Ltbp1L is focally induced in embryonic mammary mesenchyme, demarcates the ductal luminal lineage and is upregulated during involution.

INTRODUCTION: Latent TGFß binding proteins (LTBPs) govern TGFß presentation and activation and are important for elastogenesis. Although TGFß is well-known as a tumor suppressor and metastasis promoter, and LTBP1 is elevated in two distinct breast cancer metastasis signatures, LTBPs have not been studied in the normal mammary gland. METHODS: To address this we have examined Ltbp1 promoter activity throughout mammary development using an Ltbp1L-LacZ reporter as well as expression of both Ltbp1L and 1S mRNA and protein by qRT-PCR, immunofluorescence and flow cytometry. RESULTS: Our data show that Ltbp1L is transcribed coincident with lumen formation, providing a rare marker distinguishing ductal from alveolar luminal lineages. Ltbp1L and Ltbp1S are silent during lactation but robustly induced during involution, peaking at the stage when the remodeling process becomes irreversible. Ltbp1L is also induced within the embryonic mammary mesenchyme and maintained within nipple smooth muscle cells and myofibroblasts. Ltbp1 protein exclusively ensheaths ducts and side branches. CONCLUSIONS: These data show Ltbp1 is transcriptionally regulated in a dynamic manner that is likely to impose significant spatial restriction on TGFß bioavailability during mammary development. We hypothesize that Ltbp1 functions in a mechanosensory capacity to establish and maintain ductal luminal cell fate, support and detect ductal distension, trigger irreversible involution, and facilitate nipple sphincter function.

Parathyroid hormone-related protein specifies the mammary mesenchyme and regulates embryonic mammary development.

Parathyroid Hormone related Protein (PTHrP) is a critical regulator of mammary gland morphogenesis in the mouse embryo. Loss of PTHrP, or its receptor, PTHR1, results in arrested mammary buds at day 15 of embryonic development (E15). In contrast, overexpression of PTHrP converts the ventral epidermis into hairless nipple skin. PTHrP signaling appears to be critical for mammary mesenchyme specification, which in turn maintains mammary epithelial identity, directs bud outgrowth, disrupts the male mammary rudiment and specifies the formation of the nipple. In the embryonic mammary bud, PTHrP exerts its effects on morphogenesis, in part, through epithelial-stromal crosstalk mediated by Wnt and BMP signaling. Recently, PTHLH has been identified as a strong candidate for a novel breast cancer susceptibility locus, although PTHrP's role in breast cancer has not been clearly defined. The effects of PTHrP on the growth of the embryonic mammary rudiment and its invasion into the dermis may, in turn, have connections to the role of PTHrP in breast cancer.

Parathyroid hormone-related protein activates Wnt signaling to specify the embryonic mammary mesenchyme.

Parathyroid hormone-related protein (PTHrP) regulates cell fate and specifies the mammary mesenchyme during embryonic development. Loss of PTHrP or its receptor (Pthr1) abolishes the expression of mammary mesenchyme markers and allows mammary bud cells to revert to an epidermal fate. By contrast, overexpression of PTHrP in basal keratinocytes induces inappropriate differentiation of the ventral epidermis into nipple-like skin and is accompanied by ectopic expression of Lef1, ß-catenin and other markers of the mammary mesenchyme. In this study, we document that PTHrP modulates Wnt/ß-catenin signaling in the mammary mesenchyme using a Wnt signaling reporter, TOPGAL-C. Reporter expression is completely abolished by loss of PTHrP signaling and ectopic reporter activity is induced by overexpression of PTHrP. We also demonstrate that loss of Lef1, a key component of the Wnt pathway, attenuates the PTHrP-induced abnormal differentiation of the ventral skin. To characterize further the contribution of canonical Wnt signaling to embryonic mammary development, we deleted ß-catenin specifically in the mammary mesenchyme. Loss of mesenchymal ß-catenin abolished expression of the TOPGAL-C reporter and resulted in mammary buds with reduced expression of mammary mesenchyme markers and impaired sexual dimorphism. It also prevented the ectopic, ventral expression of mammary mesenchyme markers caused by overexpression of PTHrP in basal keratinocytes. Therefore, we conclude that a mesenchymal, canonical Wnt pathway mediates the PTHrP-dependent specification of the mammary mesenchyme.

Parathyroid hormone-related protein is not required for normal ductal or alveolar development in the post-natal mammary gland.

PTHrP is necessary for the formation of the embryonic mammary gland and, in its absence, the embryonic mammary bud fails to form the neonatal duct system. In addition, PTHrP is produced by the breast during lactation and contributes to the regulation of maternal calcium homeostasis during milk production. In this study, we examined the role of PTHrP during post-natal mammary development. Using a PTHrP-lacZ transgenic mouse, we surveyed the expression of PTHrP in the developing post-natal mouse mammary gland. We found that PTHrP expression is restricted to the basal cells of the gland during pubertal development and becomes expressed in milk secreting alveolar cells during pregnancy and lactation. Based on the previous findings that overexpression of PTHrP in cap and myoepithelial cells inhibited ductal elongation during puberty, we predicted that ablation of native PTHrP expression in the post-natal gland would result in accelerated ductal development. To address this hypothesis, we generated two conditional models of PTHrP-deficiency specifically targeted to the postnatal mammary gland. We used the MMTV-Cre transgene to ablate the floxed PTHrP gene in both luminal and myoepithelial cells and a tetracycline-regulated K14-tTA;tetO-Cre transgene to target PTHrP expression in just myoepithelial and cap cells. In both models of PTHrP ablation, we found that mammary development proceeds normally despite the absence of PTHrP. We conclude that PTHrP signaling is not required for normal ductal or alveolar development.

A mouse transgenic approach to induce ß-catenin signaling in a temporally controlled manner.

Although constitutive murine transgenic models have provided important insights into ß-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated ß-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of ß-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89ß-catenin responder transgenic mouse. Crossed with the MTB transgenic effector mouse, which targets the expression of the reverse tetracycline transactivator (rtTA) to the mammary epithelium, we demonstrate that the stabilized (and activated) form of ß-catenin (ΔN89ß-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment. Furthermore, we show that transgene-derived ΔN89ß-catenin elicits significant mammary epithelial proliferation and precocious alveologenesis in the virgin doxycycline-treated MTB/TETO-ΔN89ß-catenin bitransgenic. Remarkably, deinduction of TETO-ΔN89ß-catenin transgene expression (through doxycycline withdrawal) results in the reversal of these morphological changes. Importantly, continued activation of the TETO-ΔN89ß-catenin transgene results in palpable mammary tumors (within 7-9 months) in the doxycycline-treated virgin MTB/TETO-ΔN89ß-catenin bigenic but not in the same bitransgenic without doxycycline administration. Collectively, these mammary epithelial responses to ΔN89ß-catenin expression agree with previous reports using conventional transgenesis and therefore confirm that ΔN89ß-catenin functions as expected in this doxycycline-responsive bigenic system. In sum, our mammary gland studies demonstrate "proof-of-principle" for using the TETO-ΔN89ß-catenin transgenic responder to activate (and then de-activate) ß-catenin signaling in any tissue of interest in a spatiotemporal specific fashion.

Analysis of gene expression in PTHrP-/- mammary buds supports a role for BMP signaling and MMP2 in the initiation of ductal morphogenesis.

Parathyroid hormone-related protein (PTHrP) acts on the mammary mesenchyme and is required for proper embryonic mammary development. In order to understand PTHrP's effects on mesenchymal cells, we profiled gene expression in WT and PTHrP(-/-) mammary buds, and in WT and K14-PTHrP ventral skin at E15.5. By cross-referencing the differences in gene expression between these groups, we identified 35 genes potentially regulated by PTHrP in the mammary mesenchyme, including 6 genes known to be involved in BMP signaling. One of these genes was MMP2. We demonstrated that PTHrP and BMP4 regulate MMP2 gene expression and MMP2 activity in mesenchymal cells. Using mammary bud cultures, we demonstrated that MMP2 acts downstream of PTHrP to stimulate ductal outgrowth. Future studies on the functional role of other genes on this list should expand our knowledge of how PTHrP signaling triggers the onset of ductal outgrowth from the embryonic mammary buds.

MMTV-Wnt1 and -DeltaN89beta-catenin induce canonical signaling in distinct progenitors and differentially activate Hedgehog signaling within mammary tumors.

Canonical Wnt/beta-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-DeltaN89beta-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin transgenes induce tumors with different phenotypes. Using axin2/conductin reporter genes we show that MMTV-Wnt1 and MMTV-DeltaN89beta-catenin activate canonical Wnt signaling within distinct cell-types. DeltaN89beta-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18(+)ER(-)PR(-)CD24(high)CD49f(low) profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14(+) basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14(+)/p63(+) subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-DeltaN89beta-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.

The pattern of beta-catenin responsiveness within the mammary gland is regulated by progesterone receptor.

Experiments involving beta-catenin loss- and gain-of-function in the mammary gland have decisively demonstrated the role of this protein in normal alveologenesis. However, the relationship between hormonal and beta-catenin signaling has not been investigated. In this study, we demonstrate that activated beta-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of DeltaN89beta-catenin. Cells at ductal tips are inherently beta-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers beta-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by beta-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling.

Beta-catenin and Tcfs in mammary development and cancer.

Beta-catenin regulates cell-cell adhesion and transduces signals from many pathways to regulate the transcriptional activities of Tcf/Lef DNA binding factors. Gene ablation and transgenic expression studies strongly support the concept that beta-catenin together with Lef/Tcf factors act as a switch to determine cell fate and promote cell survival and proliferation at several stages during mammary gland development. Mice expressing the negative regulator of Wnt/beta-catenin signaling (K14-Dkk) fail to form mammary buds, and those lacking Lef-1 show an early arrest in this process at stage E13.5. Stabilized deltaN89beta-catenin initiates precocious alveologenesis during pubertal development, and negative regulators of endogenous beta-catenin signaling suppress normal alveologenesis during pregnancy. Stabilized beta-catenin induces hyperplasia and mammary tumors in mice. Each of the beta-catenin-induced phenotypes is accompanied by upregulation of the target genes cyclin D1 and c-myc. Cyclin D1, however, is dispensable for tumor formation and the initiation of alveologenesis but is essential for later alveolar expansion.