Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells Sonia M. Rosenfield1, Emma T. Bowden2, Shani Cohen-Missner1, Krissa A. Gibby1, Virginie Ory1, Ralf T. Henke1, Anna T. Riegel1, Anton Wellstein1* 1 Lombardi Cancer Center, Georgetown University, Washington, District of Columbia, United States of America, 2 MedImmune, Gaithersburg, Maryland, United States of America Abstract 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. Citation: Rosenfield SM, Bowden ET, Cohen-Missner S, Gibby KA, Ory V, et al. (2012) Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells. PLoS ONE 7(10): e47876. doi:10.1371/journal.pone.0047876 Editor: John P. Lydon, Baylor College of Medicine, United States of America Received February 7, 2012; Accepted September 24, 2012; Published October 15, 2012 Copyright: 2012 Rosenfield et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants awarded to AW from the NCI/NIH (RO1 CA101811); to Shani Cohen-Missner from the Pharmaceutical Manufacturers Association (Fellowship); to Sonia M. Rosenfield from the Department of Defense Breast Cancer Research Program (Predoctoral Grant: BC083308). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. Wellstein is named as an inventor on Georgetown University owned patents related to pleiotrophin and ALK. Dr. Bowden is an employee at Medimmune Gaithersburg. This does not alter the authors9 adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: wellstea@georgetown.edu Introduction The heparin-binding growth factor pleiotrophin (PTN; also known as HB-GAM or HARP; reviewed in [1]) is a 15 kDa secreted protein named for its pleiotrophic effects on different cell types. In mice, PTN and its tyrosine kinase receptor ALK are prominently expressed during embryonic and early postnatal development of the central and peripheral nervous system [2,3,4,5,6]. In addition to neuronal functions, PTN contributes to the epithelial-mesenchymal interactions in organs undergoing branching morphogenesis and in bone formation [7,8,9,10,11,12]. In the adult, PTN and ALK expressions are downregulated in most tissues, with residual expression in the nervous system [4,13]. An increase in PTN expression has been observed in the uterus during gestation, in the human placenta and during bone repair, inflammatory diseases and in human cancers [2,13,14,15,16,17,18,19]. The biological effects of PTN-mediated signaling include neurite outgrowth and repair, angiogenesis and mitogenesis of fibroblasts, endothelial and some tumor cell lines [19,20,21,22,23]. PTN signaling can induce the activation of both the MAPK and PI3 kinase pathways in epithelial cells [24] and a direct role for PTN in mitogenesis and anti-apoptotic signaling was previously reported [19,25,26,27,28]. PTN activity has recently been shown to stimulate the differentiation and expansion of neuronal stem cells, osteoprogenitors and bone marrow stem cells [29,30,31]. The PTN protein was first purified from the supernatants of human breast cancer cells [32]. PTN mRNA expression has been shown in different estrogen receptor negative breast cancer cell lines and in primary breast cancers derived from different patients [19]. In NIH3T3 fibroblasts, PTN leads to transformation, whereas overexpression of a dominant negative, mutant PTN protein reverses the transformed phenotype of breast and other cancer cell lines [26,33]. PTN has also been shown to be an angiogenic factor during tumor growth and a promoter of invasion and metastasis in different tumor types including breast cancers [19,22,34,35]. A role for PTN-mediated signaling through the receptor tyrosine kinase ALK as well as the receptor phosphatase PTPRz has been suggested in breast cancers [36,37,38]. In normal human breast tissues, PTN is expressed in the epithelial, myoepithelial and endothelial cells [39]. In the mouse mammary gland, PTN expression has been described in both PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47876