Nulliparous CCAAT/Enhancer Binding Proteinδ (C/EBPδ) Knockout Mice Exhibit Mammary Gland Ductal Hyperlasia

Abstract

CCAAT/Enhancer binding proteins (C/EBPs) are a family of nuclear proteins that function in the control of cell growth, death, and differentiation. We previously reported that C/EBPδ plays a key role in mammary epithelial cell G₀ growth arrest. In this report, we investigated the role of C/EBPδ in mammary gland development and function using female mice homozygous for a targeted deletion of C/EBPδ (C/EBPδ –/–). C/EBPδ –/– females develop normally and exhibit normal reproductive and lactational performance. Adult nulliparous C/EBPδ –/– females, however, exhibit mammary epithelial cell growth control defects. The mean number of mammary ductal branches is significantly higher in adult nulliparous C/EBPδ –/– females compared with C/EBPδ +/+ (wild-type control) females (66.8 ± 5.2 vs 42.9 * 6.3 branch points/field, P < 0.01). In addition, the mean total mammary gland cellular volume occupied by epithelium is significantly higher in adult nulliparous C/EBPδ –/– females compared with C/EBPδ +/+ controls (29.0± 1.4 vs 20.4 ± 1.3, P < 0.001). Our results showed that the BrdU labeling index was significantly higher in mammary epithelial cells from nulliparous C/EBPδ –/– females compared with C/EBPδ +/+ controls during the proestrus/estrus (4.55 ± 0.70 vs 2.14 ± 0.43, P < 0.01) and metestrus/diestrus (6.92 ± 0.75 vs 3.98 ± 0.43 P < 0.01) phases of the estrus cycle. In contrast, the percentage of mammary epithelial cells undergoing apoptosis during both phases of the estrus cycle did not differ between C/EBPδ –/– and C/EBPδ +/+ females. The increased epithelial cell content and proliferative capacity was restricted to the nulliparous C/EBPδ –/– females as no differences in mammary gland morphology, ductal branching or total epithelial content were observed between multiparous C/EBPδ –/– and C/EBPδ +/+ females. These results demonstrate that C/EBPδ plays a novel role in mammary epithelial cell growth control that appears to be restricted to the nulliparous mammary gland.

Keywords

C/EBPδ mammary epithelial cell involution estrus cycle nulliparous

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References

Ford

, Pardee

. Cancer and the cell cycle. J Cell Biochem 33: 166–172, 1999.

Swale

, Quinn

. Tumor suppressor genes. Exp Dermatol 25: 231–235, 2000.

Hanahan

, Weinberg

. The hallmarks of cancer. Cell 100: 57–70, 2000.

Hennighausen

, Robinson

. Think globally, act locally: The making of a mouse mammary gland. Genes Dev 12: 449–455, 1998.

Richert

, Schwertfeger

, Ryder

, Anderson

. An atlas of mouse mammary gland development. J Mamm Gland Biol Neopl 5: 227–241, 2000.

Andres

A-C

, Strange

. Apoptosis in the estrous and menstrual cycles. J Mamm Gland Biol Neopl 4: 221–228, 1999.

Schedin

, Mitrenga

, Kaeck

. Estrous cycle regulation of mammary epithelial cell proliferation, differentiation, and death in the Sprague-Dawley rat: A model for investigating the role of estrous cycling in mammary carcinogenesis. J Mamm Gland Biol Neopl 5: 211–225, 2000.

Hennighausen

, Robinson

, Wagner

K-U

, Liu

. Developing a mammary gland is a Stat affair. J Mamm Gland Biol Neopl 2: 365–372, 1997.

O'rourke

, Yuan

, DeWille

. CCAAT/enhancer-binding proteindelta (C/EBP delta) is induced in growth-arrested mouse mammary epithelial cells. J Biol Chem 272: 6291–6296, 1997.

10.

O'rourke

, Newbound

, Hutt

, DeWille

. C/EBPδ regulates mammary epithelial cell G₀ growth arrest and apoptosis. J Biol Chem 274: 16582–16589, 1999.

11.

O'rourke

, Hutt

, DeWille

. Transcriptional regulation of the C/EBPδ in G₀ growth arrested mouse mammary epithelial cells. Biochem Biophys Res Commun 262: 696–701, 1999.

12.

Hutt

, O'rourke

, DeWille

. Signal transducer and activator of transcription 3 activates CCAAT enhancer-binding protein δ gene transcription in G₀ growth-arrested mouse mammary epithelial cells and in involuting mouse mammary gland. J Biol Chem 275: 29123–29131, 2000.

13.

Hutt

, DeWille

. Oncostatin M induces growth arrest of mammary epithelium via a CCAAT/enhancer-binding proteinδ-dcpendent pathway. Mol Cell Ther 1: 601–610, 2002.

14.

Gigliotti

, DeWille

. Lactation status influences expression of CCAAT/enhancer binding protein isoform mRNA in the mouse mammary gland. J Cell Physiol 174: 232–239, 1998.

15.

Gigliotti

, DeWille

. Local signals induce CCAAT/enhancer binding proteinδ (C/EBPδ) and C/EBPβ mRNA expression in the involuting mouse mammary gland. Breast Cancer Res Treat 58: 57–63, 1999.

16.

Johnson

, Williams

. CCAAT/enhancer binding proteins. In: Tronche

, Yaniv

. Eds. Liver Gene Expression. New York: Raven Publishing, pp 231–258, 1994.

17.

Jung

, Yi

Y-W

, Kim

, Shong

, Hong

S-S

, Lee

H-S

, Bae

. Regulation of Gadd45γ expression by C/EBP. Eur J Biochem 267: 6180–6187, 2000.

18.

Slomiany

, D'arigo

, Kelly

, Kurtz

. C/EBPα inhibits cell growth via direct repression of E2F-DP-mediated transcription. Mol Cell Biol 20: 5986–5997, 2000.

19.

Chien

P-L

, Riley

, Chen-Kiang

, Lee

W-H

. Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6. Proc Natl Acad Sci 93: 465–469, 1996.

20.

Timchenko

, Wilde

, Darlington

. C/EBPα regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice. Mol Cell Biol 19: 2936–2945, 1999.

21.

Harris

, Albrecht

, Nakanishi

, Darlington

. CCAAT/Enhancer-binding proteinα cooperates with p21 to inhibit cyclin-dependent kinase-2 activity and induces growth arrest independent DNA binding. J Biol Chem 276: 29200–29209, 2001.

22.

Sterneck

, Tessarollo

, Johnson

. An essential role for C/EBPβ in female reproduction. Genes Dev. 11: 2153–2162, 1997.

23.

Robinson

, Johnson

, Hennighausen

, Sterneck

. The C/EBPβ transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland. Genes Dev 12: 1907–1916, 1998.

24.

Seagroves

, Krnacik

, Raught

, Gay

, Burgess-Beusse

, Darlington

, Rosen

. C/EBPβ but not C/EBPα. is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes Dev 12: 1917–1928, 1998.

25.

Tanaka

, Yoshida

, Kishimoto

, Akira

. Defective adipocyte differentiation in mice lacking the C/EBPβ and/or C/EBPδ gene. EMBO J 16: 7432–7443, 1997.

26.

Sterneck

, Paylor

, Jackson-Lewis

, Libbey

, Przedborski

, Tessarollo

, Crawley

, Johnson

. Selectively enhanced contextual fear conditioning in mice lacking the transcriptional regulator CCAAT/enhancer binding protein delta. Proc Natl Acad Sci USA 95: 10908–10913, 1998.

27.

Allen

. The oestrus cycle in mouse. Am J Anat 30: 297–371, 1922.

28.

Guenette

, Corbeil

, Leger

, Wong

, Mezl

, Mooibroek

, Tenniswood

. Induction of gene expression during involution of the lactating mammary gland of the rat. J Mol Endocinol 12: 47–60, 1994.

29.

Flint

, Tonner

, Allan

. Insulin-like growth factor binding proteins: IGF-dependent and -independent effects in the mammary gland. J Mamm Gland Biol Neoplasia 5: 65–73, 2000.

30.

Zeps

, Bentel

, Papadimitriou , Dawkins

HJS

. Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle: Association with ERα and ERβ status. J Histochem Cytochem 47: 1323–1330, 1999.

31.

Brisken

, Park

, Vass

, Lydon

, O'malley

, Weinberg

. A paracrine role for the epithelial progesterone receptor in mammary gland development. Proc Natl Acad Sci USA 95: 5076–5081, 1998.

32.

Hagihara

, Wu-Peng

, Funabashi

, Kato

, Pfaff

. Nucleic acid sequence and Dnase hypersensitive sites of the 5′ region of the mouse progesterone receptor gene. Biochem Biophys Res Commun 205: 1093–1101, 1994.

33.

Brisken

, Heineman

, Chavarria

, Elenbaas

, Tan

, Dey

, McMahan

, Weinberg

. Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling. Genes & Dev. 14: 650–654, 2000.

34.

Sympson

, Talhouk

, Alexander

, Chin

, Bissell

, Werb

. Targeted expression of stromelysin-1 in mammary gland provides evidence for a role of proteinases in branching morphogenesis and the requirement for an intact basement membrane for tissue-specific gene expression. J Cell Biol 125: 681–693, 1994.

35.

Ball

. The development of the terminal end bud in the prepubertalpubertal mouse mammary gland. Anat Record 250: 459–464, 1998.

36.

Darlington

, Ross

, MacDougal

. The role of C/EBP genes in adipocyte differentiation. J Biol Chem. 273: 30057–30060, 1998.

37.

Porter

, Krop

, Nasser

, Sgroi

, Kaelin

, Marks

, Riggins

, Polyak

. A SAGE (Serial Analysis of Gene Expression) view of breast tumor progression. Cancer Res 61: 5697–5702, 2001.

38.

Polyak

. Molecular alterations in ductal carcinoma in situ of the breast. Curr Opin Oncol 14: 92–96, 2002.

39.

Herman

, Merlo

, Mao

, Lapidus

, Issa

JPJ

, Davidson

, Sidransky

, Baylin

. Inactivation of the CDKN2/p16MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55: 4525–4530, 1995.

40.

Sanford

. Characterization of CCAAT/Enhancer binding proteins (C/EBPs) in human prostatic cell lines. Proc Am Assoc Canc Res 42: A5309, 2001.

41.

Russo

, Russo

. Hormonal approach to breast cancer prevention. J Cell Biochem Suppl 34: 1–6, 2000.

42.

Guzman

, Yang

, Rajkumar

, Thordarson

, Chen

, Nandi

. Hormonal prevention of breast cancer: Mimicking the protective effect of pregnancy. Proc Natl Acad Sci USA 96: 2520–2525, 1999.