* Department of Pharmacology and the Environmental Toxicology Center, University of Wisconsin Medical School, Madison, Wisconsin 53706, and
Department of Biology, University of South Florida, Tampa, Florida 33620
Received June 3, 2004; accepted July 28, 2004
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ABSTRACT |
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Key Words: CYP1B1; CYP1A1; AhR; ARNT; ECM; branching morphogenesis.
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INTRODUCTION |
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The P450 cytochromes (CYPs), CYP1B1 and CYP1A1, are predominately associated with the conversion of PAHs to carcinogenic dihydrodiol epoxides (Guengerich and Shimada, 1998). CYP1B1 is largely expressed extra-hepatically in steroidogenic cells or steroid-responsive tissues, including the breast, cervix, uterus, ovary, endometrium, and prostate, which are key target tissues for carcinogenesis (Bhattacharyya et al., 1995
; Murray et al., 2001
; Muskhelishvili et al., 2001
; Savas et al., 1994
). Conversely, CYP1A1 is predominately expressed in the liver in rodents after induction. A predominance in constitutive expression of CYP1B1 relative to CYP1A1 has been observed breast carcinoma cell lines and in cultured primary breast epithelia and stromal fibroblasts (Eltom et al., 1998
; Larsen et al., 1998
; Spink et al., 1998
).
Constitutive and PAH-induced CYP1B1 and CYP1A1 expression is regulated via the aryl hydrocarbon receptor (AhR). The AhR is a member of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family of proteins (Denison and Nagy, 2003). The AhR, which is located in the cytosol in an inactive complex, translocates to the nucleus upon ligand activation. Nuclear complex formation with the aryl hydrocarbon nuclear regulatory factor (ARNT) and multiple coactivators/corepressors initiates interaction with numerous dioxin-responsive genes involved in cell cycle control and cell signaling processes (Gu et al., 2000
).
In vivo, a significant increase in AhR mRNA and protein expression is associated with 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumors and surrounding stroma (Trombino et al., 2000). A physiological role for AhR has been demonstrated in AhR-/-mice, which show altered age-related development of numerous organs, including the liver, heart, spleen, stomach, skin, prostate, uterus, and mammary gland (Fernandez-Salguero et al., 1997
; Hushka et al., 1998
; Lin et al., 2002
). In the mammary gland, AhR deficiency results in a 50% decrease in terminal end buds and stunted epithelial branching (Hushka et al., 1998
). Conversely, prenatal exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent ligand of the AhR, has been shown to substantially increase the number of terminal end buds in the offspring, thereby increasing their susceptibility to mammary carcinogenesis (Brown et al., 1998
).
The development of the mammary gland is dependent on branching morphogenesis (Silberstein, 2001a). Branching occurs through direct adhesion interactions between the epithelia and the underlying ECM, via basally located integrin receptors (Fata et al., 2004
), which facilitate signal transduction processes (Silberstein, 2001b
). ECM proteins in the EHS tumor extract, Matrigel, and floating collagen I gels facilitate the organization of rat mammary epithelial cells (RMEC) into tubular structures that resemble the in vivo mammary ductal structure (Novaro et al., 2003
; Simian et al., 2001
). These rat mammary organoids are composed of luminal and myoepithelial cells, fibroblasts, and an endogenous basement membrane, thus providing an appropriate model of paracrine and autocrine regulation of branching morphogenesis within the mammary gland.
The lesions identified in the AhR/ mouse model suggest that AhR regulates processes related to cellular proliferation and vascular homeostasis. Genetic differences in rats, which determine PAH-induced mammary cancer, also produce large differences in ductal development that are reproduced when mammary organoids are cultured on Matrigel in vitro (Benton et al., 1999). We have tested the hypothesis that ECM proteins exert selective interactions with RMEC, which affect AhR and ARNT regulation. We have shown that ECM adhesion interactions substantially determine AhR functionality, which mediates CYP expression. We have used these genetic and ECM manipulations to show that these changes in AhR and ARNT functionality are substantially co-regulated by factors that also mediate branching morphogenesis, but are not dependent on this process.
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MATERIALS AND METHODS |
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Animals and tissues. All animal studies were completed in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Virgin female Wistar Furth (WF) and Wistar Kyoto (WK) rats (5055 days of age) used in the in vivo studies were purchased from Harlan Sprague Dawley (Madison, WI). Rats were administered vehicle control (canola oil) or ß-NF (60 mg/kg body weight) by ip injection for three consecutive days. Rats were sacrificed 24 h after the final injection by CO2 anesthetization and the mammary gland tissue removed for microsomal isolation. Virgin female WF and WK rat mammary glands (5055 days of age) used in the in vitro studies were purchased from Harlan Bioproducts for Science, Inc. (Madison, WI) and cultured as described below.
ECM-preparation and coating of cell culture dishes. Matrigel and growth factor-reduced Matrigel (GFR-Matrigel) were applied to six-well plates using the thin-gel method for the culture of cells on the top of the gel, according to the manufacturer's protocol. The culture plates were coated at 4°C on the day of the RMEC preparation, during the time period that the tissue was subject to the collagenase digestion. Matrigel was evenly applied to the wells at a volume of 50 µl/cm2 of growth surface, or 500 µl/well of a six-well plate. The plates were placed at 37°C until the plating of the cells.
A thin layer of laminin was applied to six-well plates at 4°C, 24 h prior to the cell preparation, according to the manufacturer's protocol. The laminin was diluted with cell culture media to a concentration of 100 µg/ml and 1 ml was added to each well for a final concentration of 10 µg/cm2.
Collagen IV was applied as a thin layer to the six-well plates according to the manufacturer's protocol. The collagen IV was diluted with 0.05N HCl to a concentration of 100 µg/ml and 1 ml was added to each well for a final concentration of 10 µg/cm2.
The thin-layer collagen I was plated at 4°C, 24 h prior to the cell preparation as per manufacturer's instructions. The collagen I was diluted with 0.02N acetic acid to a concentration of 50 µg/ml and 2 ml were added to each well for a final concentration of 10 µg/cm2.
Collagen I was plated as a thick gel according to the protocol of the Keely laboratory (Wozniak et al., 2003). Cells were plated as a suspension in the gelled ECM at the end of the cell preparation. All solutions and the six-well plates were maintained on ice and work proceeded as quickly as possible. Immediately prior to cell plating, 9.5 ml DMEM/F12, 9.5 ml neutralizing solution (100 mM Hepes in 2X PBS, pH 7.3), and 11.5 ml collagen I (1.3 mg/ml final concentration) were mixed, in order. RMEC, in a volume of 1.2 ml, were added and mixed rapidly. The cell/collagen I suspension was plated at a volume of 2 ml/well. The volume of collagen I was sufficient to cover two six-well plates. The plates were incubated at 37°C for 3060 min, until the collagen I had formed a thick gel. Once firm, 2 ml of complete media with 5% FBS was added per well. The gelled collagen I, as plated, represents the adherent gel. The floating gel is achieved by rimming the edge of each well with a pasteur pipette to free the gel.
Preparation of rat mammary cells for cell culture. Rat mammary fibroblasts (RMF) and RMEC were prepared following the protocols of the Ip laboratory (Hahm et al., 1990), with minor modification. Briefly, the lower six abdominal/anogenital mammary glands were excised from virgin female rats and placed in sterile DMEM/F12 on ice. In a sterile hood, the glands were finely minced with a scalpel and resuspended in a digestion solution (DMEM/F12 (1:1), pH 7.2, supplemented with 0.2% (w/v) collagenase (type III), 5% FBS, 50 µg/ml gentamycin, 10,000 IU/ml penicillin/10,000 µg/ml streptomycin). This mixture was incubated in a shaking incubator (220 rpm) at 37°C for 1.5 h. At the end of this incubation, 375 U/ml DNase I was added and the mixture incubated an additional 10 min. Undigested tissue was allowed to settle for 23 min and the dispersed cells decanted to a sterile 50 ml tube, and centrifuged at 500 x g for 5 min to pellet the cells. Fat was removed by aspiration and the cell/organoid pellet was resuspended in fresh medium (DMEM/F12, pH 7.2, supplemented with 10% FBS and 50 µg/ml gentamycin, 10,000 IU/ml penicillin/10,000 µg/ml streptomycin) and centrifuged at 500 x g for 5 min. The cells were washed a total of three times. The final pellet was resuspended in 10 ml fresh medium and organoids were filtered through a sterile Nytex membrane (60 micron pore size). The flow through, which contained the fibroblasts, was collected and centrifuged at 500 x g for 5 min. The resulting fibroblast cell pellet was resuspended in fresh fibroblast medium (DMEM/F12 (1:1), pH 7.2, supplemented with 10% FBS, 50 µg/ml gentamycin, 10,000 IU/ml penicillin/10,000 µg/ml streptomycin), and plated in 175 cm2 flasks. Fresh media was applied to these cells 30 min-post fibroblastic attachment, to remove cellular debris. RMF were passaged two times to remove any contaminating epithelial cells before experiments were performed on these cells. The RMEC were washed from the membrane and collected onto a 175 cm2 dish and incubated in a humidified atmosphere of 5% CO2/95% air at 37°C for 30 min to allow for the removal of residual fibroblasts. The organoids were collected, pelleted, and resuspended in complete mammary epithelial cell medium (DMEM/F12 [1:1], pH 7.2, supplemented with 5% FBS, 10 µg/ml insulin [low zinc], 1 µg/ml progesterone, 1 µg/ml hydrocortisone, 5 µg/ml transferrin, 10 ng/ml epidermal growth factor, 1 µg/ml prolactin, 5 µM ascorbic acid, 50 µg/ml gentamycin, 1 mg/ml fatty acid-free bovine serum albumin [BSA]). Organoids were plated onto plastic, Matrigel, laminin, collagen IV, and collagen I (thin) coated dishes, as indicated. Plating to the gelled collagen was completed as described above. Following the overnight incubation, fresh, serum-free complete mammary epithelial medium was added to the attached organoids. Cultures were treated with vehicle control (DMSO) or TCDD (109 M) for 24 h prior to harvesting.
Cells were harvested by trypsinization, collagenase digestion, or Matrisperse depolymerization, as indicated below. Cells were released from the Matrigel ECM by the addition of 2 ml of the BD Cell Recovery Solution, Matrisperse. Plates were incubated at 4°C on a platform shaker until depolymerization of the ECM was complete. Cells cultured on thin layers of laminin, collagen IV, and collagen I were washed three times with ice cold 1X PBS and harvested by trypsinization. The cells cultured on the thick collagen I gel were harvested by collagenase digestion. Collagenase (Type 3) was resuspended in DMEM/F12 to a concentration of 10 mg/ml. The collagenase (800 µl/well) was added and the plates were incubated at 37°C until the collagen I was fully digested. The cells from each respective treatment were collected by centrifugation. The resultant cell pellets were washed three times with ice cold 1X PBS. The final pellets were resuspended in cell lysis buffer (50 µl/well; 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM sodium orthovanadate, 10 µl/ml protease inhibitors, and 1 mM PMSF), frozen on dry ice, and stored at 80°C. Prior to assaying protein concentration, the lysates were thawed on ice and sonicated (310 s pulses at 50% power) using a sonicator cell disrupter (Heat Systems-Ultrasonics, Inc., model W185F, Plainview, NY). Insoluble material was removed from the lysates by centrifugation at 14000 rpm for 5 min at 4°C. The supernatant was stored at 80°C. Total cell lysate protein concentrations were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions, using bovine serum albumin as a standard.
Treatment of cultured RMEC with the Arg-Gly-Asp (RGD) peptide. Active cyclo(-Arg-Gly-Asp-D-Phe-Val) (cRGDDFV) and inactive, control cyclo(-Arg-Ala-Asp-D-Phe-Val) (cRADDFV) peptides were resuspended, to a stock concentration of 1.7 mM, in ddH2O and methanol, respectively. Peptides were diluted in cell culture media at a concentration of 1 µM and mixed with cells prior to plating on Matrigel. Fresh peptide was added at each media change.
Preparation of microsomal protein from tissues and cells. Microsomal protein was prepared from tissue samples as previously described (Larsen et al., 1998). Microsomal protein concentration was determined using the BCA Protein Assay Kit (Pierce, Rockford, IL) according to the manufacturer's protocol, using bovine serum albumin as a standard.
Immunoblot analysis. Microsomal proteins and total cell lysates were separated by SDS-PAGE (7.5% acrylamide) and immunoblot analysis completed as previously described (Larsen et al., 1998). Primary antibodies used in these studies include affinity-purified antibodies to recombinant mouse CYP1B1 (Savas et al., 1997
) and rat CYP1A1 (Larsen et al., 1998
), AhR and ARNT (generously provided by Dr. Richard Pollenz), ß-catenin (generously provided by Dr. Patty Keely), E-cadherin (BD Transduction Laboratories, # 610182), EH (generous gift from Dr. Charles Kasper), and actin (Sigma, #A2066). Immunoreactive proteins were visualized by the Enhanced Chemiluminescence (ECL) method of detection (Amersham Corp., San Diego, CA), according to the manufacturer's instructions.
In vitro PAH metabolism assay. RMECs were cultured in six-well plates and treated with TCDD (109 M) or 0.1% DMSO (control) for 24 h. Microsomes were isolated as described above. Microsomal incubations contained 200 µg of microsomal protein. Microsomal metabolism was completed as previously described (Larsen et al., 1998).
Scanning electron microscopy. RMEC were cultured on cover slips in chamber slides for five days prior to analysis. Cells were fixed by incubation for 1 h at room temperature in 1 ml of fixative composed of 0.1 M sodium phosphate, pH 7.4, 1% glutaraldehyde, 5% tannic acid. Following fixation, cells were dehydrated through a series of incubations (5 min each): 15% EtOH, 30% EtOH, 50% EtOH, 70% EtOH, 80% EtOH, 90% EtOH, 95% EtOH, and three incubations of 100% EtOH. The cells were then subjected to critical point drying using a Samdri 780A critical point dryer (Rockville, MD). The cells were subsequently analyzed using a Hitachi S-570 SEM equipped with a lanthanum hexaboride emitter (LaB6, Pleasanton, CA).
Immunoquantitation. Quantitation of the immuno-detectable proteins was completed using the Kodak Image Station 440CF. Electronically scanned images were quantitated using the Kodak Digital Science 1D Image Analysis software (Version 3.0).
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RESULTS |
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However, in spite of this inherent variability, basal and induced CYP1B1 levels were elevated an average of 3.2-(p < 0.01) and 1.8-fold (p < 0.01), respectively, on Matrigel relative to plastic. TCDD-induced CYP1A1 expression was an average of 5.7-fold (p < 0.01) higher on Matrigel relative to plastic (Fig. 2C).
Strains Demonstrate Large Differences in Branching Morphogenesis, but Not CYP Expression
WF and WK rats have been shown to differ substantially in the formation of ductal structures on Matrigel, in vivo (Benton et al., 1999). Figure 3A depicts a typical RMEC culture on Matrigel. Branching morphogenesis can be described in terms of at least three phases. Initially, small protrusions develop from the organoid mass (Fig. 3B). The protrusions elongate into extensions, which often mature to the extent that their length exceeds the diameter of the organoid. In the third phase of growth, the extensions mature into branched structures. We cultured WF and WK RMEC on Matrigel and confirmed that branching morphogenesis on Matrigel was substantially higher in the WK than the WF strain (Fig. 4A). In order to provide a semi-quantitative analysis of these results, we have defined a branch as an extension that protrudes at least as long as the diameter of the organoid (Fig. 3B). In eight organoids examined, 42 branched structures were observed in the WK strain, while the WF strain developed only 24 extensions, which projected a distance that was further than the diameter of the organoid body. We have used these differences to test whether changes in CYP expression parallel the observed differences in branching morphogenesis. In contrast to the differences in the extent of branching, CYP1B1 expression (basal and TCDD-induced) and TCDD-induced CYP1A1 were not substantially different between the strains. Figure 4B depicts a representative immunoblot, while Figure 4C provides the immunoquantitation of two independent experiments. The data in Figure 4C has been quantitated relative to constitutive CYP1B1 expression in the WK strain as equal to one. This absence of strain differences in CYP1B1 and CYP1A1 expression in cells cultured on Matrigel suggests that the difference in the in vivo microsomal CYP expression presented in Figure 1A arises from higher proportions of stromal fibroblasts (CYP1B1-rich) in WF mammary tissue and epithelia (CYP1A1-rich) in WK tissue.
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Extracellular Matrix Rapidly Enhances AhR and ARNT Expression, Which Mediate Increases in CYP1B1 and CYP1A1 Expression
Our laboratory is keenly interested in studying the role of AhR and ARNT regulation in development and cellular differentiation. Since these studies specifically address mammary gland development, using branching morphogenesis as a morphological endpoint, the remaining analyses have been completed using the epithelial-rich, highly morphogenic WK RMECs as our model system. We examined the association of branching morphogenesis to ECM-regulated AhR and ARNT expression by determining the levels of AhR and ARNT expression in WK RMEC in the initial 24 h of culture on the Matrigel substratum. While cell proliferation was apparent on plastic within 24 h, branching morphogenesis on Matrigel was not apparent until day 2 (Fig. 5A). AhR and CYP1B1 levels were near the lower limit of detectability in the freshly digested organoids (0 h), while a low level of ARNT expression was clearly visible. AhR and ARNT levels were substantially elevated on Matrigel (in two independent experiments), in parallel with a more modest increase in basal CYP1B1 expression within 24 h and prior to formation of any branched structures (Fig. 5B). The increase in AhR and ARNT expression on plastic was substantially less than on Matrigel. These data indicate that these Matrigel-induced changes precede branching morphogenesis and do not depend on this morphology change. Constitutive CYP1A1 expression was not detectable under any of the assay conditions (data not shown).
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Matrigel and Collagen I (Gel) Are the Preferred ECMs for Rat Mammary CYP1B1 and CYP1A1 Expression
In order to investigate the role of the individual ECM components in the regulation of AhR, ARNT, CYP1B1, and CYP1A1 expression, we cultured the WK RMEC on purified preparations of the major Matrigel components; laminin and collagen IV, as well as collagen I, each deposited as a thin layer on plastic. Matrigel was the only substratum in this group to elicit branching morphogenesis (Fig. 7A). Cells cultured on thin films of laminin, collagen I, and collagen IV remained in a "flattened" conformation, morphologically resembling cells proliferating on plastic. These thin-layer ECM deposits failed to substantially elevate AhR, ARNT, or TCDD induction of CYP1A1 to the magnitude observed on Matrigel (Fig. 7B). Basal and TCDD-induced CYP1B1 was consistently expressed on all substratum examined.
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We suspected that the physical characteristics of the ECM surface were critical for branching morphogenesis. Collagen I can be applied to culture dishes in two distinct formulations: as a rigid, thin film, as described above (Fig. 7), or as a thick gel within which the cells can be suspended, thereby assuming three-dimensional characteristics. We, therefore, tested thick collagen I gels, which mediate the establishment of apicobasal polarity and differentiation in breast epithelia (Bissell and Bilder, 2003), in our WK RMEC model to further examine AhR-mediated regulation in relation to branching morphogenesis. In these experiments, the thick collagen I preparation was examined under conditions in which the ECM remained adherent to the culture surface or was released from contact with the culture vessel, thereby increasing the flexibility of the culture surface. Within three to five days in culture, the flexible, floating collagen I gel mediated limited organization of the primary RMEC into branched structures resembling those formed on Matrigel (Fig. 8A). In 10 organoids examined, 23 branched structures were observed in which the length of the extension exceeded the diameter of the organoid. Formation of these structures was consistently slower than on Matrigel, which generated 46 branched structures. We failed to detect even a single branch, which extended further than the diameter of the organoid body, when cells were cultured on the adherent collagen I gel (Fig. 8A).
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A cRGD Peptide-Integrin Antagonist Attenuates RMEC Branching Morphogenesis on Matrigel, without Attenuating AhR, ARNT, or CYP Expression
Previous experiments have implicated cell-cell adhesion interactions as an important aspect of AhR regulation (Cho et al., in press; Sadek and Allen-Hoffmann, 1994
). Cell-ECM adhesion is mediated by integrins, which bind to the ECM via short peptide sequences present on the matrix proteins. The mammary integrins,
2ß1 and
3ß1, recognize the tri-peptide Arg-Gly-Asp (RGD), which is present on the basement membrane components fibronectin, vitronectin, laminin, entactin, and the collagens (Gurrath et al., 1992
; Ruoslahti, 1996
). WK RMEC were cultured on Matrigel in the presence of the active cyclic Arg-Gly-Asp-D-Phe-Val peptide (cRGDDFV) or an inactive, control peptide, cyclic Arg-Ala-Asp-D-Phe-Val (cRADDFV). We have used the cyclic peptides, since the combination of cyclization and the addition of D-Phe (DF) as a hydrophobic binding element following the RGD sequence enhances its binding effectiveness (Gurrath et al., 1992
). Figure 9A demonstrates that the inhibited cell-ECM interaction mediated by the cRGDDFV peptide (1 µM) substantially inhibited branching morphogenesis, while the inactive peptide did not interfere with the characteristic branching of the cells. Semi-quantitative analysis of nine organoids per treatment demonstrated 46, 40, and 11 branched structures, which were generated from culture on Matrigel or cRAD-treated, and cRGD-treated cells, respectively. We predicted that AhR expression would decline in parallel with decreased cell-ECM contact, but were surprised to observe that AhR expression was insensitive to the cRGDDFV peptide (Fig. 9B). Similarly, the active peptide did not substantially affect ARNT and CYP expression. E-cadherin and ß-catenin expression was insensitive to the magnitude of branching morphogenesis, suggesting that cell-cell contacts are still effectively formed under the more limited branching conditions.
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DISCUSSION |
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We clearly distinguish the effects of ECM on branching morphogenesis from the effects on AhR, ARNT, and CYP regulation, even though they likely share common regulatory processes. Broadly, the regulation of AhR and ARNT expression is associated with ECM interactions that promote cell-cell and cell-ECM adhesion. Although branching morphogenesis may depend on these same initial adhesion events, branching is clearly limited by additional interactions, which do not affect AhR and ARNT expression. This is shown by the increases in AhR and ARNT within 24 h on Matrigel, which precede branching. We show that this coordinated expression of AhR and ARNT occurs independently of large differences in the magnitude of branch formation modeled by a variety of experimental conditions, including genetic differences (WK vs. WF), ECM characteristics (Matrigel, collagen formulation), EGF stimulation, and selective adhesion inhibition by the cyclic RGD peptide.
Branching morphogenesis involves the restructuring of epithelial tissues and cellular extrusion through an extensive network composed of layers of myoepithelial cells, basement membrane, and periductal stroma (Wiseman and Werb, 2002). Mammary organoids retain luminal epithelia, myoepithelia, basement membrane, and stromal fibroblasts, organized with the polarity and tight junctions of the in vivo ducts, thus providing an appropriate in vitro model for studying branching morphogenesis (Simian et al., 2001
). We have reproduced previous studies, which have shown that RMEC undergo branching morphogenesis when cultured on Matrigel, where laminin and collagen IV are the main components. We show, however, that this branched organization is not observed on plastic or on thin layers of collagen I, collagen IV, or laminin. These results imply that branching morphogenesis is not merely dependent on the presence of selective factors within the specific ECM preparations. The rapid elevation of AhR and ARNT on Matrigel, prior to branching morphogenesis, is indicative of a relatively direct ECM-mediated response in conjunction with this initial organization. This early organizational interaction with the Matrigel stimulates AhR activity, as evidenced by DMBA metabolism analyses. Matrigel-mediated increases in DMBA metabolic activity, which correspond closely to the changes in CYP expression, are indicative of the formation of functional proteins.
We compared Matrigel which is composed of laminin, collagen IV, heparin sulfate proteoglycans, enactin, nidogen, and growth factors, including EGF, bFGF, IGF-1, TGF-ß, and PDGF, to GFR-M, which is a refined matrix preparation, containing reduced levels of several growth factors, including EGF (less than 0.5 ng/ml), IGF-1, PDGF, TGF-ß, and heparan sulfate proteoglycans. CYP expression was not substantially different with the GRF-M relative to the complete preparation, further emphasizing that although growth factors, including EGF, substantially enhance RMEC branching morphogenesis, CYP expression is far more dependent on the interaction initiated by the ECM.
Studies have shown that the capacity of the cell to deform the surface through integrin/ECM complexes is a key to migration, which occurs during branching morphogenesis. The Keely laboratory has shown that T47D cells contract the floating collagen I gels as branching progresses, a process which is not possible on the adherent gels (Wozniak et al., 2003). We provide for a distinction between these very early (within 24 h) reorganizational adhesion events, marked by AhR/ARNT and CYP increases that are comparable on floating and adherent collagen I gel or Matrigel, and the subsequent branching morphogenesis response. The elevation of E-cadherin and ß-catenin, key components of cell-cell adhesion complexes, parallels AhR/ARNT enhancement, even when branching morphogenesis does not ensue (i.e., adherent collagen I gel), suggesting that appropriate cell-cell interactions have been established during this early time period and that adhesion interactions are key to this regulatory mechanism. Indeed, previous studies have shown that murine breast epithelia establish the appropriate apicobasal polarity on floating collagen I gels by day 2 (Bissell and Bilder, 2003
). A different process of reorganization, migration, and proliferation occurs on the less deformable thin films, probably due to effects on integrin clustering (Bissell and Bilder, 2003
; Bissell et al., 2003
; Novaro et al., 2003
). The parallel expression of ß-catenin and AhR is also notable because ß-catenin has been shown to positively regulate AhR transcription (Chesire et al., 2004
) and to be associated with enhanced AhR activity when activated by loss of adhesion (Cho et al., in press
).
Cell-ECM adhesion is mediated by integrins and plays a critical role in the regulation of cellular morphology and function. Mammary cells express 1ß1,
2ß1,
3ß1,
6ß1, and
6ß4 heterodimers (Novaro et al., 2003
). Integrins bind to ECM via short peptide sequences present on the matrix proteins. The RGD peptide blocks cell-ECM contact by interfering with integrin binding with basement membrane components, including fibronectin, vitronectin, laminin, entactin, and collagens (Gurrath et al., 1992
; Ruoslahti, 1996
). The sustained AhR expression observed in the presence of the active cRGDDFV peptide (decreased cellular adhesion) further demonstrates an early, selective link between ECM-adhesion and AhR expression that precedes extensive branch formation.
Previous work, in multiple cell types, has shown that AhR nuclear translocation and activation can be stimulated by manipulations of cell adhesion, specifically a decrease in cell-cell contacts (Cho et al., in press; Sadek and Allen-Hoffmann, 1994
). In these studies, AhR protein was rapidly and similarly down-regulated during activation by either TCDD or loss of adhesion. However, no evidence has been previously presented for cross-talk to ARNT, under these conditions (Eltom et al., 1999
). The present studies demonstrate that AhR and ARNT respond in parallel to multiple changes in cell adhesion. The co-regulation that we have observed in these experiments likely reflects a parallel response to common regulatory factors for each protein, which are very sensitive to the changes in cellular adhesion.
WF and WK rats demonstrate remarkable differences with respect to DMBA- and ENU-induced mammary cancer, which have been linked to genetic differences in susceptibility rather than generation of reactive metabolites (Lan et al., 2001). Nevertheless, these strains also show substantial in vivo differences in the relative expression of CYP1A1 and CYP1B1. We present evidence that the strain-selective expression of PAH-induced CYP1B1 and CYP1A1 in, respectively, WF and WK rats may be due to the differences in the extent of ductal development, which has previously been reported (Benton et al., 1999
). The mammary glands of the WK rats show more ductal proliferation in vivo and express higher levels of differentiation markers (i.e., casein production) than the WF counterparts. The enhanced AhRmediated ß-NF induction of CYP1A1 in WK rats is reflective of this more extensive ductal formation in vivo, since CYP1A1 induction is selectively seen in RMEC that form branched structures on Matrigel and gelled Collagen I in vitro. The elevated CYP1B1 levels observed in microsomes isolated from WF rats quite closely replicate the ratios from the cultured fibroblasts. It is likely that the less extensive ductal structure in WF mammary glands yields a higher proportion of the microsomes derived from stromal cells.
Previous work in our laboratory with Sprague Dawley rats has shown that although only CYP1B1 is expressed in RMF, RMEC express both CYP1B1 and CYP1A1 (Christou et al., 1995). We have confirmed that isolated WK RMF selectively express CYP1B1, and not CYP1A1. CYP1B1 is induced through the AhR to high levels. We have shown that the WK RMEC cultured on plastic, with rigorous removal of stromal fibroblasts, exhibit very low basal CYP1B1 expression, which is TCDD-inducible. Likewise, culture on plastic yields low levels of CYP1A1 induction. There is strong evidence that stromal fibroblasts modulate hormonal responses in endocrine tissues, such as the mammary gland (Haslam and Woodward, 2003
), uterus (Wegner and Carson, 1992
), and prostate (Chung et al., 1991
). Upon hormonal stimulation, fibroblasts respond by releasing growth factors and ECM proteins, which regulate epithelial cell ductal formation and growth. In view of the effects of these ECM proteins on AhR, ARNT, and CYP expression, it is likely that fibroblast secretions modulate the RMEC expression of these proteins, coincident with other expression patterns. This will be an important area of future studies in this laboratory. The retention of CYP1B1 in less differentiated cells, potentially including mammary stem cells, which are likely targets for cancer initiation, adds to the importance of the regulation of this CYP. The establishment of this in vitro rat mammary model and the identification of this novel linkage between AhR/ARNT-mediated CYP1B1 expression and early cell-ECM interactions have important implications for cancer initiation.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at University of Wisconsin, Department of Pharmacology, 1300 University Ave., Room 2640 MSC, Madison, WI 53706. Fax: (608) 262-1257. E-mail: jefcoate{at}wisc.edu.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Bhattacharyya, K. K., Brake, P. B., Eltom, S. E., Otto, S. A., and Jefcoate, C. R. (1995). Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1-Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation. J. Biol. Chem. 270, 1159511602.
Bissell, M. J., and Bilder, D. (2003). Polarity determination in breast tissue: Desmosomal adhesion, myoepithelial cells, and laminin 1. Breast Cancer Res. 5, 117119.[CrossRef][ISI][Medline]
Bissell, M. J., Rizki, A., and Mian, I. S. (2003). Tissue architecture: The ultimate regulator of breast epithelial function. Current Opinion Cell Biol. 15, 753762.[CrossRef][ISI][Medline]
Brown, N. M., Manzolillo, P. A., Zhang, J. X., Wang, J., and Lamartiniere, C. A. (1998). Prenatal TCDD and predisposition to mammary cancer in the rat. Carcinogenesis 19, 16231629.[Abstract]
Chesire, D. R., Dunn, T. A., Ewing, C. M., Luo, J., and Isaacs, W. B. (2004). Identification of aryl hydrocarbon receptor as a putative Wnt/ß-catenin pathway target gene in prostate cancer cells. Cancer Res. 64, 25232533.
Christou, M., Savas, U., Schroeder, S., Shen, X., Thompson, T., Gould, M. N., and Jefcoate, C. R. (1995). Cytochromes CYP1A1 and CYP1B1 in the rat mammary gland: Cell-specific expression and regulation by polycyclic aromatic hydrocarbons and hormones. Mol. Cell. Endocrinol. 115, 4150.[CrossRef][ISI][Medline]
Cho, Y. C., Zheng, W., and Jefcoate, C. R. (2004). Disruption of cell-cell contact maximally but transiently activates AhR-mediated transcription in 10T1/2 fibroblasts. Toxicol. Appl. Pharmacol. October issue.
Chung, L. W., Gleave, M. E., Hsieh, J. T., Hong, S. J., and Zhau, H. E. (1991). Reciprocal mesenchymal-epithelial interaction affecting prostate tumour growth and hormonal responsiveness. Cancer Surveys 11, 91121.[ISI][Medline]
Denison, M. S., and Nagy, S. R. (2003). Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43, 309334.[CrossRef][ISI][Medline]
Eltom, S. E., Larsen, M. C., and Jefcoate, C. R. (1998). Expression of CYP1B1 but not CYP1A1 by primary cultured human mammary stromal fibroblasts constitutively and in response to dioxin exposure: Role of the Ah receptor. Carcinogenesis 19, 14371444.[Abstract]
Eltom, S. E., Zhang, L., and Jefcoate, C. R. (1999). Regulation of cytochrome P-450 (CYP) 1B1 in mouse Hepa-1 variant cell lines: A possible role for aryl hydrocarbon receptor nuclear translocator (ARNT) as a suppressor of CYP1B1 gene expression. Mol. Pharmacol. 55, 594604.
Fata, J. E., Werb, Z., and Bissell, M. J. (2004). Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes. Breast Cancer Res. 6, 111.[ISI][Medline]
Fernandez-Salguero, P. M., Ward, J. M., Sundberg, J. P., and Gonzalez, F. J. (1997). Lesions of aryl-hydrocarbon receptor-deficient mice. Vet. Pathol. 34, 605614.[Abstract]
Gammon, M. D., Santella, R. M., Neugut, A. I., Eng, S. M., Teitelbaum, S. L., Paykin, A., Levin, B., Terry, M. B., Young, T. L., Wang, L. W., Wang, Q., Britton, J. A., Wolff, M. S., Stellman, S. D., Hatch, M., Kabat, G. C., Senie, R., Garbowski, G., Maffeo, C., Montalvan, P., Berkowitz, G., Kemeny, M., Citron, M., Schnabel, F., Schuss, A., et al. (2002). Environmental toxins and breast cancer on Long Island. I. Polycyclic aromatic hydrocarbon DNA adducts. Cancer Epidemol. Biomarkers Prev. 11, 677685.
Gu, Y. Z., Hogenesch, J. B., and Bradfield, C. A. (2000). The PAS superfamily: Sensors of environmental and developmental signals. Annu. Rev. Pharmacol. Toxicol. 40, 519561.[CrossRef][ISI][Medline]
Guengerich, F. P., and Shimada, T. (1998). Activation of procarcinogens by human cytochrome P450 enzymes. Mutation Res. 400, 201213.[ISI][Medline]
Gurrath, M., Muller, G., Kessler, H., Aumailley, M., and Timpl, R. (1992). Conformation/activity studies of rationally designed potent anti-adhesive RGD peptides. Eur. J. Biochem. 210, 911921.[Abstract]
Hahm, H. A., Ip, M. M., Darcy, K., Black, J. D., Shea, W. K., Forczek, S., Yoshimura, M., and Oka, T. (1990). Primary culture of normal rat mammary epithelial cells within a basement membrane matrix. II. Functional differentiation under serum-free conditions. In Vitro Cell. Develop. Biol. 26, 803814.[ISI][Medline]
Haslam, S. Z., and Woodward, T. L. (2003). Epithelialcell-stromal interactions and steroid hormone action in normal and cancerous mammary gland. Breast Cancer Res. 5, 208215.[CrossRef][ISI][Medline]
Hushka, L. J., Williams, J. S., and Greenlee, W. F. (1998). Characterization of 2,3,7,8-tetrachlorodibenzofuran-dependent suppression and AH receptor pathway gene expression in the developing mouse mammary gland. Toxicol. Appl. Pharmacol. 152, 200210.[CrossRef][ISI][Medline]
Lan, H., Kendziorski, C. M., Haag, J. D., Shepel, L. A., Newton, M. A., and Gould, M. N. (2001). Genetic loci controlling breast cancer susceptibility in the Wistar-Kyoto rat. Genetics 157, 331339.
Larsen, M. C., Angus, W. G. R., Brake, P. B., Eltom, S. E., Sukow, K. A., and Jefcoate, C. R. (1998). Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: Role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism. Cancer Res. 58, 23662374.[Abstract]
Lin, T. M., Ko, K., Moore, R. W., Simanainen, U., Oberley, T. D., and Peterson, R. E. (2002). Effects of aryl hydrocarbon receptor null mutation and in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on prostate and seminal vesicle development in C57BL/6 mice. Toxicol. Sci. 68, 479487.
Murray, G. I., Melvin, W. T., Greenlee, W. F., and Burke, M. D. (2001). Regulation, function, and tissue-specific expression of cytochrome P450 CYP1B1. Annu. Rev. Pharmacol. Toxicol. 41, 297316.[CrossRef][ISI][Medline]
Muskhelishvili, L., Thompson, P. A., Kussewitt, D. F., Wang, C., and Kadlubar, F. F. (2001). In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues. J. Histochem. Cytochem. 49, 229236.
Novaro, V., Roskelley, C. D., and Bissell, M. J. (2003). Collagen-IV and laminin-1 regulate estrogen receptor expression and function in mouse mammary epithelial cells. J. Cell Sci. 116, 29752986.
Rundle, A., Tang, D. L., Hibshoosh, H., Estabrook, A., Schnabel, F., Cao, W. F., Grumet, S., and Perera, F. P. (2000). The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer. Carcinogenesis 21, 12811289.
Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12, 697715.[CrossRef][ISI][Medline]
Russo, J., Hu, Y., Yang, X., and Russo, I. (2000). Developmental, cellular, and molecular basis of human breast cancer. J. Natl. Cancer Inst. Monogr. 27, 1737.[Medline]
Russo, J., Hu, Y.F., Silva, I. D. C. G., and Russo, I. H. (2001). Cancer risk related to mammary gland structure and development. Microscopy Res. Tech. 52, 204223.[CrossRef][ISI]
Sadek, C. M., and Allen-Hoffmann, B. L. (1994). Suspension-mediated induction of Hepa 1c1c7 Cyp1a-1 expression is dependent on the Ah receptor signal transduction pathway. J. Biol. Chem. 269, 3150531509.
Savas, Ü., Bhattacharyya, K. K., Christou, M., Alexander, D. L., and Jefcoate, C. R. (1994). Mouse cytochrome P-450EF, representative of a new 1B subfamily of cytochrome P-450s-cloning, sequence determination and tissue expression. J. Biol. Chem. 269, 1490514911.
Savas, Ü., Carstens, C-P., and Jefcoate, C. R. (1997). Biological oxidations and P450 reactions. Recombinant mouse CYP1B1 expressed in Escherichia coli exhibits selective binding by polycyclic aromatic hydrocarbons and metabolism, which parallels C3H10T1/2 cell microsomes, but differs from human recombinant CYP1B1. Arch. Biochem. Biophys. 347, 181192.[CrossRef][ISI][Medline]
Schmidt, J. V., and Bradfield, C. A. (1996). Ah receptor signaling pathways. Annu. Rev. Cell Dev. Biol. 12, 5589.[CrossRef][ISI][Medline]
Silberstein, G. B. (2001a). Postnatal mammary gland morphogenesis. Microsc. Res. Tech. 52, 155162.[CrossRef][ISI][Medline]
Silberstein, G. B. (2001b). Tumour-stromal interactions. Role of the stroma in mammary development. Breast Cancer Res. 3, 218223.[CrossRef][ISI][Medline]
Simian, M., Hirai, Y., Navre, M., Werb, Z., Lochter, A., and Bissell, M. J. (2001). The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 128, 31173131.[ISI][Medline]
Spink, D. C., Spink, B. C., Cao, J. Q., DePasquale, J. A., Pentecost, B. T., Fasco, M. J., Li, Y., and Sutter, T. R. (1998). Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells. Carcinogenesis 19, 291298.[Abstract]
Trombino, A. F., Near, R. I., Matulka, R. A., Yang, S., Hafer, L. J., Toselli, P. A., Kim, D. W., Rogers, A. E., Sonenshein, G. E., and Sherr, D. H. (2000). Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1 gene transcripts in a rat model of mammary tumorigenesis. Breast Cancer Res. Treatment 63, 117131.[CrossRef][ISI][Medline]
Wegner, C. C., and Carson, D. D. (1992). Mouse uterine stromal cells secrete a 30-kilodalton protein in response to coculture with uterine epithelial cells. Endocrinology 131, 25652572.[Abstract]
Wiseman, B. S., and Werb, Z. (2002). Stromal effects on mammary gland development and breast cancer. Science 296, 10461049.
Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J., and Keely, P. J. (2003). ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix. J. Cell Biol. 163, 583595.