Dynamic regulation of the Stra13/Sharp/Dec bHLH repressors in mammary epithelium.

Mammary gland development is a dynamic process involving cyclical proliferation, cellular differentiation, and cell death. In this study, we have determined that expression of the Stra13/Sharp/Dec basic helix-loop-helix (bHLH) family is dynamically regulated in mammary epithelium. In cultured HC11 cells, epidermal growth factor (EGF) treatment rapidly induces Stra13 protein accumulation, which is blocked by the synthetic glucocorticoid, dexamethasone. Neither the induction of Stra13 by EGF nor its repression by dexamethasone correlates with changes in Stra13 mRNA levels. During mouse mammary gland development in vivo, Stra13 is highly expressed in epithelial ducts during puberty, and strongly induced in both ducts and alveoli during early involution, while the related Sharp-1 gene is highly expressed only during late stages of involution. Together, these data indicate that Stra13/Dec/Sharp-family bHLH repressors are dynamically regulated during mammary gland development and may function to regulate apoptosis in this tissue.     

Adhesion molecules intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and B7 are not expressed by epithelium in normal or inflamed colon.

The molecular mechanisms regulating IFN-gamma production have yet to be well characterized. We describe here how treatment of activated cultures of peripheral blood mononuclear cells (PBMC) with the phosphotyrosine phosphatases (PTP) inhibitor sodium ortho vanadate results in greatly enhanced IFN-gamma production. Conversely, cellular proliferation of the same cultures is profoundly inhibited by treatment with vanadate, while the expression of IL-2R and DR molecules on activated lymphocytes remains substantially unmodified. Increased IFN-gamma production, but not inhibition of cellular proliferation, was also observed in mitogen-activated vanadate-treated Jurkat cells. On the other hand, IFN-gamma production induced in cultures of PBMC treated or not with vanadate, was strongly inhibited by incubation with the protein tyrosine kinase (PTK) inhibitor herbimycin A. As a result of the inhibited phosphatase activity, substrates for PTK become hyperphosphorylated on tyrosine residues, as shown by Western blot analysis of cell lysates from cultures of PBMC treated with vanadate. We suggest that the tyrosine phosphorylation pathway plays a role in regulating IFN-gamma production.     

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland.

Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with beta-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation.     

Differential mammary morphogenesis along the anteroposterior axis in Hoxc6 gene targeted mice.

Mammary epithelial cell proliferation, branching, and differentiation span from the appearance of the mammary bud in midgestation through to the cycling mammary gland in adulthood. Here, we show that females homozygous for a targeted disruption of the Hoxc6 homeobox gene produce thoracic mammary glands that are slightly under-developed at birth and completely cleared of epithelium by adulthood, and inguinal mammary ducts that are dilated and fail to regress in response to ovariectomy. Mammary buds are detected in E12.5 Hoxc6 homozygous embryos. However, in newborn Hoxc6 homozygous females, branching ductal structures and fat pad development are reduced. Whole-mount and histologic analyses of mammary glands from adult Hoxc6 homozygous females show the absence of mammary epithelium in thoracic glands and dilated ducts in inguinal glands at 100% penetrance. Histologic analysis of inguinal mammary glands from ovariectomized Hoxc6 homozygous females demonstrates no signs of the expected regression of epithelium, suggesting that these glands are not responsive to the loss of ovarian hormone signals. We further observe repression of Hoxc6 expression specifically within mammary stroma by estrogen and progesterone. Hoxc6 homozygous mice also exhibit a homeotic transformation of the second thoracic vertebra into the first (T2 to T1 conversion with 60% penetrance), corresponding to both the gene's anterior boundary of expression and the most extreme appearance of mammary defects. The position-specific phenotypes observed and the potential role for Hoxc6 in mediating hormone-regulated ductal expansion and regression in the adult female are discussed.     

Effect of stage of lactation and milk accumulation on mammary cell differentiation in lactating bats.

Mammary cell differentiation was measured in lactating pipistrelle bats (Pipistrellus pipistrellus) by assay of key enzyme activities, and by determination of protein and lactose synthesis rates in short-term tissue cultures. By these criteria, mammary cell differentiation did not change significantly with stage of lactation, but depended on the extent to which the gland was filled with milk. Key enzyme activities and in vitro synthesis rates were significantly higher in glands suckled immediately before tissue collection, compared with contralateral glands that were engorged with milk. This indicates that mammary cell differentiation in the lactating bat is regulated locally within each gland by a mechanism sensitive to milk accumulation, to the extent that, unlike other species, this obscures any underlying effect of stage of lactation.     

Mutant Brca2/p53 mice exhibit altered radiation responses in the developing mammary gland

Appropriate balance between proliferation and apoptosis is critical for mammary gland development and is often altered during tumorigenesis. Carcinogens like radiation induce DNA damage and activate protective responses such as cell cycle arrest and apoptosis. We used mice carrying Brca2(-/-) and/or p53(-/-) mutations to evaluate the individual and combined effects of these genes on cell proliferation and apoptosis in the developing mammary gland. Mice were exposed to 5Gy of radiation or chamber exposure (controls) followed by injection with BrdU. Mammary glands were collected 6 h post-radiation exposure and evaluated for proliferation (BrdU) and apoptosis (TUNEL) in terminal end buds (TEB) and ducts. Under control conditions, the Brca2 mutation reduced proliferation and apoptosis in TEB but not ducts, whereas the p53 mutation reduced apoptosis in TEB and ducts but did not influence proliferation. Despite these alterations in proliferation and/or apoptosis, neither mutation, either individually or combined, significantly altered the overall balance between the two as measured by the proliferation to apoptosis ratio (growth index). Following irradiation, the Brca2 mutation had no significant effect on proliferation or apoptosis, whereas the p53 mutation resulted in reduced apoptosis in TEB and ducts but did not significantly influence proliferation. Neither mutation by itself altered the growth index in the TEB after irradiation although combined Brca2/p53 mutation caused significantly increased proliferation, reduced apoptosis, and an elevated growth index in TEB and ducts. These results reveal both independent and collaborative growth regulatory roles for Brca2 and p53 under normal and adverse environmental conditions. Additionally, we demonstrate the importance of gene-environment interactions by showing that Brca2- and p53-deficient mice can compensate for their genetic deficiencies under control conditions but not after exposure to radiation. We also demonstrate distinct spatial differences in the cellular functions of Brca2 and p53 and show that combined mutation of both genes is more detrimental than loss of either gene alone.     

Adipose tissue: a vital in vivo role in mammary gland development but not differentiation.

Development and differentiation of the mammary gland occurs by means of critical stromal-epithelial interactions. Although many studies have attempted to understand these complex interactions, it has been difficult to demonstrate the essential role of adipose tissue in the development and function of the mammary gland. By using the A-ZIP/F-1 transgenic mice lacking in white adipose tissue (WAT), we have studied the role of adipocytes in mammary gland development and differentiation. In the absence of WAT, rudimentary mammary anlagen form but are unable to grow and branch normally, resulting in a few, short, severely distended ducts. However, during pregnancy, a tremendous amount of epithelial cell division and alveolar cell formation occurs even in the absence of adipocytes, illustrating that adipose tissue is not required for mammary gland differentiation. Mammary gland transplantation revealed that epithelial cells from these transgenic mice possess the potential for normal growth and differentiation when placed into a normal stromal environment. These experiments clearly demonstrate that the absence of adipocytes in the mammary gland results in disruption of stromal-epithelial interactions that prevent normal mammary gland development. The rudimentary epithelial anlage, however, contain mammary stem cells, which are fully capable of alveolar differentiation.     

Androgen dependent mammary gland virilism in rats given the selective estrogen receptor modulator LY2066948 hydrochloride

A selective estrogen receptor modulator (SERM) is a nonsteroidal compound with tissue specific estrogen receptor (ER) agonist or antagonist activities. In animals, SERMs may produce morphologic changes in hormonally-sensitive tissues like the mammary gland. Mammary glands from female rats given the SERM LY2066948 hydrochloride (LY2066948) for 1 month at >or= 175 mg/kg had intralobular ducts and alveoli lined by multiple layers of vacuolated, hypertrophied epithelial cells, resembling in part the morphology of the normal male rat mammary gland. We hypothesized that these SERM-mediated changes represented an androgen-dependent virilism of the female rat mammary gland. To test this hypothesis, the androgen receptor antagonist flutamide was co-administered with LY2066948 (175 mg/kg) to female rats for 1 month. Female rats given SERM alone had hyperandrogenemia and the duct and alveolar changes described here. Flutamide cotreatment did not affect serum androgen levels but completely blocked the SERM-mediated mammary gland change. In the mouse, a species that does not have the sex-specific differences in the mammary gland observed in the rat, SERM treatment resulted in hyperandrogenemia but did not alter mammary gland morphology. These studies demonstrate that LY2066948 produces species-specific, androgen-dependent mammary gland virilism in the female rat.     

Targeted expression of activated Rac3 in mammary epithelium leads to defective postlactational involution and benign mammary gland lesions

Rac3, a novel member of the Rho subfamily of the small GTPases, is frequently activated in cultured breast cancer cells and has been shown to mediate its effect via the p21-activated kinase (Pak) pathway. In order to evaluate these findings in vivo, we generated transgenic mice that express human constitutively active V12Rac under the control of the mouse mammary tumor virus (MMTV) promoter, which targets the transgene expression to the mammary epithelium. V12Rac3 expression could be detected during the first pregnancy, and the transgenic mammary gland tissues displayed an elevated Pak1 phosphorylation. Although milk proteins, beta-casein and whey acidic protein were expressed and milk fat globules accumulated normally during pregnancy, 60% of transgenic mothers failed to nurse their pups. Surprisingly, although full lactational differentiation was never achieved in transgenic mice, gland involution was incomplete. For 5 days after weaning, involution was normal, but thereafter, epithelial islands characteristic of this early stage of involution persisted for months. The apoptotic index decreased after 5 days, and these glands were associated with increased p38 MAPK phosphorylation. Nine months postpartum, the transgenic mammary glands still demonstrated a large amount of persistent epithelial islands and abnormally large ducts with lymphocyte infiltration, whereas the tissues of non-transgenic controls had returned to their normal 'virgin-like' phenotype. These data show that sustained activation of Rac3 in the mammary epithelium leads to impaired mammary gland physiology and results in the formation of mammary gland lesions.     

Diet‐induced obesity disrupts ductal development in the mammary glands of nonpregnant mice

Mammary glands develop postnatally in response to the hypothalamic‐pituitary‐gonadal axis. Obesity‐induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high‐fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin. Developmental Dynamics 238:1092–1099, 2009. © 2009 Wiley‐Liss, Inc.     

Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.     

Transforming growth factor-beta inhibits lactogenic hormone induction of beta-casein expression in HC11 mouse mammary epithelial cells

HC11 mouse mammary epithelial cells can undergo a limited functional differentiation in terms of beta-casein synthesis in response to the combined action of dexamethasone and prolactin. Transforming growth factor-beta (TGF-beta) can inhibit beta-casein expression in HC11 cells in a dose-dependent manner. This effect is reversible and specific as shown by comparison with the effect of other growth factors. TGF-beta also inhibits DNA synthesis of HC11 cells. These findings suggest a possible role of TGF-beta as an inhibitor of functional differentiation in the mammary gland.     

Cell size and shape changes in the myoepithelium of the mammary gland during differentiation

We have studied changes in myoepithelial cell size and shape during different stages of mouse mammary gland differentiation by using the fluorescent probe for actin NBD-phallacidin. Pieces of mammary tissue were fixed, mounted on slides, permeabilized with cold acetone (-20 degrees C), and then treated with nitrobenzoxadiazole-phallacidin. Myoepithelial cells lining ducts of glands at all stages of development are spindle-shaped structures oriented parallel to the long axis of the duct at the base of the luminal epithelium. In virgin animals, myoepithelial cells also occur as linear tracts oriented parallel to the long axis of small projections along the sides of ducts and terminal end buds. In early pregnancy, small stellate-shaped cells begin to appear around presumptive secretory units. By late pregnancy, larger star-shaped units of intense fluorescence appear at the base of alveoli. During lactation, both cell bodies and cell processes further enlarge as these interlacing stellate-shaped cells encompass the expanded alveoli. In regressing glands, cell size decreases and the processes appear to retract. Although alveoli are virtually absent in the multipartate resting gland, myoepithelial cells remain around lateral buds of ducts. These myoepithelial cells have two distinct shapes: small star-shaped cells capping the buds and spindle-shaped cells oriented parallel to the long axis of the buds. A comparison of myoepithelial cell shape in virgin mice and nulliparous women indicates a more developed cell in the human gland at this stage of development. Intact segments of mammary gland combined with NBD-phallacidin as a probe for actin provide an ideal system for future studies of the control of myoepithelial cell size and shape and their influence on cell functions.     

Suppression of epithelial apoptosis and delayed mammary gland involution in mice with a conditional knockout of Stat3

Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, Stat3 is specifically activated. To address the function of this signaling molecule in mammary epithelial apoptosis, we have generated a conditional knockout of Stat3 using the Cre-lox recombination system. Following weaning, a decrease in apoptosis and a dramatic delay of involution occurred in Stat3 null mammary tissue. Involution is normally associated with a significant increase in IGFBP-5 levels. This was observed in control glands, but not in the absence of Stat3. IGFBP-5 has been suggested to induce apoptosis by sequestering IGF-1 to casein micelles, thereby inhibiting its survival function. Our findings suggest that IGFBP-5 is a direct or indirect target for Stat3 and its upregulation is essential to normal involution. No marked differences were seen in the regulation of Stat5, Bcl-x(L), or Bax in the absence of Stat3. Precocious activation of Stat1 and increases in levels of p53 and p21 occurred and may act as compensatory mechanisms for the eventual initiation of involution observed in Stat3 null mammary glands. This is the first demonstration of the importance of a Stat factor in signaling the initiation of physiological apoptosis in vivo.     

Targeted expression of GLI1 in the salivary glands results in an altered differentiation program and hyperplasia

Hedgehog (Hh) signaling is a regulator of salivary gland morphogenesis, but its role in postnatal glands has only recently begun to be addressed. To examine the effects of deregulated Hh signaling in the salivary gland, we expressed the Hh effector protein GLI1, in salivary epithelial cells using both cytokeratin 5 and mouse mammary tumor virus (MMTV) transgenic systems. Ectopic pathway activation resulted in restrained acinar differentiation, formation of cystic lesions, and prominent appearance of ductal structures. Moreover, induced expression of GLI1 aids the formation of hyperplastic lesions, which closely resemble GLI1-induced changes in murine skin and mammary glands, suggesting that GLI1 targets cells with similar characteristics in different tissues. Furthermore, GLI1-expressing salivary epithelial cells are actively dividing, and GLI1-induced lesions are proliferative, an incident accompanied by enhanced expression of the Hh target genes, cyclin D1, and Snail. GLI1-induced salivary lesions regress after transgene withdrawal and become histologically normalized. Taken together, our data reveal the ability of GLI1 to modulate salivary acinar differentiation and to promote proliferation of ductal epithelial cells.     

Keratinocyte Growth Factor Causes Cystic Dilation of the Mammary Glands of Mice: Interactions of Keratinocyte Growth Factor, Estrogen, and Progesterone In Vivo

Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically noted cysts are actually ducts that are dilated along much of their length. The histology of the mammary glands of KGF-treated mice is similar to the histology of fibrocystic disease in the buman female breast. The response in mice differs significantly from the appearance of the mammary glands in KGF-treated rats in which ductal epithelial proliferation is most prominent. Estrogen and progesterone when administered in combination but not alone cause the development of numerous endbuds in the mouse mammary gland. KGF in estrogen- and progesterone-pretreated mice causes the growth of dilated ducts, hyperplastic epithelium within ducts and endbuds, and a fibrous metamorphosis of periductal adipose tissue. The mammary epithelial hyperplasia caused by KGF is rapidly reversible in both mice and rats after cessation of KGF treatment. The spectrum of KGF-, estrogen-, and progesterone-induced mammary histopathology in mice provides a model for the study of fibrocystic and hyperplastic breast disease.     

Overexpression of human Cripto-1 in transgenic mice delays mammary gland development and differentiation and induces mammary tumorigenesis

Overexpression of Cripto-1 has been reported in several types of human cancers including breast cancer. To investigate the role of human Cripto-1 (CR-1) in mammary gland development and tumorigenesis, we developed transgenic mice that express the human CR-1 transgene under the regulation of the whey acidic protein (WAP) promoter in the FVB/N mouse background. The CR-1 transgene was detected in the mammary gland of 15-week-old virgin WAP-CR-1 female mice that eventually developed hyperplastic lesions. From mid-pregnancy to early lactation, mammary lobulo-alveolar structures in WAP-CR-1 mice were less differentiated and delayed in their development due to decreased cell proliferation as compared to FVB/N mice. Early involution, due to increased apoptosis, was observed in the mammary glands of WAP-CR-1 mice. Higher levels of phosphorylated AKT and MAPK were detected in mammary glands of multiparous WAP-CR-1 mice as compared to multiparous FVB/N mice suggesting increased cell proliferation and survival of the transgenic mammary gland. In addition, more than half (15 of 29) of the WAP-CR-1 multiparous female mice developed multifocal mammary tumors of mixed histological subtypes. These results demonstrate that overexpression of CR-1 during pregnancy and lactation can lead to alterations in mammary gland development and to production of mammary tumors in multiparous mice.     

Vanadate-Induced Inhibition of BCR-Triggered Apoptosis is Coupled with Tyrosine Phosphorylation and Induction of G2M Growth Arrest in Ramos-BL B Cells

The regulation of the tyrosine phosphorylation of key signaling molecules by tyrosine kinases and phosphatases is essential for BCR-triggered signaling cascades during B cell selection process. We used the non-selective tyrosine phosphatase inhibitor vanadate to study the importance of the late regulation of the tyrosine phosphorylation for BCR-triggered G1 growth arrest and apoptosis in Ramos-BL B cells. Vanadate induces G2M growth arrest in a dose-dependent manner and prevents BCR-triggered apoptosis. Vanadate-induced upregulation of the tyrosine phosphorylation is concomitant with increased expression of cyclin B and inhibition of caspase-3 activation and PARP cleavage. The anti-apoptotic effect of vanadate was observed even when added up to 6 hours after the treatment of Ramos-BL B cells with anti-IgM. Vanadate increases BCR-triggered tyrosine phosphorylation of the cytosolic tyrosine phosphatases, SHP-1 and SHP-2 after 24 hours. Co-stimulation with anti-CD40 prevents anti-IgM-triggered tyrosine phosphorylation of these phosphatases and up-regulates the expression of SHP-1. We conclude that the regulation of the tyrosine phosphatase activity is indispensable for BCR-triggered execution of the apoptosis in Ramos-BL B cells.