Cyclic AMP regulates formation of mammary epithelial acini in vitro

Pavel I. Nedvetsky^a, Sang-Ho Kwon^a, Jayanta Debnath^b, and Keith E. Mostov^a,c,1

^aDepartment of Anatomy, University of California, San Francisco, San Francisco, CA 94143-2140 ^bDepartment of Pathology, University of California, San Francisco, San Francisco, CA 94143-2140 ^cDepartment of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143-2140

Monitoring Editor: Alpha Yap

Abstract: 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.

¹Address correspondence to: Keith E. Mostov (keith.mostov{at}ucsf.edu).

Abbreviations: 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 • CPT-cAMP, 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