Age-Related Changes in the Epithelial and Stromal Compartments of the Mammary Gland in Normocalcemic Mice Lacking the Vitamin D3 Receptor

The vitamin D3 receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation and involution. To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17β-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment

Effect of VDR ablation on serum calcium, estrogen, and estrogen responsive tissues in aging WT and VDR KO mice.

<p>A. Serum calcium levels in aging WT and VDR KO mice were equivalent prior to (2 and 8 months) and throughout the aging time course (12–16 months). B. Serum estrogen was measured by radioimmunoassay in WT and VDR KO mice at 2, 8, 12, 14, and 16 months of age. There was a significant reduction in serum estrogen by 12 months of age in the VDR KO mice that persisted through 16 months of age. *Statistically significant by Students t test, WT vs. VDR KO, n = 8–10 p<0.05. C. Representative hematoxylin and eosin Y stained sections of the ovary and the uterus, an estrogen responsive tissue. VDR KO tissues show signs of atrophy, likely due to incomplete follicle formation in the ovary and thus decreasing serum estrogen and inducing atrophy in the uterus compared to WT ovary supporting follicle formation and a responsive and robust uterus. Scale bar: 200 µm.</p

Morphological and quantitative assessment of inguinal mammary glands from 12, 14, and 16 month old WT and VDR KO mice.

<p>A. (Left Panels) Glands from VDR KO mice show decreased secondary and tertiary branching, signs of ductal thickening (ectasia), and large lymph nodes as the adipose tissue begins to atrophy with age. In contrast, WT glands show an extensive array of secondary and tertiary branches resting in a vast fat pad of plentiful adipose tissue. (Middle Panels) Glands at higher magnification from VDR KO mice show reduced secondary and tertiary branching and areas of darkly stained lesions along primary ducts (arrows). (Right Panels) Histological sections stained with hematoxylin and eosin Y show clusters of inflammatory cells along the primary ducts within glands of VDR KO mice compared to WT glands. B. Primary, secondary and tertiary branch points were quantitated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016479#s4" target="_blank">materials and methods</a>. Primary branch counts showed a significant decrease between WT and KO glands at 16 months whereas secondary and tertiary branches were significantly decreased in glands from VDR KO mice at 14 and 16 months compared to WT control mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. C. Whole mounts from VDR WT and KO mice were utilized to measure the area of mammary fat pads using AxioVision software. Fat pad area was calculated by tracing around the exterior of the ductal branches of the mammary fat pads. At 14 and 16 months of age, there is a significant reduction in the mammary fat pads of VDR KO mice compared to WT controls. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05. D. Whole mounts from VDR WT and KO mice were utilized to measure the mammary lymph nodes. Using AxioVision software, the exterior of the glandular lymph node was traced to calculate the area. At all time points, lymph nodes within glands from VDR KO mice were significantly enlarged compared to lymph nodes from WT mice. *Statistically significant by Students t test, WT vs. VDR KO, n = 10–12 p<0.05.</p

Adipose morphology and indirect calorimetric assessment throughout the aging of VDR WT and KO mice.

<p>A. Histological sections stained with hematoxylin and eosin Y show the small multi-locular adipocytes in glands of 16 month VDR KO mice compared to the large uni-locular fat cells in WT mice. B. Serum leptin was assessed by ELISA. There was a significant reduction in serum leptin by 8 months of age in the VDR KO mice that persisted through 16 months of age as a result of WT mice experiencing a progressive elevation in serum leptin beginning at 6 months and peaking at 16 months. C. Indirect calorimetric measurements were conducted every three months (3–12 months) in VDR WT and KO mice. Average oxygen consumption (VO₂) was trending higher by 6 months in VDR KO mice and was significantly elevated at 9 and 12 months compared to VDR WT mice. n = 6 p<0.05. Energy expenditure (Heat Kcal/hr/mouse) was similar to VO₂ in that VDR KO mice experience a significant elevation in energy expenditure by 9 and 12 months. n = 6 p<0.05. Respiratory exchange ratio (RQ), which infers the energy source utilized by the animal, was equivalent between VDR WT and KO mice throughout the aging time course (3–12 months).</p

Expression of the VDR in aging mammary glands of WT mice.

<p>A. Real Time PCR for VDR gene expression in aging mammary glands (12, 14, and 16 months) derived from wild type (WT) mice were compared to mature 10 week (10wk) pubertal and late pregnancy mammary glands. Data are expressed relative to 18S RNA (Relative VDR Expression) and represent mean ± s.e.m. of triplicate runs. B. Western blot of VDR protein expression in aging mammary glands (12 and 14 months) from WT mice, which is undetectable in VDR KO glands. C. Formalin fixed sections of mammary gland from 12, 14, and 16 month old WT mice were subjected to immunohistochemistry with a monoclonal antibody directed against VDR. A representative stained section of a 12 month WT gland shows positive staining in the mammary epithelium, stroma and adipose, a pattern that is similar to pubertal mammary gland expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016479#pone.0016479-Zinser2" target="_blank">[15]</a>. VDR positive cells appear brown against the blue hematoxylin counterstain.</p

Stromal smooth muscle actin and cell junction expression in glands from WT and VDR KO mice.

<p>A. Elevated level of smooth muscle actin staining in VDR KO glands compared to VDR WT control sections. SMA positive cells appear brown against the blue hematoxylin counterstain. B. Western blot for cell junction markers E-cadherin, β-catenin, and occludin to assess changes in the epithelial cell junctions that may account for the increase in secretion within the VDR KO ducts. E-cadherin, β-catenin, and occludin expression were equivalent at all time points during aging development between VDR WT and KO mammary glands.</p