Gertraud W. Robinson

Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation.

ABSTRACT This study explored the functions of the signal transducers and activators of transcription 5a and 5b (referred to as Stat5 here) during different stages of mouse mammary gland development by using conditional gene inactivation. Mammary gland morphogenesis includes cell specification, proliferation and differentiation during pregnancy, cell survival and maintenance of differentiation throughout lactation, and cell death during involution. Stat5 is activated by prolactin, and its presence is mandatory for the proliferation and differentiation of mammary epithelium during pregnancy. To address the question of whether Stat5 is also necessary for the maintenance and survival of the differentiated epithelium, the two genes were deleted at different time points. The 110-kb Stat5 locus in the mouse was bracketed with loxP sites, and its deletion was accomplished by using two Cre-expressing transgenic lines. Loss of Stat5 prior to pregnancy prevented epithelial proliferation and differentiation. Deletion of Stat5 during pregnancy, after mammary epithelium had entered Stat5-mediated differentiation, resulted in premature cell death, indicating that at this stage epithelial cell proliferation, differentiation, and survival require Stat5.

Information networks in the mammary gland

Unique developmental features during puberty, pregnancy, lactation and post-lactation make the mammary gland a prime object to explore genetic circuits that control the specification, proliferation, differentiation, survival and death of cells. Steroids and simple peptide hormones initiate and carry out complex developmental programmes, and reverse genetics has been used to define the underlying mechanistic connections.

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.

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.

Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development

During the development of the vertebrate head, cranial neural crest cells migrate into the branchial arches to form many of the structures of the facial skeleton. These cells follow defined developmental pathways and their fates are determined early. We have isolated and characterized the murine Distal-less homeobox gene Dlx-3 and have performed a comparative analysis of Dlx-3 and Dlx-2 expression during craniofacial development. In contrast to Dlx-2 and other vertebrate Distal-less genes, Dlx-3 is not expressed in the central nervous system and is expressed in a highly restricted region of the branchial arches. Dlx-2 and -3 display temporal and spatial differences in expression in the arches and their derivatives. In later development, these two genes are expressed in both complementary and partially overlapping domains in regions whose development is dependent on epithelial-mesenchymal interactions, such as the developing middle and inner ear, teeth and whisker follicles. The differential expression of Dlx genes in the branchial region suggests that they play key roles in craniofacial patterning and morphogenesis.

Prolactin signaling in mammary gland development.

It has now been over 60 years since Riddle et al. (1) purified a hormone from the anterior pituitary gland, which stimulated milk secretion in the mammary gland of virgin rabbits. They named it prolactin (PRL). Since then, the synergistic approaches of biochemistry, physiology, molecular biology, and cell biology have unveiled several molecular switches in the PRL signaling cascade (Fig. 1). Loss-of-function studies in the mouse have now provided clear insight into the biology of two components of the PRL pathway. A mandatory role for the prolactin receptor (PRLR) and for the signal transducer and activator of transcription (Stat) 5a in mammopoiesis and lactogenesis was established (2, 3). Although Stat5a is in the line of fire of many signals such as PRL, growth hormone (GH), and several cytokines, its absence in vivo reveals an unexpected level of specificity.

Loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration

Growth hormone controls many facets of a cells biology through the transcription factors Stat5a and Stat5b (Stat5). However, whole body deletion of these genes from the mouse does not provide portentous information on cell‐specific cytokine signaling. To explore liver‐specific functions of Stat5, the entire Stat5 locus was deleted in hepatocytes using Cre‐mediated recombination. Notably, Stat5‐mutant mice developed fatty livers and displayed impaired proliferation of hepatocytes upon partial hepatectomy (PHx). Loss of Stat5 led to molecular consequences beyond the reduced expression of Stat5 target genes, such as those encoding suppressor of cytokine signaling 2 (SOCS2), Cish, and insulin‐like growth factor 1 (IGF‐1). In particular, circulating growth hormone levels were increased and correlated with insulin resistance and increased insulin levels. Aberrant growth hormone (GH)‐induced activation of the transcription factors Stat1 and Stat3 was observed in mutant livers. To test whether some of the defects observed in liver‐specific Stat5 deficient mice were due to aberrant Stat1 expression and activation, we generated Stat1−/− mice with a hepatocyte‐specific deletion of Stat5. Concomitant loss of both Stat5 and Stat1 restored cell proliferation upon PHx but did not reverse fatty liver development. Thus the molecular underpinnings of some defects observed in the absence of Stat5 are the consequence of a deregulated activation of other signal transducers and activators of transcription (STAT) family members. Conclusion: Aberrant cytokine‐Stat5 signaling in hepatocytes alters their physiology through increased activity of Stat1 and Stat3. Such cross‐talk between different pathways could add to the complexity of syndromes observed in disease. (HEPATOLOGY 2007.)

Acute Myeloid Leukemia-Associated Mkl1 (Mrtf-a) Is a Key Regulator of Mammary Gland Function

ABSTRACT Transcription of immediate-early genes—as well as multiple genes affecting muscle function, cytoskeletal integrity, apoptosis control, and wound healing/angiogenesis—is regulated by serum response factor (Srf). Extracellular signals regulate Srf in part via a pathway involving megakaryoblastic leukemia 1 (Mkl1, also known as myocardin-related transcription factor A [Mrtf-a]), which coactivates Srf-responsive genes downstream of Rho GTPases. Here we investigate Mkl1 function using gene targeting and show the protein to be essential for the physiologic preparation of the mammary gland during pregnancy and the maintenance of lactation. Lack of Mkl1 causes premature involution and impairs expression of Srf-dependent genes in the mammary myoepithelial cells, which control milk ejection following oxytocin-induced contraction. Despite the importance of Srf in multiple transcriptional pathways and widespread Mkl1 expression, the spectrum of abnormalities associated with Mkl1 absence appears surprisingly restricted.

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

The Wnt/β-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 β-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the β-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 β-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 β-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 β-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 β-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to β-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.