[19,74] and in chemically-induced rodent models where parity was seen to have a protective effect against tumorigenesis [19]. Thus, it is tempting to speculate that the downregulation of PTN, an anti- differentiation factor, has a critical role in this protective effect. PTN mRNA expression in the mammary epithelial cells has been previously suggested [41] but its function in these cells was unclear. Treatment with the anti-PTN blocking antibody in vivo indicates that PTN inhibits the expansion of the epithelial ductal tree and the activation of the ERK1/2 signaling pathway during pubertal development of the mammary gland. Three-dimensional cultures of MECs treated with the PTN blocking antibody and expression analysis of markers associated with progenitor mam- mary epithelial cells such as CD29, CD49f, SCA-1 and CD10 indicate that PTN activity maintains the mammary epithelial cells in an undifferentiated state. A model for the cooperation between CD10 and CD29 in maintaining the homeostasis of mammary epithelial stem cells by inhibiting differentiation has been proposed where CD29 is responsible for keeping the stem cells anchored to the extra-cellular matrix and CD10 for creating a gradient of cleaved protein with differentiation activity [65]. The mRNA expression profile in the progenitor cell markers suggests that the anti-PTN antibody treated MECs have a more differentiated phenotype than the control cells and are more similar to the MEC at passage #1. The fact that the anti-PTN antibody treated MECs formed a higher number of mammospheres with correct apical polarity and laminin basement membrane deposition than control MECs in 3D cultures suggests that PTN inhibits polarization of MECs. PTN expression has been shown in neuronal progenitor cells and its activity enhances progenitor cells expansion and differentiation in different tissues [29,31,68]. A recent study has shown that PTN deficiency in the stroma both maintains the hematopoietic stem cell (HSCs) population during serial transplantation and increases their repopulating activity by promoting cell proliferation and differentiation and altering gene and protein expression of stem and progenitors cells [75]. That PTN could have opposing effects depending on dose and expression of different receptors has been previously proposed [75]. Treatment with the anti-PTN blocking antibody of mammary epithelial cells grown on a plastic surface showed that endogenous PTN sustains cell motility and invasion (Figure 4B to F). This finding seems to be in contradiction with the in vivo studies where treatment with the anti-PTN blocking antibody indicates that PTN inhibits the expansion of the epithelial ductal tree during pubertal development of the mammary gland (Figure 6). Overall our findings suggest that PTN has opposing effects depending on the cell surroundings. Blocking PTN increases MECs sphere potential by stimulating better cell-matrix adhesion in MECs grown in 3D. We propose that PTN disrupts MECs adhesion to a plastic surface resulting in sustained cell migration and invasion while in vivo this effect is overcome by the interaction with the surrounding stroma. Our study shows that PTN is a regulator of progenitor cells in the mammary gland and that its expression maintains the mammary epithelial cells in an undifferentiated state while blocking its activity causes an expansion and differentiation of mammary epithelial cells. We proposed that the increased in maturation of the mammary gland and in mammosphere formation of the anti- PTN antibody treated MECs is due to an expansion of progenitor epithelial cells that are farther committed in the epithelial lineage that have appropriate epithelial polarization and cell attachment to the ECM, a more differentiated phenotype and higher expression of the protease CD10. Asselin-Labat et al. [76] suggest that the paracrine expression of factors from the luminal epithelial hormonal responsive cells is important in keeping the homeostasis PTN in the Mammary Gland of the mammary epithelial progenitor cells [76]. We suggest that PTN is one of these regulatory factors of progenitor cells that are expressed by the mammary epithelial cells under hormonal stimuli. Conclusion PTN appears to inhibit differentiation of mammary epithelial cells and maintain their progenitor phenotype that is evidenced during mammary gland development as well as in isolated mammary epithelial cells in culture. It is tempting to speculate that PTN downregulation observed with multiparity is protective towards breast cancer, possibly by reducing the immature precursor population susceptible to malignant transformation and by expanding differentiated epithelial cells. Materials and Methods Ethics Statement All procedures using animals were approved by the Georgetown University IACUC and were conducted according to the NIH guidelines for the care and use of laboratory animals. Mice C57BL/6, Balb/c and FVB mice were purchased from the Jackson Laboratory. All procedures using animals were approved by the Georgetown University IACUC and were conducted according to the NIH guidelines for the care and use of laboratory animals. RNA detection Mammary glands or brains were isolated from female balb/c mice. Involution days are given relative to the weaning of 21 day old pups with mammary glands from exbreeders harvested .4 weeks after weaning. All experiments were performed using combined abdominal and thoracic mammary glands. Tissues were homogenized in RNA-STAT, and the RNA was isolated via a standard chloroform extraction procedure. For experiments requiring tissue fractionation, mammary glands were finely minced at 4uC and then incubated in DMEM Hams F12 media (Gibco, BRL) supplemented with 0.15% collagenase (type A, Boehringer Mannheim) for 1 hour at 37uC. After collagenase digestion, glands were passed through cheesecloth (Baxter) and filtered through a cell strainer using a 70 mm mesh (Falcon). The filtrate was centrifuged for 5 minutes at 5006g (stromal cell fraction). Both the filtrate pellet and the filtered collected fraction (epithelial organoids) were resuspended in RNA-STAT and processed for RNA extraction as described above. Northern blots were performed as previously described [19]. RNA blots of 5 mg total RNA/lane were hybridized with P32 labeled probes primed from full length cDNA for mouse Keratin 18 [77] or mouse vimentin [78] or human beta actin (Clontech). In Situ Hybridization (ISH) for PTN mRNA was done with digoxigenin labeled riboprobes as previously described [79]. For quantitative real-time PCR (qRT-PCR) RNA from thoracic mammary glands or from mammospheres was used. Mammo- spheres were harvested from three-dimensional cultures and Matrigel was removed as described elsewhere [80]. Reverse transcription (RT) of 1 mg of total RNA was performed using iScriptTM gene expression was quantified using SYBR green (BioRad). Primers were: PTN (Forward) TGGAGAATGGCAGTGGAGTGTGT PTN (Reverse) TGGTACTTGCACTCAGCTCCAAACT K18 (Forward) CAAGTCTGCCGAAATCAGGGACG PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47876