Treatment with the anti-PTN antibody inhibited MECs migration (Physique 4D; p<0

Treatment with the anti-PTN antibody inhibited MECs migration (Physique 4D; p<0.001). 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 usually 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 AKBA elucidate its function in cultured mammary Itga9 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 growth and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In 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 hippocampal activity [5], [6]. Previous data suggest a temporal regulation of PTN expression during pregnancy and a permanent downregulation 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 mammary epithelial cells (MECs) start undergoing lobular-alveolar differentiation. We also found that blocking PTN activity caused enhanced maturation of the mammary gland accompanied by AKBA 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 produced on plastic and that blocking PTN activity caused increased number of mammospheres due to a more polarized structural business 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 usually highly regulated during pregnancy and reduced 30-fold by day 15 with the ALK receptor regulated in parallel reduced 100-fold by day 15 (Physique 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 usually characterized by proliferating ductal epithelial cells. To determine which cells mostly express and secrete PTN, mRNA expression was analyzed by hybridization as well as cell fractionation followed by Northern blot (Physique 2). hybridization for PTN mRNA supports the downregulation during pregnancy and shows AKBA major expression in the epithelial compartment of the mammary gland (Physique 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.