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Magic roundabout, a tumor endothelial marker: Expression and signaling
BBRC Biochemical and Biophysical Research Communications 332 (2005) 533–541 www.elsevier.com/locate/ybbrc
Magic roundabout, a tumor endothelial marker: Expression and signaling q Pankaj Seth a, Yanfeng Lin a,1, Jun-ichi Hanai a,1, Venkatesha Shivalingappa a, Mabel P. Duyao b, Vikas P. Sukhatme a,* a
Renal Division and Center for Study of the Tumor Microenvironment, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA b Ardais Corporation, One Ledgemont Center, Lexington, MA 02421, USA Received 15 March 2005 Available online 5 May 2005
Abstract Molecular signals that guide blood vessels to specific paths are not fully deciphered, but are thought to be similar to signals that mediate neuronal guidance. These cues are not only critical for normal blood vessel development, but may also play a major role in tumor angiogenesis. In this study, we have demonstrated the tumor endothelial specific expression of a Robo family member, magic roundabout (MRB), functionally characterized its role in endothelial cell migration and defined a signaling pathway that might mediate this function. We show that MRB is differentially over-expressed in tumor endothelial cells versus normal adult endothelial cells in numerous solid tumors. Moreover, over-expression of MRB in endothelial cells activates MRB in a ligand-independent fashion, and activation of MRB via Slit2, a putative ligand, results in inhibition of VEGF and FGF induced migration. We also demonstrate that MRB induced inhibition of endothelial migration is partially mediated by the Ras–Raf–Mek–Erk signaling pathway. We therefore hypothesize that expression of MRB is involved in regulating the migration of endothelial cells during tumor angiogenesis. 2005 Elsevier Inc. All rights reserved. Keywords: Tumor endothelium; Magic roundabout; FAK; SLIT2
Recent work has implicated a number of guidance molecules that are involved in complex neuronal networking, wherein neurons are guided to appropriate destinations based on extracellular cues [1,2]. These molecules include members of roundabout (Robo) family, the ephrins, the semaphorins, and the notch family [3–8]. A series of molecular events guide neurons by
q Abbreviations: EC, endothelial cells; VEGF, vascular endothelial growth factor; HUVECs, human umbilical vascular endothelial cells; MRB, magic roundabout; sMRB, soluble magic roundabout; FAK, focal adhesion kinase; ERK, extracellular signal-regulated kinase; TEM, tumor endothelial marker. * Corresponding author. Fax: +1 617 667 7843. E-mail address: [email protected] (V.P. Sukhatme). 1 Equal contributions.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.250
utilizing a combination of attractive and repulsive cues, and these events may be important in controlling cell migration as well. Indeed, the molecular signals that guide endothelial cells to specific paths are not fully deciphered, but they are thought to be similar to signals that mediate neuronal guidance [9]. These signals are critical for normal blood vessel development. They may also play a major role in tumor angiogenesis, during which certain molecular switches turn on a quiescent endothelial cell to an active proliferating and migrating pro-angiogenic state, thereby regulating tumor progression. Therefore, understanding molecular signals that regulate tumor angiogenesis may lead to insights into our understanding of tumor progression. The Robo family consists of Robo1, Robo2, Robo3, and Robo4, the latter also known as magic roundabout
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(MRB). Robos 1, 2, and 3 are highly expressed in the nervous system and undetectable in the vascular system [10]. All known Robo family members have a large extracellular domain composed of five immunoglobulin and three fibronectin motifs except Robo4, which has only two immunoglobulin and two fibronectin motifs, and diverges significantly from the other Robos [11]. Slits are the cognate ligands for Robos and currently three Slit family members are known in humans [7,12]. The Robo/Slit interaction in Drosophila causes growth cone repulsion and ensures that commissural axons that have already crossed the midline do not cross the midline again [13]. In silico database mining previously identified MRB as a Robo family member gene selectively expressed in endothelial cells in vitro [9]. Since roundabout family members are involved in repulsive signaling in neurons, we hypothesized that MRB might mediate repulsive guidance of endothelial cells. In this study, we have isolated a human cDNA for MRB, demonstrated tumor endothelial-specific expression of MRB in multiple human tumor tissues, functionally characterized its role in endothelial cell migrationm and defined a signaling pathway that might mediate this function. While this manuscript was in preparation, another group identified Slit2 as a putative ligand for MRB and demonstrated the activation of MRB upon Slit2 binding, which in turn resulted in inhibition of endothelial cell migration via MRB [10].
Materials and methods Materials. Human vascular endothelial growth factor (VEGF), primary human umbilical vein endothelial cells (HUVECs), and primary human microvascular endothelial cells (HMVECs) were obtained from Cambrex Bio Sciences (Walkersville, MD). EGM-MV bullet kit and endothelial cell basic medium (EBM) were from Cambrex Bio Sciences. Mouse monoclonal antibody against the M2-FLAG epitope was purchased from Sigma (St. Louis, MO). Transwell plates were from BD Biosciences (San Diego, CA), Calcein-AM was from Molecular Probes (Eugene, OR), expression vector pET was from Novagen (San Diego, CA), Biomax-30K ultrafree filters were from Millipore (Waltham, MA), and type I collagen-coated plates were from BD Biosciences (Bedford, MA). Cell culture and transfections. HUVECs were cultured as described previously [14]. These cells were grown on 30 lg/ml collagen-coated dishes in endothelial growth medium-2 with EGM-MV bullet kit containing 5% fetal bovine serum with 12 lg/ml bovine brain extract, 1 lg/ml hydrocortisone, and 1 lg/ml GA-1000. HUVECs (passage 2– 4) that were 80% confluent were used for most experiments. Plasmids expressing Slit2 and various MRB constructs were transfected into the 293 T cell line with Fugene 6 (Roche, Indianapolis, IN). Serum-free conditioned medium expressing Slit2 or soluble MRB (sMRB, a construct expressing only the MRB extracellular domain-see below) was collected 56 h after transfection and concentrated with Biomax-30K ultrafree filters. RNA isolation and RT-PCR. Poly(A)+ mRNA from different cell lines was isolated using the lMACS mRNA isolation kit (Miltenyi Biotech, Auburn, CA). Briefly, cells were lysed in binding buffer, fol-
lowing which, the lysate was mixed with oligo(dT) microbeads. The mixture was separated on a lMACS column in the magnetic field of a lMACS separator. The lMACS column was washed and poly(A)+ mRNA was eluted. Poly(A)+mRNA was used to synthesize cDNA by Omniscript Reverse transcriptase (Qiagen,Valencia, CA). Specific primer sets for MRB (forward primer: 5 0 -gctgcagtcactggtgctggagctgg-3 0 and reverse primer: 5 0 -ggtcccgggcatc-cgcccccagccg-3 0 ) and b-actin were used for amplification of the respective transcripts by PCR. The primer sets for Robo1 amplification were used as described previously [15]. In situ hybridization. A fragment of human MRB (hMRB) cDNA (corresponding to nucleotides 60–825) was PCR-amplified from EST clone # CSOD1075YP13 (Invitrogen, Carlsbad, CA) and cloned into the cloning vector pZero1.0 (Invitrogen). The plasmid was linearized, and digoxigenin (DIG)-labeled antisense and sense transcripts were generated using DIG RNA labeling reagents and T7/Sp6 polymerase from an in vitro transcription kit (Roche, Indianapolis, IN). In situ analysis was performed by a non-radioactive method. Frozen sections were fixed with 4% paraformaldehyde (PFA), permeabilized with proteinase K, blocked with ISH solution (Dako, Carpinteria, CA), and incubated with RNA probes (100 ng/ml) overnight at 55 C. After washing twice in 2· SSC and then once in TNE buffer [10 mM Tris– HCl (pH 7.5), 500 mM NaCl, and 1 mM EDTA], sections were incubated at 37 C with RNase A/T1 cocktail (Ambion, Austin, TX) diluted 1:35 in TNE buffer. Slides were washed twice in 2· SSC/50% deionized formamide (American Bioanalytical, Natick, MA) and then once with 0.1· SSC at 55 C. Before immunodetection, tissues were treated with peroxidase blocking reagent and blocked with 1% blocking reagent (Roche; DIG Nucleic Acid Detection kit) containing purified, non-specific rabbit immunoglobulins (Dako). A horseradish peroxidase-rabbit anti-DIG antibody (Dako) was used to catalyze the deposition of biotin tyramide (GenPoint kit; Dako) for signal amplification. Further amplification was achieved by adding horseradish peroxidase-rabbit anti-biotin (Dako), biotin tyramide, and then alkaline phosphatase rabbit anti-biotin (Dako). Signal was detected with the alkaline phosphatase substrate, Fast Red TR/Naphthol AS-MX (Sigma Chemical, St. Louis, MO). All sections were exposed for 10–20 min in the dark. Cells were counterstained with hematoxylin and mounted with Crystal/Mount (Biomeda, Foster City, CA). We have assessed four different tissue sections from three separate tumor tissue derived from three patients. All the tumor samples examined were positive for MRB expression with anti-sense probes and negative with sense probes. Antibody generation. Polyclonal anti-MRB antibody was generated against the extracellular domain of hMRB. A fragment of hMRB corresponding to amino acids 47–206 was PCR-amplified and cloned into the pET-15b expression vector (Novagen). Fusion protein was expressed in bacteria and purified according to manufacturerÕs protocol. This MRB-histidine fusion protein was used as an immunogen to generate rabbit polyclonal antibody. This polyclonal antibody was affinity purified using bacterially expressed MRB protein as an affinity ligand. Immunohistochemistry and immunofluorescence. Immunohistochemical analysis was performed on frozen human placental sections. Briefly, sections were blocked with 1% BSA and incubated overnight with anti-MRB antibody. Following the washes after primary antibody incubation, tissue sections were incubated with HRP-conjugated secondary anti-rabbit antibody. Color reaction was developed by DAB substrate and sections were mounted in mounting solution. For immunofluorescence analysis of MRB expression, frozen sections were blocked with 1% BSA for 1 h, followed by incubation with antibody against MRB, 1:10 dilution in 0.3% BSA overnight at room temperature. After the primary antibody incubation, slides were washed with PBS twice and incubated with anti-human CD-31, 1:100 dilution in 0.3% BSA (PharMingen, San Diego, CA) for 12 h. The slides were washed with PBS thrice for 5 min. Following these washes, slides were incubated with TRITC conjugated anti-rabbit secondary antibody (recognizes primary MRB antibody). Following the incubation, slides
P. Seth et al. / Biochemical and Biophysical Research Communications 332 (2005) 533–541 were washed twice, 5 min each with PBS. Slides were then incubated with FITC-conjugated secondary antibody (recognizes primary CD-31 antibody). After the incubation, slides were washed with PBS thrice for 5 min each and mounted with 80% glycerol. The tissue sections were obtained from Ardais (Lexington, MA) and tumor sections had more than 80% tumor by H&E staining. In all, we have assessed five tissue sections from three separate tumor tissues. Cloning of MRB and adenoviral expression. For constitutive expression of hMRB in mammalian cells, a cDNA encoding human MRB was generated by PCR amplification from EST clone # CS0D1075YP13 (Invitrogen), the region corresponding to nucleotide 60–3093 with a C-terminal-FLAG tag and subcloned into pCDNA 3.1 (Invitrogen). The cDNA was sequenced to confirm the identity of the clone. For the generation of recombinant adenovirus, hMRB was PCR-amplified using pcDNA-hMRB plasmid as a template and cloned into pShuttle–CMV [16]. Vector pShuttle–CMV was used for recombination and adenovirus generation using the Ad-Easy system [17] Similarly an adenoviral expression construct for sMRB expressing the extracellular domain of hMRB was generated by PCR amplifying the extracellular domain from pcDNA-hMRB corresponding to nucleotides 1–1370 with a C-terminal Myc epitope tag, cloned into the pATrack–CMV vector [18], and used for adenovirus generation using Ad-Easy system [17]. Western blot analysis. 293 T cells were transfected using Fugene 6 (Roche, Indianapolis, IN) and HUVECs were infected with adenoviral constructs. Twenty-four hours following the transfection in 293 T cells or 20 h after adenoviral infection in HUVECs, the cells were washed with phosphate-buffered saline and solubilized with SDS lysis buffer. Total cellular proteins were separated by SDS–PAGE, electrotransferred to PDVF membranes, immunoblotted with anti-FLAG tag antibody (Sigma Chemicals, St. Louis, MO) or with anti-MRB antibody, and detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). For Western blot analysis of focal adhesion kinase (FAK) and phospho FAK, HUVECs were infected with MRB and control GFP adenovirus, cells were washed after 6 h and allowed to grow for an additional 24 h. Cells were harvested with trypsin, incubated in 15 ml Falcon tube at 37 C for 1 h, spread on a type I collagen-coated plate (BD Biosciences), and incubated for 0, 30, 60, and 120 min. For experiments using Slit2 and sMRB, conditioned medium harvested from 293 T cells expressing Slit2, sMRB or control GFP was used alone or in combination in a 1:1 ratio. HUVECs were incubated with conditioned medium for 6 h, and harvested with trypsin, and incubated in 15 ml Falcon tube with the same conditioned medium. Following incubation, cells were spread on type I collagen plates and incubated with the same conditioned medium for 30 min. Cell lysates were prepared for Western blot analysis as described above. Proliferation assay. HUVECs at passage 2–4 maintained in EGM2MV were grown to 70% confluency, infected with adenovirus for 4 h, after which they were washed and allowed to grow for another 24 h. Cells were then harvested with trypsin and resuspended at a cell density of a 2 · 103 cells/well in a 96-well plate for the assays. Cells were cultured for another 48 h. Ten microliters of WST1 reagent (Roche) was added into each well, and color was allowed to develop for 2 h and measured at a wavelength of 450 nm. Migration assay. HUVECs at passages 2–4 maintained in EGM2MV were grown to 70% confluency, infected with adenovirus for 4 h, after which they were washed and allowed to grow for another 15– 17 h. Cells were then harvested with trypsin and resuspended to the cell density required for the migration assay. The assay was performed on gelatin-coated polycarbonate membranes (Transwell). Briefly, HUVECs were infected with adenovirus harboring full-length MRB, sMRB or GFP as described above. Cells were then seeded (7.2 · 105 cells/ml) in DMEM with 0.5% BSA in the top chamber. Cell migration was assayed in the absence and presence of 0.9 ml DMEM with or without 10 ng/ml VEGF or 10 ng/ml FGF or 5% FBS in the bottom chamber. Cells were allowed to migrate for 10 h. Cells were
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fixed, stained with Giemsa solution, and the upper surface of each membrane was scraped with a cotton swab. Cells that had reached the lower surface of the membrane (migrated cells) were counted in 20 random fields using a light microscope. For migration assay using conditioned medium, HUVECs were pretreated with conditioned medium containing Slit2, sMRB, or control GFP either alone or in combination in a 1:1 ratio. The conditioned medium was harvested from 293 T cells expressing Slit2, sMRB, or control GFP. Following the pretreatment, HUVECs (7.2 · 105 cells/ml) in DMEM were seeded on gelatin-coated polycarbonate membranes (Transwell) and the assay was performed as above. For analyzing the effect of MEK-DD expression on VEGF or FGF induced migration, HUVECs were infected with the MRB adenovirus as described above, 24 h later, cells were infected with an MEK-DD adenovirus and cultured for an additional 24 h. The migration assay was performed as before. All the assays were performed in triplicate.
Results MRB expression shows it to be a tumor endothelial marker To demonstrate that MRB expression is restricted to endothelial cells, we analyzed the expression of MRB in nine different cell lines by RT-PCR analysis. MRB expression was only evident in two endothelial cell lines, HUVECs and HMVECs but not in numerous epithelial (colon, kidney), fibroblast, or muscle-derived cell lines (Fig. 1A). These in vitro data do not address the question of endothelial specific expression in vivo or whether expression might be restricted to sites of active angiogenesis. We therefore began by performing in situ analysis for MRB gene expression in placenta, an organ in which active angiogenesis is occurring and where trophoblasts are converting to a vascular phenotype (pseudovasculogenesis) [19]. Our in situ hybridization analysis confirmed our hypothesis that indeed MRB is abundantly expressed in placental endothelial cells as well as in trophoblasts undergoing pseudovasculogenesis (data not shown). Given these data and the known similarities between neo-vascular processes in placenta and in tumors, we asked whether MRB might be a tumor endothelial marker (TEM). Our in situ analysis of tumor samples from lung, liver, kidney, and a metastasis of unknown origin (Fig. 1B) suggests that MRB RNA expression is abundant in tumor endothelial cells and is specific to endothelium, as it colocalizes with CD-31 expression, a known endothelial marker [20]. We acknowledge that MRB expression does not exactly colocalize with CD-31, this may be in part due to expression of MRB in immature vessels that lack CD-31 expression. To validate our in situ findings and to demonstrate that MRB expression is highly abundant in tumor endothelium as compared to normal endothelium, we generated a polyclonal antibody against the extracellular domain of MRB. The antibody was affinity purified
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Fig. 1. RT-PCR analysis of MRB expression in cell lines and in situ hybridization analysis of MRB in human placenta and tumor tissue. (A) RTPCR analysis for MRB and b-actin expression. Source of RNA: lane 1 (HUVECs), lane 2 (HMVECs). Lanes 3–9 represent IMR90 (fibroblast), DLD (colon cancer), podocytes (kidney), C2–C12 (myocyte), IMCD (kidney), 786-0 (kidney cancer), and 293 T cell lines. (B) In situ hybridization analysis and CD-31 immunostaining of MRB in metastasis of unknown origin, liver carcinoma (C), lung cancer (D), and kidney cancer (E). All the images are taken at 40· magnification. Higher magnification is shown in insets (top left hand corner).
for Western blot and immunohistochemical analysis. We have tested the specificity of this polyclonal antibody by staining 293 T cells expressing MRB versus cells expressing GFP (data not shown). Using this purified antibody, we analyzed MRB expression in a variety of tumor and normal human tissues. Our immuno-colocalization analysis for the expression of MRB and CD-31 on normal and cancer human tissues from lung, kidney, liver, and metastatic melanoma (Fig. 2) demonstrates that MRB expression is significantly upregulated in tumor endothelium and is expressed at relatively lower levels in normal adult, presumably quiescent, endothelial cells in vivo. MRB-mediated signaling results in inhibition of endothelial cell migration Based on sequence similarity to neuronal Robo family members, it was reasonable to hypothesize that MRB expression might regulate repulsive signaling in endothelial cells. We utilized a modified Boyden chamber assay, in which cells were placed in the upper chamber and test factors such as FGF, VEGF, and FBS were placed in the lower chamber. Migration of cells was quantified by the number of cells that migrated into the lower chamber. We investigated the migration of HUVECs expressing MRB compared to migration of cells expressing soluble MRB (sMRB) or a control GFP. The expression of each construct was verified by Western blot analysis of infected HUVECs (Fig. 3B). As expected, VEGF-, FGF-, and FBS-stimulated HUVECs expressing control GFP migrated
Fig. 2. Immunohistochemical analysis of MRB. Immunofluorescence colocalization of MRB and CD-31, a known endothelial marker in (A), metastatic melanoma, cancerous, and normal tissue sections derived from (B) kidney, (C) lung, and (D) liver. All the images are taken at 40· magnification.
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Fig. 3. MRB expression inhibits endothelial cell migration. (A) HUVECs expressing control GFP, MRB, or sMRB were stimulated with 10 ng/ml VEGF for the migration assay. (B) The expression of GFP, MRB, and sMRB was checked with anti-GFP and anti-MRB antibodies. (C) HUVECs expressing control GFP or MRB were stimulated with 10 ng/ml FGF. (D) Proliferation assay for HUVECs expressing GFP or MRB.
12-fold more than non-stimulated cells, whereas cells expressing MRB migrated only 4-fold more and expression of soluble MRB did not inhibit migration (Figs. 3A, C, and data not shown). Moreover, inhibition in migration was more pronounced in FGF induced HUVECs. However, the rate of cell proliferation was unaffected (Fig. 3D), suggesting that cell viability was not compromised by MRB expression. These in vitro experiments indicate that MRB expression alone is sufficient to block migration in response to various promigratory factors; moreover, it suggests that activation of the MRB receptor when over-expressed on the cell surface can occur via a ligand-independent mechanism in a manner similar to Robo1 and Robo2 [21]. It has also been suggested that Slit2, which is known to be expressed by endothelium in vivo [22], may act as a putative ligand for MRB [11]. To assess the role of Slit2 in endothelial biology, as also recently done by Park et al. [10], we repeated the migration assay with HUVECs as described above, but with conditioned medium containing Slit2, derived from 293 T cells transfected with a Slit2 expression vector. HUVECs fail to migrate in response to VEGF in the presence of Slit2 and this inhibition of migration was rescued by addition of sMRB (Fig. 4A). The expression of sMRB did not effect cell migration as recently shown by Suchting et al. [23]. This
may be the result of differences in functional assays. The expression of Slit2 and sMRB in 293 T cell conditioned medium was confirmed by Western blot analysis (Fig. 4B). These results suggest that Slit2 is signaling via MRB as Robo1 was undetectable in HUVECs by RTPCR (Fig. 4C) and as previously described [10]. These data are in discrepancy with those of Wang et al. [15] and may be due to differences in the endothelial cells used for their study. MRB signaling intersects with the MEK/ERK pathway Cytoskeletal rearrangement and reorganization are essential features of endothelial cell migration [24]. Regulation of VEGF- and FGF-stimulated migration of endothelial cells is coordinated by separate sets of signaling components [25]. FGF initiates migration by activation of Ras that results in activation of ERK and subsequently leads to phosphorylation of FAK [26– 28], whereas VEGF binds to its receptor, VEGFR2, regulating cell migration via the PI3 kinase/AKT pathway [29,30]. ERK has been implicated in migration of numerous cell types and ERK pathway inhibitors U0126 and PD98059 inhibit migration in response to VEGF (partially), FGF, and FBS [31,32]. Moreover, a dominant negative ERK mutant or inhibition of ERK by an anti-sense strategy also inhibits cell migration
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Fig. 4. Slit2 mediates inhibition of endothelial cell migration by interaction with MRB. (A) HUVECs were incubated with 0.2% FBS conditioned medium derived from 293 T cells expressing GFP, Slit2, or sMRB for 4 h. Cells pretreated with Slit2 showed inhibition of migration when stimulated with 10 ng/ml VEGF compared to cells pretreated with control or sMRB; moreover, combined incubation of Slit2 and sMRB conditioned medium in a 1:1 ratio abrogated the inhibition of Slit2 induced migration. (B) Western blot analysis of conditioned media expressing Slit2 and sMRB used for HUVEC pretreatment. The anti-myc antibody recognizes Slit2 and anti-MRB antibody recognizes sMRB. (C) RT-PCR analysis of Robo1 and Robo4 (MRB) in HUVECs.
[32,33]. Therefore, these studies suggest that the ERK signaling pathways play a significant role in the regulation of migration. The phosphorylation of endogenous ERK and FAK is markedly downregulated in cells expressing MRB (Fig. 5A, lower panel) versus cells expressing control GFP (Fig. 5A, upper panel). These results indicate that over-expression of MRB leads to a ligand-independent activation, similar to studies with Robo1 [21], and may account for inhibition of migra-
tion by suppression of ERK and FAK phosphorylation. Since Slit2 inhibits migration of endothelial cells, which can be rescued by sMRB (Fig. 4), we speculated that molecular signals involved in the Slit2 (ligand dependent) and MRB over-expression (ligand independent) situations might be similar, and that Slit2 may inhibit migration by dephosphorylation of FAK. As shown in Fig. 5B, Slit2 indeed causes FAK dephosphorylation and this effect can be abrogated by sMRB.
Fig. 5. MRB/Slit2 activates FAK. (A) HUVECs expressing MRB and control GFP were plated on collagen-coated plates and removed from these plates at 0, 30, 60, and 120 min. Cell lysates were analyzed by Western blot analysis with antibodies specific for FAK, phospho FAK, and phospho ERK. (B) HUVECs were incubated with 0.2% FBS conditioned medium derived from 293 T cells expressing GFP, Slit2, or sMRB either alone or in combination (1:1 ratio of conditioned medium) for 6 h. Cells were harvested and plated on collagen-coated plates for 30 min (as determined by previous experimental set, see A) and Western blot analysis was performed with antibodies specific for phospho FAK and actin (loading control).
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Fig. 6. Functional rescue of MRB induced inhibition of migration by MEK-DD expression. (B) HUVECs expressing control GFP, MRB, MEKDD/MRB, and MEK-DD/GFP were stimulated with 10 ng/ml VEGF for the migration assay. (A) HUVECs expressing control GFP, MRB, MEKDD/MRB, and MEK-DD/GFP were stimulated with 10 ng/ml FGF for the migration assay. (C) Percentage functional rescue of MRB-mediated inhibition of HUVECs by MEK-DD with VEGF or FGF stimulation.
To demonstrate that migration inhibition of MRB expressing HUVECs is a specific effect resulting from inhibition of ERK and FAK phosphorylation, we have shown that over-expressing MEK-DD, a dominant active form of MEK, can override MRBÕs inhibitory effect on FGF-stimulated HUVECs (Figs. 6A and C). However, MEK-DD expression can only partially rescue MRBÕs inhibitory effects on VEGF-stimulated HUVEC migration (Fig. 6B), suggesting that MRB feeds its inhibitory signals predominantly via the ERK pathway. Since MEK-DD can only partially rescue MRB-mediated inhibition for VEGF-stimulated HUVECs, signals other than ERK, e.g., the PI3K pathway may contribute to MRB inhibitory signaling.
Discussion Tumor vasculature is an attractive target for tumor therapy. To exploit such a strategy requires the identification of functional or molecular differences between tumor and normal vessels [34]. For example, it is well known that tumor vessels tend to be tortuous, are leaky, and have less pericyte coverage than normal vessel. However, the molecular differences have been less well
characterized, though some recent progress has been made in this regard [35]. We have studied the expression of MRB by in situ hybridization, by immunohistochemistry, and by co-localization to CD-31 in numerous solid tumor and normal tissues, extending the data of Huminiecki et al. [11]. Our findings suggest that MRB is indeed a tumor endothelial specific marker. The methods we have employed to assess MRB expression are only semi-quantitative, but our data suggest that MRB expression is upregulated in tumors in a manner comparable to or more strikingly than other established markers such as VEGFR2, a(v)b3 integrin or various TEMS [35]. MRB expression during development occurs in endothelium and in the adult it appears to be minimally expressed except at sites of active angiogenesis or pseudovasculogenesis (placenta) as well as in pathological angiogenesis, for example, in tumor endothelium. Given that MRB is a surface protein, this differential expression makes it a most attractive target for directing cytotoxic therapies to tumor vasculature. The functional role of MRB in tumor angiogenesis remains unknown. Since angiogenesis is a composite process of multiple steps, we initially asked whether MRB over-expression in HUVECs affected proliferation of these cells, but found this not to be the case. Taking a
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clue from the neuronal literature in the area of axonal guidance, in which Robos are involved in signaling repulsive cues, we asked whether there might be an effect of MRB on endothelial cell migration. Our results point to an inhibitory effect mediated by MRB, in view of its ability to antagonize VEGF, FGF, and FBS (data not shown) induced migration. Moreover, our data on the ability of Slit2 to block VEGF induced migration of HUVECs support the notion that Slit2 is a putative ligand for MRB, as was also recently shown by MRBSlit2 pull-down assays [10]. This ligand had the same effect on endothelial cell migration as did MRB over-expression, an effect that was blocked by sMRBs [10] used soluble Robo1, instead of sMRB and showed similar findings. Since it was known that ERK plays a central role in cell migration, we explored whether MRB might downregulate signaling through this pathway. We showed that ERK and FAK phosphorylation was decreased by MRB over-expression or by Slit2 treatment (the specificity of these effects was ascertained by the fact that coincubation with sMRB blocked the latterÕs effects). These data are similar to the recent findings of Ganju et al. [36], who noted that Slit2 induced inhibition of phosphorylation of FAK in breast cancer cells. Moreover, over-expression of MEK-DD, a dominant active form of MEK, can override MRBÕs inhibitory effect on FGF-stimulated HUVEC migration but only partially rescues MRBÕs inhibitory effect on VEGF-stimulated migration. Hence, we would like to suggest that MRB induced inhibitory signals occur partially via Ras–Raf–Mek signaling pathway to modulate cytoskeletal events critical for migration. There appear to be discrepant findings on the expression and role of the Slit-Robo system in endothelial cells. Wang et al. [15] find Robo1 expression in HUVECs and show that Slit2 promotes endothelial migration via interaction with Robo1. Interestingly, Park et al. [10] and our data show that Robo4 may be the only Robo family member expressed on endothelial cells and that it inhibits endothelial migration. It is therefore conceivable that Slit2 signals via Robos with opposing effects and that the net effect depends on the relative expression of Robo1/4 in the endothelial cell under investigation. How can the action of MRB on endothelial cell migration be reconciled with its expression at the site of active angiogenesis? The so-called resolution phase of angiogenesis, in which, for example, the pro-angiogenic molecule TGF-b is thought to participate, includes processes of decreased endothelial proliferation and migration. It is unknown whether MRB mediates proor anti-angiogenic signals or what the functional role of MRB in vivo in tumor angiogenesis is. Experiments aimed at addressing this question are currently underway.
Acknowledgments We thank Marc Tessier-Lavigne, Stanford University, for Slit2 expression construct, and Hirofumi Hamada, Sapporo Medical University, for FAK adenovirus. This project is supported by a research training grant (T32 DK07199) to V.P.S.
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Magic roundabout, a tumor endothelial marker: Expression and signaling q Pankaj Seth a, Yanfeng Lin a,1, Jun-ichi Hanai a,1, Venkatesha Shivalingappa a, Mabel P. Duyao b, Vikas P. Sukhatme a,* a
Renal Division and Center for Study of the Tumor Microenvironment, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA b Ardais Corporation, One Ledgemont Center, Lexington, MA 02421, USA Received 15 March 2005 Available online 5 May 2005
Abstract Molecular signals that guide blood vessels to specific paths are not fully deciphered, but are thought to be similar to signals that mediate neuronal guidance. These cues are not only critical for normal blood vessel development, but may also play a major role in tumor angiogenesis. In this study, we have demonstrated the tumor endothelial specific expression of a Robo family member, magic roundabout (MRB), functionally characterized its role in endothelial cell migration and defined a signaling pathway that might mediate this function. We show that MRB is differentially over-expressed in tumor endothelial cells versus normal adult endothelial cells in numerous solid tumors. Moreover, over-expression of MRB in endothelial cells activates MRB in a ligand-independent fashion, and activation of MRB via Slit2, a putative ligand, results in inhibition of VEGF and FGF induced migration. We also demonstrate that MRB induced inhibition of endothelial migration is partially mediated by the Ras–Raf–Mek–Erk signaling pathway. We therefore hypothesize that expression of MRB is involved in regulating the migration of endothelial cells during tumor angiogenesis. 2005 Elsevier Inc. All rights reserved. Keywords: Tumor endothelium; Magic roundabout; FAK; SLIT2
Recent work has implicated a number of guidance molecules that are involved in complex neuronal networking, wherein neurons are guided to appropriate destinations based on extracellular cues [1,2]. These molecules include members of roundabout (Robo) family, the ephrins, the semaphorins, and the notch family [3–8]. A series of molecular events guide neurons by
q Abbreviations: EC, endothelial cells; VEGF, vascular endothelial growth factor; HUVECs, human umbilical vascular endothelial cells; MRB, magic roundabout; sMRB, soluble magic roundabout; FAK, focal adhesion kinase; ERK, extracellular signal-regulated kinase; TEM, tumor endothelial marker. * Corresponding author. Fax: +1 617 667 7843. E-mail address: [email protected] (V.P. Sukhatme). 1 Equal contributions.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.250
utilizing a combination of attractive and repulsive cues, and these events may be important in controlling cell migration as well. Indeed, the molecular signals that guide endothelial cells to specific paths are not fully deciphered, but they are thought to be similar to signals that mediate neuronal guidance [9]. These signals are critical for normal blood vessel development. They may also play a major role in tumor angiogenesis, during which certain molecular switches turn on a quiescent endothelial cell to an active proliferating and migrating pro-angiogenic state, thereby regulating tumor progression. Therefore, understanding molecular signals that regulate tumor angiogenesis may lead to insights into our understanding of tumor progression. The Robo family consists of Robo1, Robo2, Robo3, and Robo4, the latter also known as magic roundabout
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(MRB). Robos 1, 2, and 3 are highly expressed in the nervous system and undetectable in the vascular system [10]. All known Robo family members have a large extracellular domain composed of five immunoglobulin and three fibronectin motifs except Robo4, which has only two immunoglobulin and two fibronectin motifs, and diverges significantly from the other Robos [11]. Slits are the cognate ligands for Robos and currently three Slit family members are known in humans [7,12]. The Robo/Slit interaction in Drosophila causes growth cone repulsion and ensures that commissural axons that have already crossed the midline do not cross the midline again [13]. In silico database mining previously identified MRB as a Robo family member gene selectively expressed in endothelial cells in vitro [9]. Since roundabout family members are involved in repulsive signaling in neurons, we hypothesized that MRB might mediate repulsive guidance of endothelial cells. In this study, we have isolated a human cDNA for MRB, demonstrated tumor endothelial-specific expression of MRB in multiple human tumor tissues, functionally characterized its role in endothelial cell migrationm and defined a signaling pathway that might mediate this function. While this manuscript was in preparation, another group identified Slit2 as a putative ligand for MRB and demonstrated the activation of MRB upon Slit2 binding, which in turn resulted in inhibition of endothelial cell migration via MRB [10].
Materials and methods Materials. Human vascular endothelial growth factor (VEGF), primary human umbilical vein endothelial cells (HUVECs), and primary human microvascular endothelial cells (HMVECs) were obtained from Cambrex Bio Sciences (Walkersville, MD). EGM-MV bullet kit and endothelial cell basic medium (EBM) were from Cambrex Bio Sciences. Mouse monoclonal antibody against the M2-FLAG epitope was purchased from Sigma (St. Louis, MO). Transwell plates were from BD Biosciences (San Diego, CA), Calcein-AM was from Molecular Probes (Eugene, OR), expression vector pET was from Novagen (San Diego, CA), Biomax-30K ultrafree filters were from Millipore (Waltham, MA), and type I collagen-coated plates were from BD Biosciences (Bedford, MA). Cell culture and transfections. HUVECs were cultured as described previously [14]. These cells were grown on 30 lg/ml collagen-coated dishes in endothelial growth medium-2 with EGM-MV bullet kit containing 5% fetal bovine serum with 12 lg/ml bovine brain extract, 1 lg/ml hydrocortisone, and 1 lg/ml GA-1000. HUVECs (passage 2– 4) that were 80% confluent were used for most experiments. Plasmids expressing Slit2 and various MRB constructs were transfected into the 293 T cell line with Fugene 6 (Roche, Indianapolis, IN). Serum-free conditioned medium expressing Slit2 or soluble MRB (sMRB, a construct expressing only the MRB extracellular domain-see below) was collected 56 h after transfection and concentrated with Biomax-30K ultrafree filters. RNA isolation and RT-PCR. Poly(A)+ mRNA from different cell lines was isolated using the lMACS mRNA isolation kit (Miltenyi Biotech, Auburn, CA). Briefly, cells were lysed in binding buffer, fol-
lowing which, the lysate was mixed with oligo(dT) microbeads. The mixture was separated on a lMACS column in the magnetic field of a lMACS separator. The lMACS column was washed and poly(A)+ mRNA was eluted. Poly(A)+mRNA was used to synthesize cDNA by Omniscript Reverse transcriptase (Qiagen,Valencia, CA). Specific primer sets for MRB (forward primer: 5 0 -gctgcagtcactggtgctggagctgg-3 0 and reverse primer: 5 0 -ggtcccgggcatc-cgcccccagccg-3 0 ) and b-actin were used for amplification of the respective transcripts by PCR. The primer sets for Robo1 amplification were used as described previously [15]. In situ hybridization. A fragment of human MRB (hMRB) cDNA (corresponding to nucleotides 60–825) was PCR-amplified from EST clone # CSOD1075YP13 (Invitrogen, Carlsbad, CA) and cloned into the cloning vector pZero1.0 (Invitrogen). The plasmid was linearized, and digoxigenin (DIG)-labeled antisense and sense transcripts were generated using DIG RNA labeling reagents and T7/Sp6 polymerase from an in vitro transcription kit (Roche, Indianapolis, IN). In situ analysis was performed by a non-radioactive method. Frozen sections were fixed with 4% paraformaldehyde (PFA), permeabilized with proteinase K, blocked with ISH solution (Dako, Carpinteria, CA), and incubated with RNA probes (100 ng/ml) overnight at 55 C. After washing twice in 2· SSC and then once in TNE buffer [10 mM Tris– HCl (pH 7.5), 500 mM NaCl, and 1 mM EDTA], sections were incubated at 37 C with RNase A/T1 cocktail (Ambion, Austin, TX) diluted 1:35 in TNE buffer. Slides were washed twice in 2· SSC/50% deionized formamide (American Bioanalytical, Natick, MA) and then once with 0.1· SSC at 55 C. Before immunodetection, tissues were treated with peroxidase blocking reagent and blocked with 1% blocking reagent (Roche; DIG Nucleic Acid Detection kit) containing purified, non-specific rabbit immunoglobulins (Dako). A horseradish peroxidase-rabbit anti-DIG antibody (Dako) was used to catalyze the deposition of biotin tyramide (GenPoint kit; Dako) for signal amplification. Further amplification was achieved by adding horseradish peroxidase-rabbit anti-biotin (Dako), biotin tyramide, and then alkaline phosphatase rabbit anti-biotin (Dako). Signal was detected with the alkaline phosphatase substrate, Fast Red TR/Naphthol AS-MX (Sigma Chemical, St. Louis, MO). All sections were exposed for 10–20 min in the dark. Cells were counterstained with hematoxylin and mounted with Crystal/Mount (Biomeda, Foster City, CA). We have assessed four different tissue sections from three separate tumor tissue derived from three patients. All the tumor samples examined were positive for MRB expression with anti-sense probes and negative with sense probes. Antibody generation. Polyclonal anti-MRB antibody was generated against the extracellular domain of hMRB. A fragment of hMRB corresponding to amino acids 47–206 was PCR-amplified and cloned into the pET-15b expression vector (Novagen). Fusion protein was expressed in bacteria and purified according to manufacturerÕs protocol. This MRB-histidine fusion protein was used as an immunogen to generate rabbit polyclonal antibody. This polyclonal antibody was affinity purified using bacterially expressed MRB protein as an affinity ligand. Immunohistochemistry and immunofluorescence. Immunohistochemical analysis was performed on frozen human placental sections. Briefly, sections were blocked with 1% BSA and incubated overnight with anti-MRB antibody. Following the washes after primary antibody incubation, tissue sections were incubated with HRP-conjugated secondary anti-rabbit antibody. Color reaction was developed by DAB substrate and sections were mounted in mounting solution. For immunofluorescence analysis of MRB expression, frozen sections were blocked with 1% BSA for 1 h, followed by incubation with antibody against MRB, 1:10 dilution in 0.3% BSA overnight at room temperature. After the primary antibody incubation, slides were washed with PBS twice and incubated with anti-human CD-31, 1:100 dilution in 0.3% BSA (PharMingen, San Diego, CA) for 12 h. The slides were washed with PBS thrice for 5 min. Following these washes, slides were incubated with TRITC conjugated anti-rabbit secondary antibody (recognizes primary MRB antibody). Following the incubation, slides
P. Seth et al. / Biochemical and Biophysical Research Communications 332 (2005) 533–541 were washed twice, 5 min each with PBS. Slides were then incubated with FITC-conjugated secondary antibody (recognizes primary CD-31 antibody). After the incubation, slides were washed with PBS thrice for 5 min each and mounted with 80% glycerol. The tissue sections were obtained from Ardais (Lexington, MA) and tumor sections had more than 80% tumor by H&E staining. In all, we have assessed five tissue sections from three separate tumor tissues. Cloning of MRB and adenoviral expression. For constitutive expression of hMRB in mammalian cells, a cDNA encoding human MRB was generated by PCR amplification from EST clone # CS0D1075YP13 (Invitrogen), the region corresponding to nucleotide 60–3093 with a C-terminal-FLAG tag and subcloned into pCDNA 3.1 (Invitrogen). The cDNA was sequenced to confirm the identity of the clone. For the generation of recombinant adenovirus, hMRB was PCR-amplified using pcDNA-hMRB plasmid as a template and cloned into pShuttle–CMV [16]. Vector pShuttle–CMV was used for recombination and adenovirus generation using the Ad-Easy system [17] Similarly an adenoviral expression construct for sMRB expressing the extracellular domain of hMRB was generated by PCR amplifying the extracellular domain from pcDNA-hMRB corresponding to nucleotides 1–1370 with a C-terminal Myc epitope tag, cloned into the pATrack–CMV vector [18], and used for adenovirus generation using Ad-Easy system [17]. Western blot analysis. 293 T cells were transfected using Fugene 6 (Roche, Indianapolis, IN) and HUVECs were infected with adenoviral constructs. Twenty-four hours following the transfection in 293 T cells or 20 h after adenoviral infection in HUVECs, the cells were washed with phosphate-buffered saline and solubilized with SDS lysis buffer. Total cellular proteins were separated by SDS–PAGE, electrotransferred to PDVF membranes, immunoblotted with anti-FLAG tag antibody (Sigma Chemicals, St. Louis, MO) or with anti-MRB antibody, and detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL). For Western blot analysis of focal adhesion kinase (FAK) and phospho FAK, HUVECs were infected with MRB and control GFP adenovirus, cells were washed after 6 h and allowed to grow for an additional 24 h. Cells were harvested with trypsin, incubated in 15 ml Falcon tube at 37 C for 1 h, spread on a type I collagen-coated plate (BD Biosciences), and incubated for 0, 30, 60, and 120 min. For experiments using Slit2 and sMRB, conditioned medium harvested from 293 T cells expressing Slit2, sMRB or control GFP was used alone or in combination in a 1:1 ratio. HUVECs were incubated with conditioned medium for 6 h, and harvested with trypsin, and incubated in 15 ml Falcon tube with the same conditioned medium. Following incubation, cells were spread on type I collagen plates and incubated with the same conditioned medium for 30 min. Cell lysates were prepared for Western blot analysis as described above. Proliferation assay. HUVECs at passage 2–4 maintained in EGM2MV were grown to 70% confluency, infected with adenovirus for 4 h, after which they were washed and allowed to grow for another 24 h. Cells were then harvested with trypsin and resuspended at a cell density of a 2 · 103 cells/well in a 96-well plate for the assays. Cells were cultured for another 48 h. Ten microliters of WST1 reagent (Roche) was added into each well, and color was allowed to develop for 2 h and measured at a wavelength of 450 nm. Migration assay. HUVECs at passages 2–4 maintained in EGM2MV were grown to 70% confluency, infected with adenovirus for 4 h, after which they were washed and allowed to grow for another 15– 17 h. Cells were then harvested with trypsin and resuspended to the cell density required for the migration assay. The assay was performed on gelatin-coated polycarbonate membranes (Transwell). Briefly, HUVECs were infected with adenovirus harboring full-length MRB, sMRB or GFP as described above. Cells were then seeded (7.2 · 105 cells/ml) in DMEM with 0.5% BSA in the top chamber. Cell migration was assayed in the absence and presence of 0.9 ml DMEM with or without 10 ng/ml VEGF or 10 ng/ml FGF or 5% FBS in the bottom chamber. Cells were allowed to migrate for 10 h. Cells were
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fixed, stained with Giemsa solution, and the upper surface of each membrane was scraped with a cotton swab. Cells that had reached the lower surface of the membrane (migrated cells) were counted in 20 random fields using a light microscope. For migration assay using conditioned medium, HUVECs were pretreated with conditioned medium containing Slit2, sMRB, or control GFP either alone or in combination in a 1:1 ratio. The conditioned medium was harvested from 293 T cells expressing Slit2, sMRB, or control GFP. Following the pretreatment, HUVECs (7.2 · 105 cells/ml) in DMEM were seeded on gelatin-coated polycarbonate membranes (Transwell) and the assay was performed as above. For analyzing the effect of MEK-DD expression on VEGF or FGF induced migration, HUVECs were infected with the MRB adenovirus as described above, 24 h later, cells were infected with an MEK-DD adenovirus and cultured for an additional 24 h. The migration assay was performed as before. All the assays were performed in triplicate.
Results MRB expression shows it to be a tumor endothelial marker To demonstrate that MRB expression is restricted to endothelial cells, we analyzed the expression of MRB in nine different cell lines by RT-PCR analysis. MRB expression was only evident in two endothelial cell lines, HUVECs and HMVECs but not in numerous epithelial (colon, kidney), fibroblast, or muscle-derived cell lines (Fig. 1A). These in vitro data do not address the question of endothelial specific expression in vivo or whether expression might be restricted to sites of active angiogenesis. We therefore began by performing in situ analysis for MRB gene expression in placenta, an organ in which active angiogenesis is occurring and where trophoblasts are converting to a vascular phenotype (pseudovasculogenesis) [19]. Our in situ hybridization analysis confirmed our hypothesis that indeed MRB is abundantly expressed in placental endothelial cells as well as in trophoblasts undergoing pseudovasculogenesis (data not shown). Given these data and the known similarities between neo-vascular processes in placenta and in tumors, we asked whether MRB might be a tumor endothelial marker (TEM). Our in situ analysis of tumor samples from lung, liver, kidney, and a metastasis of unknown origin (Fig. 1B) suggests that MRB RNA expression is abundant in tumor endothelial cells and is specific to endothelium, as it colocalizes with CD-31 expression, a known endothelial marker [20]. We acknowledge that MRB expression does not exactly colocalize with CD-31, this may be in part due to expression of MRB in immature vessels that lack CD-31 expression. To validate our in situ findings and to demonstrate that MRB expression is highly abundant in tumor endothelium as compared to normal endothelium, we generated a polyclonal antibody against the extracellular domain of MRB. The antibody was affinity purified
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Fig. 1. RT-PCR analysis of MRB expression in cell lines and in situ hybridization analysis of MRB in human placenta and tumor tissue. (A) RTPCR analysis for MRB and b-actin expression. Source of RNA: lane 1 (HUVECs), lane 2 (HMVECs). Lanes 3–9 represent IMR90 (fibroblast), DLD (colon cancer), podocytes (kidney), C2–C12 (myocyte), IMCD (kidney), 786-0 (kidney cancer), and 293 T cell lines. (B) In situ hybridization analysis and CD-31 immunostaining of MRB in metastasis of unknown origin, liver carcinoma (C), lung cancer (D), and kidney cancer (E). All the images are taken at 40· magnification. Higher magnification is shown in insets (top left hand corner).
for Western blot and immunohistochemical analysis. We have tested the specificity of this polyclonal antibody by staining 293 T cells expressing MRB versus cells expressing GFP (data not shown). Using this purified antibody, we analyzed MRB expression in a variety of tumor and normal human tissues. Our immuno-colocalization analysis for the expression of MRB and CD-31 on normal and cancer human tissues from lung, kidney, liver, and metastatic melanoma (Fig. 2) demonstrates that MRB expression is significantly upregulated in tumor endothelium and is expressed at relatively lower levels in normal adult, presumably quiescent, endothelial cells in vivo. MRB-mediated signaling results in inhibition of endothelial cell migration Based on sequence similarity to neuronal Robo family members, it was reasonable to hypothesize that MRB expression might regulate repulsive signaling in endothelial cells. We utilized a modified Boyden chamber assay, in which cells were placed in the upper chamber and test factors such as FGF, VEGF, and FBS were placed in the lower chamber. Migration of cells was quantified by the number of cells that migrated into the lower chamber. We investigated the migration of HUVECs expressing MRB compared to migration of cells expressing soluble MRB (sMRB) or a control GFP. The expression of each construct was verified by Western blot analysis of infected HUVECs (Fig. 3B). As expected, VEGF-, FGF-, and FBS-stimulated HUVECs expressing control GFP migrated
Fig. 2. Immunohistochemical analysis of MRB. Immunofluorescence colocalization of MRB and CD-31, a known endothelial marker in (A), metastatic melanoma, cancerous, and normal tissue sections derived from (B) kidney, (C) lung, and (D) liver. All the images are taken at 40· magnification.
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Fig. 3. MRB expression inhibits endothelial cell migration. (A) HUVECs expressing control GFP, MRB, or sMRB were stimulated with 10 ng/ml VEGF for the migration assay. (B) The expression of GFP, MRB, and sMRB was checked with anti-GFP and anti-MRB antibodies. (C) HUVECs expressing control GFP or MRB were stimulated with 10 ng/ml FGF. (D) Proliferation assay for HUVECs expressing GFP or MRB.
12-fold more than non-stimulated cells, whereas cells expressing MRB migrated only 4-fold more and expression of soluble MRB did not inhibit migration (Figs. 3A, C, and data not shown). Moreover, inhibition in migration was more pronounced in FGF induced HUVECs. However, the rate of cell proliferation was unaffected (Fig. 3D), suggesting that cell viability was not compromised by MRB expression. These in vitro experiments indicate that MRB expression alone is sufficient to block migration in response to various promigratory factors; moreover, it suggests that activation of the MRB receptor when over-expressed on the cell surface can occur via a ligand-independent mechanism in a manner similar to Robo1 and Robo2 [21]. It has also been suggested that Slit2, which is known to be expressed by endothelium in vivo [22], may act as a putative ligand for MRB [11]. To assess the role of Slit2 in endothelial biology, as also recently done by Park et al. [10], we repeated the migration assay with HUVECs as described above, but with conditioned medium containing Slit2, derived from 293 T cells transfected with a Slit2 expression vector. HUVECs fail to migrate in response to VEGF in the presence of Slit2 and this inhibition of migration was rescued by addition of sMRB (Fig. 4A). The expression of sMRB did not effect cell migration as recently shown by Suchting et al. [23]. This
may be the result of differences in functional assays. The expression of Slit2 and sMRB in 293 T cell conditioned medium was confirmed by Western blot analysis (Fig. 4B). These results suggest that Slit2 is signaling via MRB as Robo1 was undetectable in HUVECs by RTPCR (Fig. 4C) and as previously described [10]. These data are in discrepancy with those of Wang et al. [15] and may be due to differences in the endothelial cells used for their study. MRB signaling intersects with the MEK/ERK pathway Cytoskeletal rearrangement and reorganization are essential features of endothelial cell migration [24]. Regulation of VEGF- and FGF-stimulated migration of endothelial cells is coordinated by separate sets of signaling components [25]. FGF initiates migration by activation of Ras that results in activation of ERK and subsequently leads to phosphorylation of FAK [26– 28], whereas VEGF binds to its receptor, VEGFR2, regulating cell migration via the PI3 kinase/AKT pathway [29,30]. ERK has been implicated in migration of numerous cell types and ERK pathway inhibitors U0126 and PD98059 inhibit migration in response to VEGF (partially), FGF, and FBS [31,32]. Moreover, a dominant negative ERK mutant or inhibition of ERK by an anti-sense strategy also inhibits cell migration
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Fig. 4. Slit2 mediates inhibition of endothelial cell migration by interaction with MRB. (A) HUVECs were incubated with 0.2% FBS conditioned medium derived from 293 T cells expressing GFP, Slit2, or sMRB for 4 h. Cells pretreated with Slit2 showed inhibition of migration when stimulated with 10 ng/ml VEGF compared to cells pretreated with control or sMRB; moreover, combined incubation of Slit2 and sMRB conditioned medium in a 1:1 ratio abrogated the inhibition of Slit2 induced migration. (B) Western blot analysis of conditioned media expressing Slit2 and sMRB used for HUVEC pretreatment. The anti-myc antibody recognizes Slit2 and anti-MRB antibody recognizes sMRB. (C) RT-PCR analysis of Robo1 and Robo4 (MRB) in HUVECs.
[32,33]. Therefore, these studies suggest that the ERK signaling pathways play a significant role in the regulation of migration. The phosphorylation of endogenous ERK and FAK is markedly downregulated in cells expressing MRB (Fig. 5A, lower panel) versus cells expressing control GFP (Fig. 5A, upper panel). These results indicate that over-expression of MRB leads to a ligand-independent activation, similar to studies with Robo1 [21], and may account for inhibition of migra-
tion by suppression of ERK and FAK phosphorylation. Since Slit2 inhibits migration of endothelial cells, which can be rescued by sMRB (Fig. 4), we speculated that molecular signals involved in the Slit2 (ligand dependent) and MRB over-expression (ligand independent) situations might be similar, and that Slit2 may inhibit migration by dephosphorylation of FAK. As shown in Fig. 5B, Slit2 indeed causes FAK dephosphorylation and this effect can be abrogated by sMRB.
Fig. 5. MRB/Slit2 activates FAK. (A) HUVECs expressing MRB and control GFP were plated on collagen-coated plates and removed from these plates at 0, 30, 60, and 120 min. Cell lysates were analyzed by Western blot analysis with antibodies specific for FAK, phospho FAK, and phospho ERK. (B) HUVECs were incubated with 0.2% FBS conditioned medium derived from 293 T cells expressing GFP, Slit2, or sMRB either alone or in combination (1:1 ratio of conditioned medium) for 6 h. Cells were harvested and plated on collagen-coated plates for 30 min (as determined by previous experimental set, see A) and Western blot analysis was performed with antibodies specific for phospho FAK and actin (loading control).
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Fig. 6. Functional rescue of MRB induced inhibition of migration by MEK-DD expression. (B) HUVECs expressing control GFP, MRB, MEKDD/MRB, and MEK-DD/GFP were stimulated with 10 ng/ml VEGF for the migration assay. (A) HUVECs expressing control GFP, MRB, MEKDD/MRB, and MEK-DD/GFP were stimulated with 10 ng/ml FGF for the migration assay. (C) Percentage functional rescue of MRB-mediated inhibition of HUVECs by MEK-DD with VEGF or FGF stimulation.
To demonstrate that migration inhibition of MRB expressing HUVECs is a specific effect resulting from inhibition of ERK and FAK phosphorylation, we have shown that over-expressing MEK-DD, a dominant active form of MEK, can override MRBÕs inhibitory effect on FGF-stimulated HUVECs (Figs. 6A and C). However, MEK-DD expression can only partially rescue MRBÕs inhibitory effects on VEGF-stimulated HUVEC migration (Fig. 6B), suggesting that MRB feeds its inhibitory signals predominantly via the ERK pathway. Since MEK-DD can only partially rescue MRB-mediated inhibition for VEGF-stimulated HUVECs, signals other than ERK, e.g., the PI3K pathway may contribute to MRB inhibitory signaling.
Discussion Tumor vasculature is an attractive target for tumor therapy. To exploit such a strategy requires the identification of functional or molecular differences between tumor and normal vessels [34]. For example, it is well known that tumor vessels tend to be tortuous, are leaky, and have less pericyte coverage than normal vessel. However, the molecular differences have been less well
characterized, though some recent progress has been made in this regard [35]. We have studied the expression of MRB by in situ hybridization, by immunohistochemistry, and by co-localization to CD-31 in numerous solid tumor and normal tissues, extending the data of Huminiecki et al. [11]. Our findings suggest that MRB is indeed a tumor endothelial specific marker. The methods we have employed to assess MRB expression are only semi-quantitative, but our data suggest that MRB expression is upregulated in tumors in a manner comparable to or more strikingly than other established markers such as VEGFR2, a(v)b3 integrin or various TEMS [35]. MRB expression during development occurs in endothelium and in the adult it appears to be minimally expressed except at sites of active angiogenesis or pseudovasculogenesis (placenta) as well as in pathological angiogenesis, for example, in tumor endothelium. Given that MRB is a surface protein, this differential expression makes it a most attractive target for directing cytotoxic therapies to tumor vasculature. The functional role of MRB in tumor angiogenesis remains unknown. Since angiogenesis is a composite process of multiple steps, we initially asked whether MRB over-expression in HUVECs affected proliferation of these cells, but found this not to be the case. Taking a
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clue from the neuronal literature in the area of axonal guidance, in which Robos are involved in signaling repulsive cues, we asked whether there might be an effect of MRB on endothelial cell migration. Our results point to an inhibitory effect mediated by MRB, in view of its ability to antagonize VEGF, FGF, and FBS (data not shown) induced migration. Moreover, our data on the ability of Slit2 to block VEGF induced migration of HUVECs support the notion that Slit2 is a putative ligand for MRB, as was also recently shown by MRBSlit2 pull-down assays [10]. This ligand had the same effect on endothelial cell migration as did MRB over-expression, an effect that was blocked by sMRBs [10] used soluble Robo1, instead of sMRB and showed similar findings. Since it was known that ERK plays a central role in cell migration, we explored whether MRB might downregulate signaling through this pathway. We showed that ERK and FAK phosphorylation was decreased by MRB over-expression or by Slit2 treatment (the specificity of these effects was ascertained by the fact that coincubation with sMRB blocked the latterÕs effects). These data are similar to the recent findings of Ganju et al. [36], who noted that Slit2 induced inhibition of phosphorylation of FAK in breast cancer cells. Moreover, over-expression of MEK-DD, a dominant active form of MEK, can override MRBÕs inhibitory effect on FGF-stimulated HUVEC migration but only partially rescues MRBÕs inhibitory effect on VEGF-stimulated migration. Hence, we would like to suggest that MRB induced inhibitory signals occur partially via Ras–Raf–Mek signaling pathway to modulate cytoskeletal events critical for migration. There appear to be discrepant findings on the expression and role of the Slit-Robo system in endothelial cells. Wang et al. [15] find Robo1 expression in HUVECs and show that Slit2 promotes endothelial migration via interaction with Robo1. Interestingly, Park et al. [10] and our data show that Robo4 may be the only Robo family member expressed on endothelial cells and that it inhibits endothelial migration. It is therefore conceivable that Slit2 signals via Robos with opposing effects and that the net effect depends on the relative expression of Robo1/4 in the endothelial cell under investigation. How can the action of MRB on endothelial cell migration be reconciled with its expression at the site of active angiogenesis? The so-called resolution phase of angiogenesis, in which, for example, the pro-angiogenic molecule TGF-b is thought to participate, includes processes of decreased endothelial proliferation and migration. It is unknown whether MRB mediates proor anti-angiogenic signals or what the functional role of MRB in vivo in tumor angiogenesis is. Experiments aimed at addressing this question are currently underway.
Acknowledgments We thank Marc Tessier-Lavigne, Stanford University, for Slit2 expression construct, and Hirofumi Hamada, Sapporo Medical University, for FAK adenovirus. This project is supported by a research training grant (T32 DK07199) to V.P.S.
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