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L Hennighausen

Publications and source records attributed to L Hennighausen.

At least 19 recordsLinked to original sources
PubMed2001

Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium.

Functional development of mammary epithelium during pregnancy depends on prolactin signaling. However, the underlying molecular and cellular events are not fully understood. We examined the specific contributions of the prolactin receptor (PrlR) and the signal transducers and activators of transcription 5a and 5b (referred to as Stat5) in the formation and differentiation of mammary alveolar epithelium. PrlR- and Stat5-null mammary epithelia were transplanted into wild-type hosts, and pregnancy-mediated development was investigated at a histological and molecular level. Stat5-null mammary epithelium developed ducts but failed to form alveoli, and no milk protein gene expression was observed. In contrast, PrlR-null epithelium formed alveoli-like structures with small open lumina. Electron microscopy revealed undifferentiated features of organelles and a perturbation of cell-cell contacts in PrlR- and Stat5-null epithelia. Expression of NKCC1, an Na-K-Cl cotransporter characteristic for ductal epithelia, and ZO-1, a protein associated with tight junction, were maintained in the alveoli-like structures of PrlR- and Stat5-null epithelia. In contrast, the Na-Pi cotransporter Npt2b, and the gap junction component connexin 32, usually expressed in secretory epithelia, were undetectable in PrlR- and Stat5-null mice. These data demonstrate that signaling via the PrlR and Stat5 is critical for the proliferation and differentiation of mammary alveoli during pregnancy.

Animals↗
PubMed2001

Essential role of Stat5 for IL-5-dependent IgH switch recombination in mouse B cells.

IL-5 stimulation of CD38-activated murine splenic B cells induces mu-gamma1 CSR at the DNA level leading to a high level of IgG1 production. Further addition of IL-4 in the system enhances IL-5-dependent mu-gamma1 CSR. Although some of the postreceptor signaling events initiated by IL-5 in activated B cells have been characterized, the involvement of Stat in IL-5 signaling has not been thoroughly evaluated. In this study, we examined the activation of Stat5 and activation-induced cytidine deaminase (AID) in CD38-activated murine splenic B cells by IL-5. The role of Stat5a and Stat5b in IL-5-induced mu-gamma1 CSR and also IgG1 and IgM production was documented, as IL-5 does not act on CD38-stimulated splenic B cells from Stat5a(-/-) and Stat5b(-/-) mice. Expression levels of CD38-induced germline gamma1 transcripts and AID in Stat5a(-/-) and Stat5b(-/-) B cells upon IL-5 stimulation were comparable to those of wild-type B cells. The impaired mu-gamma1 CSR by Stat5b(-/-) B cells, but not by Stat5a(-/-) B cells, was rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells induced mu-gamma1 CSR leading to IgG1 production. Analysis of cell division cycle number of wild-type B cells revealed that mu-gamma1 CSR was observed after five or six cell divisions. Stat5a(-/-) and Stat5b(-/-) B cells showed similar cell division cycles, but they did not undergo mu-gamma1 CSR. Our data support the notion that both Stat5a and Stat5b are essential for IL-5-dependent mu;-gamma1 CSR and Ig secretion; however, their major target may not be AID. Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

ADP-ribosyl Cyclase↗
PubMed2001

Role of serine phosphorylation of Stat5a in prolactin-stimulated beta-casein gene expression.

Milk production remains suppressed in mammals during late pregnancy despite high levels of lactogenic polypeptide hormones. At parturition, associated with a precipitous fall in circulating progesterone, rising glucocorticoid levels synergize with prolactin to initiate copious milk production. This synergy is mediated at least in part through the coordinated activation of glucocorticoid receptors and transcription factor Stat5, particularly Stat5a. Here we show that two proline-juxtaposed serine residues within the transactivation domain of Stat5a are phosphorylated in the mammary gland during late gestation and lactation, and that these phosphorylation sites inhibit the transcriptional activity of Stat5a in the absence of glucocorticoid receptor costimulation. Specifically, transfection assays revealed that phosphorylation of residues S725 and S779 of Stat5a cooperatively suppressed prolactin-stimulated transcription from the beta-casein promoter in both COS-7 kidney and MCF-7 mammary cells. This suppression was associated with shortened duration and reduced amplitude of nuclear DNA binding activity of wild type Stat5a relative to that of the serine phosphorylation-defective Stat5 mutant. However, costimulation of glucocorticoid receptors completely reversed the suppressive effect of Stat5a serine phosphorylation on beta-casein gene transcription. We propose that serine phosphorylation within the transactivation domain may limit the activity of Stat5a in the absence of proper coactivation by glucocorticoid receptors.

Amino Acid Sequence↗
PubMed2001

Functional mammary gland development and oncogene-induced tumor formation are not affected by the absence of the retinoblastoma gene.

Loss of cell cycle regulation in mammary epithelium results in impaired mammary gland development and neoplasia. We investigated the consequences of the absence of pRb in mammary epithelial cells during normal development and in mice that express an oncogene in the mammary epithelium. Since pRb-deficiency results in embryonic lethality, we transplanted pRb-null mammary anlagen into wild hosts. pRb-deficient mammary epithelia were capable of functional differentiation in term animals and they regenerated a differentiated gland even after multiple pregnancies. In serial transplantations no significant differences were found in outgrowth of pRb-deficient and wild type epithelia indicating that the absence of pRb does not lead to transformation. Likewise the effect of a TGFbeta1 transgene was not altered in the absence of pRb. The susceptibility of mammary epithelium to form tumors was assessed in three different models. No differences in tumor incidence were found between wild type and Rb +/- WAP-int3, MMTV-PyMT transgenic and Brcal-/- epithelia. These results demonstrate that the absence of pRb does not affect normal mammary gland development and tumorigenesis in three different mouse models investigated and suggest that loss of more than one member of the pRb pathway is required to induce mammary tumors.

Animals↗
PubMed2001

Structure of the mouse Stat 3/5 locus: evolution from Drosophila to zebrafish to mouse.

Signal transducers and activators of transcription (Stat) are transcription factors that can be activated by many cytokines. While Drosophila contains only one Stat (d-Stat), mammals contain seven, with STATs 3, 5a, and 5b being the closest functional relatives. To understand the evolutionary relationship between d-Stat and vertebrate STATs 3 and 5, we isolated, sequenced, and analyzed the zebrafish Stat3 (z-Stat3) gene and a 500-kb region spanning mouse chromosome 11, 60.5 cM containing three Stat genes (m-Stats). Within this region we identified the genes encoding m-Stats 3, 5a, and 5b, Cnp1, Hcrt/Orexin, Ptrf, GCN5, mDj11, and four new genes. The 5' ends of the m-Stat5a and m-Stat5b genes are juxtaposed to each other, and the 3' ends of the m-Stat3 and Stat5a genes face each other. While the m-Stat5a and m-Stat3 genes have one promoter each, which are active in many tissues, the m-Stat5b gene acquired two distinct promoters. The distal promoter is expressed ubiquitously, and transcription from the proximal promoter is restricted to liver, muscle, and mammary tissue. Through a comparison of exon-intron boundaries from the m-Stat3, m-Stat5a, and m-Stat5b, z-Stat3, and d-Stat genes, we deduced their evolutionary relationship. We propose that the Stat3 and Stat5 lineages are derived from the duplication of a common primordial gene and that d-Stat is a part of the Stat5 lineage.

Animals↗
PubMed2001

Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects.

Prolactin (Prl)-induced phosphorylation of Stat (signal transducer and activator of transcription) 5 is considered a key event in functional mammary development and differentiation. We now demonstrate that not only Prl, but also growth hormone (GH) and epidermal growth factor (EGF), can activate Stat5 in mammary tissue. We investigated the roles of these hormones in mammary development using mice in which the respective receptors had been inactivated. Although Prl receptor (PrlR)-null mice are infertile, we were able to maintain pregnancies in a few mice by treatment with progesterone. Mammary tissue in these mice was severely underdeveloped and exhibited limited differentiation as assessed by the phosphorylation status of Stat5 and the expression of milk protein genes. PrlR +/- mice showed impaired mammary development and alveolar differentiation during pregnancy, which corresponded with reduced phosphorylation levels of Stat5a and 5b, and impaired expression of milk protein genes. Development of the glands in these mice was arrested at around day 13 of pregnancy. While Prl activated Stat5 only in the epithelium, GH and EGF activated Stat5 preferentially in the stroma. To assess the relevance of the GH receptor (GHR) in the mammary gland, we transplanted GHR-null epithelium into cleared fat pads of wild-type mice. These experiments demonstrated that the GHR in the epithelium is not required for functional mammary development. Similarly, the EGFR in the epithelium is not required for alveolar development. In contrast, epithelial PrlR is required for mammary development and milk protein gene expression during pregnancy. Although GH is not required for alveolar development, we were able to demonstrate its lactogenic function in cultured mammary epithelium from PrlR-null mice. However, ductal development in GHR-null mice was impaired, supporting the notion that GH signals through the stromal compartment. Our findings demonstrate that GH, Prl, and EGF activate Stat5 in separate compartments, which in turn reflects their specific roles in ductal and alveolar development and differentiation.

Animals↗
PubMed2001

The Stat3/5 locus encodes novel endoplasmic reticulum and helicase-like proteins that are preferentially expressed in normal and neoplastic mammary tissue.

The signal transducers and activators of transcription (STAT) 5 and 3 are critical for mammary alveolar development during pregnancy and remodeling during involution. In the mouse, STAT3, STAT5a, and STAT5b are encoded by adjacent genes on chromosome 11 (60.5 cM). To identify additional genes in the Stat3/5 locus that may participate in normal and neoplastic development of the mammary gland, we have cloned and sequenced 500 kb and searched for genes preferentially expressed in mammary tissue. We identified six known genes and cloned two new genes, termed D11Lgp1 and D11Lgp2. Both genes are most highly expressed in normal mammary tissue and mammary tumors from several transgenic mouse models. LGP1 consists of 532 and 530 amino acids in mouse and human, respectively (88% similarity). A region in the carboxy-terminal half of LGP1 has limited homology with Arabidopsis thaliana GH3-like proteins. Immunofluorescence studies demonstrated that LGP1 is located in the nuclear envelope and the endoplasmic reticulum. LGP2 is a cytoplasmic protein of 678 amino acids.

Amino Acid Sequence↗
PubMed2001

Conditional deletion of the bcl-x gene from mouse mammary epithelium results in accelerated apoptosis during involution but does not compromise cell function during lactation.

In the mammary gland Bcl-x is the most abundant cell survival factor from the Bcl-2 family. Since Bcl-x null mice die around day 12 of embryogenesis, the relevance of this protein in organ development and function is poorly understood. In erythroid cells bcl-x gene expression is controlled by cytokines and the transcription factor Stat5 (signal transducer and activator of transcription). However, we identified that bcl-x RNA levels in mammary tissue from prolactin receptor- and Stat5-null mice were indistinguishable from wild type mice. We have proposed that Bcl-x might control the survival of mammary epithelial cells throughout pregnancy, lactation, and the early stages of involution, and we have now tested this hypothesis through the conditional deletion of the bcl-x gene from mouse mammary epithelium. Conditional (floxed) bcl-x alleles were excised from alveolar cells during pregnancy using a Cre transgene under the control of the whey acidic protein gene promoter. Deletion of the bcl-x gene from the entire epithelial compartment (ducts and alveoli) was achieved by expressing Cre-recombinase under control of the mouse mammary tumor virus long terminal repeat. The absence of Bcl-x did not compromise proliferation and differentiation of mammary ductal and alveolar epithelial cells in virgin mice and during pregnancy and lactation. However, epithelial cell death and tissue remodeling were accelerated in the bcl-x conditional knockout mice during the first stage of involution. Concomitant deletion of the bax gene did not significantly modify the Bcl-x phenotype. Our results suggest that Bcl-x is not essential during mammopoiesis, but is critical for controlled apoptosis during the first phase of involution.

Alleles↗
PubMed2001

Experimental mouse genetics-- answering fundamental questions about mammary gland biology.

Contemporary gene-targeting techniques now make it possible to alter specific genes in the genome. As a result, a plethora of mouse models have been generated that allow researchers to dissect cell-signaling pathways involved in mammary gland development and in breast cancer. But what have we learned so far? What do these models teach us? This review presents a global picture of how the analyses and comparison of individual knockout mouse models provide important insights into basic mammary gland biology. Particular emphasis is placed upon what is currently known about the signaling pathways involved in the establishment of the mammary ductal tree, and its subsequent proliferation at pregnancy and lactation. In addition to these well-established pathways, we address recent data that describe the role of lesser-known genes in the development of the mammary epithelium.

Animals↗
PubMed2001

Signaling pathways in mammary gland development.

Unlike most other organs, development of the mammary gland occurs predominantly after birth, under the control of steroid and peptide hormones. Once the gland is established, cycles of proliferation, functional differentiation, and death of alveolar epithelium occur repeatedly with each pregnancy. Although it is unique in this respect, the signaling pathways utilized by the gland are shared with other cell types, and have been tailored to meet the needs of this secretory tissue. Here we discuss the signaling pathways that have been adopted by the mammary gland for its own purposes, and the functions they perform.

Animals↗
PubMed2001

Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice.

Cre-loxP based gene deletion approaches hold great promise to enhance our understanding of molecular pathways controlling mammary development and breast cancer. We reported earlier the generation of transgenic mice that express the Cre recombinase under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). These mice have become a valuable research tool to delete genes specifically in the mammary gland, other secretory organs, and the female germline. We have now characterized in depth the expression of the MMTV-Cre transgene using the ROSA26-lox-Stop-lox-LacZ reporter strain to determine the temporal and spatial activation of Cre on the level of single cells. Our results show that MMTV-mediated Cre-activation is restricted to specific cell types of various secretory tissues and the hematopoietic system. Secondly, the timing of Cre expression varies between tissues and cell types. Some tissues express Cre during embryonic development, while other selected cell types highly activate Cre around puberty, suggesting a strong influence of steroid hormones on the transcriptional activation of the MMTV-LTR. Thirdly, Cre expression in the female germline is restricted to individual mouse lines and is therefore dependent on the site of integration of the transgene. Information provided by this study will guide the researcher to those cell types and developmental stages at which a phenotype can be expected upon deletion of relevant genes.

Animals↗
PubMed2000

The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins, are preferentially expressed during lactation.

Animals↗
PubMed2000

Linear and cooperative signaling: roles for Stat proteins in the regulation of cell survival and apoptosis in the mammary epithelium.

The mammary epithelium undergoes cyclical periods of cellular proliferation, differentiation and regression. These processes are under the control of the hormones secreted during pregnancy, lactation and involution. Signaling pathways have been identified that connect the hormonal stimuli with the transcription of genes responsible for the determination of the cellular fate. The kinetics of induction and deinduction have suggested that cytokine-activated Stat proteins play a crucial role. Stat5 is strongly activated towards the end of pregnancy, persists in an activated state during pregnancy and is rapidly inactivated after cessation of suckling. Stat3 activation is hardly detectable during lactation, but is strongly induced at the onset of involution. The phenotypes of mice in which these genes have been inactivated through homologous recombination corroborate some of the functional assignments deducted from the activation pattern. Stat3 activation seems to be a driving force in the induction of apoptosis early in the involution period.

Animals↗
PubMed2000

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting.

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Animals↗
PubMed2000

Transforming growth factor alpha and mouse models of human breast cancer.

Transforming growth factor alpha (TGFalpha) is a principal molecule in the normal and neoplastic development of the mammary gland. Binding of TGFalpha to the epidermal growth factor receptor (EGFR), activates the EGFRs' endogenous tyrosine kinase activity and stimulates growth of the epithelium in the virgin and pregnant mouse mammary gland. TGFalpha expression can be detected in breast cancer cells in vivo and in vitro and overexpression can elicit partial transformation or immortalized human and rodent mammary epithelial cells. Despite evidence implicating TGFalpha in the development of mammary neoplasia, the actual mechanism of TGFalpha-induced transformation is unclear. Transgenic mouse models targeting heterologus TGFalpha to the mammary gland have established TGFalpha overexpression can induce hyperproliferation, hyperplasia and occasional carcinoma. These transgenic studies demonstrated a facilitating, proliferative role for TGFalpha in the development of neoplasia and implicated several oncogenes that can cooperate with TGFalpha to transform the mammary epithelium. From studies of EGFR signaling pathways, inhibitory and modulating agents such as anti-EGFR antibodies and specific kinases inhibitors have been used to block the action of this pathway and prevent the development of TGFalpha-induced neoplasia and tumor formation. Studies in Stat5a knockout mice have established that the JAK2/Stat5a pathway can facilitate the survival of the mammary epithelium and can impact the progression of TGFalpha-mandated mammary tumorigenesis. Together these experiments indicate that TGFalpha and the EGFR signaling pathway are potentially amenable to therapies for treatment of human breast disease.

Animals↗