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Vol. 20, Issue 10, 2572-2581, May 15, 2009
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*Department of Cancer Biology, Department of Medicine, Division of Rheumatology and Immunology, and Department of Cell and Developmental Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, 37232
Submitted April 11, 2008;
Revised February 23, 2009;
Accepted March 17, 2009
Monitoring Editor: Joan Brugge
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Watson and Khaled, 2008). This process, termed branching morphogenesis, is most active during puberty. In response to hormonal stimuli, terminal end buds (TEBs) form at the tips of the ducts and invade into the surrounding stroma. New primary ducts then form by bifurcation of the TEBs and secondary side-branches sprout laterally from the trailing ducts. This process is reiterated through branching and tissue remodeling until the entire mammary fat pad is filled with a ductal tree in the virgin gland. During pregnancy, the mammary epithelium undergoes differentiation and expands drastically to meet the demand of milk production throughout lactation. After weaning, the mammary epithelium regresses through a process of programmed cell death.
Mammary gland branching morphogenesis is regulated by endocrine hormones and local paracrine interaction between the developing epithelial ducts and their adjacent mesenchymal stroma. Although the mediators of the complex interaction in mammary gland development are not fully characterized, receptor tyrosine kinases (RTKs) are among the critical regulators of branching morphogenesis (Sternlicht et al., 2006). Hepatocyte growth factor/scatter factor (HGF/SF), a mesenchymal-derived mitogen and morphogen, induces branching morphogenesis through its receptor c-Met, which is expressed on mammary epithelial cells (reviewed in Kamalati et al., 1999; Pollard, 2001). More recently, expression of multiple Eph family RTKs has been reported in the mammary gland (reviewed in Andres and Ziemiecki, 2003). However, their role in branching morphogenesis remains to be investigated.
The Eph RTK family is the largest family of RTKs identified in the genome, with at least 15 receptors and 9 ligands identified in vertebrates (reviewed in Murai and Pasquale, 2003; Brantley-Sieders and Chen, 2004). The family is subdivided into class A and class B based on homology and binding affinity for two distinct types of membrane-anchored ephrin ligands. Class B receptors generally bind to class B ephrins that are attached to the cell membrane by a transmembrane-spanning domain, while A class receptors normally interact with glycosyl-phosphatidylinositol (GPI)-linked class A ephrins, although interclass binding does occur among certain family members (reviewed in Murai and Pasquale, 2003; Brantley-Sieders and Chen, 2004). These molecules function in cell–cell communication during embryogenesis to regulate angiogenic remodeling processes, axon guidance, and tissue boundary formation (reviewed in Poliakov et al., 2004; Pasquale, 2005). In adult organisms, members of this RTK family have been linked to tumor progression and neovascularization (reviewed in Brantley-Sieders and Chen, 2004).
The first Eph receptors discovered in mammary gland are EphB4 (myk-1) and EphA2 (myk-2; Andres et al., 1994). The EphB4 receptor is expressed predominantly on myoepithelial cells surrounding the ducts and alveoli, whereas its cognate ligand, ephrin-B2, is expressed complementarily in luminal epithelial cells. Expression of both ligand and receptor is estrogen-dependent (Nikolova et al., 1998). More recently, a genome-wide transcript analysis identified EphA2 receptor and ephrin-B1 as the only two Eph molecules that are enriched in TEBs relative to ducts (Kouros-Mehr and Werb, 2006). Functionally, overexpression of EphB4 in mammary epithelial cells in MMTV-EphB4 transgenic mice disrupts the patterning of the normal mammary ductal tree, induces angiogenesis, accelerates tumor formation, and promotes metastasis when coexpressed with MMTV-Neu in bigenic mice (Munarini et al., 2002). EphA2 receptor overexpression has been associated with many types of cancer, including breast cancer (reviewed in Kinch and Carles-Kinch, 2003; Brantley-Sieders and Chen, 2004; Ireton and Chen, 2005). In addition, EphA2 has been shown to regulate HGF-induced Madin-Darby canine kidney (MDCK) cell branching morphogenesis in three-dimensional (3D) collagen gels (Miao et al., 2003). However, the role of EphA2 in mammary gland development remains unknown.
In this study we investigated the role of EphA2 in mammary gland branching morphogenesis in EphA2-deficient mice. We found that loss of EphA2 inhibits the proliferation of the mammary epithelium and delays ductal branching necessary for complete fat pad filling. At the cellular level, EphA2-deficiency resulted in marked reduction of branching in response to HGF stimulation. This defect, at least in part, is due to misregulation of Rho family GTPase function. These results suggest that EphA2 is required for mammary gland branching morphogenesis in vivo. Taken together, these data demonstrate a positive role for EphA2 in proliferation and branching morphogenesis of normal mammary epithelium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2005) were identified by PCR analysis of genomic DNA from tail biopsy using the following primers: 5'-GGG TGC CAA AGT AGA ACT GCG-3' (forward), 5'-GAC AGA ATA AAA CGC ACG GGT G-3' (neo), and 5'-TTC AGC CAA GCC TAT GTA GAA AGC-3' (reverse).
Reagents
Antibodies used include anti-EphA2 (SC-924 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz, CA; 5 µg/ml, Zymed Laboratories, Burlingame, CA; D7 clone 2 µg/ml, Upstate Biotechnology, Lake Placid, NY), anti-Ephrin-A1 (clone P1 1:200, Immunex, Seattle, WA), normal rabbit IgG (Santa Cruz Biotechnology), anti-β-tubulin (1:500, Sigma-Aldrich, St. Louis, MO), biotinylated anti-proliferating cell nuclear antigen (PCNA; 1:500, BD Biosciences, San Jose, CA). Avidin peroxidase (ABC) reagents were purchased from Molecular Probes (Eugene, OR). 4',6-Diamidino-2 phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich. Liquid 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate kit was purchased from Zymed Laboratories. Recombinant murine HGF was purchased from R&D Systems (Minneapolis, MN).
Whole-Mount Mammary Gland Analyses
Whole-mount hematoxylin staining of mammary glands was performed by taking no. 4 inguinal mammary glands and fixing in 10% buffered Formalin (Fisher Scientific, Pittsburgh, PA; SF93-4) overnight at 4°C. The glands were washed in acetone, equilibrated into 100% ethanol, and stained in Mayer's hematoxylin solution (VWR Scientific, West Chester, PA) for 1 h at room temperature, light protected. After staining the glands were destained in tap water and then further destained in 50% ethanol acidified with hydrochloric acid at a 0.05 M final concentration. The glands were then dehydrated in a graded ethanol series followed by xylenes and mounted on slides for photodocumentation.
Proliferation, Apoptosis, and Immunohistochemistry Assays
Proliferation and apoptosis in mammary gland in situ were assessed by PNCA immunohistochemistry or TUNEL analysis, as previously described (Brantley-Sieders et al., 2008). For immunohistochemistry, sections were de-waxed, rehydrated, and subjected to thermal antigen retrieval in citrate buffer (2 mM citric acid, 10 mM sodium citrate, pH 6.0) using a PickCell Laboratories 2100 Retriever (Leiden, The Netherlands) according to the manufacturer's instructions. Sections were incubated with primary anti-EphA2 antibody (5 µg/ml, Zymed Laboratories), anti-ephrin-A1 antibody (1:200, Immunex), or control rabbit IgG (5 µg/ml, Santa Cruz Biotechnology) overnight at 4°C, followed by 1-h room temperature incubation with biotinylated anti-rabbit antibodies (BD Biosciences). Sections were then treated incubated with avidin-peroxidase (Vector Laboratories, Burlingame, CA), followed by DAB substrate, counterstained with hematoxylin (Fisher Scientific) and mounted.
Mammary Fat Pad Clearing and Transplantation
Fat pad clearing and transplantation of EphA2-deficient mammary tissue into wild-type hosts, as well as wild-type tissue into EphA2-deficient hosts, was performed as described previously (Brantley et al., 2001). Briefly, the endogenous epithelium was surgically removed from the right no.4 inguinal mammary gland of 3-wk-old mice by excising the portion of the fat pad between the nipple and the lymph node, which contains the endogenous epithelium rudiment. A small portion (
2 mm2) of donor tissue from 6-wk-old female mammary glands was engrafted into the remaining fat pad. Mammary glands harboring transplanted tissue were harvested 8 wk after transplantation and processed for whole-mount staining as described above. Engraftment of exogenous mammary epithelium was verified by radial outgrowth of epithelium, versus glands contaminated with endogenous epithelium that grows directionally from the nipple toward the body cavity.
Isolation and Culture of Primary Mammary Epithelial Cells
Primary mouse mammary epithelial cells (PMECs) were isolated from FVB wild-type and EphA2-deficient FVB female mice and cultured as follows: mammary glands were collected under sterile conditions and digested at 37°C for 4 h in 3 mg/ml collagenase A (Boehringer Mannheim, Indianapolis, IN; 103578) in PBS (pH 7.4), 100 U/ml hyaluronidase (Sigma; H-4272), and 1:1000 dilution of Fungizone (Invitrogen, Carlsbad, CA; 15290-018). The cell suspension was first plated on bacterial Petri dishes for 3–5 h to separate epithelial cells from fibroblasts, which adhere to Petri dishes. The epithelium-enriched cell suspension was then plated on dishes coated with collagen (Vitrogen, Cohesion Technologies, Palo Alto, CA) in 0.02 N acetic acid, washed with PBS before addition of cell culture media consisting of serum-free DMEM-F12 (50:50; Invitrogen), 5 ng/ml estrogen (Sigma; E-4389), 1 ng/ml progesterone (Sigma; P-7556), 5 ng/ml EGF (Sigma; E-4127), and 5 µg/ml insulin (Sigma; I-1882), and cultured at 37°C in 5% CO2.
In Vitro Branching Morphogenesis Assays
HGF-induced branching of PMECs isolated from wild-type or EphA2-deficient female mice was scored in 3D mammosphere culture using a modification of previously described methods (Debnath et al., 2003). Briefly, PMECs were trypsinized, and 100,000 cells were plated on a thin layer of growth factor–reduced Matrigel (BD Biosciences) in eight-well chamber slides (BD Falcon, Oxnard, CA). Cultures were maintained in normal PMEC medium supplemented with 2% growth factor-reduced Matrigel and 5% FCS in the presence or absence of 20 ng/ml recombinant murine HGF (R&D Systems) for 5 d, changing the media after 48 h. Cultures were photographed on day 5 using an Olympus CK40 inverted microscope with digital camera (Melville, NY), and branching was scored by counting branches in four independent 10x photographs per culture condition. For some experiments, PMECs were infected with 108 pfu/ml recombinant adenoviruses harboring constitutively active RhoA (Q63L, Cell Biolabs, San Diego, CA), EphA2, or control β-galactosidase (Brantley-Sieders et al., 2008) 24 h before mammosphere culture. For ROCK inhibition studies, PMECs were pre-treated with 0, 0.1 µM, 1 µM, or 10 µM Y27632 ROCK inhibitor (Calbiochem, San Diego, CA) or vehicle control for 1 h before mammosphere culture, and cultures were treated again after 48 h for 1 h. All experiments were performed three times with 4 independent cultures per condition in each experiment.
Rho Kinase Assay
Primary mammary epithelial cells were plated in 6 well plates and stimulated with HGF (25 ng/ml) for 15 min upon reaching 60% confluency. Cells were harvested in a 1.0% Triton X-100 lysis buffer consisting of 50 mM Tris-HCl, pH 8.0, 1.0% Triton X-100, 1 mM PMSF, 5 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM sodium orthovanadate. Harvested cells were sonicated and cleared lysates were assayed for ROCK activity using the CycLex Rho-Kinase Assay (ROCK Assay) Kit (MBL International, Woburn, MA) according to manufacturer's protocol. Briefly, 10 µL of each sample was incubated with a substrate corresponding to the C terminus of recombinant Myosin Binding Subunit of myosin phosphatase (MBS). The phosphorylated form of threonine 696 of the MBS was detected by an HRP conjugated detection antibody AF20, coupled with TMB color reaction. Absorbance was measured at 450 nm, which reflects relative amount of Rho-kinase activity. For some experiments, PMECs were infected with 108 pfu/ml recombinant adenoviruses harboring EphA2, or control β-galactosidase (Brantley-Sieders et al., 2008) 48 h prior the ROCK assay.
In Situ Rho/Rac Activity Detection Assays
Detection of GTP-bound Rho and Rac in mammary gland sections was performed as described previously (Muraoka et al., 2003). Briefly, paraffin-embedded tumor sections were rehydrated, treated with 0.01% trypsin (Cellgro; Mediatech, Herndon, VA) for 5 min, and blocked for 30 min in MOM (mouse-on-mouse) diluent (Molecular Probes). Sections were incubated with purified Pak-1–binding domain–glutathione S-transferase (PBD-GST), rhotekin-binding domain GST (RBD-GST) fusion protein, or GST control in MOM diluent containing 10 mM MgCl2 for 15 min at room temperature. Binding of GST fusion proteins was detected using an anti-GST antibody (Santa Cruz Biotechnology) followed by anti-rabbit Cy3. Sections were counterstained with DAPI and photographed, and binding of GST fusion proteins was quantified based on Cy3+ pixel area using NIH ImageJ software (http://rsb.info.nih.gov/ij/).
Statistical Analyses
Statistical analysis of developmental and in vitro studies were performed using two-tailed, paired Student's t tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting that this receptor–ligand pair may regulate epithelial morphogenesis.
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3.5 wk, the rudimentary mammary epithelium anlagen undergoes rapid proliferation and invades the stroma by directional growth and branching, giving rise to the characteristic mammary epithelial tree that populates the entire mammary fat pad at 10 wk of age. In contrast, the EphA2-deficient animals exhibit severe growth retardation (Figure 2). This defect is more prominent at 5 and 6 wk of age, with reduced branching activity and decreased numbers of TEBs (Figure 2, A–D). In addition, outgrowth of the mammary epithelial tree was also retarded in these animals (Figure 2, A and D). In mature animals at 12 wk, 50% of the EphA2-deficient animals exhibited a fully formed mammary ductal tree (Figure 2D). Failure of the mammary epithelium to fully penetrate the mammary fat pad persists in 30% of EphA2-deficient MMTV-Neu animals at 8 mo (Brantley-Sieders et al., 2008). We did not observe any defects in expansion of the mammary epithelium during pregnancy, in function during lactation, or in apoptosis during postlactational involution in EphA2-deficient animals relative to controls (data not shown).
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). In particular, HGF promotes ductal outgrowth and tubule formation in the mammary gland (Pollard, 2001). We thus analyzed the role of EphA2 in HGF-induced mammary epithelial cell branching morphogenesis. As shown in Figure 4, primary mammary epithelial cells form spheroid structures in growth factor reduced Matrigel, and EphA2-deficient cultures display diminished branching relative to wild-type control cells. In response to HGF stimulation, wild-type spheroids undergo extensive remodeling and branching. In contrast, EphA2-deficient spheroids fail to undergo branching morphogenesis, displaying significantly fewer branches relative to wild-type cells in response to HGF stimulation. Overexpression of wild-type EphA2 receptor in primary mammary epithelial cells via adenovirus transduction not only rescued phenotypes in EphA2-deficient cells, but also drastically enhanced branching morphogenesis in wild-type cells. Taken together, these data suggest that EphA2 receptor is required for HGF-induced branching morphogenesis.
Increased RhoA Activity in EphA2-deficient Cells Inhibits Mammary Epithelial Cell Branching
We next investigated the molecular mechanisms through which EphA2 regulates mammary epithelial cell branching morphogenesis. Dynamic regulation of the actin cytoskeleton is critical in a number of cellular processes including cell migration and branching morphogenesis. RhoA GTPases are key regulators of actin stress fiber formation and are necessary for cell migration (Ridley, 2001a,b; Connolly et al., 2002). Moreover, Ewald et al. (2008) recently reported that inhibition of ROCK kinase, a downstream effector of RhoA, results in hyperbranched mammary epithelium in organoid cultures, suggesting that RhoA is crucial for proper branching morphogenesis in mammary epithelial development. To investigate whether RhoA GTPase is involved in HGF/EphA2 induced branching of mammary epithelial cells, we measured ROCK kinase activity in wild-type and EphA2-deficient mammary epithelium. As shown in Figure 5A, HGF stimulation of primary mammary epithelial cells for 15 min induced ROCK kinase activity in wild-type cells. Interestingly, the basal level of ROCK activity is markedly increased in EphA2-deficient cells, and this level does not change in response to HGF stimulation. Experiments in which we restored expression of EphA2 with adenoviral infection EphA2-deficient cells rescued the phenotype by reducing ROCK activity to near wild-type levels, though levels of ROCK activity remained similar in wild-type cells upon adenoviral EphA2 overexpression (Figure 5A). To determine the level of active RhoA GTPase in vivo, we performed effector-binding assays on mammary gland tissue sections in situ (Muraoka et al., 2003). Consistent with results from ROCK assay in vitro, RhoA activity is markedly increased in EphA2-deficient mammary gland epithelium in 6-wk-old mice, compared with that in wild-type control littermates, as judged by GST-rhotekin binding detected by anti-GST antibodies (Figure 5B). As a balance of RhoA and other small Rho GTPases activities (such as Rac1 and Cdc42) often determines the biological outcome in branching morphogenesis, we assayed Rac1 and Cdc42 activity in situ using GST-Pak proteins. We did not detect any significant levels of Rac1 and Cdc42 activities in the 6-wk-old mammary gland sections (Figure 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, their role in mammary gland development remains poorly understood. Using EphA2-null animals, we demonstrated for the first time that EphA2 receptor function is required for mammary gland branching morphogenesis. Loss of EphA2 resulted in decreased penetration of mammary epithelium into the fat pad, reduced proliferation of epithelial cells, and inhibition of mammary epithelial branching. In addition, HGF-induced mammary epithelial cell migration and branching morphogenesis was significantly reduced in EphA-deficient cells, compared with that in wild-type cells. These results suggest that EphA2 receptor acts as a positive regulator in mammary gland development.
Other studies, however, suggest that Eph receptors may inhibit mammary gland morphogenesis. Overexpression of EphB4 in mammary epithelium under the control of MMTV promoter/enhancer led to less branching activity, reduced alveolar buds, and a decrease in proliferation of mammary epithelial cells (Munarini et al., 2002). Although not observed in mammary epithelium, this phenotype is reminiscent of the effect of EphA receptor activation seen in MDCK cells in response to HGF-induced branching morphogenesis in collagen gels (Miao et al., 2003). In this model, costimulation of MDCK cells with ephrin-A1 and HGF inhibited sprouting and induced the collapse of preexisting branches. These results suggest that high levels of Eph receptor signaling above the endogenous level in epithelial cells, either by overexpression of receptor or exogenous stimulation with a large dose of ephrin ligand, may inhibit branching morphogenesis. However, low levels of endogenous EphA2 receptor are required for proper mammary gland development in vivo based on our data.
Epithelial branching morphogenesis is a fundamental biological process underlying the development of many organs. In breast tissue, epithelial branching morphogenesis is driven by endocrine hormonal stimuli that elicit local paracrine interactions between the developing epithelial ducts and their adjacent mesenchymal stroma. Cytokines and growth factors, such as HGF, FGF, TGF-β, and amphiregulin are among molecules that are critical in local regulation of branching morphogenesis (Pollard, 2001; Sternlicht et al., 2006). A common pathway activated downstream of these signaling molecules is the Rho family of small GTPases. These proteins cycle between an inactive, GDP-bound state and an active, GTP-bound state, regulated by guanine nucleotide exchange factors (GEFs) that exchange GDP for GTP and GTPase activating proteins (GAPs) that promote hydrolysis of GTP to GDP (Ridley, 2001a,b). Recently, RhoA, Rac-1, and Cdc42 GTPases have emerged as critical mediators of Eph signal transduction, as association of several Eph RTK family members with GEFs concomitant with activation of Rho family GTPases has been observed in a variety of cell types. Interactions between Eph RTKs and the GEFs ephexin, intersectin, and kalarin link Eph-mediated activation of Rho family GTPases to growth-cone collapse (ephexin) or dendritic spine morphogenesis (intersectin and kalarin) in neuronal patterning during embryogenesis (reviewed in Murai and Pasquale, 2003; Brantley-Sieders and Chen, 2004; Pasquale, 2005). More recently, Eph signaling through Rho family GTPases has been linked to vascular remodeling and angiogenesis (Brantley-Sieders et al., 2004; Hunter et al., 2006). In MDCK epithelial cells, Miao et al. (2003) reported that ephrin-A1 has no effect on RhoA but inhibits Rac1 activation induced by HGF. Here we show that EphA2-dependent RhoA activity modulates HGF-induced branching morphogenesis in mammary epithelial cells. First, loss of EphA2 led to a constitutive higher level of RhoA GTPase activity, which is insensitive to HGF stimulation (Figure 5, A and B). Interestingly, Rac1 activity did not appear to be significantly altered in EphA2-deficient mice, indicating that RhoA activity is a primary target regulated by the EphA2 receptor (Figure 5C). Furthermore, a ROCK kinase inhibitor, Y27632, rescued branching defects in EphA2-deficient cells in 3D culture (Figure 6), functionally linking EphA2-dependent RhoA activity and branching morphogenesis.
Regulation of Rho GTPase in mammary epithelium is complex. For example, targeted disruption of p190-B RhoGAP inhibits mammary epithelial ductal outgrowth, and heterozygous female mice display reduced proliferation within TEBs and delayed outgrowth of mammary ducts (Chakravarty et al., 2000). Because deletion of this GAP, which negatively regulates Rho, results in elevated Rho activity, these data are consistent with our findings that elevated RhoA function in virgin mammary epithelium is associated with decreased proliferation and ductal outgrowth in EphA2-deficient mammary epithelium. Interestingly, overexpression of the same RhoGAP in mammary epithelium elevates branching in transgenic animals, though ductal elongation is still delayed (Vargo-Gogola et al., 2006). These data suggest that disruption of Rho activity in mammary epithelium, either positive or negative, has a profound impact on epithelial morphogenesis in vivo. This hypothesis is consistent with the apparent differential regulation of Rho by EphA2 signaling in normal versus transformed mammary epithelium, which is influenced by cooperative signaling pathways. In the present study, loss of EphA2 results in elevated RhoA activity in mammary epithelium that is accompanied by suppression of ductal outgrowth in vivo. Moreover, impaired HGF-mediated branching in primary mammary epithelial cells derived from EphA2-deficient mice is rescued by inhibition of the downstream effector ROCK in vitro. These data are consistent with reported hyperbranching of mammary epithelial organoid cultures upon treatment with the same ROCK inhibitor (Ewald et al., 2008). In the normal mammary development, EphA2 may serve to restrict levels of active RhoA, enabling HGF to maintain levels of activity that promote the proper balance between branching and ductal outgrowth necessary for normal epithelial morphogenesis. By contrast, elevated EphA2 expression in normal mammary epithelial cells via adenoviral transduction enhances branching. These data are consistent with reports that EphA2 overexpression in nontransformed MCF10A cells confers malignant transformation and tumor forming potential in vivo (Zelinski et al., 2001; Carles-Kinch et al., 2002), and overexpression in 4T1 mouse mammary adenocarcinoma cells enhances tumor progression and metastasis in vivo (Fang et al., 2005). In MMTV-Neu mice, loss of EphA2 diminishes tumorigenesis and metastasis, as well as reducing levels of active RhoA (Brantley-Sieders et al., 2008). Thus, overexpression of EphA2, as well as cooperation with other oncogenic pathways in the context of mammary epithelial neoplasia, appears to enhances RhoA.
Interestingly, we did observe diminished RhoA activity in EphA2-deficient mammary epithelium relative to wild-type controls in 5-wk-old mice (data not shown), during which time the epithelium is still actively growing and branching. This observation suggests that EphA2 might also regulate Rho activity differentially at different stages of mammary epithelial development and/or malignant progression. For example, in normal mammary epithelial cells, ephrin-A ligands, such as ephrin-A1, may engage EphA2 receptors on adjacent cells and disrupt activation of Rho GTPase, thus alleviating inhibition of epithelial branching morphogenesis. We previously reported that EphA2 physically and functionally interacts with ErbB2 and that coexpression of ErbB2 was sufficient to induce phosphorylation of EphA2 in the absence of exogenous ephrin-A1 ligand. Moreover, EphA2 deficiency results in impaired Rho activation in MMTV-Neu tumors and diminished motility of primary tumor cells (Brantley-Sieders et al., 2008). Cell motility in EphA2-deficient MMTV-Neu tumor cells was rescued by overexpression of activated RhoA. Thus, EphA2 receptor activation by ErbB2 and/or other receptor tyrosine kinases might enhance Rho activity, cell motility, and malignancy in the context of cancer cells in which weakened cell–cell contacts could impair interaction between EphA2 and endogenous ligands. This might also be true in 5-wk-old mammary glands, in which active growth might also diminish cell–cell contact and interaction between EphA2 and ephrin-A1 on adjacent luminal epithelial cells. It will be of great interest to investigate differential regulation of Rho family GTPases by EphA2 in normal mammary epithelium versus mammary adenocarcinoma.
In conclusion, we provide the first evidence for EphA2 receptor regulation of normal growth and branching morphogenesis in normal mammary epithelial development. Based on reciprocal transplantation and primary epithelial culture studies, EphA2 function specifically in mammary epithelium, rather than mesenchymal stroma, appears to mediate these processes. Our data suggest that EphA2 receptor functions downstream of HGF to regulate mammary epithelial branching through inhibition of RhoA GTPase.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Dana M. Brantley-Sieders (dana.brantley{at}vanderbilt.edu)
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REFERENCES |
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