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

J Cell Biochem. 2001;82(3):357-70. doi: 10.1002/jcb.1167.

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/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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Breast / cytology
  • Breast / metabolism*
  • CCAAT-Enhancer-Binding Protein-beta / genetics
  • CCAAT-Enhancer-Binding Protein-beta / metabolism*
  • CCAAT-Enhancer-Binding Proteins / genetics
  • Caseins / genetics
  • Cell Cycle
  • Cell Differentiation
  • Cell Division
  • Cell Line
  • Epithelial Cells / metabolism*
  • Female
  • Gene Expression Regulation*
  • Lactation
  • Mammary Neoplasms, Animal / metabolism*
  • Mammary Neoplasms, Animal / pathology
  • Mice
  • Mice, Transgenic
  • Pregnancy
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Transcription Factor CHOP
  • Transcription Factors / genetics

Substances

  • CCAAT-Enhancer-Binding Protein-beta
  • CCAAT-Enhancer-Binding Proteins
  • Caseins
  • Ddit3 protein, mouse
  • Protein Isoforms
  • RNA, Messenger
  • Transcription Factors
  • Transcription Factor CHOP