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Endocrinology Vol. 147, No. 10 4772-4780
Copyright © 2006 by The Endocrine Society

Peroxisome Proliferator-Activated Receptor {alpha} Activation during Pregnancy Severely Impairs Mammary Lobuloalveolar Development in Mice

Qian Yang, Reiko Kurotani, Atsushi Yamada, Shioko Kimura and Frank J. Gonzalez

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Frank J. Gonzalez, Building 37, Room 3106B, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. E-mail: fjgonz{at}helix.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To identify the potential functions of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) in skin development, transgenic mice were generated to target constitutively activated PPAR{alpha} (VP16PPAR{alpha}) to the stratified epithelia by use of the keratin K5 promoter. In addition to marked alterations in epidermal development, the transgenic mice had a severe defect in lactation during pregnancy resulting in 100% pup mortality. In this study, the alteration of mammary gland development in these transgenic mice was investigated. The results showed that expression of the VP16PPAR{alpha} transgene during pregnancy resulted in impaired development of lobuloalveoli, which is associated with reduced proliferation and increased apoptosis of mammary epithelia. Mammary epithelia from transgenic mice also showed a significant reduction in the expression of ß-catenin and a down-regulation of one of its target genes, cyclin D1, which is thought to be required for lobuloalveolar development. Furthermore, upon PPAR{alpha} ligand treatment, similar effects on lobuloalveolar development were observed in wild-type mice, but not in PPAR{alpha}-null mice. These findings suggest that PPAR{alpha} activation has a marked influence in mammary lobuloalveolar development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARs) are members of the nuclear hormone receptor superfamily and function as classic ligand-responsive transcription factors that participate in many physiological processes (1). Three isoforms of PPARs ({alpha}, ß/{delta}, and {gamma}) were identified in frogs, mice, nonhuman primates, and humans (2, 3, 4). Upon binding to their ligands, PPARs undergo conformational changes that allow corepressor release and coactivator recruitment, heterodimerization with RXR and selective binding to a degenerate direct repeat hexameric nucleotide sequence separated by one base (direct repeat 1, DR1), also called peroxisome proliferator-responsive element (2). Peroxisome proliferator-responsive elements are found in the promoter regions of several genes that are mainly involved in lipid storage, transport, and metabolism (5, 6, 7).

PPAR{alpha} is expressed in many tissues requiring, under certain physiological conditions, fatty acids as an energy source such as liver, kidney, and heart (8, 9). However, PPAR{alpha} has also been identified in rodent and human keratinocytes (8, 10, 11), and a number of PPAR{alpha} ligands were shown to alter epidermal homeostasis (12, 13, 14, 15). To further investigate the role of PPAR{alpha} in epithelial tissues, a transgenic mouse line was generated in which a constitutively activated PPAR{alpha} is expressed in the basal layer of the epidermis under control of the bovine keratin K5 promoter and the tetracycline regulatory system (16). In addition to the marked alterations in epidermal development, the transgenic mice had a severe defect in lactation during pregnancy and resulting in newborn mortality which is consistent with keratin 5 promoter expression in the myoepithelium of the mammary gland (17, 18, 19). However, the information on the involvement of PPAR{alpha} in mammary development is limited. All three PPARs are expressed in both stromal adipocytes and epithelial cells in rodent mammary tissues (20), and the levels of PPAR{alpha} and PPAR{gamma} mRNAs decrease during pregnancy and lactation, whereas the PPARß remains relatively unchanged (20). In rodent mammary tumor models, PPAR ligands were reported to reduce tumor incidence and progression (21, 22, 23, 24). However, there is no evidence that PPAR{alpha} activation results in impaired mammary development and lactation. Targeted disruption of PPAR{alpha} has revealed no reported phenotype associated with mammary gland development (25) suggesting that PPAR{alpha} is not required for mammalian development in the mouse model; however, this does not exclude a role for activated receptor.

The current study revealed that activation of PPAR{alpha} during pregnancy impairs mammary gland development and results in a defect of lactation and mortality of the pups. To this end, the transgenic mice with constitutively activated PPAR{alpha} under the control of K5 promoter were used to assess mammary gland development at various stages of pregnancy (16). The consequences of exposure of wild-type (Wt) or PPAR{alpha}-null mice to PPAR{alpha} ligand during pregnancy were also examined. The results revealed severe defects in lobuloalveolar development upon activation of PPAR{alpha} during pregnancy and suggest that lactation and neonatal survival are adversely affected by activation of PPAR{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Mice targeting constitutively activated PPAR{alpha} in epidermis and other stratified epithelia, under the control of the keratin K5 promoter were recently generated (16). Briefly, the potent viral transcriptional activator VP16 was fused to the mouse PPAR{alpha} cDNA to create a transcription factor that constitutively activates PPAR{alpha} target genes in the absence of ligand. The single-transgenic mice were generated with the VP16PPAR{alpha} cDNA driven by the tetracycline response element (TREVP16PPAR{alpha}) (Fig 1AGo). Double-transgenic (designated Tg) mice were generated by breeding TREVP16PPAR{alpha} mice with transgenic mice (K5-tTA) expressing tTA under the control of keratin 5 promoter (26) to reconstitute the tetracycline responsive regulatory system (27, 28). TREVP16PPAR{alpha} mice and K5-tTA mice behaved similar to Wt mice throughout the study. Therefore, mice with these three genotypes were grouped together as control littermates unless otherwise specified.


Figure 1
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  		FIG. 1. Generation of transgenic mice and analysis of transgene expression. A, Schematic representation of the 7-kb construct used for generating TREVP16PPAR{alpha} transgenic mice. PminhCMV, Minimal human cytomegalovirus promoter. B, VP16 immunostaining in mammary gland. Sections of Wt glands and Tg glands in the absence of dox were stained with anti-VP16 antibody. C, PPAR{alpha} immunostaining in mammary gland. Sections of Wt glands and Tg glands in the absence or in the presence of dox were stained with anti-PPAR{alpha} antibody. In both B and C, positive cells were visualized with DAB (brown, shown by red arrows). Nuclei were counter-stained with hematoxylin (blue). Bars, 125 µm. D, Northern blot analysis of RNA from d 1 of parturition. The expression of the VP16PPAR{alpha} transgene was observed only in Tg animals in the absence of dox.

 
The mice were maintained under a standard 12-h light/12-h dark cycle with water and chow provided ad libitum. Handling was in accordance with animal study protocols approved by the National Cancer Institute Animal Care and Use Committee. Females were housed with males and checked daily for presence of vaginal plugs. The day that the vaginal plug was found was designated as d 0.5 of pregnancy. Pregnant mice were then individually housed for the remainder of the study. Virgin mice were not housed with male mice. To characterize the effect of PPAR{alpha} ligand on mammary gland development through pregnancy, Wt and PPAR{alpha}-null mice were given pelleted mouse chow containing 0.1% (wt/wt) Wy-14,643 (Bio-Serv, Frenchton, NJ) from d 0.5, 7.5, 12.5, or 15.5 during pregnancy and killed on parturition d 1. Some double-transgenic mice were administered doxycycline (dox; 200 mg/kg; Bio-Serv) in the diet to regulate expression of the transgene VP16PPAR{alpha}.

To assess cell proliferation, mice were injected ip with 0.25 mg 5-bromo-2'-deoxyuridine (BrdU) per gram of body weight 2 h before killing by overexposure to carbon dioxide. The mammary glands were removed. Tissues not used for histology were snap frozen in liquid nitrogen and stored at –80 C until further analysis.

Hormone analysis
Blood was collected from mouse suborbital veins and separated by a serum separator tube (Becton Dickinson, Franklin Lakes, NJ). The concentrations of serum estradiol and progesterone were determined using commercial ELISA kits (Alpha Diagnostic, San Antonio, TX).

Mammary gland whole mount
Preparation of mammary glands was previously described (http:mammary.nih.gov/methodcd/methodcd.html). Briefly, the fourth abdominal mammary glands were collected on the indicated day of pregnancy. One gland from each animal was spread onto a glass slide under weight, fixed in Carnoy’s fixative solution, stained with carmine alum, and permanently mounted in Permount (Fisher Scientific, Pittsburgh, PA).

Hematoxylin and eosin (H&E) staining and immunohistochemistry
Another gland from each mouse was fixed in 10% formaldehyde and embedded in paraffin. Sections (4 µM) were mounted on glass slides (Superfrost/plus; A. Daigger & Company, Inc., Vernon Hills, IL), deparaffinized by xylene, dehydrated by graded ethanol, and processed for histology. For histology, tissues were stained with H&E.

Immunostaining for mouse PPAR{alpha}, proliferating cell nuclear antigen (PCNA; Santa Cruz Biotech, Santa Cruz, CA), VP16 tag (Abcam, Cambridge, MA), and cyclin D1 (Lab Vision, Fremont, CA) was performed using polyclonal antibodies. Each primary antibody was detected by ABC-kit (Vector Laboratory Inc., Burlingame, CA). Immunostaining for BrdU (DakoCytomation, Carpinteria, CA) and ß-catenin (BD Bioscience, San Jose, CA) was performed using monoclonal antibody, which was labeled with biotin by ARK kit (DakoCytomation) before application to the tissues. In brief, sections were washed in PBS and were incubated in 0.3% hydrogen peroxide in 100% methanol for 30 min at room temperature. The sections were then incubated in citrate buffer (pH 6.0) at 100 C for 10 min. After washing in PBS, the sections were blocked in PBS containing 5% skim milk at room temperature for 30 min. The sections were then rinsed and incubated sequentially in primary antibody (diluted 1:100 in PBS containing 1% BSA) for 2 h at room temperature, biotinylated goat antirabbit IgG (diluted 1:50 in PBS containing 1% BSA) for 30 min (when polyclonal antibody was used as the primary antibody), and avidin-biotinylated peroxidase complex (Vector Laboratory Inc.) in PBS for 30 min. The bound antibody was visualized by 3,3'-diaminobenzidine (DAB) as a peroxidase substrate. Sections were rinsed in water, counterstained with hematoxylin (Sigma), dehydrated, and mounted in permanent mounting medium.

Proliferation and apoptosis assays
Proliferation assays were performed as described previously by monitoring the incorporation of BrdU injected 2 h before killing (29). Proliferating cells were quantitated as number of brown color (i.e. BrdU incorporated) cells out of the total hematoxylin-stained cells (blue nuclei).

Apoptosis in mammary glands was analyzed by using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (Promega Corporation, Madison, WI) as previously described (30). For both proliferation and apoptosis assays, only luminal epithelial cells were included in these counts. At least 2000 cells from three animals were counted for each group at each time-point.

Quantitative real-time PCR and Northern blot analysis
Total RNA was isolated by mechanical disruption of mammary tissue in TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The concentration of RNA was determined by spectrophotometry. cDNA was synthesized from an equivalent amount of total RNA from each sample using Superscript first-strand synthesis system (Invitrogen). Primers were designed for real-time PCR using the Primer Express software (Applied Biosystems, Foster City, CA). The sequence and GenBank accession number for the forward and reverse primers used to quantify mRNA were: ß-casein (NM_009972) forward: 5'-GGATGTGCTCCAGGCTAAAGTTC-3' and reverse: 5'-TGTTTTGTGGGACGGGATTG-3', whey acidic protein (WAP) (NM_011709) forward: 5'-CCATTGAGGGCACAGAGTGTATC-3' and reverse: 5'-TTGACAGGAGTTTTGCGGGTC-3'. Real-time reactions were carried out using SYBR Green PCR master mix (AB Applied Biosystems, Warrington, UK) using the ABI PRISM 7900 HT sequence detection system (AB Applied Biosystems). The following conditions were used for PCR: 95 C for 15 sec, 94 C for 10 sec, 60 C for 30 sec, and 72 C for 30 sec, in 45 cycles. Relative expression levels of mRNA were normalized to GAPDH and analyzed for statistical significance with Student’s t test.

Northern blot analysis was carried out as described previously (31). Briefly, 10 µg of total RNA was electrophoresed on a 1.0% agarose gel containing 0.22 M formaldehyde, transferred to a nylon membrane, and cross-linked by exposure to UV light. The previously described cDNA probes (PPAR{alpha} and cyclin D1) were used for Northern blotting and 18S as a loading control (32). Membranes were hybridized in ULTRAhyb buffer (Ambion, Austin, TX) with random primer 32P-labeled cDNA probes following the manufacturer’s protocol, and washed with salt/detergent solution using standard procedures.

Statistical analysis
All of the values are expressed as the mean ± SD. All of the data were analyzed by paired or unpaired Student’s t test for significant differences between the mean values of each group. A value of P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and regulation of the VP16PPAR{alpha} transgene in mammary gland
The transgene used to express constitutively activated PPAR{alpha} in the epidermis and other stratified epithelia, under control of the keratin K5 promoter is shown in Fig. 1AGo (16). Immunohistochemical analysis of mammary glands revealed that in contrast to the absence of VP16PPAR{alpha} proteins in Wt mammary glands, VP16PPAR{alpha}, as detected by use of a VP16 antibody that detects the fusion protein, was expressed in the basal epithelial cells and not in the luminal epithelial cells of Tg mice in the absence of dox (Fig. 1BGo). However, the expression of PPAR{alpha} revealed that, in contrast to no detectable PPAR{alpha} in Wt mice, PPAR{alpha} proteins were detected in the luminal epithelial cells of Tg mice in the absence of dox, suggesting that endogenous PPAR{alpha} may have been up-regulated in these cells (Fig. 1CGo). The PPAR{alpha} induced in mammary epithelial cells was repressed in the presence of dox (Fig. 1CGo), which suppresses expression of the VP16PPAR{alpha} transgene as shown by Northern blot analysis of mammary glands, in which the VP16PPAR{alpha} transgene was only expressed in Tg mice in the absence of dox (Fig. 1DGo). Northern blot analysis also revealed that mammary glands in Wt dams expressed very low levels PPAR{alpha}. It should also be noted that the expression of endogenous PPAR{alpha} is highly induced by VP16PPAR{alpha} (Fig. 1DGo), a result that is consistent with expression of VP16PPAR{alpha} as revealed by immunohistochemistry.

Impaired lobuloalveolar development in Tg mammary glands
In a previous study, impaired lactation was observed in Tg dams. All pups produced by Tg dams could not obtain adequate milk and thus died within 2 d of birth. Wt pups cross-fostered with Tg dams exhibited the same extent of lethality. However, as expected, when dams received dox from the diet, the impaired phenotype was rescued, demonstrating that expression of the VP16PPAR{alpha} transgene led to a defect in the lactation (16).

Whole-mount analysis of mammary glands was used to examine the development of mammary gland during pregnancy. At d 12.5 during pregnancy, the Tg dams completed normal ductal development with the usual ductal structure and branching compared with Wt dams (Fig. 2AGo). In late pregnancy (18.5 d), the density of alveoli in Tg dams was lower than in Wt controls and was most pronounced in the day just after parturition (L1) (Fig. 2AGo). As expected, this phenotypic defect could be reversed after treatment with dox through the diet (data not shown), indicating that it was caused by expression of VP16PPAR{alpha} during pregnancy. Therefore, the expression of VP16PPAR{alpha} in mammary glands appeared to inhibit mammary lobuloalveolar development.


Figure 2
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  		FIG. 2. Impaired lobuloalveolar development in the transgenic mammary glands during pregnancy. A, Morphological observation by whole mount. Wt and Tg mammary glands in the absence of dox at different times during pregnancy from d 12.5 to d 1 of parturition (L1) were compared. Note, despite continued side branching, alveolar development in Tg glands was severely hypoplastic as compared with Wt controls. B, Histological analyses by H&E staining. Wt and Tg glands in the absence of dox at L1 were compared. Top panels are low magnification of glands. Note there is a scarce distribution of alveoli in Tg glands. Middle panels are medium magnification of glands. Note some alveolar structure was disrupted in Tg glands (representative shown by red arrow). Bottom panels are high magnification of glands. Note there are accumulated lipid droplets in Tg glands (representative shown by red arrow). Bars, A, 1.25 mm; B, top, 1.25 mm; middle, 250 µm; bottom, 125 µm. C, Relative expression of milk protein genes. Expression of ß-casein and WAP mRNAs were analyzed using total RNA extracts from Wt and Tg glands in the absence of dox at L1 by quantitative real-time PCR.

 
H&E staining confirmed the failure of proper lobuloalveolar development in Tg mammary glands. At L1, the stroma of Wt dams is typically packed with well-extended alveolar lobules (Fig. 2BGo). However, the Tg dams at L1 showed an unevenly spaced and condensed distribution of the lobuloalveoli compared with Wt dams. The numbers of alveoli in Tg dams were also lower than Wt controls. High magnification of mammary glands showed that some of the typical luminal structure was disrupted in Tg dams. In addition, more remnants of lipid droplets are noted in the alveoli of Tg dams as compared with Wt dams. The accumulation of lipid in Tg alveoli was also observed before parturition, e.g. d 18.5 (data not shown), indicating that this is not due to residual milk not removed by the pups. The expression of milk protein genes increased dramatically during pregnancy. At d 12.5 during pregnancy, ß-casein mRNA is clearly expressed, whereas very weak expression of WAP mRNA was observed at this stage. In late pregnancy (18.5 d) and during lactation, both ß-casein and WAP mRNAs were highly expressed. However, the expression of ß-casein and WAP mRNAs was not significantly changed in Tg dams compared with Wt controls at these stages (Fig. 2CGo), suggesting that the defect in lactation may not be due to lack of milk production. As expected, both estradiol and progesterone serum levels were not changed in Tg dams (data not shown), which rules out hormone-deficiency as a cause of the defect in lobuloalveolar development. Together with the specificity of the K5 promoter (26), these data indicated that the defect seen in Tg mice was intrinsic to the mammary epithelium.

Reduced proliferation and increased apoptosis in Tg mice
To determine whether the defect in mammary lobuloalveoli development was the result of reduced cell proliferation or increased cell death, BrdU incorporation and TUNEL assays were performed. The incorporation of BrdU into DNA was detected by immunohistochemistry (Fig. 3AGo) and the proliferation index was calculated as a percentage of BrdU-positive alveolar cells per total epithelial cells (Fig. 3BGo). The results showed that the proliferation rate of Tg mammary epithelium in the absence of dox was dramatically decreased compared with Wt or Tg epithelial cells in the presence of dox. Similar results were obtained by detection of PCNA, a marker for S phase cells. The number of PCNA-positive cells in the Tg mammary epithelium was also dramatically lower than in the Wt or Tg cells in the presence of dox (Fig. 3CGo). These results strongly suggest that PPAR{alpha} signaling plays an important role in proliferation of the lobuloalveolar epithelium in response to pregnancy signals. In addition, the apoptotic cells were increased in mammary epithelial cells from Tg dams in the absence of dox compared with Wt or Tg dams in the presence of dox as revealed by TUNEL assay (Fig. 3Go, D and E), that may also contribute to the observed hypoplastic development of lobuloalveoli.


Figure 3
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  		FIG. 3. Impaired epithelial proliferation in transgenic mammary glands. A, BrdU incorporation and detection. BrdU was administered to Wt and Tg mice in the absence or in the presence of dox at L1, and the mammary glands were removed 2 h later. BrdU incorporated into mammary glands was detected by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). B, BrdU labeling indices. A total of 300–400 epithelial cell nuclei were examined per section of Wt and Tg glands. The values represent the average fraction of BrdU-positive epithelial cells per total number of epithelial cells of three different mice, P < 0.001. Note Tg glands without dox had decreased numbers of BrdU-positive cells compared with Wt controls or Tg in the presence of dox. C, PCNA detection. PCNA was detected in mammary glands of Wt and Tg glands in the absence or in the presence of dox at L1. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox had decreased numbers of PCNA-positive cells compared with Wt or Tg with dox glands. D, TUNEL assay. Paraffin sections of mammary glands at L1 from Wt and Tg glands in the absence or in the presence of dox were subjected to TUNEL analysis. Positive cells were visualized with ABC (brown, representative shown by red arrow). Nuclei were counter-stained with a hematoxylin (blue). E, Percentage of apoptotic cells in mammary epithelial cells. A total of 300–400 epithelial cell nuclei were examined per section. The values represent the average fraction of TUNEL-positive epithelial cells per total number of epithelial cells of three different mice, P < 0.001. Note Tg glands without dox displayed increased numbers of apoptotic cells compared with Wt or Tg with dox glands. Bars, 125 µm.

 
Decreased expression of cyclin D1 and ß-catenin in Tg mice
The reduced proliferation and, in particular, the reduced PCNA expression in transgenic mammary epithelial cells suggested that expression of cell cycle regulators were altered upon expression of the VP16PPAR{alpha} transgene. Cyclin D1 is a critical component of the core cell cycle machinery. Immunohistochemical analysis revealed that, in the absence of dox, only 15.6% of Tg mammary epithelial cells were cyclin D1 positive vs. 85.5% of Wt cells or 80.2% of Tg in the presence of dox (Fig. 4AGo). However, by Northern blotting, expression of cyclin D1 gene from whole mammary gland at L1 was only slightly decreased upon expression of the VP16PPAR{alpha} transgene compared with Wt dams at L1 (Fig. 4BGo). This suggests that changes in cyclin D1 may be largely confined to epithelial cells, but not other cells that are constituents of the mammary gland. These results suggest that the proliferation defect in Tg mammary epithelium may be due, at least in part, to the reduced cyclin D1 expression in mammary epithelial cells.


Figure 4
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  		FIG. 4. Defect in the ß-catenin-cyclin D1 pathway in transgenic mouse mammary glands. A, Immunohistochemistry of cyclin D1. Cyclin D1 was detected in Wt and Tg mammary glands in the absence or in the presence of dox at L1 by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox displayed reduced levels of cyclin D1 expression compared with Wt glands or Tg with dox. B, Northern blot analysis of cyclin D1 expression in Wt and Tg whole mammary glands in the absence of dox. Average relative expression levels are indicated below with arbitrary value. C, Immunohistochemistry of ß-catenin. ß-Catenin was detected in Wt and Tg mammary glands in the absence or in the presence of dox at L1 by immunohistochemistry. Positive cells were visualized with DAB (brown, representative shown by red arrow). Nuclei were counter-stained with hematoxylin (blue). Note Tg glands without dox showed reduced levels of ß-catenin compared with high levels of cytoplasmic ß-catenin expression in Wt or Tg with dox glands. Bars, A, 125 µm; C, 250 µm.

 
Because cyclin D1 has been identified as a bona fide target gene of the Wnt/ß-catenin signaling pathway, the possible inhibitory effects of VP16PPAR{alpha} in Wnt signaling were examined. Even though high levels of ß-catenin in the membrane of mammary epithelia at L1 were consistently found in Wt or Tg glands in the presence of dox, the Tg mice in the absence of dox showed relatively lower levels of ß-catenin at L1 (Fig. 4CGo). Therefore, the inhibitory effects of VP16PPAR{alpha} on alveolar development may be through the Wnt/ß-catenin-cyclin D1 pathway.

Suppression of mammary lobuloalveolar development by the PPAR{alpha} ligand, Wy-14,643
To determine whether the impaired lobuloalveolar development during pregnancy by expression of the VP16PPAR{alpha} transgene can be reproduced by PPAR{alpha} ligands, both Wt and PPAR{alpha}-null female impregnated mice were treated with PPAR{alpha} ligand Wy-14,643 from different time points during pregnancy and killed on L1. Treatment of Wt mice from d 0.5 during pregnancy resulted in embryo lethality (n = 10) as revealed by no embryos at later stages of pregnancy. However, treatment of Wt mice from d 7.5 yielded no embryonic lethality. As expected, profound suppression of mammary lobuloalveolar development was observed. Specifically, the density and the size of alveoli in Wy-14,643 treated dams were highly decreased when compared with untreated controls (Figs. 2AGo and 5AGo). This effect was gradually weaker by shorter treatment times, e.g. from d 12.5 or 15.5 during pregnancy (Fig. 5AGo). In contrast, treatment of PPAR{alpha}-null mice with Wy-14,643 from d 7.5 during pregnancy did not cause significant morphological changes of the mammary glands (Fig. 5AGo). H&E staining confirmed this conclusion. Thus, in Wt mice, treatment with Wy-14,643 from d 7.5 resulted in smaller alveoli, and this effect was gradually weaker by shorter treatment times, whereas PPAR{alpha}-null mice did not demonstrate different morphology from untreated Wt mice (Fig. 5BGo). Interestingly, increased lipid droplets were observed in Wy-14,643-treated PPAR{alpha}-null mice (Fig. 5BGo). These data further establish that activation of PPAR{alpha} results in impaired lobuloalveolar development during pregnancy.


Figure 5
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  		FIG. 5. Impaired lobuloalveolar development in Wt mouse mammary glands upon Wy-14,643 treatment during pregnancy. Pregnant Wt and PPAR{alpha}-null female mice were treated with Wy-14,643 from the indicated time, and the mammary glands were removed at L1. A, Morphological observation by whole mount. Note lobuloalveolar development was inhibited by Wy-14,643 in Wt mice in a time-dependent manner, but not in PPAR{alpha}-null animals. B, Histological analyses by H&E staining. Note the small alveoli observed in Wt mice upon Wy-14,643 treatment from d 7.5, but not in PPAR{alpha}-null mice. PPAR{alpha}-null mice have accumulated lipid droplets in the lumen. Bars, 125 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Functional differentiation of the mammary gland is a crucial step in the reproductive cycle of mammals. Distinct steps of mammary epithelium differentiation and proliferation take place during puberty, pregnancy, and lactation (33). PPAR{alpha} is not necessary for mammary gland development as PPAR{alpha}-null mice have normal mammary gland development during pregnancy and exhibit normal nursing behavior; however, activation of PPAR{alpha} during pregnancy resulted in impaired lobuloalveolar development that probably contributed to the severe defect in lactation and pup death observed in this transgenic mouse line (16). Similar effects on lobuloalveolar development were also found in Wt mouse mammary glands upon PPAR{alpha} ligand treatment, but not in mammary glands from PPAR{alpha}-null mice. There was also increased pup mortality in Wy-14,643-treated Wt mice that could be due to the alterations in mammary gland development, modified suckling behavior, or subtle changes in hormonal levels as a result of drug treatment. Because the physiological expression of PPAR{alpha} is dramatically decreased in mammary gland during pregnancy and lactation (20), it is not surprising that activation of PPAR{alpha} alters the development of mammary glands during these reproductive changes.

Expression of the VP16PPAR{alpha} transgene was found in myoepithelial cells, the target site of the keratin 5 promoter consistent with previous studies using same promoter (19), whereas endogenous PPAR{alpha} was induced in the luminal epithelial cells of the transgenic mice. These results suggest that interactions may exist between the two mammary epithelial cell layers, and suggest the presence of a paracrine signal. In Wt mammary glands, PPAR{alpha} expression is not detected by immunohistochemistry either with or without Wy-14,643 treatment probably due to its low-level expression and the lack of sufficient sensitivity of the technique in detecting low abundance nuclear proteins. However, by use of quantitative real-time PCR, PPAR{alpha} mRNA was readily detected in Wt mice; these levels were only minimally increased by Wy-14,643 treatment (data not shown). Wy-14,643 treatment presented the same mammary gland phenotypes as found in the VP16PPAR{alpha} transgenic mice, suggesting that the level of PPAR{alpha} expression does not necessarily correlate with the activation of PPAR{alpha} target genes. A paracrine signal from myoepithelial cells where the VP16PPAR{alpha} transgene is expressed, may affect the luminal epithelial cells of transgenic mice, resulting in enhanced expression and activation of the endogenous PPAR{alpha}. Indeed, autoregulation of human PPAR{alpha} gene by PPAR{alpha} was reported (34). The VP16PPAR{alpha} may induce myoepithelial cells to produce a paracrine factor that interferes with alveolar development, or suppresses the production of paracrine factors such as receptor activator of nuclear factor {kappa}B (RANK) ligand, Wnt4, and IGF-II, which have been shown to be important for alveolar development (35, 36, 37). In this regard, it is interesting to note that RANK ligand, RANK, or CCAAT/enhancer-binding protein ß act in a signaling pathway that also involves cyclin D1 (35, 38), and mice null for these genes failed to develop alveolar structures (35, 39). In this context, it was reported that activating PPAR{alpha} signaling inhibits CCAAT/enhancer-binding protein ß or nuclear factor {kappa}B signaling (40, 41). Changes in these pathways may affect PPAR{alpha} signaling. Similar to the current observation in mammary glands, induction of endogenous PPAR{alpha} in VP16PPAR{alpha} transgenic mice was also previously observed in skin and tongue (16). This suggests that the same mechanism may exist in stratified epithelial cells. The precise mechanism for the effect of the expression of VP16PPAR{alpha} in myoepithelial cells on the expression of endogenous PPAR{alpha} in luminal epithelial cells remains to be determined.

It is well known that PPAR{alpha} is involved in the control of lipid transport and metabolism. In mouse skin, activation of PPAR{alpha} by the VP16PPAR{alpha} transgene or PPAR{alpha} ligand resulted in up-regulation of adipose differentiation-related protein (adipophilin in human) and fasting-induced adipose factor, which play important roles in the uptake and accumulation of lipids (16, 42). Up-regulation of adipose differentiation-related protein is thought be responsible for the formation of lipid droplets in many cell types. The accumulated lipid droplets observed in transgenic mammary lumen suggest that the same mechanism might exist in this tissue. The increased lipid droplets were also observed in PPAR{alpha}-null dams in the present study. This may be due to alterations in lipoprotein metabolism as previously observed in PPAR{alpha}-null mice (43). However, PPAR{alpha}-null dams exhibited normal lobuloalveolar development. The accumulated lipid droplets in mammary epithelial cells were also observed in mice with constitutive activation of Akt in mammary glands, whereas these dams exhibit normal lobuloalveolar development (44). Thus, the elevated lipid droplets in the VP16PPAR{alpha} transgenic mice may not influence lobuloalveolar development. However, the lipid droplets in transgenic mice may influence nursing because decreased nursing was observed in transgenic mice with constitutive activation of Akt in mammary glands due to increased lipid content in the milk (39). Previous cross-fostering experiments excluded suckling defects by pups (16). Thus, it is possible that the increase in milk viscosity by changes in milk composition may also result in a defect in milk ejection in the VP16PPAR{alpha} transgenic mice. Further studies are needed to determine this possibility.

The marked decrease in transgenic mammary epithelial cell proliferation during pregnancy might directly cause the observed phenotypes in transgenic mammary gland. The decreased proliferation of mammary epithelial cells is consistent with the effects of VP16PPAR{alpha} on keratinocytes, which also caused an attenuation of cell proliferation (16). In addition, the increased apoptosis in these cells may also contribute to the reduced alveoli in transgenic dams.

The cyclin D1 gene is a target of the Wnt signaling pathway, which is activated by ß-catenin and T-cell factor/lymphoid enhancer factor (45, 46). In the case of adipocytes that express high levels of PPAR{gamma} and ß-catenin, adipogenesis is dependent on the balance between PPAR{gamma} and ß-catenin signaling (47). ß-Catenin functions as a promoter of preadipocyte growth and proliferation through cyclin D1, and also functions as a potent inhibitor of adipogenesis (48). Recently, the cross-regulation of Wnt/ß-catenin/Tcf ligands and associated transcription factors with members of the nuclear receptor family has emerged as a clinically and developmentally important area of endocrine cell biology (49). Indeed, a dramatic reduction in expression of cyclin D1 and ß-catenin was observed in mammary epithelial cells of transgenic dams. However, to our knowledge, no PPAR{alpha} target genes that directly regulate the cell cycle have been identified, although ligand activation of PPAR{alpha} in liver was reported to increase the expression of cyclin D1 (32). In this context, a dramatic reduction of cyclin D1 was also observed previously in keratinocytes from VP16PPAR{alpha} transgenic mice (16), suggesting that the same mechanism may exist. Interestingly, a mouse model with a targeted mutation in cyclin D1 or with silenced ß-catenin signaling also exhibit a very similar defect in mammary gland development as the VP16PPAR{alpha} model, including no effect on ductal side branching, but specific inhibition of alveolar development with compromised lactation resulting in pup death (50, 51, 52, 53). This suggests the effects of activated PPAR{alpha} on mammary alveolar development may be through interference with the ß-catenin-cyclin D1 pathway. Thus, a mechanistically reciprocal interplay of inhibitory signals may occur between PPAR{alpha} and Wnt/ß-catenin signaling. However, how PPAR{alpha} signaling regulates Wnt/ß-catenin signaling in mammary epithelium requires further studies.

Although PPAR{alpha} ligands induce pleiotropic responses in rodents, results from VP16PPAR{alpha} transgenic mice indicate that the impaired alveoli development is due to activation of PPAR{alpha} in mammary glands. In addition, PPAR{alpha} ligand treatment of dams also caused embryonic lethality at an early stage of pregnancy. To our knowledge, the effect of PPAR{alpha} ligands exposure on lactation in humans has not been studied. Thus, epidemiological data on the effects of PPAR{alpha} ligands on breast development and function may be warranted in view of the current study. In contrast, the development of mammary gland is complex and unique. Determining the contribution of PPAR{alpha} signaling to mammary epithelial cell proliferation and its influence on Wnt/ß-catenin signaling in cell cycle regulation may also be of value in understanding mammary gland biology.


   Footnotes
 
This research was supported in part by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.

Disclosure summary: all authors have nothing to disclose.

First Published Online July 20, 2006

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; DAB, 3,3'-diaminobenzidine; dox, doxycycline; H&E, hematoxylin and eosin; PCNA, proliferating cell nuclear antigen; PPAR, peroxisome proliferator-activated receptor; RANK, receptor activator of nuclear factor {kappa}B; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; WAP, whey acidic protein; Wt, wild type.

Received April 6, 2006.

Accepted for publication July 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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