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

Although constitutive murine transgenic models have provided important insights into β-catenin signaling in tissue morphogenesis and tumorigenesis, these models are unable to express activated β-catenin in a temporally controlled manner. Therefore, to enable the induction (and subsequent de-induction) of β-catenin signaling during a predetermined time-period or developmental stage, we have generated and characterized a TETO-ΔN89β-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 β-catenin (ΔN89β-catenin) is expressed only in the presence doxycycline-activated rtTA in the mammary epithelial compartment.

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.

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)).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.