epithelial cells and adipocytes [40,41]. Marzan et al. showed that PTN expression in differentiated adipocytes of the mammary gland was regulated by the retinoid acid receptor (RAR) and that PTN activity in the adipocytes was involved in the migration of epithelial ductal cells [40]. PTN mRNA expression has also been reported in mammary epithelial ducts by in situ hybridization [41]. However, the function of PTN in mammary epithelial cells is still unexplored. Finally, no mammary gland phenotype was described in PTN knock-out mice though they have an increased hippo- campal activity [5,6]. Previous data suggest a temporal regulation of PTN expression during pregnancy and a permanent downreg- ulation of this growth factor in the mammary gland induced by parity [42]. A protective effect of early parity from breast cancer has been suggested by epidemiological studies [43,44] as well as from carcinogen-induced breast cancer models in rats [45,46,47]. A better understanding of PTN function and regulation during mammary gland development could help to understand the role of PTN during breast cancer development and progression. Here we show that PTN expression is regulated in mouse mammary glands both temporally and spatially during pregnancy and is affected by multiparity. A 30-fold downregulation of PTN expression was observed during mid-pregnancy when the mam- mary epithelial cells (MECs) start undergoing lobular-alveolar differentiation. We also found that blocking PTN activity caused enhanced maturation of the mammary gland accompanied by activation of the ERK1/2 signaling pathway in the epithelial compartment of the mammary gland. We show that PTN activity sustains motility and invasion of MECs grown on plastic and that blocking PTN activity caused increased number of mammo- spheres due to a more polarized structural organization shown by laminin deposition and a more differentiated phenotype as indicated by the expression of progenitor cell markers CD29, CD49f, SCA-1 and CD10. Results Temporal and spatial expression of PTN in the mouse mammary gland during pregnancy PTN mRNA is highly regulated during pregnancy and reduced 30-fold by day 15 with the ALK receptor regulated in parallel reduced 100-fold by day 15 (Figure 1A,B). In agreement with a previous report [42], PTN and its receptor ALK mRNA levels were not affected during the first 10 days of gestation when the mammary gland is characterized by proliferating ductal epithelial cells. To determine which cells mostly express and secrete PTN, mRNA expression was analyzed by in situ hybridization as well as cell fractionation followed by Northern blot (Figure 2). In situ hybridization for PTN mRNA supports the downregulation during pregnancy and shows major expression in the epithelial compartment of the mammary gland (Figure 2A). Also, mammary glands from virgin mice were digested with collagenase to isolate epithelial cells from the glands. Northern blots showed Keratin 18 expression in the epithelial fraction and detectable expression of PTN only in this fraction. Actin and vimentin showed similar expression in both of the fractions (Figure 2B). The PTN protein is released from cells and bound to heparin sulfate-containing proteoglycans in the extracellular matrix [32]. Immunohistochemistry and Immunofluorescence shows PTN protein staining in the mammary duct epithelium and in cultured primary mammary epithelial cells (MECs) (Figure 3 and 4A). In the mammary tissue staining, PTN protein is detected preferen- tially in duct epithelia though stromal tissues also show immuno- reactivity very likely due to secreted PTN that is stored locally (Figure 3). In cultured MECs co-localization with DAPI and PTN in the Mammary Gland Figure 1. PTN and ALK mRNA expression in mouse mammary glands during pregnancy. Mammary glands from mice that were virgin, pseudopregnant (PsPreg), at different days of pregnancy or after weaning ( = involution). Quantitative, real-time PCR was used. Pseudo- pregnants denotes 12 days since plug date. Pups were removed from the mother at 21 days of age and involution followed thereafter. Expression is given relative to keratin 18. Mean 6 SE (n = 4; PsPreg n = 2). **P,0.01; ***P,0.001, by ANOVA. doi:10.1371/journal.pone.0047876.g001 Phalloidin shows the PTN protein mostly in the extracellular matrix (Figure 4A). Together with the in situ hybridization for PTN mRNA, these data suggest that PTN is mostly expressed in the epithelial cells of the mammary gland, released from there and stored in the tissues. Hormonal changes induced by breeding have been linked to changes in the mammary gland composition [48] but there was no statistically significant difference in PTN mRNA expression levels between mammary gland of virgin and pseudopregnant mice. Also, after the first pregnancy, PTN and ALK expression returned to pre-pregnancy levels (Figure 1). However, after repeated pregnancies, we saw a 8-fold downregulation of PTN mRNA in the mammary gland of exbreeders compared to virgins (mean 6 SEM qPCR cycle: virgin, 24.9660.34; exbreeders, 21.9860.57; n = 4–5 age-matched mice per group; P = 0.004, t-test). Our findings are in agreement with D’Cruz et al. [42] who previously described permanent changes induced by parity in gene expression of growth factors such as PTN and suggested an increased differentiation state of the mammary gland after multiparity. We propose that the decrease in PTN expression in the mammary gland during mid-pregnancy and after repeated pregnancies is due to increased differentiation of mammary epithelial cells and a loss of undifferentiated epithelial cells (see below). PTN activity in primary mammary epithelial cells (MECs) Since PTN expression was detected mostly in the mammary epithelial cells, we studied its potential contribution using a PTN blocking antibody in cultured primary mammary epithelial cells (MECs; Figure 4,5). We first investigated whether PTN activity regulates MECs growth using the real-time monitoring electric impedance approach (Figure 4B,C). Treatment with PTN blocking antibody or stimulation with PTN recombinant protein did not change the MECs proliferation index (Figure 4B). The addition of bFGF, another growth factor, enhanced MECs proliferation (Figure 4C). This suggests that MECs growth was still responsive to growth factors stimulation. These findings were confirmed by Cell Titer Glow assay, another standard procedure to monitor cell proliferation (data not shown). As mentioned in the Introduction, PTN has been shown to be a promoter of invasion and metastasis in different tumor types including breast cancers [19,22,34,35]. Real-time monitoring electric impedance shows PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47876