Model for HGF-mediated regulation of EphA2 in mammary epithelial branching morphogenesis. HGF produced by mesenchymal cells within mammary stroma binds to c-Met receptor, expressed on mammary epithelium. Through activation of EphA2 receptor function by an as yet unidentified mechanism, Rho activity is then down-regulated to promote branching morphogenesis of the developing mammary epithelium during puberty.

EphA2 deficiency inhibits proliferation but has no effect on apoptosis. (A) PCNA immunohistochemistry in mammary gland tissue sections from wild-type and EphA2-deficient animals. Arrowheads indicate PCNA+ nuclei. Scale bars, 50 μm. (B) Proliferation was assessed by quantification of nuclear staining for PCNA in tissue sections. A significant reduction in the percentage of PCNA-positive nuclei relative to total nuclei was observed in KO EphA2 mammary glands, compared with WT controls (p < 0.05). (C) No significant change in the percentage of apoptotic nuclei, as assessed by TUNEL assay, was observed in KO mammary glands relative to WT controls. Quantification of the percentage of PCNA+ or TUNEL+ nuclei was performed by analyzing >10 independent animals per developmental stage per genotype. Five random 20× fields/tissue section were photographed for PCNA and TUNEL per each independent animal/genotype. Statistical analyses were performed using two-tailed, paired Student's t tests.

EphA2 receptor tyrosine kinase and ephrin-A1 ligand are expressed in luminal epithelial cells in virgin mammary gland tissue. (A) Immunohistochemical staining in virgin mammary tissue sections prepared from wild-type (WT) 6-wk-old virgin female mice revealed EphA2 protein expression on the surface of luminal epithelial cells (arrowheads), but no apparent expression in myoepithelial cells/fibroblasts surrounding ducts (arrows) or in fatty tissue (*). Staining specificity, as well as EphA2 deficiency, was confirmed by probing mammary tissue sections prepared from age-matched EphA2-deficient (KO) virgin female mice. (B) EphA2 protein deficiency in KO mice versus WT was also confirmed by immunoblot analysis of protein lysates prepared from whole mammary gland tissue. Uniform loading was confirmed by reprobing blots for expression of tubulin. (C) We also observed expression of ephrin-A1, the primary ligand for EphA2 receptor, in luminal epithelial cells (arrowheads), versus surrounding stromal cells (arrows) and fat (*) in tissue sections prepared from 6-wk-old wild-type virgin female mice. Staining specificity was validated by probing tissue with control rabbit IgG (rIgG). Scale bars, 10 μm.

Increased RhoA activity in EphA2-deficient mammary epithelial cells. (A) Rho kinase activity was measured by an immune-based kinase assay, as described in Materials and Methods. Rho kinase activity was significantly increased in EphA2-deficient mammary epithelial cells (p < 0.05; two-tailed, paired student's t test). Data are a representation of three independent experiments. (B) Rho GTPase activities were analyzed in 6-wk-old mammary glands in situ by incubating tissue sections with GST-rhotekin or GST-Pak for the detection of RhoA and Rac1/Cdc42 activities, respectively; followed by detection using a Cy3-conjugated anti-GST antibody. Scale bars, 50 μm. Rho activity was quantified based on Cy3-positive pixel area using NIH ImageJ software. Quantification of Rho activity was performed in four 40× fields/section in tissue sections from three independent wild-type or EphA2-deficient mammary animals. Statistical analyses were performed using two-tailed, paired Student's t tests.

EphA2-deficiency impairs normal development and architecture of the mammary epithelial tree. (A) Whole-mount hematoxylin staining of number 4 inguinal mammary glands collected from WT and KO FVB female animals 5 and 6 wk after birth. Scale bar, 200 μm. Quantification of numbers of terminal end buds (B) and branching points (C) in mammary gland whole mounts from 4- and 5-wk-old mice. (D) Mammary gland whole mounts prepared from KO EphA2 and WT EphA2 animals at 6 wk of age and 12 wk of age were analyzed for epithelial penetration into the fat pad by measuring the distance between the lymph node to the tips of the epithelial tree in gland. Quantification of TEBs, branching points, and the degree of epithelial penetration through the mammary fat pad was performed by analyzing whole-mount preparations from >10 independent animals per developmental stage per genotype. Statistical analyses were performed using two-tailed, paired Student's t tests.

EphA2 activity is required in mammary epithelium for optimal branching in vivo, as well as for HGF-induced epithelial cell branching morphogenesis in Matrigel. (A) To determine if defective epithelial branching in EphA2-deficient (KO) mice was due to loss of EphA2 function in epithelium versus stroma, we transplanted KO mammary tissue into the cleared fat pads of WT female mice, as well as WT mammary tissue into the cleared fat pads of KO female mice. Photomicrographs display two independent whole-mount preparations of mammary fat pads harboring transplanted tissue. Although WT epithelium displayed robust branching in KO stroma 8-wk after transplantation, branching of KO epithelium was significantly diminished in WT hosts (p < 0.05; two-tailed, paired Student's t test). Scale bar, 200 μm. Quantification of branching was performed by analyzing whole-mount preparations from five independent transplants/condition. (B) Primary mammary epithelial cells isolated from WT or KO animals were transduced with adenoviruses expressing EphA2 (Ad-EphA2) or control LacZ (Ad-LacZ) and plated on growth-factor–reduced Matrigel with or without HGF for 5 d and photographed. HGF enhances branching morphogenesis in WT, but not KO, mammary epithelial cells. The defects in KO cells was rescued by reexpressing wild-type EphA2 receptor. Scale bar, 25 μm. (C) Branching morphogenesis was quantified by counting the number of branches per photograph in four independent samples per culture condition in three independent experiments. Statistical analyses were performed using two-tailed, paired Student's t tests.

EphA2-dependent branching morphogenesis is dependent on RhoA activity. (A) Primary mammary epithelial cells isolated from WT or KO animals were transduced with adenoviruses expressing a constitutively activated RhoA (CA-Rho) or control LacZ. Cells were plated on growth-factor reduced Matrigel with 20 ng/ml HGF for 5 d and photographed. Branching in KO LacZ control cells was significantly diminished relative to WT LacZ control cells in response to HGF (* p < 0.05; two-tailed, paired Student's t test). Expression of the Q63L RhoA inhibited HGF-induced branching morphogenesis in WT cells. Scale bar, 25 μm. (B) Branching morphogenesis was quantified by counting the number of branches per photograph in four independent samples per culture condition in three independent experiments. Statistical analyses were performed using two-tailed, paired Student's t tests. (C) Expression of wild-type EphA2, CA-Rho, and β-galactosidase via adenovirus transduction was confirmed by immunoblot analysis. (D) Branching was also assessed in WT and KO primary mammary epithelial cells in the presence or absence of 20 ng/ml HGF and upon treatment with the Rho kinase inhibitor 10 mM Y27632. As observed previously, WT, but not KO, cells displayed elevated branching in response to HGF (p < 0.05 WT untreated vs. WT + HGF). Branching was elevated in both WT and KO cells in the presence of Y27632. Although the addition of HGF enhanced Y27632-mediated branching in WT cells, adding HGF had no effect on Y27632-mediated branching in KO cells (p < 0.05 WT vs. WT Y27632 and WT Y27632 vs. Y27632 + HGF; p < 0.05 KO vs. KO Y27632; two-tailed, paired Student's t tests). (E) The differential effects of HGF Rho-dependent branching for WT versus KO cells was confirmed by a dose response assay in which cultures were treated with 0, 0.1, 1, or 10 μM Y27632 in the presence or absence of HGF.