Title Cyclic AMP regulates formation of mammary epithelial acini in vitro Permalink

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus. INTRODUCTION Epithelial organ morphogenesis requires the formation of tubular or acinar structures composed of a sheet of epithelial cells surrounding a hollow lumen. While these epithelial structures usually have a simple architecture, a single layer of cells enclosing a luminal space, they are formed by a variety of mechanisms in different organs and species (Hogan and Kolodziej, 2002; Lubarsky and Krasnow, 2003). Three-dimensional culture of MCF10A mammary epithelial cells has contributed significantly to our understanding of such architecture (Debnath et al., 2003; Debnath and Brugge, 2005). When cultured on a reconstituted extracellular matrix (ECM), MCF10A cells form acini via a stereotypical sequence of events (Debnath and Brugge, 2005). Initially, a single cell proliferates to form a solid spheroid with an outer layer of polarized cells contacting the ECM, and an inner core of cells lacking ECM contact. The lack of ECM engagement with integrin receptors, and the consequent down-regulation of epidermal growth factor receptor (EGFR) and extracellular signal–regulated kinase (ERK) signaling (Reginato et al., 2003) predispose the cells to detachment-induced apoptosis, commonly termed anoikis (Frisch and Screaton, 2001; Reginato et al., 2003). The resulting imbalance between prosurvival and proapoptotic signals induces proapoptotic BH3-only family proteins Bcl-2 interacting mediator of cell death (BIM) and Bcl-2 modifying factor (Bmf), causing inner cell death and formation of hollow lumen-containing acini (Reginato et al., 2005, 2003; Schmelzle et al., 2007). The MCF10A system has also been particularly important in studies on several aspects of carcinogenesis, such as lumen filling (Muthuswamy et al., 2001; Debnath et al., 2002; Zhan et al., 2008). Remarkably, in this model, cells are routinely cultured in the presence of cholera toxin, which raises intracellular cyclical AMP (cAMP) concentrations; however, the role of cAMP during acinar morphogenesis remains unclear. In this study, we examined the role of cAMP in MCF10A acini formation. Initially, cAMP accelerated polarization of outer cells in developing acini and Monitoring Editor Alpha Yap University of Queensland Received: Feb 1, 2012 Revised: May 30, 2012 Accepted: May 30, 2012 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E12-02-0078) on June 6, 2012. Address correspondence to: Keith E. Mostov (keith.mostov@ucsf.edu). © 2012 Nedvetsky et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society of Cell Biology. Abbreviations used: ANOVA, analysis of variance; BIM, Bcl-2 interacting mediator of cell death; Bmf, Bcl-2 modifying factor; Bnz-cAMP, N6-benzoyl-cAMP; cAMP, cyclic AMP; CPT-2′-O-Me-cAMP, 8-(4-chlorophenylthio)-2′-O-methyl-cAMP; CPTcAMP, 8-(4-chlorophenylthio)-cAMP; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; ECM, extracellular matrix; EGFR, epidermal growth factor receptor; ERK, extracellular signal–regulated kinase; EtBr, ethidium bromide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HMEC, human mammary epithelial cells; HPV, human papilloma virus; NMuMG, normal murine mammary gland; pERK, phosphorylated ERK; PKA, protein kinase; qPCR, quantitative PCR; RNAi, RNA interference; ROI, region of interest; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.