The intracellular domain of ErbB4 induces differentiation of mammary epithelial cells

Differentiation of mammary epithelium in vivo requires signaling through prolactin- and ErbB4/HER4-dependent mechanisms; how these pathways intersect is unknown. We show herein that HC11 mouse mammary cells undergo ErbB4-dependent lactational differentiation. Prolactin and the ErbB4 ligand HB-EGF each induced STAT5A activation, expression of lactogenic differentiation markers, and lumen formation in three-dimensional Matrigel cultures in HC11 cells. ErbB4 undergoes ligand-dependent transmembrane domain cleavage at Val-675, releasing a soluble 80-kDa intracellular domain (s80(HER4)) that localizes to nuclei; the physiological relevance of s80(HER4) is unknown. A HER4(V675A) mutant abolishing transmembrane cleavage impaired STAT5A activity, lactogenic gene expression, and lumen formation. Kinase-dead HER4(KD) was neither cleaved nor able to induce differentiation of HC11 cells. Without treating HC11 cells with prolactin or HB-EGF, s80(HER4) (expressed from a cDNA construct) localized to the nucleus, activated STAT5A, and induced three-dimensional lumen formation. Nuclear localization of exogenous s80(HER4) required intact kinase activity of s80(HER4), as did activation of STAT5A. In contrast, nuclear localization of s80(HER4) and STAT5A activation did not require the 16-amino acid region of the ErbB4 intracellular domain specific to the Cyt-1 isoform of ErbB4, and absent in the Cyt-2 isoform. These results suggest that s80(HER4) formation contributes to ErbB4-dependent differentiation of mammary epithelial cells.     

Heregulin-α促进小鼠妊娠期乳腺上皮细胞的增殖/分化及β酪蛋白的分泌

为阐明heregulin-α（HRG-α）对乳腺发育的调控作用及其机理,采用组织培养、毛细管电泳、Western印迹、ELISA等方法,首次对小鼠妊娠期HRG-α对乳腺形态发育、β酪蛋白表达和分泌、Rab3A蛋白表达以及HRG-α诱导的信号转导途径中信号分子的磷酸化状态的影响进行了系统研究．结果表明,在妊娠期,HRG-α能够促进STAT5、p42/p44、p38和PKC的磷酸化以及Rab3A蛋白表达,刺激乳腺上皮细胞的增殖和分化,增加并维持β酪蛋白的表达和分泌．     

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.     

Expression and function of heregulin-α and its receptors in the mouse mammary gland

Heregulin-α (HRGα) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGα and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGα and its receptors ErbB2 and ErbB3. We also determined the role of HRGα in the development of mammary glands, β-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGα signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGα, indicating that HRGα positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGα enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of β-casein. During lactation, HRGα enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of β-casein in mammary epithelial cells. During involution, HRGα induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of β-casein, resulting in increased levels of β-casein in mammary epithelial cells.     

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.     

Prolactin and epidermal growth factor stimulate adipophilin synthesis in HC11 mouse mammary epithelial cells via the PI3-kinase/Akt/mTOR pathway

The aim of the present study is to estimate the role played by cortisol, prolactin (PRL) and epidermal growth factor (EGF) in the synthesis of adipocyte differentiation-related protein (ADRP) as compared to the well-studied regulation of? β-casein synthesis by these hormones in the mammary epithelial cell line HC11. This comparison between a cytoplasmic lipid droplet-associated protein, which is strictly specific to both lipid accumulation and secretion by lactating mammary epithelial cells, and an archetypal milk protein is useful for evaluating the extent to which a mechanistic relationship exists between biosynthesis, transport and secretion of these two major milk components. We found that cortisol inhibits PRL-stimulated ADRP synthesis, as opposed to its known stimulating effect on β-casein synthesis. The involvement of PRL and EGF in ADRP synthesis was explored by means of a battery of inhibitors. The Jak2 inhibitor AG490 provoked a stimulation of ADRP synthesis whereas it totally suppressed that of β-casein. The use of AG1478, a specific inhibitor of EGF receptor phosphorylation, or of PD98059, a specific MEK inhibitor, revealed that the Ras/Raf/MEK/ERK1/2 pathway has no significant influence on ADRP levels. Inhibition of JNK was also ineffective. In contrast, incubation of the cells with SB 203580, a specific inhibitor of p38, slightly stimulated ADRP synthesis and induced a proportional dose-response inhibition of PRL-induced β-casein synthesis. Finally, cell treatment with wortmannin or LY294002 revealed that both PRL and EGF positively regulate ADRP and β-casein synthesis through PI3-kinase signaling. Because both the Akt inhibitor MK-2206 and the mTOR inhibitor rapamycin provoked a strong diminution of PRL-induced synthesis of the two proteins, and because oleate induced phosphorylation of Akt, we concluded that, in the mammary epithelial cell line HC11, the PI3-kinase/Akt/mTOR signaling pathway strongly participates in β-casein synthesis and is a main regulator of ADRP expression.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Gap junctions mediate STAT5-independent β-casein expression in CID-9 mammary epithelial cells

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent β-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed β-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed β-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced β-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed β-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.     

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub‐populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi‐potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell‐enriched population showed the most pronounced deregulation of migration‐associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.     

Image-based evaluation of the molecular events underlying HC11 mammary epithelial cell differentiation

We have developed an image-based technique for signal pathway analysis, target validation, and compound screening related to mammary epithelial cell differentiation. This technique used the advantages of optical imaging and the HC11-Lux model system. The HC11-Lux cell line is a subclone of HC11 mammary epithelial cells transfected stably with a luciferase construct of the β-casein gene promoter (p-344/-1βc-Lux). The promoter activity was imaged optically in real time following lactogenic induction. The imaging signal intensity was closely correlated with that measured using a luminometer following protein extraction (R = 0.99, P < 0.0001) and consistent with the messenger RNA (mRNA) level of the endogenous β -casein gene. Using this technique, we examined the roles of JAK2/Stat5A, Raf-1/MEK/MAKP, and PI3K/Akt signal pathways with respect to differentiation. The imaging studies showed that treatment of the cells with epidermal growth factor (EGF), AG490 (JAK2-specific inhibitor), and LY294002 (PI3K-specific inhibitor) blocked lactogenic differentiation in a dose-dependent manner. PD98059 (MEK-specific inhibitor) could reverse EGF-mediated differentiation arrest. These results indicate that these pathways are essential in cell differentiation. This simple, sensitive, and reproducible technique permits visualization and real-time evaluation of the molecular events related to milk protein production. It can be adopted for high-throughput screening of small molecules for their effects on mammary epithelial cell growth, differentiation, and carcinogenesis.     

Conditional repression of STAT5 expression during lactation reveals its exclusive roles in mammary gland morphology, milk-protein gene expression, and neonate growth

The role of Stat5 in maintaining adequate lactation was studied in Stat5a(-/-) mice expressing a conditionally suppressed transgenic STAT5 in their mammary glands. This system enables distinguishing STAT5's effects on lactation from its contribution to mammary development during gestation. Females were allowed to express STAT5 during their first pregnancy. After delivery, STAT5 levels were manipulated by doxycycline administration and withdrawal. In two lines of genetically modified mice, the absence of STAT5 expression during the first 10 days of lactation resulted in a decrease of 29% or 41% in newborn weight gain. The STAT5-dependent decrease in growth was recoverable, but not completely reversible, particularly when STAT5 expression was omitted for the first 4 days of lactation. Within the first 10 days of STAT5-omitted lactation, alveolar occupancy regressed by 50% compared to that measured at delivery. By Day 10, only 18% of the fat-pad area was involved in milk production. The alveolar regression caused by 4 days of STAT5 deficiency was reversible, but neonate growth remained delayed. STAT5 deficiency resulted in reduced estrogen receptor α and connexin 32 gene expression, accompanied by delayed induction of both anti- and pro-apoptotic Bcl-2 family members. An increase in Gata-3 expression may reflect an attempt to maintain alveolar progenitors. A decrease of 39% and 23% in WAP and α-lactalbumin expression, respectively, with no associated effects on β-casein, also resulted from lack of STAT5 expression in the first 10 days of lactation. This deficiency enhances the major effect of alveolar regression on delayed weight gain in newborns.     

Prolactin-mediated regulation of lipid biosynthesis genes in vivo in the lactating mammary epithelial cell

Prolactin (PRL) is known to play an essential role in mammary alveolar proliferation in the pregnant mouse, but its role in lactation has been more difficult to define. Genetic manipulations that alter expression of the PRL receptor and its downstream signaling molecules resulted in developmental defects that may directly or indirectly impact secretory activation and lactation. To examine the in vivo role of PRL specifically in lactation, bromocriptine (BrCr) was administered every 8 h to lactating mice on the second day postpartum, resulting in an ~95% decrease in serum PRL levels. Although morphological changes in secretory alveoli were slight, by 8 h of BrCr, pup growth was inhibited significantly. Phosphorylated STAT5 fell to undetectable levels within 4 h. Decreased milk protein gene expression, β-casein, and α-lactalbumin, was observed after 8 h of treatment. To assess mammary-specific effects on lipid synthesis genes, we isolated mammary epithelial cells (MECs) depleted of mammary adipocytes. Expression of genes involved in glucose uptake, glycolysis, pentose phosphate shunt, de novo synthesis of fatty acids, and biosynthesis of triacylglycerides was decreased up to 19-fold in MECs by just 8 h of BrCr treatment. Glands from BrCr-treated mice showed a twofold reduction in intracellular cytoplasmic lipid droplets and a reduction in cytosolic β-casein. These data demonstrate that PRL signaling regulates MEC-specific lipogenic gene expression and that PRL signals coordinate the milk synthesis and mammary epithelial cell survival during lactation in the mouse.     

EGF Modulates Expression of STAT5 in Mammary Epithelial Cells

HC11 mouse mammary epithelial cells are capable of differentiatingin vitro.By growing cells in EGF-containing medium, and upon confluence withdrawing EGF, these cells become competent at responding to lactogenic hormone treatment and expressing milk proteins. We found that during proliferation and at confluence STAT5A and STAT5B proteins were expressed at equal levels or with STAT5B being predominant. In competent cells, expression levels of STAT5A and STAT5B increased markedly with STAT5A now being the predominant form, an expression pattern resembling the expression patterns of STAT5 proteins seen during mammary gland differentiationin vivo.This suggests that EGF has a suppressive effect on STAT5 expression, in particular, STAT5A, which we conclude to be mediated through ras/raf/MEK/MAPK pathway and to a lesser extent through a PI3-kinase-mediated pathway. Furthermore, we also found that EGF regulated a nuclear phosphatase capable of dephosphorylating tyrosine-phosphorylated STAT5. Our data show that HC11 cells have retained the expression patterns of STAT5 proteins seenin vivo.This makes HC11 cells useful for studying molecular mechanisms regulating expression of STAT factors and their participation in differentiation processes of mammary gland.