PTN in the Mammary Gland Figure 8. PTN effect on cultured mammary epithelial cell (MEC) differentiation. (A, B) Effect of treatment of MECs with a blocking anti-PTN antibody. (A) Progenitor marker mRNA, (B) Keratin 18 (K18), Vimentin (VM) and PTN mRNA expression were measured by quantitative, real-time PCR. The effect of continuous passaging of MECs 6 anti-PTN is shown relative to passage #1 MECs. Data are means 6 SE; n = 4 and 2 independent experiments for VM and K18 respectively; **P ,0.01; ***P ,0.001 by ANOVA. MECs passage#1 expression relative to Actin: CD29, 3.5; CD49f, 5.6; CD10, 6.6; SCA-1, 2.3; K18, 2.9; VM, 5.3; PTN, 12.2. Control MECs passage#3 expression relative to Actin: CD29, 2.9; CD49f, 5.1; CD10, 9; SCA-1, 3.8; K18, 2.9; VM, 5.5; PTN, 10.3. Treated MECs passage#3 expression relative to Actin: CD29, 3.5; CD49f, 5.4; CD10, 7.5; SCA-1, 3.2; K18, 2.9; VM, 5.9; PTN, 10.9. (C) Expression of K18, VM and PTN in mammary fibroblasts relative to MECs passage #1. Expression of K18, VM and PTN in mammary fibroblasts relative to Actin: PTN, 15.7; K18, 15.9; VM, 3. doi:10.1371/journal.pone.0047876.g008 during pregnancy. Using different, independent approaches we show here that PTN is predominately expressed in epithelial cells in the virgin mammary gland. The tissue analysis was also validated in isolated mammary epithelial cells (MECs) harvested in parallel with isolated fibroblasts showing a .10 fold higher expression of PTN in the MECs than in fibroblasts (Figure 8C) and expression of PTN protein at the cell membrane (Figure 4A). PTN expression remained unaltered during the first 10 days of pregnancy but was downregulated 30-fold during late pregnancy when the mammary gland is proliferating and differentiating in preparation for lactation. A downregulation of PTN expression during late pregnancy was surprising to us because the epithelial ducts are still proliferating and PTN has previously been shown to be a growth factor for different types of cells [19]. We surmise that PTN expression is sustained by the first wave of proliferation of the epithelial cells during early pregnancy when the cells are still in an undifferentiated state but downregulated during the second wave of proliferation in late pregnancy when the epithelial cells are undergoing lobular-alveolar differentiation. There is only one known PTN homologue, midkine (MK), that shares 50% protein identity [69], has a similar activity profile [1] but an approximately 10-fold lower affinity for the ALK receptor when compared with PTN [70,71]. In Northern blots MK was below detection in virgin, midgestation, or lactating mammary glands (data not shown) although data mining of published cDNA array studies suggests a regulation of MK in the mammary gland during pregnancy (NCBI GEO GDS2843). We conclude from our functional data and a comparison with MK that PTN is the predominant ligand of this growth factor family in the mammary gland. PTN was found to be a ligand that binds to the extracellular domain of the tyrosine kinase receptor ALK [72]. Others have described that the protein tyrosine phosphatase receptor f1 (PTPRf1) is involved in the control of the PTN/ALK signaling [73]. Marzan et al. showed expression of PTPRf1 in HC11, a mammary epithelial cell line, and suggested a role for the interaction of PTN and PTPRf1 in the migration of the mammary epithelial cells [40]. Here we describe a striking parallel regulation of PTN and ALK expression in the mammary gland. This suggests to us that ALK constitutes a major signaling pathway utilized by PTN during mammary gland development. When comparing PTN expression among nulliparous, unipa- rous, multiparous and pseudopregnant mice, a significant decrease in PTN mRNA expression level was detected in the mammary gland of multiparous versus nulliparous mice but not in pseudopregnant mice. These findings suggest a regulation of PTN expression due to gestational hormones and a decrease in PTN expression possibly due to a loss in mammary epithelial progenitor cells by repeated pregnancies. Full term pregnancy has been demonstrated as a protective factor against the risk of breast cancer [47]. Studies in rodent mammary glands and in the human breast suggest that the differentiation status induced during repeat pregnancies contributes to this protective effect. We suggest that permanent changes in the expression of local regulatory factors such as PTN, may be important in the progression to this protected state. In fact, at least one other study has implicated the downregulation of a variety of growth-promoting factors including PTN, amphiregulin and IGF-1 due to parity as a cause for the protective effects [42]. Furthermore, we have seen that PTN is overexpressed in approximately 60% of human breast carcinoma PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47876