| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
Address all correspondence and requests for reprints to: JoEllen Welsh, University of Notre Dame, Galvin Life Science Building, Notre Dame, Indiana 46556. E-mail: jwelsh3{at}nd.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
hydroxylase (CYP27B1), which produces 1,25-(OH)2D are present in mammary gland and dynamically regulated during pregnancy, lactation, and involution. Furthermore, we show that mice lacking VDR exhibit accelerated lobuloalveolar development and premature casein expression during pregnancy and delayed postlactational involution compared with mice with functional VDR. The delay in mammary gland regression after weaning of VDR knockout mice is associated with impaired apoptosis as demonstrated by reductions in terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling staining, caspase-3 activation and Bax induction. Under the conditions used in this study, VDR ablation was not associated with hypocalcemia, suggesting that altered mammary gland development in the absence of the VDR is not related to disturbances in calcium homeostasis. Furthermore, in the setting of normocalcemia, VDR ablation does not affect milk protein or calcium content. These studies suggest that the VDR contributes to mammary cell turnover during the reproductive cycle, and its effects may be mediated via both endocrine and autocrine signaling pathways. Unlike many mammary regulatory factors that exert transient, stage-specific effects, VDR signaling impacts on mammary gland biology during all phases of the reproductive cycle. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Although best known for its role in maintenance of calcium homeostasis, 1,25-(OH)2D also regulates epithelial cell proliferation, differentiation and apoptosis. 1,25-(OH)2D modulates casein expression and branching morphogenesis in mouse mammary organ culture (7, 9) and induces differentiation of normal and transformed mammary epithelial cells in vitro (12, 13, 14). Vitamin D deficiency is associated with reduced serum prolactin and decreased milk protein synthesis (15); however, it is not clear whether these effects are due to reduced vitamin D signaling or secondary to hypocalcemia. During pregnancy and lactation, the production of 1,25-(OH)2D from its precursor, 25hydroxyvitamin D [25(OH)D] increases to meet calcium requirements for fetal mineralization and milk production (16). Conversion of 25(OH)D to 1,25-(OH)2D is catalyzed by the 25(OH)D 1-hydroxylase (CYP27B1), a type I mitochondrial cytochrome P450 oxidase expressed in kidney and several extra-renal tissues, including epidermis, prostate, colon, and placenta (17). Despite identification of the VDR in mammary gland, the impact of this receptor on glandular proliferation, differentiation, and apoptosis during pregnancy, lactation, or involution has been largely unexplored.
In the present study, we have examined expression of VDR and the 25(OH)D 1-hydroxylase during pregnancy, lactation, and involution and used VDR null mice to assess whether disruption of vitamin D signaling in the setting of normocalcemia affects lobuloalveolar outgrowth, secretory differentiation, milk composition or glandular involution in vivo. We provide evidence that VDR ablation is associated with precocious lobuloalveolar development and casein expression during pregnancy, increased milk secretion during lactation, and delayed apoptotic regression during involution. In conjunction with our previous studies demonstrating accelerated mammary gland development during puberty of VDR null mice, our data support the concept that the vitamin D signaling pathway participates in development and remodeling of the mammary gland.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-Hydroxylase Expression during Pregnancy, Lactation, and Involution
). Compared with the low levels detected in glands from 6- to 10-wk-old virgin mice, VDR mRNA in mammary gland increased approximately 50-fold by late pregnancy. The peak level of VDR mRNA expression was observed during lactation, when VDR expression was significantly higher than any other developmental period (P < 0.05, ANOVA). VDR mRNA expression in lactating mammary gland approached that in kidney, and was higher than that in intestine. For mammary gland, similar profiles were obtained with both 18S and cytokeratin 18 normalizing genes, indicating that the observed changes in VDR expression are not solely due to altered epithelial content of the gland during the reproductive cycle. These changes in VDR expression during pregnancy and lactation are consistent with reports that lactogenic hormones up-regulate VDR in normal mammary gland and nontransformed mammary cells in vitro (12, 18). Within 2 d of weaning, VDR gene expression declined rapidly in association with the first phase of involution. An increase in VDR mRNA was observed during the second phase of involution (3–4 d after weaning), but only in samples normalized against 18S, suggesting that this increase may reflect VDR up-regulation in nonepithelial cells.
|
-a) and in differentiated secretory alveolar cells during lactation (not shown). By 4 d after weaning, VDR protein was detectable in the epithelial cells undergoing apoptosis as well as in the stromal cells that participate in remodeling the extracellular matrix during involution (Fig. 1B-b).
To assess whether murine mammary gland has the potential to produce 1,25-(OH)2D, the ligand for VDR, we assessed 25(OH)D 1-hydroxylase mRNA by real-time PCR and protein by immunohistochemistry and Western blotting. As demonstrated in Fig. 1C, 1-hydroxylase mRNA was low but detectable in virgin glands from wild-type (WT) mice and increased approximately 20-fold by d 9 of pregnancy (P < 0.05, 10 wk vs. TM9). The peak expression of 1-hydroxylase mRNA, detected in early pregnancy, was greater than that detected in intestine but much lower than that detected in kidney. By Western blotting, we identified the 1-hydroxylase protein in murine mammary gland as a single band at the expected molecular mass of 56 kDa that comigrated with the 1-hydroxylase from MCF-7 and T47D breast cancer cells (Fig. 1D). The 1-hydroxylase protein was expressed at comparable levels in mammary glands from WT and VDR knockout (KO) mice during pregnancy and lactation. Expression of 1-hydroxylase protein was confirmed by immunohistochemistry (Fig. 1E), which demonstrated specific cytosolic staining consistent with the enzyme’s mitochondrial location at high levels in kidney tubules (Fig. 1E-b) and lower levels in selected cells of the developing alveolar buds during pregnancy (Fig. 1E-d). No nonspecific staining was detected in either kidney (Fig. 1E-a) or mammary gland (Fig. 1E-c).
VDR Ablation Is Associated with Precocious Lobuloalveolar Development during Pregnancy
To assess whether VDR impacts on pregnancyinduced growth and differentiation, inguinal mammary glands removed from WT and VDR KO females 9 and 16 d after mating were assessed by whole mount technique and histological examination. Glands from WT mice exhibited stage-appropriate development, with limited branching and alveolar bud development on primary ducts during early pregnancy (Fig. 2 A and C, arrows) and more extensive branching and alveolar bud production by d 16 of pregnancy (Fig. 2E). In comparison to WT mice, glands from VDR KO mice examined on d 9 of pregnancy were characterized by more extensive side branching and a higher density of mature lobuloalveolar units (Fig. 2, B and D, arrows). The increased branching and alveolar budding arising from the ducts as a result of VDR ablation is reasonable given that VDR is highly expressed in the ducts of WT animals (Fig. 1B-a, arrow). Glandular development in VDR KO mice at d 9 of pregnancy was roughly comparable to that detected in WT mice at d 16 of pregnancy (compare Fig. 2, D and E). At pregnancy d 16, VDR KO mice continued to display accelerated lobuloalveolar development with larger alveolar clusters and secretions in dilated lumens (Fig. 2F). Because estrogen and progesterone are critical determinants of ductal side-branching and lobuloalveolar development, we assessed estrogen receptor (ER) and progesterone receptor (PR) expression by immunohistochemistry; however, there were no differences in localization or expression of PR (Fig. 2, G and H) or ER (data not shown) as a function of VDR status during pregnancy.
|
Effect of VDR Ablation on Glandular Development and Milk Production during Lactation
Secretory differentiation during lactation is associated with milk synthesis and represents a period of extremely high calcium demand. We thus examined glandular development and milk production in females after 5 and 10 d of lactation, with litter sizes equalized at six pups per lactating mother. No obvious impairments were detected in either whole-mounts or hematoxylin and eosin (H&E)-stained glands from VDR KO mice (Fig. 3A, a and b), indicating that lack of the VDR did not grossly impair lactation. Rather, milk-filled alveolar structures were consistently larger in VDR KO mice than in WT mice at the same stage of lactation. To determine whether VDR ablation altered milk volume or composition, milk was quantitatively collected from lactating WT and VDR KO females after dosing with oxytocin as described in Materials and Methods. As seen in Fig. 3B, VDR KO mice secreted almost two times more milk by volume in response to exogenous oxytocin than did WT mice; however, there were no differences in protein or calcium concentration of milk from WT and VDR KO mice (Fig. 3B). Western blotting confirmed that VDR ablation did not alter the amount of either ß-casein (29 kDa) or 
-casein (26 kDa) in milk (Fig. 3C). These data indicate that, in the setting of normocalcemia, the VDR is not required for protein secretion or calcium transport into milk, but VDR deficiency may modulate the accumulation or oxytocin-stimulated milk release during lactation.
|
, serum calcium increased during pregnancy and declined during lactation; however, these changes were comparable in WT and VDR KO mice, suggesting that the high-calcium diet is sufficient to prevent hypocalcemia in VDR KO mice even in a setting of increased calcium demand.
|
) and progesterone (Fig. 4C) increase during pregnancy, and prolactin increases during lactation (Fig. 4D), no differences in any of these hormones were detected between WT and VDR KO mice. Thus, the accelerated lobuloalveolar outgrowth observed in VDR KO mice was not secondary to alterations in circulating estradiol, progesterone, or prolactin.
Mammary Gland Regression after Weaning Is Delayed in VDR KO Mice
Involution of the mammary gland after weaning represents a period of intense apoptotic activity associated with remodeling of the tissue back to the quiescent state. Because 1,25-(OH)2D and the VDR trigger apoptosis in transformed mammary epithelial cells (21), we assessed the impact of VDR on glandular regression after weaning. To induce glandular involution, litter sizes were equalized after partuition at six pups, females lactated for 5 d, and then all pups were removed. Examination of whole-mounts indicated that glandular involution peaked on d 4 in WT mice, but on d 6 in VDR KO mice. For example, on d 4 of involution, glands from WT mice contained extensive alveolar condensation and stromal influx, with few residual alveolar structures (Fig. 5 A and C), whereas intact alveoli distended with milk predominated in glands from VDR KO females (Fig. 5, B and D). Even on d 6 of involution, alveolar breakdown was minimal, and dilated ducts containing milk persisted in VDR KO mice (Fig. 5, F and H), whereas in glands from WT mice evidence of milk breakdown, replacement of alveolar buds with condensed ductal structures and adipocyte recolonization were prevalent (Fig. 5, E and G).
|
. On d 3 of regression, the percentage of TUNEL-positive cells was significantly reduced in VDR KO mice compared with WT mice (Fig. 6C). Consistent with higher TUNEL staining in WT mice, Western blotting indicated that expression of the proapoptotic protein Bax was higher in WT mice than in VDR KO mice on d 1 of involution (Fig. 6D). Furthermore, activated caspase 3 was present as cleaved bands of 17 and 19 kDa in WT mice, beginning on d 1 of involution, but was not detected in glands from VDR KO mice on d 1 and was minimal on d 3 of involution (Fig. 6E). These data suggest that mammary gland regression is delayed by approximately 2 d in VDR KO mice, and this delay is associated with reduced apoptosis in the epithelial cell compartment.
|
), but circulating progesterone was higher in VDR KO mice compared with WT mice on d 2 and 4 post weaning (Fig. 7B). Because involution can be inhibited by elevated circulating progesterone, these findings suggest that VDR ablation may impinge on glandular regression via changes in systemic progesterone signaling.
|
, serum calcium increased significantly in WT mice, peaking at almost 18 mg/dl on d 4, the peak of glandular involution (P < 0.05, ANOVA, 10 wk vs. involution d 2 or 4). By d 6 of involution, the milk within the WT glands had been degraded and serum calcium dropped to about 13 mg/dl. During involution in VDR KO mice, serum calcium also increased beginning on d 2, but the elevation was sustained, peaking on d 6 (P < 0.05, ANOVA, 10 wk vs. involution d 2, 4, or 6) at levels higher than that of WT mice. This sustained increase in serum calcium could reflect the delayed glandular regression process in VDR KO mice because it coincided with the peak of glandular involution observed on d 6. However, our studies do not rule out the possibility that differences in serum calcium during involution of VDR KO mice may reflect altered extracellular calcium handling secondary to VDR deficiency in other tissues.
VDR Ablation Alters Mammary Gland ß-Casein Expression
We next determined whether the developmental differences observed in mammary glands from WT and VDR KO mice were associated with altered expression of ß-casein, which is selectively expressed by secretory epithelial cells (Fig. 8A). As expected, casein gene expression was not detected in glands from virgin WT mice, nor was casein expression induced in virgin mice as a result of VDR ablation. Although casein mRNA increased during pregnancy in both genotypes, casein was significantly higher in VDR KO mice than WT mice on d 9 of pregnancy. At pregnancy d 16, casein mRNA was higher than pregnancy d 9 for both WT and VDR KO mice, with no significant difference due to genotype. Casein gene expression peaked during lactation in both genotypes, with no difference in expression levels during lactation or the early stages of involution. During the second stage of involution beginning at d 4, glands from VDR KO mice exhibited significantly higher levels of ß-casein mRNA than glands from WT mice (Fig. 8B). Western blotting of mammary gland homogenates indicated that expression of both ß- and -caseins were elevated in VDR KO mice on d 16 of pregnancy and d 6 of involution (Fig. 8, C and D). Furthermore, Western blotting of glands removed on d 3 and 6 post weaning indicated that signaling through stat 5a, a major regulator of casein expression, was higher in VDR KO mice than WT mice (Fig. 8E). These data indicate precocious induction of milk production and a delay in the involution-associated decline in ß-casein in mammary glands lacking VDR and are consistent with the morphological evidence of accelerated lobuloalveolar development and delayed involution in VDR KO mice.
|
). Contrary to our hypothesis, however, glands from VDR KO mice expressed significantly higher levels of TGFß1 during puberty and pregnancy than glands from WT mice, whereas during lactation TGFß1 mRNA was lower in VDR KO mice than WT mice. As a result, TGFß1 expression increased almost 6-fold from early pregnancy to the peak of lactation in WT mice, but only 2-fold over the same period in VDR KO mice. During involution, glands from WT mice displayed a rapid increase in TGFß1 gene expression between d 2 and 3, whereas glands from VDR KO mice displayed a gradual increase which did not peak until d 6, suggesting that reduced TGFß1 gene expression may correlate with delayed apoptotic regression during postlactational involution in the VDR KO mammary gland.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Another potentially important outcome of these studies is identification of the 25(OH)D 1-hydroxylase mRNA and protein in mammary gland. Although previously thought to be exclusively expressed in kidney, this rate-limiting enzyme in the generation of 1,25-(OH)2D, the VDR ligand, has increasingly been identified in extra-renal tissues, including placenta, colon, and prostate gland (17, 23). Although functional studies are still needed to determine the significance of 1-hydroxylase in mammary gland, our data raise the possibility that bioactivation of 25(OH)D to 1,25-(OH)2D within the mammary epithelial cells could activate VDR in an autocrine or paracrine fashion.
Consistent with our findings, the VDR was recently identified as a gene whose expression increases sharply around parturition and stays high during lactation (24). These data confirm findings of Colston et al. (6) who reported increased 1,25-(OH)2D binding sites in mammary gland during pregnancy and lactation. In conjunction with our observation that VDR KO mice exhibit precocious alveolar development, these data suggest that the VDR may exert antiproliferative and/or prodifferentiating effects at the onset of alveolar formation. VDR signaling may trigger growth arrest of proliferating epithelial cells to induce differentiation before lactation, similar to the antiproliferative, prodifferentiating effects of VDR during puberty (7). Whereas VDR could exert effects locally or systemically, a direct effect of VDR on the gland is supported by previous organ culture studies in which we reported that glands from VDR KO mice exhibited increased growth and branching in response to lactogenic hormones compared with glands from WT mice and that 1,25-(OH)2D inhibited alveolar outgrowth in glands from WT mice but not in glands from VDR KO mice (7). Direct effects of 1,25-(OH)2D have also been clearly demonstrated in normal and transformed VDRpositive epithelial cells, but not in mammary cell lines from VDR KO mice (12, 13, 14, 25). Collectively, these studies suggest that the altered lobuloalveolar differentiation observed in VDR KO mice may result from loss of 1,25-(OH)2D3-VDR signaling within the mammary gland. In support of this suggestion, no changes in circulating estrogen, progesterone, or prolactin, critical regulators of glandular morphogenesis, were observed during pregnancy or lactation as a result of VDR ablation. However, we cannot not rule out the possibility that VDR in tissues other than the mammary gland and/or systemic effects might contribute to the mammary phenotype observed in VDR KO mice.
If VDR does exert direct effects within the mammary gland, the most likely mechanism would be via changes in gene expression. VDR gene regulation can reflect both ligand-dependent and ligand-independent mechanisms (26), and thus the mammary gland phenotype in VDR KO mice could be secondary to lack of either liganded or unliganded receptor. Assessment of glandular development in 25(OH)D 1-hydroxylase null mice (which lack the VDR ligand) will be necessary to distinguish between these distinct modes of action. With respect to ligand-dependent functions, however, microarray studies have identified more than 100 genes induced or repressed by 1,25-(OH)2D in VDR-positive breast cancer cells, including several known to impact on mammary gland development such as TGFß (27). In the current work, TGFß was studied as a potential VDR target gene because it regulates branching morphogenesis and alveolar differentiation, and because mice harboring a dominantnegative TGFß receptor II display precocious alveolar development (28) similar to that observed in VDR KO mice. In contrast to what would be predicted based on this phenotype similarity, however, TGFß1 gene expression was higher in glands from VDR KO mice than WT mice during early pregnancy. Thus, precocious alveolar development during early pregnancy of VDR KO mice is not simply secondary to impaired TGFß1 gene expression.
Our studies have confirmed the elevated expression of mammary gland VDR during lactation (6) but do not support the concept that VDR is required for casein production or calcium transport into milk. These data are in contrast to studies that demonstrated that vitamin D-depleted, hypocalcemic rodents displayed impaired casein production (15) but are consistent with studies of mice (29) and humans (30) that concluded that vitamin D deficiency has little effect on milk composition in the setting of normocalcemia. Although VDR ablation did not affect milk composition, we did observe an increase in secreted milk volume in response to exogenous oxytocin in VDR KO mice compared with WT mice. This novel finding suggests that lack of VDR may enhance myoepithelial cell sensitivity to oxytocin or may alter tight junction integrity; further studies are required to distinguish between these possibilities.
Another major contribution of this work is the demonstration that VDR expression remains high after weaning and that VDR ablation delays epithelial cell apoptosis during mammary gland involution. Although studies have shown that 1,25-(OH)2D induces apoptosis in breast cancer cells and tumors in a VDR-dependent fashion (25, 31, 32), the current findings provide the first evidence that VDR impacts on physiological apoptosis in vivo. Mammary gland regression after weaning proceeds in two phases: an initial, reversible stage stimulated by milk accumulation and a second irreversible stage characterized by protease activation and epithelial cell apoptosis (33). Postlactational changes in ß-casein expression were comparable in WT and VDR KO mice during the first phase of involution, but during the second phase of involution, ß-casein expression and phosphorylated stat 5a declined more rapidly in WT than in VDR KO mice. In addition, breakdown of the lobuloalveolar structures, bax induction, caspase-3 activation, and TGFß1 gene expression were all delayed in VDR KO mice compared with WT mice during this phase of involution. Because progesterone, which can inhibit both mammary gland involution (34) and TGFß1 expression (24), was elevated in serum of VDR KO mice, changes in production or turnover of this hormone in VDR KO mice could be responsible for the phenotype observed after weaning. Further studies will be required to address this issue and to identify the mechanistic basis for an effect of VDR on serum progesterone.
In summary, VDR KO mice exhibit precocious alveolar development, increased milk release, and delayed postlactational apoptotic regression in association with changes in genes related to differentiation and apoptosis. Furthermore, localization of the 25(OH)D 1-hydroxylase mRNA and protein to mammary cells suggests the possibility that 25(OH)D (an inactive, circulating vitamin D metabolite) might be converted to 1,25-(OH)2D within the mammary gland, providing a local source of ligand for the VDR. Although additional studies are needed to dissect the relative contributions of systemic vs. local factors and liganded vs. unliganded VDR in mediating the phenotype observed in VDR KO mice, these studies are the first to show that VDR modulates mammary gland morphology, function, and gene expression during pregnancy, lactation, and involution.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Analysis of Mammary Glands during Pregnancy, Lactation, and Involution
For developmental studies, six to 12 females/genotype were killed 9 or 16 d after copulation, 5 or 10 d postpartum, or up to 2 wk after pup removal after 5 d of lactation. For lactating or regressing females, litter sizes were equalized at six pups per litter right after partuition. At the time the mice were killed, one inguinal gland from each mouse was whole mounted, the contralateral inguinal gland was paraffin embedded, and the two thoracic glands were frozen for Western blotting or real-time PCR. For whole mount analysis, entire mammary glands were surgically dissected, spread on glass slides, fixed in Carnoy’s fixative, and stained overnight in Carmine Alum. Samples were dehydrated, cleared in xylene, mounted, and examined on an Olympus (Nashua, NH) SZX12 stereoscope.
For histology and immunohistochemistry, formalin-fixed mammary glands were embedded in paraffin, sectioned at 5 µm, and stained with H&E. To detect VDR, sections were incubated in 2 N HCl at 37 C for 20 min before overnight incubation with monoclonal VDR antibody (clone 9A7, Neomarkers, Fremont, CA). To detect 1-hydroxylase, slides in citrate buffer (pH 6.0) were heated in a pressure cooker for 2 min and incubated with sheep 1-hydroxylase (PC290, The Binding Site, Birmingham, UK) antibody for 1 h at 37 C and detected with the M.O.M. kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s directions. To detect ER and PR, slides in citrate buffer (pH 6.0) were heated in a pressure cooker for 15–20 min and incubated for 1 h with mouse ER (clone 6F11, Novocastra, Burlingame, CA) or PR (clone AB-7, NeoMarkers) antibodies and detected with the M.O.M. kit (Vector Laboratories) according to the manufacturer’s directions. For all antibodies, sections were counterstained with Harris modified hematoxylin (Fisher Scientific, Pittsburgh, PA).
For assessment of proliferation, BrdU incorporation was assessed in sections from mice injected with BrdU 2 h before the mice were killed. Mouse monoclonal biotinylated anti-BrdU (Zymed Laboratories Inc., San Francisco, CA) was used with the avidin-biotinylated enzyme complex technique as described (7). Apoptosis was assessed with a commercial TUNEL assay kit (Roche Diagnostics Corp., Indianapolis, IN) after optimization of pretreatment conditions and reagent dilutions to ensure that labeling was restricted to cells with apoptotic morphology. For both assays, tissues were counterstained with Harris modified hematoxylin (Fisher Scientific).
PR, BrdU incorporation, and TUNEL were quantified on photographs taken on an Olympus AX70 microscope at x40 with a Spot RT Slider camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Four fields (containing 
1000 cells) were assessed per animal, and a minimum of six animals were assessed per genotype. Data are expressed as mean ± SEM of the total number of positive cells per field and the percentage of positively stained cells.
Serum Hormone and Calcium Assays
Progesterone and 17ß-estradiol RIAs were conducted with reagent kits from DiaSorin (Stillwater, MN) and prolactin RIA was performed at the National Hormone and Peptide Program, directed by Dr. A. Parlow (Torrance, CA; parlow@humc.edu). Calcium was determined with a colorimetric assay kit (Sigma, St. Louis, MO) according to the manufacturer’s directions.
Milk Analyses
Milk was collected from ten lactating WT and VDR KO females with six pups per litter at 5 d postpartum. Pups were removed from lactating mice 4 h before an im dose of oxytocin (2 IU). Twenty minutes after oxytocin dosing, suction was applied quickly to all five sets of teats to resemble suckling activity and induce milk release. A collection tube was then placed in the vacuum chamber and suction was applied using a pulsating rhythm until milk secretion was complete. After measurement of volume, milk was diluted 25x in Laemmli buffer containing phosphatase and protease inhibitors (25) and analyzed for total protein and calcium content.
Real-Time Quantitative PCR
Total RNA was isolated from 90–150 mg of frozen mammary gland, kidney or intestine with Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA). Independent mammary gland RNA preps from five mice of each genotype were made at the time points indicated in the figure legends. After concentration and purity of the RNA was determined by spectrophotometry, total RNA was reverse transcribed with TaqMan Reverse Transcription Reagents (N808-0234, Applied Biosystems). Three independent 1.5-µg cDNA stocks were generated from each RNA sample, and each was independently analyzed in duplicate (60 ng of cDNA/well) using the TaqMan PCR Core Reagent Kit (N808-0228, Applied Biosystems) and specific primer and probe sets. Gene expression levels were normalized against 18S mRNA or cytokeratin 18 mRNA as indicated, and reported as normalized gene expression. For data presentation, duplicate values from each run were averaged, and triplicate values were then averaged to generate one value for each animal. The final data are expressed as the mean ± SE of five animals per time point.
Western Blotting
Thoracic mammary glands (100 mg) were homogenized in Laemmli buffer containing phosphatase and protease inhibitors (25), separated by SDS-PAGE, transferred to nitrocellulose, and blocked with either 3% BSA (for casein), 5% skin milk (for Bax, caspase-3, and Stat5a), or 20% skim milk (for 1-hydroxylase). Immunoblotting was performed with antibodies against ß-casein (generously provided by Dr. Itamar Barash, Institute of Animal Science, Bet-Dagan, Israel), Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), caspase-3 (Cell Signaling, Beverly, MA), total Stat5a or phospho-Stat5a (BD Transduction Laboratories), and 1-hydroxylase (The Binding Site). Milk samples, collected as described above, were diluted 25x in the same buffer, separated by SDS-PAGE (5 g protein/lane), transferred to nitrocellulose, blocked in 3% BSA overnight and incubated with ß-casein antibody. All blots were visualized by enhanced chemiluminescence using products from Pierce (Rockford, IL).
Statistical Evaluation
Data are presented as mean ± SEM, with the number of analyses for each mean indicated. Because our major objective was to assess the effect of genotype, Student’s t test was used to assess differences between WT and VDR KO mice at each time point. ANOVA was used to assess temporal changes; however, for clarity these comparisons are not shown on the individual figures but are discussed in the text. All statistical evaluations were performed with Instat software (GraphPad Software, Inc., San Diego, CA; http://www.graphpad.com), and means were considered significantly different if a P value less than 0.05 was obtained.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Abbreviations: BrdU, Bromodeoxyuridine; ER, estrogen receptor; H&E, hematoxylin and eosin; INV, involution; KO, knockout; LS, lactation; 1,25-(OH)2D, 1,25-dihydroxyvitamin D3; PR, progesterone receptor; STAT, signal transducer and activator of transcription; TM, timed mating; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling; VDR, vitamin D receptor; WT, wild-type; +/+, WT; –/–, VDR KO.
Received for publication December 4, 2003. Accepted for publication May 25, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel. Cell Biol Int 23:481–487[CrossRef][Medline]
-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors. Eur J Cancer 36:780–786
-Hydroxylase and the action of vitamin D. J Mol Endocrinol 25:141–148[Abstract]
-hydroxyvitamin D5. J Natl Cancer Inst 89:212–218NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  M. Sinotte, F. Rousseau, P. Ayotte, E. Dewailly, C. Diorio, Y. Giguere, S. Berube, and J. Brisson Vitamin D receptor polymorphisms (FokI, BsmI) and breast cancer risk: association replication in two case-control studies within French Canadian population Endocr. Relat. Cancer, December 1, 2008; 15(4): 975 - 983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. G. Tiffen, N. Omidvar, N. Marquez-Almuina, D. Croston, C. J. Watson, and R. W. E. Clarkson A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo Mol. Endocrinol., December 1, 2008; 22(12): 2677 - 2688. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H. F. Luderer, L. Lieben, C. Mathieu, and M. Demay Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice Endocr. Rev., October 1, 2008; 29(6): 726 - 776. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Valrance, A. H. Brunet, and J. Welsh Vitamin D Receptor-Dependent Inhibition of Mammary Tumor Growth by EB1089 and Ultraviolet Radiation in Vivo Endocrinology, October 1, 2007; 148(10): 4887 - 4894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Rowling, C. M. Kemmis, D. A. Taffany, and J. Welsh Megalin-Mediated Endocytosis of Vitamin D Binding Protein Correlates with 25-Hydroxycholecalciferol Actions in Human Mammary Cells J. Nutr., November 1, 2006; 136(11): 2754 - 2759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Woodrow, C. J. Sharpe, N. J. Fudge, A. O. Hoff, R. F. Gagel, and C. S. Kovacs Calcitonin Plays a Critical Role in Regulating Skeletal Mineral Metabolism during Lactation Endocrinology, September 1, 2006; 147(9): 4010 - 4021. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Toyama, Z. Zhang, H. Iwase, H. Yamashita, Y. Ando, M. Hamaguchi, M. Mizutani, N. Kondo, T. Fujita, Y. Fujii, et al. Low Expression of the Snail Gene is a Good Prognostic Factor in Node-Negative Invasive Ductal Carcinomas Jpn. J. Clin. Oncol., June 1, 2006; 36(6): 357 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Kemmis, S. M. Salvador, K. M. Smith, and J. Welsh Human Mammary Epithelial Cells Express CYP27B1 and Are Growth Inhibited by 25-Hydroxyvitamin D-3, the Major Circulating Form of Vitamin D-3 J. Nutr., April 1, 2006; 136(4): 887 - 892. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Diorio, S. Berube, C. Byrne, B. Masse, N. Hebert-Croteau, M. Yaffe, G. Cote, M. Pollak, and J. Brisson Influence of Insulin-like Growth Factors on the Strength of the Relation of Vitamin D and Calcium Intakes to Mammographic Breast Density Cancer Res., January 1, 2006; 66(1): 588 - 597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. G. Demicco, K. T. Kavanagh, R. Romieu-Mourez, X. Wang, S. R. Shin, E. Landesman-Bollag, D. C. Seldin, and G. E. Sonenshein RelB/p52 NF-{kappa}B Complexes Rescue an Early Delay in Mammary Gland Development in Transgenic Mice with Targeted Superrepressor I{kappa}B-{alpha} Expression and Promote Carcinogenesis of the Mammary Gland Mol. Cell. Biol., November 15, 2005; 25(22): 10136 - 10147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Dusso, A. J. Brown, and E. Slatopolsky Vitamin D Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Townsend, C. M. Banwell, M. Guy, K. W. Colston, J. L. Mansi, P. M. Stewart, M. J. Campbell, and M. Hewison Autocrine Metabolism of Vitamin D in Normal and Malignant Breast Tissue Clin. Cancer Res., May 1, 2005; 11(9): 3579 - 3586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Welsh Vitamin D and breast cancer: insights from animal models Am. J. Clinical Nutrition, December 1, 2004; 80(6): 1721S - 1724S. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Zinser and J. Welsh Vitamin D receptor status alters mammary gland morphology and tumorigenesis in MMTV-neu mice Carcinogenesis, December 1, 2004; 25(12): 2361 - 2372. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
