Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) 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/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta 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/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta 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/EBPbeta 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/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-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/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).
Dearth
L R
LR
Molecular, Cellular and Developmental Biology Program, Department of Veterinary Biosciences, Ohio State University Comprehensive Cancer Center, Ohio State University, Columbus, Ohio 43210, USA.
Hutt
J
J
Sattler
A
A
Gigliotti
A
A
DeWille
J
J
D000818:Animals; D001940:Breast / Q000166:cytology / Q000378:metabolism*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D002364:Caseins / Q000235:genetics; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D007774:Lactation; D015674:Mammary Neoplasms, Animal / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D011247:Pregnancy; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000235:genetics
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; D002364:Caseins; C497250:Ddit3 protein, mouse; D020033:Protein Isoforms; D012333:RNA, Messenger; D014157:Transcription Factors; D051742:Transcription Factor CHOP