The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with widespread expression throughout many tissues and cell types. The mechanism of action of AhR in regulating the transcription of its target genes is well-characterized [1
]. Prior to ligand binding, AhR is primarily cytosolic and in complex with molecular chaperones such as Hsp90, XAP2, and p23. Once activated through ligand binding, AhR translocates to the cell nucleus and dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) [4
]. The AhR/ARNT complex further associates with coregulator proteins that modulate interaction of the complex with aryl hydrocarbon receptor response elements (AhREs) found in the upstream promoter regions of various target genes [5
]. The normal physiological functions of AhR are beginning to be explored; however, much of what is known about the AhR has resulted from studies using xenobiotic ligands to activate receptor function [3
]. Inappropriate activation of AhR by exogenous ligands, such as 2,3,7,8-tetrachlorodibenzo-p
-dioxin (TCDD), has been shown to cause disruption of many cellular and developmental processes, including cell proliferation, fate determination, and differentiation [2
]. AhR, through AhRE binding, is known to regulate genes involved in drug metabolism, cell cycle, proliferation, differentiation, and other cellular processes [3
], implicating direct gene regulation as a target for disruption by xenobiotic chemicals.
The capacity of AhR agonists to cause endocrine disruption has been demonstrated in both the male and female reproductive systems, with effects observed in wildlife, experimental animals, and humans [11
]. Hormone signaling pathways may be sensitive to extremely low dose exposure to various chemicals [16
], and TCDD has been strongly implicated in disruption of estrogen-mediated signaling [17
]. There are various mechanisms by which AhR has been shown to affect estrogen receptor or other nuclear receptor signaling pathways, and these include modulation of hormone synthesis and/or metabolism, direct transcriptional regulation of nuclear receptors, stimulation of nuclear receptor degradation, as well as competition for coregulators necessary for transcriptional regulation by nuclear receptors [18
]. Additionally, as a lipophilic, persistent organic pollutant, TCDD exposure may increase over time via bioaccumulation [19
The mammary gland serves as both a likely target for bioaccumulation of xenobiotic AhR ligands, as well as a model system to study how AhR regulates many aspects of development. The mammary gland develops rudimentary ductal structures during fetal and prepubertal stages, and these structures develop further at puberty in the female. However, it is not until pregnancy that the full developmental process proceeds with extensive cell proliferation and differentiation, resulting in further ductal branching and development of the milk-producing alveolar structures. Upon the weaning of offspring, the mammary gland undergoes involution, which is characterized by widespread apoptosis [21
]. Our laboratory discovered that mice exposed to TCDD during pregnancy display impaired mammary gland differentiation and an inability to nutritionally support their offspring. Expression of the milk protein WAP gene (whey acidic protein) was also reduced in TCDD-exposed dams [22
]. In mice, the coordinated induction of milk protein genes occurs around the 9th day of pregnancy [23
], and we have found that the coordinated induction of these genes is impaired by TCDD (our unpublished data). These changes occurred in the absence of alterations in circulating estradiol, progesterone and prolactin levels [22
], suggesting that AhR modulates other pathways important for mammary gland differentiation. Further evidence that AhR ligands disrupt mammary gland differentiation during pregnancy was provided in a study using mammary gland explants from estrogen and progesterone-primed mice that were then maintained under hormonal stimulation in culture. The explants were also exposed to the AhR agonist ligand 2,3,7,8-tetrachlorodibenzofuran, which acted as a negative regulator of mammary gland growth and differentiation by suppressing development of lobuloalveolar structures [24
]. In order to determine the molecular mechanisms responsible for AhR-mediated disruption of mammary gland development and function, we have continued to study the effects of TCDD exposure in mice during pregnancy, and we have extended our studies using SCp2 mammary epithelial cells in culture.
Mammary gland development during pregnancy involves the differentiation of epithelial cells through formation of alveolar structures, cell polarization, and the production, secretion, and luminal sequestration of milk proteins [25
]. Primary mouse mammary epithelial cells and derived cell lines, cultured on extracellular matrix (ECM) in the presence of lactogenic hormones, have been shown to undergo morphological and functional changes that mirror those that occur in the mammary gland of the pregnant mouse [27
]. The SCp2 mammary epithelial cell line is a clonal derivation of cells isolated from BALB/c mice at mid-pregnancy [29
]. The original COMMA-1D cell line was heterogeneous, with only a subset of the cells expressing β-casein after the addition of ECM and lactogenic hormones [31
]. COMMA-1D cells were then enriched for ECM-responsive, β-casein producing cells that were designated CID-9 [32
]. Finally, the homogeneous SCp2 cell line was generated through expansion of a single cell-derived clone isolated from the CID-9 cell line [29
]. SCp2 cells have a distinctly epithelial morphology, express cytokeratins, and lack the fibroblast-type filament protein vimentin. These characteristics, along with the capacity for β-casein expression, suggest that the SCp2 cell clone originated from mouse mammary epithelial cells.
The differentiation potential of SCp2 cells has been well-characterized, beginning with morphological and functional response to ECM and lactogenic hormones. When cultured with lactogenic hormones, but without ECM, less than 0.1% of cells express β-casein. With the addition of ECM, more than 90% of SCp2 cells form cell aggregates and express β-casein [29
], demonstrating dependence on the addition of exogenous ECM for differentiation in culture. In contrast, the fibroblastic cell line SCg6, also derived from a CID-9 clone, did not form cell aggregates or express β-casein under any of the inductive culture conditions, indicating that a mixture of cell types was present in the parental cell line [29
]. Further analysis showed that even in permissive cultures, SCp2 cells that remained at the periphery of cell clusters did not express β-casein, underscoring the importance of cell aggregation for functional differentiation [29
As a functional endpoint for our studies, we have focused on impaired expression of the milk protein β-casein. At the molecular level, we have also found changes in expression of the cell adhesion molecule E-cadherin along with defects in activation of STAT5, a transcription factor essential for mammary gland development and lactogenesis [33
]. Our results demonstrate an in vitro
model system used to assess the effects of TCDD on mammary epithelial cell differentiation and identify TCDD-mediated defects in molecular pathways that are important for normal mammary gland function.