Biomedical subjects

Kay-Uwe Wagner

Publications and source records attributed to Kay-Uwe Wagner.

At least 19 recordsLinked to original sources

PubMed2006

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells.

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.

Animals↗

PubMed2006

Epithelial-specific and stage-specific functions of insulin-like growth factor-I during postnatal mammary development.

Postnatal development of the mammary gland requires interactions between the epithelial and stromal compartments, which regulate actions of hormones and growth factors. IGF-I is expressed in both epithelial and stromal compartments during postnatal development of the mammary gland. However, little is known about how local expression of IGF-I in epithelium or stroma regulates mammary growth and differentiation during puberty and pregnancy-induced alveolar development. The goal of this study was to investigate the mechanisms of IGF-I actions in the postnatal mammary gland and test the hypothesis that IGF-I expressed in stromal and epithelial compartments has distinct functions. We established mouse lines with inactivation of the igf1 gene in mammary epithelium by crossing igf1/loxP mice with mouse lines expressing Cre recombinase under the control of either the mouse mammary tumor virus long-terminal repeat or the whey acidic protein gene promoter. Epithelial-specific loss of IGF-I during pubertal growth resulted in deficits in ductal branching. In contrast, heterozygous reduction of IGF-I throughout the gland decreased expression of cyclins A2 and B1 during pubertal growth and resulted in alterations in proliferation of the alveolar epithelium and milk protein levels during pregnancy-induced differentiation. Reduction in epithelial IGF-I at either of these stages had no effect on these indices. Taken together, our results support distinct roles for IGF-I expressed in epithelial and stromal compartments in mediating growth of the postnatal mammary gland.

Animals↗

PubMed2005

Loss of the LIM domain protein Lmo4 in the mammary gland during pregnancy impedes lobuloalveolar development.

LMO4, a member of the LIM-only family of zinc-finger proteins, is overexpressed in a significant proportion of breast carcinomas and acts as a negative regulator of mammary epithelial differentiation. To delineate cell types within the developing mouse mammary gland that express Lmo4, we analysed different stages of mammopoiesis by immunohistochemistry. Lmo4 was found to be highly expressed in the proliferating cap cells of the terminal end bud and in the ductal and alveolar luminal cells of the mature mammary gland but was negligible or low in myoepithelial cells. To assess the physiological role of Lmo4 in the mammary gland, we generated conditionally targeted mice lacking Lmo4 in the mammary epithelium during pregnancy. Acute loss of Lmo4 in late pregnancy impaired lobuloalveolar development, accompanied by a two-fold reduction in the percentage of BrdU-positive cells. In contrast, germline loss of Lmo4 did not alter lobuloalveolar development arising from transplanted mammary anlagen, implying the existence of a compensatory mechanism in these knockout mice. Thus, the use of a conditional targeting strategy has revealed that Lmo4 is required for proper development of the mammary gland during pregnancy and indicated that Lmo4 acts as a positive regulator of alveolar epithelial proliferation.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-beta1 expression.

A parity-induced mammary population, marked by beta-galactosidase expression conditionally activated through cre-lox recombinase originates in WAP-Cre/Rosa-lox-STOP-lox-LacZ (WAP-Cre/Rosa-LacZ) female mice during pregnancy, lactation and involution. During subsequent pregnancies, these parity-induced mammary epithelial cells (PI-MEC) proliferated to produce new secretory acini composed of secretory luminal cells and myoepithelium. In serial transplantation assays, PI-MEC were able to self-renew over several transplant generations and to contribute significantly to the resulting mammary outgrowths. In limiting dilution transplantation, they proliferated to produce both luminal and myoepithelial cells, comprised both lobule-limited and duct-limited epithelial outgrowths, and differentiated into all the cellular subtypes recognized in murine mammary epithelium. TGF-beta1 expression from the whey acidic protein promoter (WAP) in triply transgenic females did not prevent the appearance of PI-MEC after pregnancy despite the absence of full lactation or their ability to proliferate and produce progeny with diverse cellular fates in situ upon subsequent pregnancies. However, in transplants from triple transgenic parous females, the WAP-TGF-beta1-positive PI-MEC did not contribute to the newly recapitulated mammary outgrowths, suggesting that they were incapable of expansive cellular proliferation (self-renewal). This result is consistent with our earlier publication that WAP-TGF-beta1 expression in mammary epithelium induces premature stem cell senescence in mammary transplants and decreases mammary cancer risk in mouse mammary tumor virus (MMTV)-infected females even after multiple pregnancies.

Animals↗

PubMed2005

Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells.

Whey acidic protein (WAP) is the principal whey protein found in rodent milk, which contains a cysteine-rich motif identified in some protease inhibitors and proteins involved in tissue modeling. The expression of the Wap gene, which is principally restricted to the mammary gland, increases more than 1,000-fold around mid-pregnancy. To determine whether the expression of this major milk protein gene is a prerequisite for functional differentiation of mammary epithelial cells, we generated conventional knockout mice lacking two alleles of the Wap gene. Wap-deficient females gave birth to normal litter sizes and, initially, produced enough milk to sustain the offspring. The histological analysis of postpartum mammary glands from knockout dams does not reveal striking phenotypic abnormalities. This suggests that the expression of the Wap gene is not required for alveolar specification and functional differentiation. In addition, we found that Wap is dispensable as a protease inhibitor to maintain the stability of secretory proteins in the milk. Nevertheless, a significant number of litters thrived poorly on Wap-deficient dams, in particular during the second half of lactation. This observation suggests that Wap may be essential for the adequate nourishment of the growing young, which triple in size within the first 10 days of lactation. Important implications of these findings for the use of Wap as a marker for advanced differentiation of mammary epithelial cells and the biology of pluripotent progenitors are discussed in the final section.

Animals↗

PubMed2005

Pregnancy and stem cell behavior.

The identification of cancer-initiating epithelial subtypes (i.e. cancer stem cells) is important for gaining a more comprehensive understanding of the process of neoplastic transformation and tumorigenesis. Since reproductive history has a major impact on breast tumorigenesis, it is reasonable to assume that pregnancy and lactation have enduring effects on the cancer susceptibility of multipotent progenitors. Using the Cre-lox technology as a tool to genetically label pregnancy-hormone-responsive cells, we identified a mammary epithelial subtype that is abundant in parous females. These pregnancy-induced mammary epithelial cells (PI-MECs) originate from differentiating cells during the first pregnancy and lactation cycle. They do not undergo apoptosis during postlactational remodeling, and they persist throughout the remainder of a female's life. In this review, we discuss the biological relevance of PI-MECs in multiparous females and their important stem cell-like features, such as self renewal, as well as their ability to produce progeny with diverse cellular fates. Using appropriate animal models, we further demonstrate that PI-MECs are cellular targets for pregnancy-enhanced mammary tumorigenesis.

Animals↗

PubMed2004

Brca1-deficient murine mammary epithelial cells have increased sensitivity to CDDP and MMS.

In this report we describe the isolation of an isogenic pair of Brca1(++) and Brca1(-/-) murine mammary epithelial cells (MMECs). These cells were isolated from Brca1 conditional knock out mice which contained loxP sites flanking exon 11 of the Brca1 gene (Brca1(fl/f1)) and then immortalized by infection with HPV-16E6 retrovirus to degrade p53 protein. Brca1(-/-) MMECs were generated by deletion of exon 11 following transduction of Brca1(fl/f1) MMECs with a retroviral vector expressing Cre recombinase. Brca1-deficiency rendered MMECs sensitive to cis-platinum (II) diamine dichloride (CDDP) and methylmethane sulfonate (MMS). The Brca1(+/+) and Brca1(-/-) MMECS is the only known pair of isogenic mammary epithelial cell lines. The understanding of the mechanisms of the CDDP sensitivity of the BRCA1-deficient mammary epithelial cells would be very important in understanding how BRCA1-deficiency plays out in tissue specific breast cancer chemotherapy. These studies support the role of BRCA1 in the CDDP-induced and MMS-induced DNA damage and repair by p53-independent pathways.

Animals↗

PubMed2004

Parity-induced mammary epithelial cells facilitate tumorigenesis in MMTV-neu transgenic mice.

Using a Cre-lox-based genetic labeling technique, we have recently discovered a parity-induced mammary epithelial subtype that is abundant in nonlactating and nonpregnant, parous females. These mammary epithelial cells serve as alveolar progenitors in subsequent pregnancies, and transplantation studies revealed that they possess features of multipotent progenitors such as self-renewal and the capability to contribute to ductal and alveolar morphogenesis. Here, we report that these cells are the cellular targets for transformation in MMTV-neu transgenic mice that exhibit accelerated mammary tumorigenesis in multiparous animals. The selective ablation of this epithelial subtype reduces the onset of tumorigenesis in multiparous MMTV-neu transgenics. There is, however, experimental evidence to suggest that parity-induced mammary epithelial cells may not be the only cellular targets in other MMTV-promoter-based transgenic strains. In particular, the heterogeneous MMTV-wnt1 lesions predominantly express the ductal differentiation marker Nkcc1 that is absent in MMTV-neu-derived tumors. Our observations support the idea that tumors originate from distinctly different epithelial subtypes in selected MMTV-promoter-driven cancer models and that diverse oncogenes might exert discrete effects on particular mammary epithelial subtypes.

Animals↗

PubMed2004

Cell cycle arrest and cell death are controlled by p53-dependent and p53-independent mechanisms in Tsg101-deficient cells.

Our previous studies have shown that cells conditionally deficient in Tsg101 arrested at the G(1)/S cell cycle checkpoint and died. We created a series of Tsg101 conditional knock-out cell lines that lack p53, p21(Cip1), or p19(Arf) to determine the involvement of the Mdm2-p53 circuit as a regulator for G(1)/S progression and cell death. In this new report we show that the cell cycle arrest in Tsg101-deficient cells is p53-dependent, but a null mutation of the p53 gene is unable to maintain cell survival. The deletion of the Cdkn1a gene in Tsg101 conditional knock-out cells resulted in G(1)/S progression, suggesting that the p53-dependent G(1) arrest in the Tsg101 knock-out is mediated by p21(Cip1). The Cre-mediated excision of Tsg101 in immortalized fibroblasts that lack p19(Arf) seemed not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G(1) arrest was not restored. Based on these findings, we propose that the p21-dependent cell cycle arrest in Tsg101-deficient cells is an indirect consequence of cellular stress and not caused by a direct effect of Tsg101 on Mdm2 function as previously suggested. Finally, the deletion of Tsg101 from primary tumor cells that express mutant p53 and that lack p21(Cip1) expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of Tsg101 for cell survival.

Animals↗

PubMed2004

Brca2 deficiency does not impair mammary epithelium development but promotes mammary adenocarcinoma formation in p53(+/-) mutant mice.

Brca2 is an important tumor suppressor associated with susceptibility to breast cancer. Although increasing evidence indicates that the primary function of Brca2 is to facilitate the repair of DNA damage via the homologous recombination pathway, how Brca2 prevents breast cancer is largely unknown. To study the role of Brca2 specifically in mammary epithelium development, we crossed mice bearing the conditionally deficient allele Brca2(flox9-10) to mouse mammary tumor virus- or whey acidic protein-Cre transgenic lines. Analysis of these animals showed that Brca2 is not required for epithelial expansion in mammary glands of pregnant mice. In addition, examination of mammary gland involution revealed normal kinetics of mammary alveolar cell apoptosis after weaning of litters. Nevertheless, Brca2-deficient mice developed mammary adenocarcinomas after a long latency (average, 1.6 years). Detailed histopathological analysis of four of these tumors demonstrated that three of them showed abnormal p53 protein expression. A mutation in the p53 gene was detected in one case. Moreover, homozygosity versus heterozygosity for the Brca2 mutation heavily skewed the tumor spectrum toward mammary adenocarcinoma development in p53(+/-) mice. Our data indicate that Brca2 is not essential for mammary epithelium development but that Brca2 deficiency and down-regulated p53 expression can work jointly to promote mammary tumorigenesis.

Adenocarcinoma↗

PubMed2004

Early onset of neoplasia in the prostate and skin of mice with tissue-specific deletion of Pten.

PTEN is a tumor suppressor gene mutated in various advanced human neoplasias, including glioblastomas and prostate, breast, endometrial, and kidney cancers. This tumor suppressor is a lipid phosphatase that negatively regulates cell survival and proliferation mediated by phosphatidylinositol 3-kinase/protein kinase B signaling. Using the Cre-loxP system, we selectively inactivated Pten in murine tissues in which the MMTV-LTR promoter is active, resulting in hyperproliferation and neoplastic changes in Pten-null skin and prostate. These phenotypes had early onset and were completely penetrant. Abnormalities in Pten mutant skin consisted of mild epidermal hyperplasia, whereas prostates from these mice exhibited high-grade prostatic intraepithelial neoplasia (HGPIN) that frequently progressed to focally invasive cancer. These data demonstrate that Pten is an important physiological regulator of growth in the skin and prostate. Further, the early onset of HGPIN in Pten mutant males is unique to this animal model and implicates PTEN mutations in the initiation of prostate cancer. Consistent with high PTEN mutation rates in human prostate tumors, these data indicate that PTEN is a critical tumor suppressor in this organ.

Animals↗

PubMed2004

Generation of a conditional knockout allele for the Janus kinase 2 (Jak2) gene in mice.

To study biologically relevant functions of the Janus kinase 2 (Jak2) in multiple cytokine and hormone receptor signal transduction pathways, we generated a conditional knockout (floxed) allele of this gene by placing loxP sites around the first coding exon of Jak2. Homozygous floxed animals developed normally and exhibited no phenotypic abnormalities. The conversion of the floxed allele into a null mutation was achieved by transmitting the targeted allele through the female germline of MMTV-Cre (line A) mice. Embryos that carry two Jak2 null alleles died around midgestation and exhibited impaired definitive erythropoiesis, which is a hallmark of Jak2 deficiency reported previously in conventional knockouts. This observation suggested that the Cre-mediated deletion of the first coding exon results in a true null mutation that is incapable of mediating signals through the erythropoietin receptor. Using mouse embryonic fibroblasts derived from Jak2 null embryos and their wildtype littermate controls, we demonstrated that Jak2-deficiency decouples growth hormone-receptor signaling from its downstream mediators, the signal transducer and activator of transcription (Stat) 5a and 5b.

Animals↗

PubMed2004

Impaired alveologenesis and maintenance of secretory mammary epithelial cells in Jak2 conditional knockout mice.

Jak2 is a hormone-receptor-coupled kinase that mediates the tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat). The biological relevance of Jak2-Stat signaling in hormone-responsive adult tissues is difficult to investigate since Jak2 deficiency leads to embryonic lethality. We generated Jak2 conditional knockout mice to study essential functions of Jak2 during mammary gland development. The mouse mammary tumor virus-Cre-mediated excision of the first coding exon resulted in a Jak2 null mutation that uncouples signaling from the prolactin receptor (PRL-R) to its downstream mediator Stat5 in the presence of normal and supraphysiological levels of PRL. Jak2-deficient females were unable to lactate as a result of impaired alveologenesis. Unlike Stat5a knockouts, multiple gestation cycles could not reverse the Jak2-deficient phenotype, suggesting that neither other components of the PRL-R signaling cascade nor other growth factors and their signal transducers were able to compensate for the loss of Jak2 function to activate Stat5 in vivo. A comparative analysis of Jak2-deficient mammary glands with transplants from Stat5a/b knockouts revealed that Jak2 deficiency also impairs the pregnancy-induced branching morphogenesis. Jak2 conditional mutants therefore resemble PRL-R knockouts more closely, which suggested that Jak2 deficiency might affect additional PRL-R downstream mediators other than Stat5a and Stat5b. To address whether Jak2 is required for the maintenance of PRL-responsive, differentiating alveolar cells, we utilized a transgenic strain that expresses Cre recombinase under regulatory elements of the whey acidic protein gene (Wap). The Wap-Cre-mediated excision of Jak2 resulted in a negative selection of differentiated alveolar cells, suggesting that Jak2 is required not only for the proliferation and differentiation of alveolar cells but also for their maintenance during lactation.

Animals↗

PubMed2003

Models of breast cancer: quo vadis, animal modeling?

Rodent models for breast cancer have for many decades provided unparalleled insights into cellular and molecular aspects of neoplastic transformation and tumorigenesis. Despite recent improvements in the fidelity of genetically engineered mice, rodent models are still being criticized by many colleagues for not being 'authentic' enough to the human disease. Motives for this criticism are manifold and range from a very general antipathy against the rodent model system to well-founded arguments that highlight physiological variations between species. Newly proposed differences in genetic pathways that cause cancer in humans and mice invigorated the ongoing discussion about the legitimacy of the murine system to model the human disease. The present commentary intends to stimulate a debate on this subject by providing the background about new developments in animal modeling, by disputing suggested limitations of genetically engineered mice, and by discussing improvements but also ambiguous expectations on the authenticity of xenograft models to faithfully mimic the human disease.

Animals↗

PubMed2003

Impaired differentiation and lactational failure of Erbb4-deficient mammary glands identify ERBB4 as an obligate mediator of STAT5.

The ERBB family of type 1 receptor tyrosine kinases and their ligands have crucial functions during mammopoiesis, but the signaling networks that ultimately regulate ERBB activity in the breast have remained elusive. Here, we show that mice with Cre-lox mediated deletions of both Erbb4 alleles within the developing mammary gland (Erbb4(Flox/Flox)Wap-Cre) fail to accumulate lobuloalveoli or successfully engage lactation at parturition owing, in part, to impaired epithelial proliferation. Analysis of the mammary differentiation factor STAT5 by immunohistochemistry and western blot revealed a complete ablation of STAT5 activation in Erbb4(Flox/Flox)Wap-Cre mammary epithelium at parturition. Consistent with disrupted STAT5 function, Erbb4(Flox/Flox)Wap-Cre mammary glands at parturition failed to express the mammary epithelial differentiation marker NPT2B. Defects in epithelial functional differentiation at parturition were accompanied by a profound reduction in expression of the STAT5-regulated milk genes casein beta and whey acidic protein. We propose that ERBB4 functions as an essential mediator of STAT5 signaling, and that loss of STAT5 activity contributes to the impaired functional differentiation of mammary glands observed in mice containing conditional Erbb4 deletions.

Animals↗

PubMed2003

Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation.

The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

Adipose Tissue↗

PubMed2003

Tsg101 is essential for cell growth, proliferation, and cell survival of embryonic and adult tissues.

Tumor susceptibility gene 101 (Tsg101) was identified in a random mutagenesis screen for potential tumor suppressors in NIH 3T3 cells. Altered transcripts of this gene have been detected in sporadic breast cancers and many other human malignancies. However, the involvement of this gene in neoplastic transformation and tumorigenesis is still elusive. Using gene targeting, we generated genetically engineered mice with a floxed allele of Tsg101. We investigated essential functions of this gene in vivo and examined whether the loss of function of Tsg101 results in tumorigenesis. Conventional knockout mice were generated through Cre-mediated excision of the first coding exon in the germ line of mouse mammary tumor virus (MMTV)-Cre transgenic mice. The complete ablation of Tsg101 in the developing embryo resulted in death around implantation. In contrast, mammary gland-specific knockout mice developed normally but were unable to nurse their young as a result of impaired mammogenesis during late pregnancy. Neither heterozygous null mutants nor somatic knockout mice developed mammary tumors after a latency of 2 years. The Cre-mediated deletion of Tsg101 in primary cells demonstrated that this gene is essential for the growth, proliferation, and survival of mammary epithelial cells. In summary, our results suggest that Tsg101 is required for normal cell function of embryonic and adult tissues but that this gene is not a tumor suppressor for sporadic forms of breast cancer.

Animals↗

PubMed2003

HIF1alpha is a critical regulator of secretory differentiation and activation, but not vascular expansion, in the mouse mammary gland.

During pregnancy the mammary epithelium and its supporting vasculature rapidly expand to prepare for lactation, resulting in dramatic changes in the micro-environment. In order to investigate the role of oxygenation and metabolism in these processes, the oxygen-responsive component of the hypoxia-inducible factor (HIF) 1 complex, HIF1alpha, was deleted in the murine mammary gland. Although vascular density was unchanged in the HIF1alpha null mammary gland, loss of HIF alpha impaired mammary differentiation and lipid secretion, culminating in lactation failure and striking changes in milk composition. Transplantation experiments confirmed that these developmental defects were mammary epithelial cell autonomous. These data make clear that HIF1alpha plays a critical role in the differentiation and function of the mammary epithelium.

Adenoviridae↗