Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

<div><p>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 <em>vivo</em>, 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.</p> </div

PTN effect on MAP kinase pathway activity in developing mammary glands.

<p>Phospho-ERK1/2 (A), phospho-p38 (B), and phospho-JNK (C) in whole mammary gland extracts from anti-PTN treated mice (n = 10) versus controls (n = 9). The ratio of phospho- to total protein was obtained from multiplex assays. Ns, not significant; *P<0.05. (D, E) Western blot analysis for pERK1/2. Representative Western blots (mouse m1 to m6) and quantitation of phospho ERK1/2 relative to total ERK1/2 in 3 independent experiments (n = 9 to 10 per group). (F) Representative Western blots of whole cell lysates from MECs treated for 24 hours with either UO126 (500 nM) alone or in combination with anti-PTN blocking antibody and quantitation of phospho ERK1/2 relative to total ERK1/2. Numbers represent the ratio of the quantification of phospho ERK1/2 to total ERK1/2. Data are means ± SE; *P<0.05.</p

PTN and ALK mRNA expression in mouse mammary glands during pregnancy.

<p>Mammary glands from mice that were virgin, pseudopregnant (PsPreg), at different days of pregnancy or after weaning ( = involution). Quantitative, real-time PCR was used. Pseudopregnants denotes 12 days since plug date. Pups were removed from the mother at 21 days of age and involution followed thereafter. Expression is given relative to keratin 18. Mean ± SE (n = 4; PsPreg n = 2). **P<0.01; ***P<0.001, by ANOVA.</p

PTN protein expression and function in mammary epithelial cells (MECs).

<p>(A) Immunofluorescence (IF) staining of MECs grown on poly-D-lysine coated slides. PTN (green), DAPI (blue), Phalloidin (red). Images are shown as merged images of anti-PTN with DAPI and anti-PTN with Phalloidin staining. Scale Bar  = 20 μm. (B, C) Real-time impedance sensing of MECs growth relative to stimulation time (20 hours) with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), or antibody elution buffer (Control) (B), bFGF or resuspension buffer (C). (D) Real-time impedance sensing of MECs migration relative to 30 hours from plating and four hours from scratching. MECs were treated with anti-PTN blocking antibody (anti-PTN), recombinant PTN (PTN), alone or in combination or antibody elution buffer (Control). (E) Real-time impedance sensing of MECs invasion relative to 30 minutes from plating. MECs were treated with anti-PTN blocking antibody (anti-PTN), or antibody elution buffer (Control) (E), recombinant PTN (PTN) or PTN resuspension buffer (Control) (F). The data are representative of at least three independent experiments done in duplicates. ***P<0.001 versus control by ANOVA.</p

Mammary gland tissue distribution of PTN mRNA.

<p>(A) In situ hybridization (ISH) with PTN mRNA antisense and sense probes. Red squares indicate the areas shown at higher magnification. Scale Bars  = 25 μm and 100 μm respectively. Red arrowheads indicate PTN mRNA expression in mammary epithelial cells. (B) Northern blot for PTN mRNA in virgin mammary glands fractionated into epithelial and stromal cells.</p

PTN effect on mammosphere formation.

<p>(A) Time course of mammosphere formation day 1, 3 and 5 without and with a blocking anti-PTN antibody ± recombinant PTN protein. Representative mammospheres that meet the threshold surface area of >900 µm² are indicated by an arrowhead. Pictures are representative of 3 independent experiments. Scale bar  = 50 µm. (B) Quantification of mammospheres (>900 μm² ) formed per field at day 5. Data are means ± SE; n = 5 fields/well; 4 wells for each condition. **P<0.01; versus anti-PTN treated cells; ***P<0.001 versus control by ANOVA. (C to F) Immunofluorescence of mammospheres at Day 5 for Ki67 (green)(C), Caspase 3 & DAPI (green & blue) (D), laminin & DAPI (green & blue) or for PTN (grey scale). Staining is shown in mammospheres treated with blocking anti-PTN antibody (anti-PTN) versus control. Scale bar = 20 µm.</p

PTN effect on cultured mammary epithelial cell (MEC) differentiation.

<p>(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 ± anti-PTN is shown relative to passage #1 MECs. Data are means ± 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.</p

PTN effect on ductal outgrowth and branching in developing mammary glands.

<p>(A) Whole mounts of representative mammary glands from mice treated with anti-PTN blocking antibody versus control. White arrows: distance of the outgrowth of a representative duct measured from the midpoint of the lymph node. Black arrowheads: branch points. Scale bar  = 1.5 mm. (B to F) Controls (n = 9 mice, white), anti-PTN (n = 10 mice, grey). (B, C) Ductal length and number of branch points with the ductal length expressed in mm and the number of branch points per mm of duct. Data are means ± SE; *P = 0.04; ** P = 0.006; n = 3 ducts per mouse. (D to F) Quantitation of Terminal Ends (TE) and Terminal End Buds (TEBs) of mammary ducts; (D) TEBs per duct, ns =  not significant; (E) TEs per duct, * P = 0.03; (F) TEBs per total number of duct ends ( = TEBs + TEs), *P = 0.027. Data are means ± SE; n = 6 ducts per mouse.</p