The Wnt Co-Receptor Lrp6 Is Required for Normal Mouse Mammary Gland Development

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland

Modulation of β-Catenin Signaling by Glucagon Receptor Activation

The glucagon receptor (GCGR) is a member of the class B G protein–coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA) pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin–mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R) and glucagon-like peptide 1 (GLP-1R) receptors. Since low-density-lipoprotein receptor–related protein 5 (Lrp5) is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter–mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1) or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations

Wnt/β-catenin Signaling in Normal and Cancer Stem Cells

The ability of Wnt ligands to initiate a signaling cascade that results in cytoplasmic stabilization of, and nuclear localization of, β-catenin underlies their ability to regulate progenitor cell differentiation. In this review, we will summarize the current knowledge of the mechanisms underlying Wnt/β-catenin signaling and how the pathway regulates normal differentiation of stem cells in the intestine, mammary gland, and prostate. We will also discuss how dysregulation of the pathway is associated with putative cancer stem cells and the potential therapeutic implications of regulating Wnt signaling

Increased <i>Lrp6</i> expression in basal-like human breast cancer.

<p>The relative expression of <i>Lrp6</i> in breast cancer samples analyzed by Affymetrix and organized into defined subgroups. Each dot represents the relative expression of <i>Lrp6</i> in one tumor sample. (A) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Richardson1" target="_blank">[30]</a>. <i>Normal</i> includes samples obtained from normal breast tissue. (B) Expression data was obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005813#pone.0005813-Herschkowitz1" target="_blank">[31]</a>. <i>Normal Breast-like</i> includes samples obtained from normal and cancerous breast tissue that exhibited expression profiles similar to that of normal breast tissue. A subset of breast cancers within the basal-like subgroup of both studies exhibit increased expression of <i>Lrp6</i>.</p

Expression pattern of <i>Lrp6</i> in the mammary gland.

<p>β<i>-gal</i> expression directed from the <i>Lrp6</i> promoter in <i>Lrp6^+/−</i> mice was used as a surrogate marker for <i>Lrp6</i> expression. We used the β<i>-gal</i> substrates X-gal and DDAOG to identify cells that express <i>Lrp6</i>. Shown in (A-B) are X-gal-stained mammary whole mounts and 5-µm sections from 2-day-old (A) and 12-week-old (B) female mice. (A) <i>Lrp6</i> promoter-driven β<i>-gal</i> expression is visible as blue staining in both the basal and luminal mammary epithelium and in the mammary fat pad of newborn mice. (B) In older mice, β<i>-gal</i>-expressing cells are primarily identified within the basal epithelial cell layer and in the mammary fat pad. The <i>arrows</i> indicate typical cells that stained blue with X-gal. No staining was detected in <i>Lrp6^+/+</i> mammary glands, which were used as negative controls for X-gal staining. (C-F) Representative FACS results of DDAOG-stained and CD24/CD49f antibody–labeled mammary epithelial cells. The β<i>-gal</i>-cleaved product of DDAOG has far-red fluorescence and was used to detect cells with <i>Lrp6</i> promoter–driven β<i>-gal</i> expression. (C) The luminal and basal cell compartments are marked by pink and blue dashed lines, respectively. (D) 80% of the DDAOG-positive cells are found within the basal epithelial cell compartment. (E-F) DDAOG gating strategy. The DDAOG gate is indicated by the black line. The <i>Lrp6^+/−</i> sample (E) has 0.33% DDAOG-positive cells; the <i>Lrp6^+/+</i> negative control sample (F) has 0.01% DDAOG-positive cells.</p

<i>Lrp6</i> is required for embryonic mammary development.

<p>(A) Carmine-stained skin pads of inguinal mammary glands at E18.5. While in the <i>Lrp6^+/+</i> mammary gland the nipple, rudimentary ductal tree, and fat pad are all normally developed, the <i>Lrp6^−/−</i> mammary gland contains a small nipple, a single ductal out-growth, and an abnormally small fat pad. <i>Dashed lines</i> indicate inguinal epithelium. (B) Oil red O staining of mammary fat pads from <i>Lrp6^+/+</i> and <i>Lrp6^−/−</i> embryos. The <i>Lrp6^−/−</i> fat pad is abnormally small compared to that of the littermate control. <i>Scale bar</i>: 0.5 mm. (C-D) X-gal-stained <i>BAT-gal</i> embryo whole mounts (C) and histology sections of mammary placode (D) at E12.5. Cells expressing BAT-gal are stained blue. (C) X-gal stains the mammary placodes of <i>BAT-gal;Lrp6^+/+</i> embryos dark blue. <i>Arrow heads</i> indicate mammary placodes number 2, 3, 4, and 5. Mammary placodes are not readily visible on X-gal-stained <i>BAT-gal;Lrp6^−/−</i> embryo whole mounts. (D) On the histological level, the mammary placodes of <i>BAT-gal;Lrp6^−/−</i> embryos are significantly smaller and exhibit fewer cells with <i>BAT-gal</i> expression than the mammary placodes of littermate controls. <i>Dashed lines</i> indicate inguinal placodes.</p

Wnt1-induced mammary tumorigenesis in <i>Lrp6^+/−</i> females.

<p>Shown in (A-B) are representative carmine stained skin pads and mammary whole mounts. (A) Skin pads collected from E18.5 <i>MMTV-Wnt1;Lrp6^+/+</i> and <i>MMTV-Wnt1;Lrp6^−/−</i> embryos. (B) Mammary whole mounts collected from adult <i>MMTV-Wnt1;Lrp6^+/+</i> and <i>MMTV-Wnt1;Lrp6^+/−</i> females. (C) The percentages of <i>MMTV-Wnt1;Lrp6^+/+</i> (<i>n</i> = 12) and <i>MMTV-Wnt1;Lrp6^+/−</i> mice (<i>n</i> = 13) that were tumor-free (as determined by weekly visual inspection and/or palpation) were plotted against the age when tumors were found. (D) Standard histopathological evaluation showed that all <i>Lrp6^+/+</i> and <i>Lrp6^+/− MMTV-Wnt1</i> tumors are moderately differentiated alveolar mammary adenocarcinomas.</p

Haploinsufficiency for <i>Lrp6</i> in postnatal mammary development.

<p>(A) Representative mammary whole-mount preparations are shown for juvenile (5-week-old) mice. The <i>arrows</i> indicate typical terminal end buds; <i>LN</i>, lymph node. (B) The result of morphometric analysis of the average number of TEBs at 5 weeks and (C) of branches per gland at 11 weeks. At least 10 animals of each genotype were analyzed for each time point. In the absence of one copy of <i>Lrp6</i>, the number of TEBs is reduced by 33% (<i>p</i> = 1.3×10⁻⁶, 2-tailed <i>t</i> test assuming unequal variances), and the number of branches per gland is reduced by 17% (<i>p</i> = 8.4×10⁻³, 2-tailed <i>t</i> test assuming unequal variances) compared with <i>Lrp6</i>^+/+ littermate controls. (D) Mammary whole mounts containing ductal colonization originating from transplanted <i>Lrp6^+/+</i> or <i>Lrp6^+/−</i> mammary epithelial cells. Also shown is a transplantation control whose inguinal fat pads were cleared of endogenous epithelium but not injected with mammary cells. (E) The inguinal mammary gland from an adult <i>Lrp6^+/−;Lrp5^−/−</i> female. The mammary gland contains a fat pad and a nipple with associated epithelium but lacks a ductal tree. <i>Box</i> indicates the nipple epithelium.</p

Lrp5 coexpression enhances glucagon and GLP1-induced β-catenin signaling.

<p>A). HEK293 cells cultured in 12-well plate were transfected with a combination of indicated plasmids (GCGR 1000 ng, Lrp5 500 ng) for 24 h and then treated with or without 50 nM GCG1-29 for 1 h. The cells were harvested and lysed, and samples were used for western blot analysis. The blot was first probed with anti-β-catenin antibody and then stripped and reprobed for anti-β-actin antibody as a loading control. B). 293STF cells cultured in 24-well plate were transfected with 100 ng of empty vector (pcDNA3.1) or GCGR plasmid along with the indicated amount of Lrp5 and 5 ng TKRlu plasmids on day 1, and then treated with or without 50 nM GCG1-29 on day 2. Cells were harvested for luciferase activity measurement on day 3. Triplicate samples were used for each treatment. *p<0.005 compared with non-treated group. 4C). 293STF cells cultured in 24-well plate were transfected with 100 ng GLP1R, 100 ng Lrp5 and 5 ng TKRlu plasmids on day 1 and then treated with the GLP1 agonist GLP1(7–36) (50 nM) or the antagonist Exendin(9–39) (50 nM) on day 2. Cells were harvested on day 3 to measure luciferase activity. Duplicate samples were used for each treatment. *p<0.05 compared with the non-treated (NT) group.</p

Blocking Lrp5/6 inhibited glucagon-induced β-catenin signaling.

<p>A). 293STF cells cultured in 24-well plate were transfected with a combination of GCGR (100 ng), Lrp5 (100 ng), Lrp5ECD (200 ng) and TKRlu (5 ng) plasmids as indicated on day 1, and then were treated with or without 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the TCF-mediated luciferase activity. Triplicate samples were used for each treatment. *p<0.005 compared with the non-treated (NT) group. ^#p<0.005 compared with the group without the Lrp5ECD transfection. B). DKK1 protein inhibits glucagon-induced β-catenin signaling. 293STF cells cultured in 24-well plate were transfected with pcDNA3.1 and GCGR plasmids (100 ng each) or GCGR and Lrp5 plasmids (100 ng each) along with 5 ng TKRlu plasmid on day 1, and then were treated with 50 nM GCG1-29±2 µg/ml DKK1 on day 2. Cells were harvested on day 3 to measure the luciferase activity. Duplicate samples were used for each treatment. *p<0.02 compared with the GCG1-29-treated group.</p