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

Abstract

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/EBPβ isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPβ 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/EBPβ LAP (“Liver‐enriched Activator Protein”) isoform is the predominant C/EBPβ protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPβ LIP (“Liver‐enriched Inhibitory Protein”) isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPβ 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/EBPβ 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/EBPβ LIP or C/EBPβ LAP did not alter HC11 growth rates. However, C/EBPβ LIP over expressing HC11 cells (LAP:LIP ratio of ∼1:1) exhibited a consistent 2–4 h delay in G₀/S phase transition. C/EBPβ LIP overexpressing HC11 cells did not express β‐casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in β‐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/EBPβ LAP isoform is the predominant C/EBPβ protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPβ LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation‐induced proteolysis of C/EBPβ LAP. Ectopic overexpression of C/EBPβ LIP (LAP:LIP ratio of ∼1:1) inhibits mammary epithelial cell differentiation (β‐casein expression). J. Cell. Biochem. 82:357–370, 2001.