Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Vimentin Is Involved in Peptidylarginine Deiminase 2-Induced Apoptosis of Activated Jurkat Cells

Peptidylarginine deiminase type 2 (PADI2) deiminates (or citrullinates) arginine residues in protein to citrulline residues in a Ca²⁺-dependent manner, and is found in lymphocytes and macrophages. Vimentin is an intermediate filament protein and a well-known substrate of PADI2. Citrullinated vimentin is found in ionomycin-induced macrophage apoptosis. Citrullinated vimentin is the target of anti-Sa antibodies, which are specific to rheumatoid arthritis, and play a critical role in the pathogenesis of the disease. To investigate the role of PADI2 in apoptosis, we generated a Jurkat cell line that overexpressed the PADI2 transgene from a tetracycline-inducible promoter, and used a combination of 12-O-tetradecanoylphorbol-13-acetate and ionomycin to activate Jurkat cells. We found that PADI2 overexpression reduced the cell viability of activated Jurkat cells in a dose- and time-dependent manner. The PADI2-overexpressed and -activated Jurkat cells presented typical manifestations of apoptosis, and exhibited greater levels of citrullinated proteins, including citrullinated vimentin. Vimentin overexpression rescued a portion of the cells from apoptosis. In conclusion, PADI2 overexpression induces apoptosis in activated Jurkat cells. Vimentin is involved in PADI2-induced apoptosis. Moreover, PADI2-overexpressed Jurkat cells secreted greater levels of vimentin after activation, and expressed more vimentin on their cell surfaces when undergoing apoptosis. Through artificially highlighting PADI2 and vimentin, we demonstrated that PADI2 and vimentin participate in the apoptotic mechanisms of activated T lymphocytes. The secretion and surface expression of vimentin are possible ways of autoantigen presentation to the immune system.     

Structures Indicative of Keratinization in Lactating Cells of Bovine Mammary Gland

Keratohyalin granule-like aggregates and tangles of filaments similar to epidermal prekeratin fibrils have been observed in lactating cells of bovine mammary epithelium. The concomitant occurrence of such structures, which are characteristic of early stages of keratinization, with typical secretory products such as casein micelles demonstrates that keratinization can take place in functional secretory cells. The observations are discussed in relation to current concepts of keratinization in epidermal and in non-epidermal epithelia.     

Proteins Deiminated by Peptidylarginine Deiminase in Mouse Uterus and Their Changes during the Estrous Cycle

Peptidylarginine deiminase (PAD) catalyzes the formation of citrullyl residues by deimination of arginyl residues of proteins in the presence of Ca²⁺. We found several deiminated proteins in mouse uterus using antibodies recognizing the citrulline residue. The levels of these proteins changed during the estrous cycle in parallel with PAD activity.     

Porphyromonas gingivalis Facilitates the Development and Progression of Destructive Arthritis through Its Unique Bacterial Peptidylarginine Deiminase (PAD)

Rheumatoid arthritis and periodontitis are two prevalent chronic inflammatory diseases in humans and are associated with each other both clinically and epidemiologically. Recent findings suggest a causative link between periodontal infection and rheumatoid arthritis via bacteria-dependent induction of a pathogenic autoimmune response to citrullinated epitopes. Here we showed that infection with viable periodontal pathogen Porphyromonas gingivalis strain W83 exacerbated collagen-induced arthritis (CIA) in a mouse model, as manifested by earlier onset, accelerated progression and enhanced severity of the disease, including significantly increased bone and cartilage destruction. The ability of P. gingivalis to augment CIA was dependent on the expression of a unique P. gingivalis peptidylarginine deiminase (PPAD), which converts arginine residues in proteins to citrulline. Infection with wild type P. gingivalis was responsible for significantly increased levels of autoantibodies to collagen type II and citrullinated epitopes as a PPAD-null mutant did not elicit similar host response. High level of citrullinated proteins was also detected at the site of infection with wild-type P. gingivalis. Together, these results suggest bacterial PAD as the mechanistic link between P. gingivalis periodontal infection and rheumatoid arthritis.     

X-Ray Fluorescence Microscopy Reveals Accumulation and Secretion of Discrete Intracellular Zinc Pools in the Lactating Mouse Mammary Gland

Background

The mammary gland is responsible for the transfer of a tremendous amount of zinc (∼1–3 mg zinc/day) from maternal circulation into milk during lactation to support the growth and development of the offspring. When this process is compromised, severe zinc deficiency compromises neuronal development and immune function and increases infant morbidity and/or mortality. It remains unclear as to how the lactating mammary gland dynamically integrates zinc import from maternal circulation with the enormous amount of zinc that is secreted into milk.

Methodology/Principal Findings

Herein we utilized X-ray fluorescence microscopy (XFM) which allowed for the visualization and quantification of the process of zinc transfer through the mammary gland of the lactating mouse. Our data illustrate that a large amount of zinc first accumulates in the mammary gland during lactation. Interestingly, this zinc is not cytosolic, but accumulated in large, discrete sub-cellular compartments. These zinc pools were then redistributed to small intracellular vesicles destined for secretion in a prolactin-responsive manner. Confocal microscopy identified mitochondria and the Golgi apparatus as the sub-cellular compartments which accumulate zinc; however, zinc pools in the Golgi apparatus, but not mitochondria are redistributed to vesicles destined for secretion during lactation.

Conclusions/Significance

Our data directly implicate the Golgi apparatus in providing a large, mobilizable zinc storage pool to assist in providing for the tremendous amount of zinc that is secreted into milk. Interestingly, our study also provides compelling evidence that mitochondrial zinc pools expand in the mammary gland during lactation which we speculate may play a role in regulating mammary gland function.     

CNN3 Regulates Trophoblast Invasion and Is Upregulated by Hypoxia in BeWo Cells

CNN3 is an ubiquitously expressed F-actin binding protein, shown to regulate trophoblast fusion and hence seems to play a role in the placentation process. In this study we demonstrate that CNN3 levels are upregulated under low oxygen conditions in the trophoblast cell line BeWo. Since hypoxia is discussed to be a pro-migratory stimulus for placental cells, we examined if CNN3 is involved in trophoblast invasion. Indeed, when performing a matrigel invasion assay we were able to show that CNN3 promotes BeWo cell invasion. Moreover, CNN3 activates the MAPKs ERK1/2 and p38 in trophoblast cells and interestingly, both kinases are involved in BeWo invasion. However, when we repeated the experiments under hypoxic conditions, CNN3 did neither promote cell invasion nor MAPK activation. These results indicate that CNN3 promotes invasive processes by the stimulation of ERK1/2 and/or p38 under normoxic conditions in BeWo cells, but seems to have different functions at low oxygen levels. We further speculated that CNN3 expression might be altered in human placentas derived from pregnancies complicated by IUGR and preeclampsia, since these placental disorders have been described to go along with impaired trophoblast invasion. Our studies show that, at least in our set of placenta samples, CNN3 expression is neither deregulated in IUGR nor in preeclampsia. In summary, we identified CNN3 as a new pro-invasive protein in trophoblast cells that is induced under low oxygen conditions.     

Prolactin and Growth Hormone Binding in Mammary and Liver Tissue of Lactating Cows

Abstract

A radioligand/receptor binding assay was developed using homologous hormones to distinguish between bovine growth hormone (bGH) and bovine prolactin (bPRL) receptors in liver and mammary tissue of lactating cows. Mammary and liver tissues were homogenized in 0.3 M sucrose and centrifuged at 100,000 x g over a 1.3 M sucrose density gradient. Membranes from the 0.3 - 1.3 M sucrose interface were incubated with 1 ng of iodinated bGH or bPRL for 20 h at 22°C in the presence of increasing concentrations of native bGH or bPRL. High affinity receptor binding sites were found for bPRL in liver and mammary tissue membranes (Ka=3.2 and 1.3 × 10⁸ 1/mol with 34 and 63 fmol receptors/mg liver and mammary membrane protein, respectively) and for bGH only in liver tissue (Ka=1.8 × 10⁹ 1/mol, 18 fmol receptors/mg membrane protein). Receptor number estimates were 3 and 11 times higher in mammary and liver tissue using a heterologous hGH system indicating that heterologous systems may overestimate the number of receptors in bovine tissue. The absence of demonstratable bGH receptors in lactating bovine mammary tissue supports in vitro results of others with isolated mammary tissue indicating that the positive effect of bGH on milk production in intact cows is via an indirect mechanism.     

Augmentation of Prolactin Release by alpha-Melanocyte Stimulating Hormone Is Possibly Mediated by Melanocortin 3-Receptors in the Mouse Anterior Pituitary Cells

Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by alpha-melanocyte stimulating hormone (alpha-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the alpha-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not alpha-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that alpha-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that alpha-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.     

小鼠乳腺细胞凋亡及瘦素对凋亡的影响

目的系统的研究小鼠乳腺发育周期中乳腺细胞凋亡情况,并阐明瘦素对乳腺细胞凋亡的影响。方法以小鼠乳腺为实验材料,采用TUNEL法系统地研究小鼠乳腺在青春期、妊娠期、泌乳期和退化期的整个发育周期中的细胞凋亡情况,并通过培养基中添加瘦素的方法研究瘦素对乳腺细胞凋亡的影响。结果在青春期50～60d、妊娠期10～16d、退化期1～10d检测到较多的细胞凋亡,其中退化期细胞凋亡最为显著。添加瘦素培养的乳腺细胞凋亡信号明显增多。结论小鼠乳腺发育不同时期细胞凋亡同结构和功能发育之间相互联系。同时通过小鼠乳腺组织体外培养的方法,证明瘦素在退化期乳腺组织中可明显诱导组织凋亡。     

Prolactin Cells: Study of the Pituitary Gland of the Roach (Leuciscus rutilus) by Immuno-histochemical Methods

Fluorescein-labelled antibodies against ovine prolactin have been used to localize the prolactin-producing cells in the pituitary gland of the roach, Leuciscus rutilus. Two methods of protein tracing have been used: the direct and the indirect. In the direct method gammaglobulin fractions of rabbit-antisera were conjugated with fluorescein-isothiocyanate. Frozen sections of the pituitary gland were treated with conjugate. Cells with a content of prolactin gave off green fluorescence. Conjugate made from normal rabbit-serum failed to give this result. Blocking-experiments were also used to control the conjugate fluorescence. In the indirect method sheep-anti-rabbit-gammaglobulin was labelled with fluorescein. First the sections of the hypophysis were treated with antiserum from rabbits. After careful rinsing the sections were exposed to the dyemarked sheep-anti-rabbit-gammaglobulin. For orientation in the pituitary gland histological staining was carried out in parallel to that by fluorescence. The investigation has shown that the erythrosinophilic cells in the rostral pars distalis contained prolactin. Since no other cells in the gland were fluorescent, it can be concluded that the eta-cells are the prolactin cells.     

Differential Glycosylation of rhLf Expressed in the Mammary Gland of Transgenic Mice

Differential glycosylation of natural hLf and rhLf from hLf-transgenic mice, which harbored a 146 Kb BAC insert that includes the intact hLf gene sequence, was studied in the present report. There were significant differences between the immunoblotting results of rhLf and natural hLf, which were denatured with nonreducing SDS sample buffer. The differences disappeared after rhLf and natural hLf samples were digested with N-glycosidase F, respectively. The results showed that there were significant differences (P < 0.01) between the glycosylation of natural hLf and rhLf that were purified, respectively, from milk samples of seven hLf-transgenic mouse lines.     

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts¹. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane². At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones^3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy². At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli^6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups ⁸. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium^9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation^12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice. Download video file.^{(54M, mov)}