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.

Targeting RANKL to a specific subset of murine mammary epithelial cells induces ordered branching morphogenesis and alveologenesis in the absence of progesterone receptor expression.

Despite support for receptor of activated NF-κB ligand (RANKL) as a mediator of mammary progesterone action, the extent to which this cytokine can functionally contribute to established progesterone-induced mammary morphogenetic responses in the absence of other presumptive effectors is still unclear. To address this uncertainty, we developed an innovative bigenic system for the doxycycline-inducible expression of RANKL in the mammary epithelium of the progesterone receptor knockout (PRKO) mouse. In response to acute doxycycline exposure, RANKL is specifically expressed in the estrogen receptor α (ER) positive/progesterone receptor negative (ER(+)/PR(-)) cell type in the PRKO mammary epithelium, a cell type that is equivalent to the ER(+)/PR(+) cell type in the wild-type (WT) mammary epithelium. Notably, the ER(+)/PR(+) mammary cell normally expresses RANKL in the WT mammary epithelium during pregnancy. In this PRKO bigenic system, acute doxycycline-induced expression of RANKL results in ordered mammary ductal side branching and alveologenesis, morphological changes that normally occur in the parous WT mouse. This mammary epithelial expansion is accompanied by significant RANKL-induced luminal epithelial proliferation, which is driven, in part, by indirect induction of cyclin D1. Collectively, our findings support the conclusion that RANKL represents a critical mediator of mammary PR action and that restricted expression of this effector to the ER(+)/PR(+) mammary cell-type is necessary for a spatially ordered morphogenetic response to progesterone.

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland.

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR(flox)) in mice. Crossing the PR(flox/flox) mouse with the ZP3-cre transgenic demonstrated that the PR(flox) allele recombines to a PR null allele (PR(d)). Mice homozygous for the recombined null PR allele (PR(d/d)) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage-specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone-responsive target sites.

The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse.

Receptor of Activated NF-kappaB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.

Transcriptional response of the murine mammary gland to acute progesterone exposure.

Our mechanistic understanding of progesterone's involvement in murine mammary morphogenesis and tumorigenesis is dependent on defining effector pathways responsible for transducing the progesterone signal into a morphogenetic response. Toward this goal, microarray methods were applied to the murine mammary gland to identify novel downstream gene targets of progesterone. Consistent with a tissue undergoing epithelial expansion, mining of the progesterone-responsive transcriptome revealed the up-regulation of functional gene classes involved in epithelial proliferation and survival. Reassuringly, signaling pathways previously reported to be responsive to progesterone were also identified. Mining this informational resource for rapidly induced genes, we identified "inhibitor of differentiation 4" (Id4) as a new molecular target acutely induced by progesterone exposure. Mammary Id4 is transiently induced during early pregnancy and colocalizes with progesterone receptor (PR) expression, suggesting that Id4 mediates the early events of PR-dependent mammary morphogenesis. Chromatin immunoprecipitation assay detecting direct recruitment of ligand occupied PR to the Id4 promoter supports this proposal. Given that Id4 is a member of the Id family of transcriptional regulators that have been linked to the maintenance of proliferative status and tumorigenesis, the establishment of a mechanistic link between PR signaling and Id4 promises to furnish a wider conceptual framework with which to advance our understanding of normal and abnormal mammary epithelial responses to progestins. In sum, the progesterone-responsive transcriptome described herein not only reinforces the importance of progesterone in mammary epithelial expansion but also represents an invaluable information resource with which to identify novel signaling paradigms for mammary PR action.

A repressive role for prohibitin in estrogen signaling.

Nuclear receptor-mediated gene expression is regulated by corepressors and coactivators. In this study we demonstrate that prohibitin (PHB), a potential tumor suppressor, functions as a potent transcriptional corepressor for estrogen receptor alpha (ERalpha). Overexpression of PHB inhibits ERalpha transcriptional activity, whereas depletion of endogenous PHB increases the expression of ERalpha target genes in MCF-7 breast cancer cells. Chromatin immunoprecipitation experiments demonstrate that PHB is associated with the estrogen-regulated pS2 promoter in the absence of hormone and dissociates after estradiol treatment. We demonstrate that PHB interacts with the repressor of estrogen receptor activity (REA), a protein related to PHB, to form heteromers and enhance the protein stability of both corepressors. Interestingly, the corepressor activity of PHB is cross-squelched by the coexpression of REA (and vice versa), suggesting that PHB and REA repress transcription only when they are not paired. We further demonstrate that coiled-coil domains located in the middle of PHB and REA are responsible for their heteromerization, stabilization, and cross-squelching actions. Finally, ablation of PHB function in the mouse results in early embryonic lethality, whereas mice heterozygous for the PHB null allele exhibit a hyperproliferative mammary gland phenotype. Our results indicate that PHB functions as a transcriptional corepressor for ERalpha in vitro and in vivo, and that its heteromerization with REA acts as a novel mechanism to limit its corepressor activity.

Targeting reverse tetracycline-dependent transactivator to murine mammary epithelial cells that express the progesterone receptor.

Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression.

Progesterone-action in the murine uterus and mammary gland requires steroid receptor coactivator 2: relevance to the human.

The importance of the progesterone receptor (PR) in female reproductive and mammary gland biology is well recognized; however, the coregulators selectively enlisted by PR have yet to be comprehensively defined in vivo. To evaluate the involvement of steroid receptor coactivator (SRC)/p160 family members in these physiological systems, a mouse model (PRCre/+SRC-2flox/flox) was generated in which SRC-2 function was ablated specifically in cell-types that express the PR. Although PRCre/+SRC-2flox/flox ovarian activity was normal, uterine function was severely compromised. Absence of SRC-2 in PR positive uterine cells led to an early block in embryo implantation, a defect not ascribed to SRC-1 or -3 knockouts. While the PRCre/+SRC-2flox/flox uterus can display a partial decidual response, removal of SRC-1 in the PRCre/+SRC-2flox/flox uterus results in a block in decidualization, confirming that uterine SRC-2 and -1 are both necessary for PR-mediated transcriptional responses which lead to complete decidualization. The absence of significant branching and alveolar morphogenesis in the hormone-treated PRCre/+SRC-2flox/flox mammary gland establishes an important role for mammary SRC-2 in cellular proliferative programs that require PR. Finally, the observation that SRC-2 is also expressed in many of the same cell-types in the human, underscores the importance of further study of this coregulator's role in both peri-implantation biology and mammary development.

Steroid receptor coactivator 2 is required for female fertility and mammary morphogenesis: insights from the mouse, relevance to the human.

Although the importance of the progesterone receptor (PR) to female reproductive and mammary gland biology is firmly established, the coregulators selectively co-opted by PR in these systems have not been clearly delineated. A selective gene-knockout approach applied to the mouse, which abrogates gene function only in cell types that express PR, recently disclosed steroid receptor coactivator 2 (SRC-2, also known as TIF-2 or GRIP-1) to be an indispensable coregulator for uterine and mammary gland responses that require progesterone. Uterine cells positive for PR (but devoid of SRC-2) were found to be incapable of facilitating embryo implantation, a necessary first step toward the establishment of the materno-fetal interface. Importantly, such an implantation defect is not exhibited by knockouts for SRC-1 or SRC-3, underscoring the unique coregulator importance of SRC-2 in peri-implantation biology. Moreover, despite normal levels of PR, SRC-1 and SRC-3, progesterone-dependent branching morphogenesis and alveologenesis fails to occur in the murine mammary gland in the absence of SRC-2, thereby establishing a critical coregulator role for SRC-2 in signaling cascades that mediate progesterone-induced mammary epithelial proliferation. Finally, the recent detection of SRC-2 in the human endometrium and breast suggests that this coregulator may represent a new clinical target for the future management of female reproductive health and/or breast cancer.

Targeting iCre expression to murine progesterone receptor cell-lineages using bacterial artificial chromosome transgenesis.

Gene-targeting in embryonic stem cells has been the dominant genetic approach when engineering mouse models to query the physiologic importance of the progesterone receptor (PR). Although these models have been instrumental in disclosing the in vivo significance of the progesterone signaling pathway, generation of such mice exacts considerable expenditure of time, effort, and expense. Considering the growing list of new PR mouse models that are urgently required to address the next questions in progestin biology, bacterial artificial chromosome (BAC) recombineering in conjunction with transgenesis was evaluated as an alternative method to accelerate the creation of these models in the future. Using this approach, we describe the generation of three PR-BAC(iCre) transgenic lines in which improved Cre recombinase (iCre) was targeted in-frame, downstream, and under the control of the PR promoter contained within a BAC transgene. Crossing with the ROSA26R revealed that the PR-BAC(iCre) transgenic expresses active iCre only in cell-lineages that express the PR. The specificity of the PR-BAC(iCre) transgene not only underscores the importance of BAC-mediated transgenesis as a quick, easy, and affordable method by which to engineer the next generation of PR mouse models, but also provides a unique opportunity to investigate transcriptional control of PR expression as well as PR structure-function relationships in vivo.

Steroid receptor coactivator 2 is essential for progesterone-dependent uterine function and mammary morphogenesis: insights from the mouse--implications for the human.

While the indispensability of the progesterone receptor (PR) in female reproduction and mammary morphogenesis is acknowledged, the coregulators preferentially recruited by PR to mediate its in vivo effects have yet to be fully delineated. To further parse the roles of steroid receptor coactivator (SRC)/p160 family members in P-dependent physiological processes, genetic approaches were employed to generate a mouse model (PR(Cre/+)SRC-2(flox/flox)) in which SRC-2 function was ablated specifically in cell-types that express the PR. Fertility evaluation revealed that while ovulation occurred normally in the PR(Cre/+)SRC-2(flox/flox) mouse, uterine function was markedly affected. Absence of SRC-2 in PR positive uterine cells contributed to an early block in embryo implantation, a phenotype not shared by knockouts for SRC-1 or -3. Although the PR(Cre/+)SRC-2(flox/flox) uterus could mount a partial decidual response, removal of SRC-1 in the PR(Cre/+)SRC-2(flox/flox) uterus resulted in a complete block in decidualization, confirming that uterine SRC-2 and -1 are both required for P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analyses revealed the absence of significant branching morphogenesis in the hormone-treated PR(Cre/+)SRC-2(flox/flox) mammary gland, reinforcing an important role for mammary SRC-2 in cellular proliferative events that require PR. Based on the above and the observation that SRC-2 is expressed in many of the uterine and mammary cell-lineages in the human as observed in the mouse, we suggest that further investigations are warranted to gain additional insights into SRC-2's involvement in normal (and possibly abnormal) uterine and mammary cellular responses to progestins.

Steroid receptor coactivator 2 is critical for progesterone-dependent uterine function and mammary morphogenesis in the mouse.

Although the essential involvement of the progesterone receptor (PR) in female reproductive tissues is firmly established, the coregulators preferentially enlisted by PR to mediate its physiological effects have yet to be fully delineated. To further dissect the roles of members of the steroid receptor coactivator (SRC)/p160 family in PR-mediated reproductive processes in vivo, state-of-the-art cre-loxP engineering strategies were employed to generate a mouse model (PR(Cre/+) SRC-2(flox/flox)) in which SRC-2 function was abrogated only in cell lineages that express the PR. Fertility tests revealed that while ovarian activity was normal, PR(Cre/+) SRC-2(flox/flox) mouse uterine function was severely compromised. Absence of SRC-2 in PR-positive uterine cells was shown to contribute to an early block in embryo implantation, a phenotype not shared by SRC-1 or -3 knockout mice. In addition, histological and molecular analyses revealed an inability of the PR(Cre/+) SRC-2(flox/flox) mouse uterus to undergo the necessary cellular and molecular changes that precede complete P-induced decidual progression. Moreover, removal of SRC-1 in the PR(Cre/+) SRC-2(flox/flox) mouse uterus resulted in the absence of a decidual response, confirming that uterine SRC-2 and -1 cooperate in P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analysis disclosed the absence of significant ductal side branching and alveologenesis in the hormone-treated PR(Cre/+) SRC-2(flox/flox) mammary gland, reinforcing an important role for SRC-2 in cellular proliferative changes that require PR. We conclude that SRC-2 is appropriated by PR in a subset of transcriptional cascades obligate for normal uterine and mammary morphogenesis and function.

Cre-mediated recombination in cell lineages that express the progesterone receptor.

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.

Revealing progesterone's role in uterine and mammary gland biology: insights from the mouse.

Apart from distinguishing the in vivo effects of progesterone (P) from those of estrogen (E), the progesterone receptor knockout (PRKO) mouse has furnished unprecedented access to novel cell-signaling paradigms, hitherto unsuspected. Along with providing new cellular principles by which P influences proliferative and differentiative programs obligate for tissue development and tumor progression, the PRKO in conjunction with transcript profiling has begun to uncover the transcriptional cascades underlying these processes. Moreover, studies on isoform-specific knockouts for PR-A (PR-AKO) and PR-B (PR-BKO) have clearly defined distinct physiological roles for the two subtypes of PR, providing essential physiological support for previous in vitro observations. Although the PR-AKO exhibits an infertility phenotype, the PR-BKO displays normal fecundity. Conversely, although normal mammary morphogenesis can manifest in the PR-AKO, pregnancy-associated mammary morphogenesis is severely impaired in the PR-BKO. By virtue of its ability to suppress E-induced and PR-B-mediated uterine and mammary proliferation, the PR-A isoform is likely to be an attractive drug target for the next generation of selective PR modulators in the treatment of uterine and mammary gland hyperplasia. Along with defining the dynamic interplay between E and P responses and physiological events mediated by PR-A and PR-B, further studies on these models should provide a broader conceptual framework for understanding abnormal progestin responses in vivo, with attendant implications for the management of female reproductive health and for the diagnosis and/or treatment of breast cancer.

Neprilysin gene transfer reduces human amyloid pathology in transgenic mice.

The degenerative process of Alzheimer's disease is linked to a shift in the balance between amyloid-beta (Abeta) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular Abeta degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-beta in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-beta deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimer's disease.

Regulation of amyloid beta-peptide levels by enzymatic degradation.

It is generally accepted that amyloid beta peptides (Abeta) play a significant role in the etiology of Alzheimer's disease. The Abeta peptides are produced by the sequential cleavage of an amyloid precursor protein by a betasecretase followed by cleavage by a gamma secretase. The clearance of beta appears to be due primarily by the action of one or more peptidases. An imbalance between the rate of synthesis and the rate of clearance of Abeta is now considered a possible contributor to the onset of Alzheimer's disease. This review focuses on peptidases that have been proposed to contribute to Abeta catabolism and discusses the evidence for their participation in Abeta peptide clearance in vivo.