- Short Report
- Open Access
Lactogenic differentiation of HC11 cells is not accompanied by downregulation of AP-2 transcription factor genes
© Jäger et al; licensee BioMed Central Ltd. 2008
- Received: 28 April 2008
- Accepted: 23 June 2008
- Published: 23 June 2008
During pregnancy the mammary epithelium undergoes a complex developmental process which culminates in the generation of the milk-secreting epithelium. Secretory epithelial cells display lactogenic differentiation which is characterized by the expression of milk protein genes, such as beta-casein or whey acidic protein (WAP). Transcription factors AP-2alpha and AP-2gamma are downregulated during lactation, and their overexpression in transgenic mice impaired the secretory differentiation of the mammary epithelium, resulting in lactation failure. To explore whether the downregulation of AP-2alpha and AP-2gamma is of functional significance for lactogenic differentiation, we analyzed the expression of the AP-2 family members during the lactogenic differentiation of HC11 mammary epithelial cells in vitro. Differentiation of HC11 cells was induced following established protocols by applying the lactogenic hormones prolactin, dexamethasone and insulin.
HC11 cells express all AP-2 family members except AP-2delta. Using RT-PCR we could not detect a downregulation of any of these genes during the lactogenic differentiation of HC11 cells in vitro. This finding was confirmed for AP-2alpha and AP-2gamma using Northern analysis. Differentiating HC11 cells displayed lower expression levels of milk protein genes than mammary glands of mid-pregnant or lactating mice.
The extent of lactogenic differentiation of HC11 cells in vitro is limited compared to mammary epithelium undergoing secretory differentiation in vivo. Downregulation of AP-2 transcription factor genes is not required for lactogenic differentiation of HC11 cells but may functionally be involved in aspects of lactogenic differentiation in vivo that are not reflected by the HC11 system.
- Mammary Epithelium
- Whey Acidic Protein
- HC11 Cell
- Milk Protein Gene
- Secretory Epithelial Cell
During pregnancy the mammary epithelium matures in a complex developmental process which culminates in the generation of alveoli containing the milk-secreting epithelium. The lactogenic differentiation of the secretory epithelial cells is characterized by the synthesis of lactose and milk fat and by the expression of milk protein genes, such as beta-casein or whey acidic protein (WAP). The regulatory networks and transcription factors controlling these developmental processes are only incompletely understood [1, 2].
Transcription factors of the AP-2 family comprise 5 members, termed AP-2alpha-epsilon (or TFAP2a-e), which control genetic programs involved in proliferation, apoptosis, and differentiation (see  for review). They share a conserved structure and control gene expression by binding to the respective promoters as homo- or heterodimers. Transcription factors AP-2alpha and -gamma have been implicated in breast cancer  and, based on overexpression in transgenic mice, in mammary development [5, 6]. AP-2alpha and -gamma are expressed in non-pregnant mammary epithelium and are downregulated in lactating mammary glands. Overexpression of AP-2alpha or -gamma has been shown to interfere with the proper secretory differentiation of mammary epithelial cells at the end of pregnancy, resulting in lactation failure [5, 6]. These findings suggest a functional role of AP-2 transcription factors in preventing lactogenesis. To date, the genetic programs mediating this effect have not been identified. To these ends, an in vitro system of lactogenic differentiation would be informative.
The murine mammary epithelial cell line HC11 represents an established in vitro system for the study of lactogenic differentiation [7, 8]. HC11 cells can be induced to undergo lactogenic differentiation by applying a mixture of the lactogenic hormones dexamethasone, insulin and prolactin (a mixture termed DIP in the following sections). In this report we analyzed AP-2 transcription factor expression in HC11 cells subjected to lactogenic differentiation conditions.
HC11 cells were grown and differentiated as described in . Hormones were obtained from Sigma (Hamburg, Germany). Routine culture was performed using RPMI 1640 medium containing 10% fetal calf serum, L-glutamine, 5 μg/ml Insulin and 10 ng/ml epidermal growth factor (EGF). For induction of differentiation, cells were grown to confluency and then kept in medium without EGF for 48 hours to induce competence. Competent cells were incubated with DIP medium (RPMI 1640 medium containing 10% FCS, L-glutamine, 100 nM dexamethasone, 5 μg/ml insulin, 5 μg/ml ovine prolactin) for 72 hours.
Oligonucleotides used for PCR
no. of cycles
Northern Blot analyses were carried out as previously described . Briefly, 15 μg of total RNA was resolved on formaldehyde agarose gels, transferred to positively charged nylon membrane (Hybond N+, Amersham, Braunschweig, Germany), hybridized to 32P-labelled DNA probes and washed under stringent conditions (0.1 × SSC/65°C). The AP-2 probes used cover nucleotides 1760–2040 from the murine AP-2alpha and 2041–2710 from the murine AP-2gamma mRNA, respectively, both sequences within the non-conserved 3'-untranslated regions. WAP, beta-casein, and GAPDH probes have been described previously .
We thank Mathilde Hau-Liersch for technical assistance. We are grateful to Jonathan Sleeman for kindly providing HC11 cells and Natascha Cremers for technical advice about culture and differentiation conditions. LP was supported by the Life Sciences and Culture exchange program Bonn. This work was supported by DFG grant to H.S. (Scho 503/6 and Scho 503/7).
- Naylor MJ, Oakes SR, Gardiner-Garden M, Harris J, Blazek K, Ho TW, Li FC, Wynick D, Walker AM, Ormandy CJ: Transcriptional changes underlying the secretory activation phase of mammary gland development. Mol Endocrinol. 2005, 19 (7): 1868-1883. 10.1210/me.2004-0254.View ArticlePubMedGoogle Scholar
- Anderson SM, Rudolph MC, McManaman JL, Neville MC: Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis!. Breast Cancer Res. 2007, 9 (1): 204-10.1186/bcr1653.PubMed CentralView ArticlePubMedGoogle Scholar
- Eckert D, Buhl S, Weber S, Jager R, Schorle H: The AP-2 family of transcription factors. Genome Biol. 2005, 6 (13): 246-10.1186/gb-2005-6-13-246.PubMed CentralView ArticlePubMedGoogle Scholar
- Pellikainen JM, Kosma VM: Activator protein-2 in carcinogenesis with a special reference to breast cancer--a mini review. Int J Cancer. 2007, 120 (10): 2061-2067. 10.1002/ijc.22648.View ArticlePubMedGoogle Scholar
- Jager R, Werling U, Rimpf S, Jacob A, Schorle H: Transcription factor AP-2gamma stimulates proliferation and apoptosis and impairs differentiation in a transgenic model. Mol Cancer Res. 2003, 1 (12): 921-929.PubMedGoogle Scholar
- Zhang J, Brewer S, Huang J, Williams T: Overexpression of transcription factor AP-2alpha suppresses mammary gland growth and morphogenesis. Dev Biol. 2003, 256 (1): 127-145. 10.1016/S0012-1606(02)00119-7.View ArticlePubMedGoogle Scholar
- Taverna D, Groner B, Hynes NE: Epidermal growth factor receptor, platelet-derived growth factor receptor, and c-erbB-2 receptor activation all promote growth but have distinctive effects upon mouse mammary epithelial cell differentiation. Cell Growth Differ. 1991, 2 (3): 145-154.PubMedGoogle Scholar
- Ball RK, Friis RR, Schoenenberger CA, Doppler W, Groner B: Prolactin regulation of beta-casein gene expression and of a cytosolic 120-kd protein in a cloned mouse mammary epithelial cell line. Embo J. 1988, 7 (7): 2089-2095.PubMed CentralPubMedGoogle Scholar
- Hebbard L, Steffen A, Zawadzki V, Fieber C, Howells N, Moll J, Ponta H, Hofmann M, Sleeman J: CD44 expression and regulation during mammary gland development and function. J Cell Sci. 2000, 113 ( Pt 14): 2619-2630.Google Scholar
- Hennighausen L, Westphal C, Sankaran L, Pittius CW: Regulation of expression of genes for milk proteins. Biotechnology. 1991, 16: 65-74.PubMedGoogle Scholar
- Aoki N, Ishii T, Ohira S, Yamaguchi Y, Negi M, Adachi T, Nakamura R, Matsuda T: Stage specific expression of milk fat globule membrane glycoproteins in mouse mammary gland: comparison of MFG-E8, butyrophilin, and CD36 with a major milk protein, beta-casein. Biochim Biophys Acta. 1997, 1334 (2-3): 182-190.View ArticlePubMedGoogle Scholar
- McManaman JL, Hanson L, Neville MC, Wright RM: Lactogenic hormones regulate xanthine oxidoreductase and beta-casein levels in mammary epithelial cells by distinct mechanisms. Arch Biochem Biophys. 2000, 373 (2): 318-327. 10.1006/abbi.1999.1573.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.