Research Article
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ECM-induced gap junctional communication enhances mammary epithelial cell differentiation Marwan E. El-Sabban1,*, Agnel J. Sfeir2, Myriam H. Daher2, Nada Y. Kalaany2, Rola A. Bassam2 and Rabih S. Talhouk2,* 1Department
of Human Morphology, Faculty of Medicine, and 2Department of Biology, Faculty of Arts and Sciences, American University of Beirut, PO Box 11-0236, Beirut, Lebanon
*Authors for correspondence (e-mail:
[email protected];
[email protected])
Accepted 7 May 2003 Journal of Cell Science 116, 3531-3541 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00656
Summary The relationship between gap junctional intercellular communication (GJIC) and mammary cell (CID-9) differentiation in vitro was explored. CID-9 cells differentiate and express β-casein in an extracellular matrix (ECM)- and hormone-dependent manner. In response to interaction with the ECM, cells in culture modulated the expression of their gap junction proteins at the transcriptional and post-translational levels. In the presence of EHS-matrix, connexins (Cx)26, 32 and 43 localized predominantly to the plasma membrane, and enhanced GJIC [as measured by Lucifer Yellow (LY) dye transfer assays] was noted. Inhibition of GJIC of cells on EHS-matrix with 18α glycyrrhetinic acid (GA) resulted in reversible downregulation of β-casein expression. In the presence of cAMP, cells cultured on plastic expressed βcasein, upregulated Cx43 and Cx26 protein levels and
Introduction Gap junctional intercellular communication (GJIC) is critical in diverse cell and tissue functions (White and Paul, 1999). Gap junctions are perceived as ‘modulators of cellular differentiation’ in several systems (Pitts et al., 1988; Paul et al., 1995; Bruzzone et al., 1996; Kumar and Gilula, 1996) and recent studies have revealed a role for connexins in stratification and differentiation of human epidermal cells (Wiszniewski et al., 2000) and lung alveolar epithelial cells (Alford and Rannels, 2001), and in bone homeostasis, promoting osteoblast differentiation (Gramsch et al., 2001; Romanello et al., 2001; Schiller et al., 2001). The basic structural component of the gap junction is connexin (Cx). Connexins constitute a family of more than 20 homologous proteins that are temporally and spatially distributed throughout the body (reviewed by Goodenough et al., 1996; Kumar and Gilula, 1996; Kidder and Mhawi, 2002). Besides humoral mediators (Hynes et al., 1997; Hennighausen et al., 1997), the extracellular matrix (ECM) has been regarded as the dominant regulator of mammary differentiation (Weaver et al., 1997; Boudreau and Bissell, 1998; Schmeichel et al., 1998; Smalley et al., 1999; Klinowska and Streuli, 2000; Hansen and Bissell, 2000). The few studies that addressed the role of cell-cell interaction in mammary differentiation (Streuli et al., 1991; Desprez et al., 1993; Alford and Taylor-Papadimitriou, 1996; Hansen and Bissell, 2000) undervalued its role as compared with the effect exerted by the ECM.
enhanced GJIC. This was reversed in the presence of 18α GA. cAMP-treated cells plated either on a non-adhesive PolyHEMA substratum or on plastic supplemented with function-blocking anti-β1 integrin antibodies, maintained β-casein expression. These studies suggest that cell-ECM interaction alone may induce differentiation through changes in cAMP levels and formation of functional gap junctions. That these events are downstream of ECM signalling was underscored by the fact that enhanced GJIC induced partial differentiation in mammary epithelial cells in the absence of an exogenously provided basement membrane and in a β1-integrin- and adhesion-independent manner. Key words: Connexins, Differentiation, ECM, GJIC, Mammary
Studies have suggested that gap junctions play a critical role in the coordinated changes through development, differentiation, maintenance and involution of the mammary gland (Monaghan et al., 1994; Monaghan et al., 1996; Pozzi et al., 1995; Yamanaka et al., 1997; Locke et al., 2000; Yamanaka et al., 2001). However, no studies have established a clear correlation between functional GJIC and mammary epithelial differentiation, either in vivo (Perez-Armendariz et al., 1995; Pozzi et al., 1995; Monaghan and Moss, 1996; Locke et al., 2000) or in vitro (Lee et al., 1991; Lee et al., 1992; Tomasetto et al., 1993; Hirschi et al., 1996; Sia et al., 1999). The CID-9 mouse mammary cell culture system, responsive to both lactogenic hormones and substrata, consists of a heterogeneous cell strain of epithelial, myoepithelial and fibroblastic cells and is a widely accepted model that mimics in vivo differentiation of mammary cells (Schmidhauser et al., 1992; Talhouk et al., 2001). To determine the role of GJIC in modulating the differentiation phenotype of mammary cells, gap junction proteins of mammary CID-9 cells were characterized and their regulation by ECM assessed. The cause-and-effect relationship between GJIC and mammary epithelial differentiation was also investigated. We demonstrate that mammary CID-9 cells express Cx26, Cx32 and Cx43 proteins, which are modulated by ECM, and that proper cellECM interaction favours GJIC. Our studies suggest CID-9 cells are capable of differentiating and expressing β-casein in
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Journal of Cell Science 116 (17)
the absence of an exogenous basement membrane in a β1integrin-independent pathway, provided the cells are coupled via functional gap junctions. Materials and Methods Materials Highest grade materials were used: 18α glycyrrhetinic acid (18α GA), 8-Br-cAMP, bovine serum albumin (BSA), insulin, Lucifer Yellow (LY), ovine hydrocortisone, ovine prolactin, polyHEMA [poly-2hydroxyethyl methacrylate], propidium iodide and Trypsin-EDTA were obtained from Sigma (St Louis, MO). Protease inhibitors (Complete™, Boehringer Mannheim, Germany). Hybond-N membrane, Rediprime kit and α-32P dCTP were from Amersham Pharmacia Biotech (Uppsala, Sweden). Affinity-purified polyclonal rabbit anti-Cx 26 (Cat# 71-0500), 32 (Cat# 71-0600) and 43 (Cat# 710700) antibodies raised against peptide portions of the cytoplasmic domains of the respective connexins were from Zymed Laboratories (San Francisco, CA). Function-blocking integrin antibody against the β1 (Ha2/5) integrin subunit was purchased as azide- and endotoxinfree from PharMingen (San Diego, CA). Enhanced chemiluminescence (ECL) and horseradish peroxidase (HRP)conjugated anti-rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA), FITC-conjugated secondary goat anti-rabbit IgG (H+L) and Prolong antifade were from Molecular Probes (Eugene, OR). Cell culture media and reagents were purchased from Gibco BRL Life Technologies (Parsley, UK). MatTek glass bottom tissue culture plates and Bio-Rad protein assay were from MatTek (Ashland, MA) and Bio-Rad (Hercules, CA), respectively. EHS-matrix growthfactor-reduced Matrigel was purchased from Collaborative Biomedical Products (Bedford, MA). CID-9 mammary cell strain, polyclonal rabbit anti-mouse milk antiserum and β-casein c-DNA inserts were provided by Mina Bissell (Lawrence Berkeley National Laboratory, Berkeley, CA, USA). Cell culture A low passage number (17 to 21) of the CID-9 mouse mammary cell strain was used throughout. Cells were grown in ‘growth medium’ consisting of Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F12 Ham (DMEM/F12) with 5% FBS, insulin (5 µg/ml) and gentamycin (50 µg/ml) in a humidified incubator (95% air 5% CO2) at 37°C. Cells were propagated by trypsinization and plated either on tissue culture plastic petri dishes or on petri dishes coated with different substrata. CID-9 cells were seeded at 3.0×106 or at 5.0×106 cells/75cm2 dish on culture dishes or dishes coated with the reconstituted basement membrane, growth-factor-reduced Matrigel, respectively. Alternatively, diluted Matrigel (1.5% vol/vol) in HBSS was dripped onto cells 24 hours after plating (Streuli et al., 1995a). Cells cultured on EHS-matrix were directly plated in differentiation or nondifferentiation media consisting of DMEM/F12 containing insulin (5 µg/ml), hydrocortisone (1 µg/ml) and either supplemented with or lacking ovine prolactin (3 µg/ml), respectively. Cells cultured on plastic or dripped with EHS-matrix were first plated in growth medium for initial cell attachment and spreading. Twenty-four hours after plating, cells were washed three times with HBSS, and the growth medium was replaced with either differentiation or nondifferentiation media. Media were changed on a daily basis. PolyHEMA, a non-adhesive substratum, was prepared using a solution of 6 mg/ml in 95% ethanol and was added to culture plates at 5.0×10–2 ml/cm2 and allowed to evaporate to dryness at 37°C. The plates were then washed twice with HBSS and CID-9 cells were plated at a concentration of 5.0×105 cells/ml and in differentiating media. Since the cells were grown in suspension, cAMP diluted in differentiating media was added to the existing media on days 1, 3
and 5 of culture. On day 6 of culture, the cells were harvested for analysis. RNA extraction and northern blot analysis Total RNA was extracted from cells at day 6 after plating as described elsewhere (Chomczynski and Sacchi, 1987). For northern analysis, 5 µg of total RNA were electrophoresis through 1% agarose/formaldehyde gel, blotted overnight onto Amersham Hybond-N membrane in 10× SSC and UV crosslinked for subsequent hybridization. β-Casein c-DNA inserts were 32PdCTP-labelled using Rediprime kit and hybridization was performed overnight at 42°C in a shaker-incubator. The blots were then washed at high stringency (0.1% SSC, 65°C) and signals were detected by fluorography. Western blot analysis Proteins were extracted by scraping the cells into lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate). The scraped cells were then sheared by passing them several times through a 21-gauge needle. Protease inhibitors were added at a concentration of 40 µl per 1 ml of lysis buffer, and the cell extracts were centrifuged. The protein content of the supernatants was determined by Bio-Rad assay and equal amounts of protein were resolved by gel electrophoresis. To detect milk proteins, the membranes were blocked overnight in a wash buffer (100 mM Tris-HCl buffer, pH 7.5, 150 mM NaCl. 0.3% Tween 20) with 2% fatty acid-free BSA. The membranes were then incubated for 1 hour in polyclonal rabbit antimouse milk antiserum at room temperature and washed three times, for 20 minutes each, to remove unbound antiserum. For Cx26, Cx32 and Cx43 proteins, membranes were blocked for 1 hour in a wash buffer [Dulbecco’s phosphate buffered saline (PBS), 0.1% Tween 20] with 3% skim milk. They were then incubated for two hours in a humid chamber in the corresponding polyclonal rabbit anti-Cx antibody at a concentration of 0.5 µg per ml of blocking buffer. Bound antibody was detected by enhanced ECL for casein, Cx32 and Cx43 immunoblots. Addition of HRP-conjugated antirabbit IgG followed by tetramethyl benzidine (TMB) was used for detection of Cx26 immunoblots. Immunohistochemistry Cultured CID-9 cells were washed three times with warm HBSS and fixed in ice-cold (–20°C) 70% ethanol overnight. Fixed cells were first rinsed twice with PBS and then incubated for 1 hour at room temperature with 3% normal goat serum. After blocking, cells were labelled for 2 hours at room temperature with rabbit anti-connexin 26, 32 and 43. This was followed by labelling with a FITC-conjugated secondary goat anti-rabbit IgG (H+L) that was incubated for 1 hour with the fixed cells. Concentrations of the primary and secondary antibodies were used as recommended by the supplier. Nuclei were then counter-stained by incubation for 3 minutes with propidium iodide at 5 µg/ml. Washing with PBS was performed twice between incubations. Finally, cells were mounted on slides and staining was preserved by addition of antifade to the stained cells, which were kept at 4°C. Cells were then observed under fluorescence microscopy (LSM 410, Zeiss, Germany). Lucifer yellow (LY) dye microinjection and scrape-loading assays For microinjection assay, CID-9 cells were cultured on MatTek glass bottom tissue culture plates. Cells were microinjected with 5% LY CH in 150 mM LiCl. The solution of LY was injected by pressure injection into cells through microelectrodes. The spread of the dye fluorescence to neighbouring cells was recorded photographically using fluorescence microscopy.
Mammary cell GJIC-dependent differentiation For scrape-loading assay, CID-9 cells were cultured on plastic or on EHS-drip, into 4-chamber polystyrene vessel tissue culture-treated glass slides. After 24 hours, medium was supplemented with 10 µM 18α GA, or 50 µM 8-Br-cAMP. The scrape-loading method was performed as described elsewhere (El-Fouly et al., 1987). Cells plated on 4-chamber vessel slides were washed three times with warm HBSS before addition of LY at 0.1% dilution in PBS. Using a scalpel, cuts were made throughout the monolayer, followed by incubation for 10 minutes at 37°C. The cells were then washed with warm HBSS and fixed with 4% formaldehyde. Slides were preserved by mounting in antifade and stored at 4°C. Observation of dye spread was recorded photographically as described earlier. 18α GA and 8-Br-cAMP treatment of CID-9 cells CID-9 cells were seeded on EHS-matrix and treated with 10 µM 18α GA on day 1 of culture. Medium supplemented with 18α GA was changed on a daily basis up to day 10 in culture. Alternatively, 18α GA was supplemented to the medium for the first 5 days in vitro and later removed from the medium for days 6-10. Trypan Blue staining was used to determine cell viability as affected by 18α GA for the duration of the treatment. Samples were counted in triplicate wells. Proteins for western blot analysis (normalized to equal cell counts) were extracted
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on day 5 and day 10 of culture. Control cells were not treated with 18α GA. CID-9 cells treated with 8-Br-cAMP were seeded in 100 mm petri dishes on tissue culture plastic. Twenty-four hours later, growth medium was replaced with differentiation medium and 8-Br-cAMP was added at a concentration of 50 µM. Medium supplemented with 8-Br-cAMP was changed on a daily basis up to day 5 of culture, when proteins for western blot analysis were extracted. Control cells were not treated with 8-Br-cAMP. Treatment of CID-9 cells with integrin function-blocking antibody CID-9 cells were seeded in 6-well plates on plastic substratum or EHS-drip. Twenty-four hours after plating, cells were washed three times with HBSS and growth medium was replaced with differentiation medium containing 100 µg/ml of the function-blocking β1 integrin antibody. The antibody was supplemented daily with the media and the cells were harvested at day 4 after plating. Quantitative analysis of β-casein, connexin expression and functional GJIC β-Casein, connexin expression and functional GJIC using LY scrapeload assays were quantified from different experiments using NIH Image 1.62 software. Quantification of βcasein and connexin expression was normalized with respect to β-actin. For LY scrape-load assays, quantification was based on measuring the integrated fluorescence intensity, at the scrape site, over an equivalent area in both control and experimental conditions. The degree of significance of variations between control and experimental values was assessed by ANOVA uni-variant test using the Graph Pad Prism software version 3.00. Where applicable, the quantification was from three different experiments.
Results ECM modulates connexin expression, localization and GJIC CID-9 cells exhibited different morphologies in vitro depending on the substratum upon which they were Fig. 1. ECM affects morphology, β-casein and connexin expression by CID-9 cells on day 6 of culture. (A) Morphology of CID-9 cells cultured in differentiation medium on (a) plastic, (b) EHS-drip and (c) EHSmatrix. (B) Northern blot analysis of βcasein, Cx43 and Cx26 by cells on plastic (Pl), EHS-drip (Ed) and EHS-matrix (Ec). β-Casein expression was only evident in the presence (+) of prolactin (Prl) and not in its absence (–), whereas Cx43 was significantly (P
