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.
Highlights
The heparin-binding growth factor pleiotrophin (PTN; 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 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)
Summary
The heparin-binding growth factor pleiotrophin (PTN; known as HB-GAM or HARP; reviewed in [1]) is a 15 kDa secreted protein named for its pleiotrophic effects on different cell types. 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]. 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 activity has recently been shown to stimulate the differentiation and expansion of neuronal stem cells, osteoprogenitors and bone marrow stem cells [29,30,31]
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