ABSTRACT
Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.
Subject(s)
Apoptosis , Extracellular Matrix/metabolism , MAP Kinase Signaling System , Mammary Glands, Animal/cytology , Animals , Apoptosis/drug effects , Cell Survival , Cells, Cultured , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Epithelial Cells/drug effects , Female , Flavonoids/pharmacology , Imidazoles/pharmacology , Mice , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Pyridines/pharmacology , p38 Mitogen-Activated Protein KinasesABSTRACT
Testosterone repressed prostate message-2 (TRPM-2)/clusterin gene expression is rapidly induced in early involution of the mouse mammary gland, after weaning, and in the rat ventral prostate, after castration. A search for involution-enhanced DNaseI footprints in the proximal mouse TRPM-2/clusterin gene promoter led to the identification and characterization (by DNase I footprinting and EMSA) of a twin nuclear factor 1 (NF1) binding element at -356/-309, relative to the proposed transcription start site; nuclear extracts from 2-day involuting mouse mammary gland showed an enhanced footprint over the proximal NF1 element; extracts from involuting prostate showed enhanced occupancy of both NF1 binding elements. Subsequent EMSA and Western analysis led to the detection of a 74-kDa NF1 protein whose expression is triggered in early involution in the mouse mammary gland; such an induced protein is not found in the involuting rat ventral prostate. This protein was not found in lactation where three other NF1 proteins of 114, 68, and 46 kDa were detected. Reiteration of the epithelial cell apoptosis associated with early mammary gland involution, in vitro, in a primary cell culture system, triggered the appearance of the 74-kDa NF1. Overlaying the cells with laminin-rich extracellular matrix suppressed the apoptosis and the expression of the 74-kDa NF1 and, in the presence of lactogenic hormones, initiated milk protein gene expression and the expression of two of the lactation-associated NF1 proteins (68 and 46 kDa). This study, thus, identifies for the first time the occurrence of a switch in expression of different members of the family of NF1 transcription factors as mammary epithelial cells move from the differentiated to the involution/apoptotic state, and it is likely that the involution-specific 74-kDa NF1 accounts for the enhanced NF1 footprint detected on the TRPM-2/clusterin promoter with extracts of mouse mammary gland.
