellos-Hoff etal. 1989). After mincing and digestion with collagenase and trypsin, epithelial cells were separated by differential low-speed centrifugation, resuspended in F-12 medium containing 10 % fetal bovine serum (both from Gibco, Grand Island, NY), and plated at 2xlOs to 5xl06cellscm"2 in culture dishes precoated with a reconstituted basement membrane extract isolated from the Englebreth-Holm-Swann (EHS) tumor (Kleinman et al. 1986). Serum was removed after 2 days of culture and cells were maintained in serum-free medium for the remainder of the experiment. Fresh medium was added every day. All cultures were carried out in the presence of the lactogenic hormones, ovine prolactin O/zgrnl"1; National Hormone and Pituitary Program, National Institutes of Health, Bethesda, MD), hydrocortisone (2figml"1) and insulin (SfigmP1) (both from Sigma Chemical Co., St Louis, MO), which were added daily. The isolated cell preparations contained >90 % epithelial cells (Seely and Aggeler, 1991), as judged by staining for cytokeratins (Lane, 1982). Immunofluorescence microscopy and in situ hybridization Pregnant and lactating mammary gland and cultured mammary epithelial cells were prepared for immunofluorescence microscopy or in situ hybridization by fixation with 2% paraformaldehyde, followed by freezing and sectioning with a Leitz cryotome, as previously described (Streuli and Bissell, 1990). Immunofluorescence microscopy was carried out using a /3-casein-specific mouse monoclonal antibody (kindly supplied by C. Kaetzel, Case Western Reserve University, Cleveland, OH), followed by biotinylated rabbit anti-mouse IgG and streptavidin-Texas Red (both from Amersham Corp., Arlington Heights, IL). Slides were counterstained with 4',6'-diamidino-2-phenyl-indole (DAPI) (Sigma) to localize cell nuclei. The distribution of /J-casein mRNA was determined by in situ hybridization, according to the method of Cox et al. (1984). A total of 540 bases of mouse /3-casein cDNA coding sequence (originally isolated by Dr Jeffrey Rosen, Baylor College of Medicine, Houston, TX) were recloned into transcription vectors (PGEM-1, Promega Corp., Madison, WI; and pT7/T3ol9, Bethesda Research Labs, Gaithersburg, MD) and high specific activity ^S-labeled riboprobes (sense and antisense, respectively) were prepared by transcription using bacte (...truncated)