Expression and function of CCAAT/enhancer binding proteinβ (C/EBPβ) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBP beta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBP beta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBP beta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBP beta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBP beta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBP beta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBP beta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBP beta LIP or C/EBP beta LAP did not alter HC11 growth rates. However, C/EBP beta LIP over expressing HC11 cells (LAP:LIP ratio of similar to1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBP beta LIP overexpressing HC11 cells did not express P-casein mRNA (mammary epithelial cel I differentiation marker) in response to lactogenic hormones. This defect in P-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBP beta LAP isoform is the predominant C/EBP beta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBP beta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBP beta LAP. Ectopic overexpression of C/EBP beta LIP (LAP:LIP ratio of similar to1:1) inhibits mammary epithelial cell differentiation (P-casein expression). (C) 2001 Wiley-Liss.