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Magic Roundabout Is a New Member of the Roundabout Receptor Family That Is Endothelial Specific and Expressed at Sites of Active Angiogenesis
Article
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
Magic Roundabout Is a New Member of the Roundabout Receptor Family That Is Endothelial Specific and Expressed at Sites of Active Angiogenesis Lukasz Huminiecki,1 Michael Gorn,1 Steven Suchting,1 Richard Poulsom,2 and Roy Bicknell1,* 1
Molecular Angiogenesis Laboratory, Cancer Research UK, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK 2 In Situ Hybridisation Service, Cancer Research UK, 44 Lincolns Inn Fields, London, WC2A 3PX, UK *To whom correspondence and reprint requests should be addressed. Fax: 44-1865-222431. E-mail: [email protected]
We have used bioinformatic data mining to identify a novel, endothelial-specific gene encoding a protein with homology to the axon guidance protein roundabout (ROBO1). The new gene has been called magic roundabout (ROBO4; GenBank acc. no. AF361473) and is smaller than other members of the roundabout gene family. Thus, in the extracellular region, magic roundabout has only two of the five immunoglobulin and two of the three fibronectin domains present in other roundabout genes. Expression of magic roundabout in vitro was detected in only endothelial cells and was greater in cells exposed to hypoxia. In situ hybridization and immunohistochemistry validated the bioinformatic prediction that magic roundabout expression would be endothelial specific in vivo. Magic roundabout expression in the adult was restricted exclusively to sites of active angiogenesis, notably tumor vessels. The identification of magic roundabout shows that the roundabout gene family extends beyond neuronal tissue and that roundabout/slit interactions are likely to have a role in angiogenesis. Key Words: cDNA cloning, endothelial, roundabout, ROBO, Gif-1, Dutt1, slit, tumor marker, gene expression
INTRODUCTION
RESULTS
We previously described a computing procedure that enables searching for tissue-specific genes and applied this to the identification of novel, endothelial-specific genes [1]. One such gene was Unigene cluster HS.111518, which showed homology to human roundabout (ROBO1). Roundabout was originally isolated from Drosophila melanogaster [2], but other family members were rapidly identified from species including Caenorhabditis elegans, zebrafish, mouse, and human [3–5]. All roundabouts are large transmembrane receptors with an extracellular domain composed of five immunoglobulin and three fibronectin motifs followed by a long cytoplasmic tail. The roundabout cognate ligands are the slits, of which three are now known in human [6–9]. In Drosophila, the slit/roundabout interaction elicits axon growth cone repulsion. Thus, roundabout expression ensures that commissural axons that have already crossed the midline do not recross it, and that other axons that stay ipsilaterally do not cross the midline at all [2,10]. In view of the previous neuronal specificity of roundabouts, the identification of an endothelial-specific roundabout is of particular significance.
Molecular cloning has enabled the isolation of a full-length cDNA clone of Unigene cluster HS.111518 that shows homology to the roundabout family of genes. We have called this gene magic roundabout (ROBO4; GenBank acc. no. AF361473). Search of the RIKEN database (http://www.riken.go.jp) identified mouse magic roundabout (Fig. 1A). The predicted molecular weight for the peptide core of human magic roundabout was 107,457 kDa. This was confirmed by in vitro translation using a rabbit reticulocyte lysate (data not shown). Figure 1A shows a multiple sequence alignment of the mouse and human magic roundabout sequences to those of mouse, rat, and human roundabout. All known roundabouts contain five immunoglobulin and three fibronectin domains in their extracellular region. In contrast, the extracellular domain (1–467) of magic roundabout is quite different, with only three immunoglobulin and two fibronectin domains (Fig. 1B). A transmembrane domain was identified (468–490) using the transmembrane predicting software PRED-TMR and using an alignment between human magic roundabout and human ROBO1 peptide sequences.
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A
FIG. 1. cDNA and structural analysis of magic roundabout. (A) (this and next page) Multiple sequence alignment of mouse and human magic roundabout cDNAs with the mouse (Dutt1), rat, and human roundabout sequences. The figure is constructed in CHROMA (http://www.lg.ndirect.co.uk/chroma/index.htm). Mouse and human magic roundabout show 75% nucleotide identity. (B) (next page) Structural comparison of human roundabout and magic roundabout. The position of the motifs in the protein sequence are as follows. ROBO1: five immunoglobulin and major histocompatability complex domains, 82–149, 184–242, 276–332, 365–430, and 469–527; three fibronectin type III domains, 561–646, 673–763, and 775–864; proline-rich region, 1187–1308. Magic roundabout: two immunoglobulin and major histocompatibility complex domains, 46–116 and 151–209; two fibronectin type III domains, 252–335 and 347–432; transmembrane region 468–490; proline-rich region, 715–772.
The translated protein contains a signal peptide sequence and four N-glycosylation sites in the extracellular domain at residues 246, 360, 388, and 395. The intracellular domain (491–1007) contains a putative proline-rich region that is homologous to regions of ROBO1 that are thought to couple to c-ABL [11]. BLAST showed that human ROBO4 lies on chromosome 11 at 11q24.2 and is composed of 18 exons. An initial RT-PCR screen detected ROBO4 expression in endothelial cells but not in cell lines such as fibroblasts (normal endometrial and FEK4), colon carcinoma (SW480 and HCT116), breast carcinoma (MDA453 and MDA468), or HeLa cells [1]. Ribonuclease protection analysis has confirmed and extended these results (Fig. 2A). ROBO4 expression was
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detected in endothelium (three different isolates), but was absent from fibroblast, carcinoma, and neuronal cells. Further analysis showed ROBO4 to be induced by hypoxia in endothelial (but not non-endothelial) cells. Thus, using two different RNase protection probes, expression was on average 5.5- and 2.6-fold higher in hypoxia for human umbilical vein (HUVEC) and human dermal microvascular endothelial (HDMEC) cells, respectively. Western analysis identified a weak band of 110 kDa in HDMEC cells that was absent from the non-endothelial cell lines (Fig. 2B). The band was more intense when the HDMEC cells were exposed to 18 hours of hypoxia, consistent with ROBO4 being a hypoxically regulated gene.
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Article
B
In situ hybridization characterized expression of ROBO4 in vivo. Expression of ROBO4 was found to be highly restricted (Table 1), with no signal detectable in many tissues including neuronal tissue. In contrast, strong expression was detected in placenta and a range of tumors including those of the brain, bladder, and colonic metastasis to the liver (Fig. 3). Expression within tumors was restricted to the tumor vasculature. Immunohistochemical staining of placenta confirmed endothelial-specific expression of the protein (Fig. 3).
DISCUSSION This work has identified a new gene encoding a protein with homology to the roundabout family of transmembrane receptor proteins. It should be noted that magic roundabout is smaller than the other roundabouts in its extracellular domain. A most striking feature of magic roundabout is its endothelial specificity of expression and even then only at sights of active angiogensis in vivo. This expression pattern
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
is unique among the roundabout genes, which were originally thought to show neuronal-specific expression but are now known to be more widely expressed [12,13]. We have failed to detect magic roundabout expression in neuronal tissue. There is increasing evidence for overlap between genes expressed in neuronal and endothelial tissue, for example, neuropilin, ephrins, and delta4 [reviewed in 14], and the roundabouts are undoubtedly another such family. It is tempting to speculate that similar genes may perform similar functions in the two cell types. Thus, as roundabout is involved in repulsive axon guidance, magic roundabout may be involved in inhibition of endothelial cell migration. The latter is known to precede differentiation of endothelial cells into tube-like vessel precursors. Slit-2 was recently shown to inhibit leukocyte chemotaxis [12], which is of interest in that leukocytes and endothelial cells are derived from a similar cell lineage. Slit is the ligand for roundabout [7]. The three known human slits show close sequence similarity and their individual roles in vivo are probably mediated by their markedly different expression patterns. Clearly divergence here lies in
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doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
A
FIG. 2. Expression of magic roundabout in vitro. (A) Ribonuclease protection analysis. Top, two probes to different regions (nucleotides 1–355 and 3333–3679) of magic roundabout were used in the analysis (left and right). RNase protection assay was performed with U6 small nuclear RNA as control (bottom). Human cell lines and primary isolates: MRC5, fibroblast cell line; MCF-7, breast carcinoma cell line; Neuro, SY-SH-5Y neuroblastoma cell line; HUVEC, umbilical vein endothelial isolate; HDMEC, dermal microvascular endothelial isolate; HMME2, mammary microvascular endothelial cell line; N, normoxia; H, hypoxia; P, proliferating. (B) Western analysis of cell lysates. A band at ~ 110 kDa corresponds to magic roundabout and was stronger in cells exposed to hypoxia for 18 hours. The experiment was repeated twice with similar results. Both anti-sera gave identical results. A western blot of -tubulin was used as a loading control.
B
the promoters. Slit-2 and slit-3 are known to be expressed by endothelium in vivo [12]. Whether slit or another molecule is the ligand for magic roundabout awaits determination. A search of CGAP SAGE libraries for magic roundabout found it only in endothelial and tumor libraries (Table 2). This is consistent with in situ hybridization results in the adult showing that expression was restricted to tumor vessels (colon metastasis to liver, ganglioglioma, bladder, and breast carcinoma). Hypoxic induction of magic roundabout (Fig. 2) could explain selective expression on tumor and not normal tissue vasculature. The lack of expression of magic roundabout in adult tissues, except sites of active angiogenesis, points to a developmental role for the gene.
TABLE 1: Expression of magic roundabout in human tissue in vivo Expression detected Placenta and umbilical cord fetal tissue (10.8 weeks menstrual age) Vessels in colorectal liver metastasis, ganglioglioma, bladder and breast carcinoma Expression not detected Adult liver, brain cerebrum and large vessels, prostate, colon, small bowel, heart, eye choroid and sclera, ovary, stomach, breast
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Selective expression of magic roundabout on tumor endothelium combined with the fact that magic roundabout is a transmembrane molecule has implications for the therapeutic targeting of the tumor vasculature. Targeting the tumor vasculature is an effective anti-cancer strategy [15], but has proven difficult to enable in human due to lack of suitable targets. Magic roundabout may well be such a target.
MATERIALS AND METHODS Cells and cell culture. MCF-7 breast carcinoma cells and MRC-5 fibroblasts were obtained from the Cancer Research UK Central Cell Services, Clare Hall, London. SY-SH-5Y neuroblastoma cells were obtained from the American Type Culture Collection, Bethesda, MD. Human dermal microvascular endothelial cells (HDMECs) were purchased from TCS Biologicals. HMME2 cells were a gift from Michael O’Hare (University College, London) [16]. All cells were cultured in Dulbecco’s modified Eagle’s medium/10% fetal calf serum in a 5% carbon dioxide atmosphere, except HDMEC, which were cultured according to the retailer’s instructions. cDNA cloning. The magic roundabout EST sequence identified in the bioinformatics search for endothelial-specific transcripts was used to isolate a cDNA of 3800 bp in length from a human heart cDNA library. A screen using genespecific primers showed the gene to be present in libraries from heart, adult and fetal brain, liver, lung, kidney, muscle, placenta, and small intestine, but absent from peripheral blood leukocytes, spleen, and testis. Highest expression was in the placental library. Ribonuclease protection analysis. A ribonuclease protection assay was carried out essentially as described [17]. Briefly, 15 g total RNA from each cell line was hybridized to probe overnight. Probe 1 was a 373-bp fragment lying
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Article
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A
D
B E
C F
FIG. 3. Expression of ROBO4 in vivo. (A–C) Expression of ROBO4 detected by in situ hybridization of a placental arteriole (A) and venule (B) (left, light field and right, dark field). (C) Immunohistochemical staining of ROBO4 in a placental arteriole. Left, von Willibrand factor control and right, ROBO4. (D–G) Expression of ROBO4 in tumor vessels. A ganglioglioma is shown at 20 (D) and 50 (E). Left, light field; right, dark field. Arrows highlight a vessel running diagonally down the section with an erythrocyte (turquoise) within it. Blood vessels were strongly positive for ROBO4 expression. A papillary bladder carcinoma is shown at 20 (F) and 50 (G). The vascular core of the papilla of the tumor is strongly positive, and the signal most probably originates from the “flat” endothelial cells indicated by the arrows.
between nucleotides 3341 and 3714 that was generated by RT-PCR. Probe 2 was the initial 355 bp of the cDNA generated by SmaI digestion. The loading control was U6 snRNA [18].
In situ hybridization analysis. A ROBO4 antisense in situ probe was generated using T3 polymerase from IMAGE EST clone 1912098 (GenBank acc. no. AI278949). The plasmid was linearized with EcoRI before probe synthesis. In situ analysis was then performed as described [19]. Preparation of rabbit anti-human magic roundabout anti-serum. Polyclonal rabbit anti-serum was raised against the following peptides coupled to keyhole limpet hemocyanin: amino acids 165–181 (LSQSPGAVPQALVAWRA) and 274–288 (DSVLTPEEVALCLEL; anti-serum 1) or peptides 311–320 (TYGYISVPTA) and 336–351 (KGGVLLCPPRPCLTPT; anti-serum 2). For western analysis, anti-serum was affinity purified on a Hi-Trap NHS-activated HP column (Amersham) to which the peptides used to raise the anti-serum 1 were coupled. Immunohistochemistry. Placental tissue was snap frozen immediately following removal by immersion in liquid nitrogen and stored at –80C. Sections (6 m) were cut using a cryotome. Sections were air dried for 12–16 hours, fixed by immersion in acetone for 2 minutes, air dried again, wrapped back to back in aluminium foil, and stored at –20C until use. Immunostaining was performed using the Vectastain elite ABC kit (Vecta Labs, UK). Polyclonal rabbit anti-human von Willibrand factor antibody (DAKO Ltd.) was used as the positive endothelial control. Sera was diluted 1:300 in 1% goat serum in TBS. The same dilution was used for immunostaining of magic roundabout. Pre-bleed serum at a 100-fold dilution in goat serum was used as negative control. Sections were rehydrated and endogenous peroxidase blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 minutes. Slides were rinsed in TBS, blocked in 10% goat serum for 20 minutes, and incubated with the primary antibody, diluted in 1% goat serum in TBS, for 1 hour. Following washing, secondary-biotinylated goat anti-rabbit antibody was applied to the
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
G
sections for 30 minutes. Following washing in TBS (3, 5 minutes each wash), slides were placed in a streptavidin-peroxidase (or streptavidin-alkaline phosphatase) complex solution 30 minutes before use. The developing reaction was stopped after 2–3 minutes by immersing the slides in cold tap water. Sections were lightly counterstained with hematoxylin before examination. Western blot analysis. Cells were released with 2 mM EDTA in PBS, collected, and centrifuged, and the pellet washed in PBS. Cells were lysed by treatment with urea. Samples were assayed and standardized for protein concentrations (BCA protein assay) before separation in a 10% polyacrylamide SDS containing gel by electrophoresis. Proteins were then transferred to Immobilon membrane before immunoblotting essentially as described [20]. Blots were blocked with buffer containing milk protein and 0.05% Tween (1 ml Tween 20% with 20 g Marvel milk protein in 400 ml PBS) and incubated overnight. Primary antiserum was diluted 1:1000 in blocking buffer and the
TABLE 2: CGAP SAGE libraries in which magic roundabout was found on the basis of gene to tag mapping Library
Tags/million tags
HDMEC
171
HDMEC + VEGF
224
Medulloblastoma
102
Glioblastoma multiforme
85
Ovary, serous adenocarcinoma
59
Glioblastoma multiforme, pooled
48
HDMEC, human dermal microvascular endothelial cells; VEGF, vascular endothelial growth factor.
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blots immersed in the solution for 1.5 hours. Blots were then washed (4) in PBS containing 0.05% Tween. The second antibody (DAKO goat anti-rabbit antibody conjugated to horseradish peroxidase) was applied at room temperature for 45 minutes. Excess antibody was washed off and immunodetection performed with an ECL Western Blotting kit. Blots were exposed to high-performance chemiluminescence films (Hyperfilm, Amersham Pharmacia).
ACKNOWLEDGMENTS We thank John Moore (Cancer Research UK, Oxford) for support and advice; Toby Hunt, Rosemary Jeffery, and Jan Longcroft (Cancer Research UK, London) for performing in situ analysis; Michael O’Hare (University College, London) for HMME2 cells; and Zsuzsanna Nagy (Department of Neuropathology, University of Oxford) for brain tissue. This work was supported by the Imperial Cancer Research Fund (Cancer Research UK). RECEIVED FOR PUBLICATION OCTOBER 19, 2001; ACCEPTED FEBRUARY 1, 2002.
REFERENCES 1. Huminiecki, L., and Bicknell, R. (2000). In silico cloning of novel endothelial-specific genes. Genome Res. 10: 1796–1806. 2. Kidd, T., et al. (1998). Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionary conserved guidance receptors. Cell 92: 205–215. 3. Zallen, J. A., Yi, S. A., and Bargmann, C. J. (1998). The conserved immunoglobulin superfamily SAX-3/Robo directs multiple aspects of axon guidance in C. elegans. Cell 92: 217–227. 4. Lee, J. S., Ray, R., and Chien, C. B. (2001). Cloning and expression of three zebrafish roundabout homologs suggest roles in axon guidance and cell migration. Dev. Dyn. 22: 216–230. 5. Yuan, S. S., Cox, L. A., Dasika, G. K., and Lee, E. Y. (1999). Cloning and functional studies of a novel gene aberrantly expressed in RB-deficient embryos. Dev. Biol. 207: 62–75.
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6. Brose, K., et al. (1999). Slit proteins bind robo receptors and have an evolutionary conserved role in repulsive axon guidance. Cell 96: 795–806. 7. Kidd, T., Bland, K. S., and Goodman, C. S. (1999). Slit is the midline repellent for the robo receptor in Drosophila. Cell 96: 785–794. 8. Itoh, A., Miyabayashi, T., Ohno, M., and Sakano, S. (1998). Cloning and expressions of three mammalian homologues of Drosophila slit suggests possible roles for Slit in the formation and maintenance of the nervous system. Mol. Brain. Res. 62: 175–186. 9. Nakayaman, M., et al. (1998). Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening. Genomics 51: 27–34. 10. Seeger, M., Tear, G., Ferres-Marco, D., and Goodman, C. S. (1993). Mutations affecting growth cone guidance in Drosophila: genes necessary for guidance toward or away from the midline. Neuron 10: 409–426. 11. Bashaw, G. J., Kidd, T., Murray, D., Pawson, T., and Goodman, C. S. (2000). Repulsive axon guidance: Abelson and Enabled play opposing roles downstream of the roundabout receptor. Cell 101: 703–715. 12. Wu, J. Y., et al. (2001). The neuronal repellant slit inhibits leukocyte chemotaxis induced by chemotactic factors. Nature 410: 948–952. 13. Xian, J., et al. (2001). Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene. Proc. Natl. Acad. Sci. USA 98: 15062–15066. 14. Shima, D. T., and Mailhos, C. (2000). Vascular developmental biology: getting nervous. Curr. Opin. Genet. Dev. 10: 536–542. 15. Burrows, F. J., and Thorpe, P. E. (1993). Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature. Proc. Natl. Acad. Sci. USA 90: 8996–9600. 16. O’Hare, M. J., et al. (2001). Conditional immortalisation of freshly isolated human mammary fibroblasts and endothelial cells. Proc. Natl. Acad. Sci. USA 98: 646–651. 17. Ausubel, F. M., et al. (1987). Current Protocols in Molecular Biology. New York, John Wiley and Sons. 18. Maxwell, P., et al. (1999). The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399: 271–275. 19. Poulsom, R., Longcroft, J. M., Jeffery, R. E., Rogers, L., and Steel, J. H. (1998). A robust method for isotopic riboprobe in situ hybridisation to localise mRNAs in routine pathology specimens. Eur. J. Histochem. 42: 121–132. 20. Brown, N. S., Jones, A., Fujiyama, C., Harris, A. L., and Bicknell, R. (2000). Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors. Cancer Res. 60: 6298–6302.
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
Magic Roundabout Is a New Member of the Roundabout Receptor Family That Is Endothelial Specific and Expressed at Sites of Active Angiogenesis Lukasz Huminiecki,1 Michael Gorn,1 Steven Suchting,1 Richard Poulsom,2 and Roy Bicknell1,* 1
Molecular Angiogenesis Laboratory, Cancer Research UK, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK 2 In Situ Hybridisation Service, Cancer Research UK, 44 Lincolns Inn Fields, London, WC2A 3PX, UK *To whom correspondence and reprint requests should be addressed. Fax: 44-1865-222431. E-mail: [email protected]
We have used bioinformatic data mining to identify a novel, endothelial-specific gene encoding a protein with homology to the axon guidance protein roundabout (ROBO1). The new gene has been called magic roundabout (ROBO4; GenBank acc. no. AF361473) and is smaller than other members of the roundabout gene family. Thus, in the extracellular region, magic roundabout has only two of the five immunoglobulin and two of the three fibronectin domains present in other roundabout genes. Expression of magic roundabout in vitro was detected in only endothelial cells and was greater in cells exposed to hypoxia. In situ hybridization and immunohistochemistry validated the bioinformatic prediction that magic roundabout expression would be endothelial specific in vivo. Magic roundabout expression in the adult was restricted exclusively to sites of active angiogenesis, notably tumor vessels. The identification of magic roundabout shows that the roundabout gene family extends beyond neuronal tissue and that roundabout/slit interactions are likely to have a role in angiogenesis. Key Words: cDNA cloning, endothelial, roundabout, ROBO, Gif-1, Dutt1, slit, tumor marker, gene expression
INTRODUCTION
RESULTS
We previously described a computing procedure that enables searching for tissue-specific genes and applied this to the identification of novel, endothelial-specific genes [1]. One such gene was Unigene cluster HS.111518, which showed homology to human roundabout (ROBO1). Roundabout was originally isolated from Drosophila melanogaster [2], but other family members were rapidly identified from species including Caenorhabditis elegans, zebrafish, mouse, and human [3–5]. All roundabouts are large transmembrane receptors with an extracellular domain composed of five immunoglobulin and three fibronectin motifs followed by a long cytoplasmic tail. The roundabout cognate ligands are the slits, of which three are now known in human [6–9]. In Drosophila, the slit/roundabout interaction elicits axon growth cone repulsion. Thus, roundabout expression ensures that commissural axons that have already crossed the midline do not recross it, and that other axons that stay ipsilaterally do not cross the midline at all [2,10]. In view of the previous neuronal specificity of roundabouts, the identification of an endothelial-specific roundabout is of particular significance.
Molecular cloning has enabled the isolation of a full-length cDNA clone of Unigene cluster HS.111518 that shows homology to the roundabout family of genes. We have called this gene magic roundabout (ROBO4; GenBank acc. no. AF361473). Search of the RIKEN database (http://www.riken.go.jp) identified mouse magic roundabout (Fig. 1A). The predicted molecular weight for the peptide core of human magic roundabout was 107,457 kDa. This was confirmed by in vitro translation using a rabbit reticulocyte lysate (data not shown). Figure 1A shows a multiple sequence alignment of the mouse and human magic roundabout sequences to those of mouse, rat, and human roundabout. All known roundabouts contain five immunoglobulin and three fibronectin domains in their extracellular region. In contrast, the extracellular domain (1–467) of magic roundabout is quite different, with only three immunoglobulin and two fibronectin domains (Fig. 1B). A transmembrane domain was identified (468–490) using the transmembrane predicting software PRED-TMR and using an alignment between human magic roundabout and human ROBO1 peptide sequences.
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved. 0888-7543/02 $35.00
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Article
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
A
FIG. 1. cDNA and structural analysis of magic roundabout. (A) (this and next page) Multiple sequence alignment of mouse and human magic roundabout cDNAs with the mouse (Dutt1), rat, and human roundabout sequences. The figure is constructed in CHROMA (http://www.lg.ndirect.co.uk/chroma/index.htm). Mouse and human magic roundabout show 75% nucleotide identity. (B) (next page) Structural comparison of human roundabout and magic roundabout. The position of the motifs in the protein sequence are as follows. ROBO1: five immunoglobulin and major histocompatability complex domains, 82–149, 184–242, 276–332, 365–430, and 469–527; three fibronectin type III domains, 561–646, 673–763, and 775–864; proline-rich region, 1187–1308. Magic roundabout: two immunoglobulin and major histocompatibility complex domains, 46–116 and 151–209; two fibronectin type III domains, 252–335 and 347–432; transmembrane region 468–490; proline-rich region, 715–772.
The translated protein contains a signal peptide sequence and four N-glycosylation sites in the extracellular domain at residues 246, 360, 388, and 395. The intracellular domain (491–1007) contains a putative proline-rich region that is homologous to regions of ROBO1 that are thought to couple to c-ABL [11]. BLAST showed that human ROBO4 lies on chromosome 11 at 11q24.2 and is composed of 18 exons. An initial RT-PCR screen detected ROBO4 expression in endothelial cells but not in cell lines such as fibroblasts (normal endometrial and FEK4), colon carcinoma (SW480 and HCT116), breast carcinoma (MDA453 and MDA468), or HeLa cells [1]. Ribonuclease protection analysis has confirmed and extended these results (Fig. 2A). ROBO4 expression was
548
detected in endothelium (three different isolates), but was absent from fibroblast, carcinoma, and neuronal cells. Further analysis showed ROBO4 to be induced by hypoxia in endothelial (but not non-endothelial) cells. Thus, using two different RNase protection probes, expression was on average 5.5- and 2.6-fold higher in hypoxia for human umbilical vein (HUVEC) and human dermal microvascular endothelial (HDMEC) cells, respectively. Western analysis identified a weak band of 110 kDa in HDMEC cells that was absent from the non-endothelial cell lines (Fig. 2B). The band was more intense when the HDMEC cells were exposed to 18 hours of hypoxia, consistent with ROBO4 being a hypoxically regulated gene.
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
Article
B
In situ hybridization characterized expression of ROBO4 in vivo. Expression of ROBO4 was found to be highly restricted (Table 1), with no signal detectable in many tissues including neuronal tissue. In contrast, strong expression was detected in placenta and a range of tumors including those of the brain, bladder, and colonic metastasis to the liver (Fig. 3). Expression within tumors was restricted to the tumor vasculature. Immunohistochemical staining of placenta confirmed endothelial-specific expression of the protein (Fig. 3).
DISCUSSION This work has identified a new gene encoding a protein with homology to the roundabout family of transmembrane receptor proteins. It should be noted that magic roundabout is smaller than the other roundabouts in its extracellular domain. A most striking feature of magic roundabout is its endothelial specificity of expression and even then only at sights of active angiogensis in vivo. This expression pattern
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
is unique among the roundabout genes, which were originally thought to show neuronal-specific expression but are now known to be more widely expressed [12,13]. We have failed to detect magic roundabout expression in neuronal tissue. There is increasing evidence for overlap between genes expressed in neuronal and endothelial tissue, for example, neuropilin, ephrins, and delta4 [reviewed in 14], and the roundabouts are undoubtedly another such family. It is tempting to speculate that similar genes may perform similar functions in the two cell types. Thus, as roundabout is involved in repulsive axon guidance, magic roundabout may be involved in inhibition of endothelial cell migration. The latter is known to precede differentiation of endothelial cells into tube-like vessel precursors. Slit-2 was recently shown to inhibit leukocyte chemotaxis [12], which is of interest in that leukocytes and endothelial cells are derived from a similar cell lineage. Slit is the ligand for roundabout [7]. The three known human slits show close sequence similarity and their individual roles in vivo are probably mediated by their markedly different expression patterns. Clearly divergence here lies in
549
Article
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
A
FIG. 2. Expression of magic roundabout in vitro. (A) Ribonuclease protection analysis. Top, two probes to different regions (nucleotides 1–355 and 3333–3679) of magic roundabout were used in the analysis (left and right). RNase protection assay was performed with U6 small nuclear RNA as control (bottom). Human cell lines and primary isolates: MRC5, fibroblast cell line; MCF-7, breast carcinoma cell line; Neuro, SY-SH-5Y neuroblastoma cell line; HUVEC, umbilical vein endothelial isolate; HDMEC, dermal microvascular endothelial isolate; HMME2, mammary microvascular endothelial cell line; N, normoxia; H, hypoxia; P, proliferating. (B) Western analysis of cell lysates. A band at ~ 110 kDa corresponds to magic roundabout and was stronger in cells exposed to hypoxia for 18 hours. The experiment was repeated twice with similar results. Both anti-sera gave identical results. A western blot of -tubulin was used as a loading control.
B
the promoters. Slit-2 and slit-3 are known to be expressed by endothelium in vivo [12]. Whether slit or another molecule is the ligand for magic roundabout awaits determination. A search of CGAP SAGE libraries for magic roundabout found it only in endothelial and tumor libraries (Table 2). This is consistent with in situ hybridization results in the adult showing that expression was restricted to tumor vessels (colon metastasis to liver, ganglioglioma, bladder, and breast carcinoma). Hypoxic induction of magic roundabout (Fig. 2) could explain selective expression on tumor and not normal tissue vasculature. The lack of expression of magic roundabout in adult tissues, except sites of active angiogenesis, points to a developmental role for the gene.
TABLE 1: Expression of magic roundabout in human tissue in vivo Expression detected Placenta and umbilical cord fetal tissue (10.8 weeks menstrual age) Vessels in colorectal liver metastasis, ganglioglioma, bladder and breast carcinoma Expression not detected Adult liver, brain cerebrum and large vessels, prostate, colon, small bowel, heart, eye choroid and sclera, ovary, stomach, breast
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Selective expression of magic roundabout on tumor endothelium combined with the fact that magic roundabout is a transmembrane molecule has implications for the therapeutic targeting of the tumor vasculature. Targeting the tumor vasculature is an effective anti-cancer strategy [15], but has proven difficult to enable in human due to lack of suitable targets. Magic roundabout may well be such a target.
MATERIALS AND METHODS Cells and cell culture. MCF-7 breast carcinoma cells and MRC-5 fibroblasts were obtained from the Cancer Research UK Central Cell Services, Clare Hall, London. SY-SH-5Y neuroblastoma cells were obtained from the American Type Culture Collection, Bethesda, MD. Human dermal microvascular endothelial cells (HDMECs) were purchased from TCS Biologicals. HMME2 cells were a gift from Michael O’Hare (University College, London) [16]. All cells were cultured in Dulbecco’s modified Eagle’s medium/10% fetal calf serum in a 5% carbon dioxide atmosphere, except HDMEC, which were cultured according to the retailer’s instructions. cDNA cloning. The magic roundabout EST sequence identified in the bioinformatics search for endothelial-specific transcripts was used to isolate a cDNA of 3800 bp in length from a human heart cDNA library. A screen using genespecific primers showed the gene to be present in libraries from heart, adult and fetal brain, liver, lung, kidney, muscle, placenta, and small intestine, but absent from peripheral blood leukocytes, spleen, and testis. Highest expression was in the placental library. Ribonuclease protection analysis. A ribonuclease protection assay was carried out essentially as described [17]. Briefly, 15 g total RNA from each cell line was hybridized to probe overnight. Probe 1 was a 373-bp fragment lying
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
Article
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
A
D
B E
C F
FIG. 3. Expression of ROBO4 in vivo. (A–C) Expression of ROBO4 detected by in situ hybridization of a placental arteriole (A) and venule (B) (left, light field and right, dark field). (C) Immunohistochemical staining of ROBO4 in a placental arteriole. Left, von Willibrand factor control and right, ROBO4. (D–G) Expression of ROBO4 in tumor vessels. A ganglioglioma is shown at 20 (D) and 50 (E). Left, light field; right, dark field. Arrows highlight a vessel running diagonally down the section with an erythrocyte (turquoise) within it. Blood vessels were strongly positive for ROBO4 expression. A papillary bladder carcinoma is shown at 20 (F) and 50 (G). The vascular core of the papilla of the tumor is strongly positive, and the signal most probably originates from the “flat” endothelial cells indicated by the arrows.
between nucleotides 3341 and 3714 that was generated by RT-PCR. Probe 2 was the initial 355 bp of the cDNA generated by SmaI digestion. The loading control was U6 snRNA [18].
In situ hybridization analysis. A ROBO4 antisense in situ probe was generated using T3 polymerase from IMAGE EST clone 1912098 (GenBank acc. no. AI278949). The plasmid was linearized with EcoRI before probe synthesis. In situ analysis was then performed as described [19]. Preparation of rabbit anti-human magic roundabout anti-serum. Polyclonal rabbit anti-serum was raised against the following peptides coupled to keyhole limpet hemocyanin: amino acids 165–181 (LSQSPGAVPQALVAWRA) and 274–288 (DSVLTPEEVALCLEL; anti-serum 1) or peptides 311–320 (TYGYISVPTA) and 336–351 (KGGVLLCPPRPCLTPT; anti-serum 2). For western analysis, anti-serum was affinity purified on a Hi-Trap NHS-activated HP column (Amersham) to which the peptides used to raise the anti-serum 1 were coupled. Immunohistochemistry. Placental tissue was snap frozen immediately following removal by immersion in liquid nitrogen and stored at –80C. Sections (6 m) were cut using a cryotome. Sections were air dried for 12–16 hours, fixed by immersion in acetone for 2 minutes, air dried again, wrapped back to back in aluminium foil, and stored at –20C until use. Immunostaining was performed using the Vectastain elite ABC kit (Vecta Labs, UK). Polyclonal rabbit anti-human von Willibrand factor antibody (DAKO Ltd.) was used as the positive endothelial control. Sera was diluted 1:300 in 1% goat serum in TBS. The same dilution was used for immunostaining of magic roundabout. Pre-bleed serum at a 100-fold dilution in goat serum was used as negative control. Sections were rehydrated and endogenous peroxidase blocked by incubation in 0.3% hydrogen peroxide in methanol for 30 minutes. Slides were rinsed in TBS, blocked in 10% goat serum for 20 minutes, and incubated with the primary antibody, diluted in 1% goat serum in TBS, for 1 hour. Following washing, secondary-biotinylated goat anti-rabbit antibody was applied to the
GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.
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sections for 30 minutes. Following washing in TBS (3, 5 minutes each wash), slides were placed in a streptavidin-peroxidase (or streptavidin-alkaline phosphatase) complex solution 30 minutes before use. The developing reaction was stopped after 2–3 minutes by immersing the slides in cold tap water. Sections were lightly counterstained with hematoxylin before examination. Western blot analysis. Cells were released with 2 mM EDTA in PBS, collected, and centrifuged, and the pellet washed in PBS. Cells were lysed by treatment with urea. Samples were assayed and standardized for protein concentrations (BCA protein assay) before separation in a 10% polyacrylamide SDS containing gel by electrophoresis. Proteins were then transferred to Immobilon membrane before immunoblotting essentially as described [20]. Blots were blocked with buffer containing milk protein and 0.05% Tween (1 ml Tween 20% with 20 g Marvel milk protein in 400 ml PBS) and incubated overnight. Primary antiserum was diluted 1:1000 in blocking buffer and the
TABLE 2: CGAP SAGE libraries in which magic roundabout was found on the basis of gene to tag mapping Library
Tags/million tags
HDMEC
171
HDMEC + VEGF
224
Medulloblastoma
102
Glioblastoma multiforme
85
Ovary, serous adenocarcinoma
59
Glioblastoma multiforme, pooled
48
HDMEC, human dermal microvascular endothelial cells; VEGF, vascular endothelial growth factor.
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Article
doi:10.1006/geno.2002.6745, available online at http://www.idealibrary.com on IDEAL
blots immersed in the solution for 1.5 hours. Blots were then washed (4) in PBS containing 0.05% Tween. The second antibody (DAKO goat anti-rabbit antibody conjugated to horseradish peroxidase) was applied at room temperature for 45 minutes. Excess antibody was washed off and immunodetection performed with an ECL Western Blotting kit. Blots were exposed to high-performance chemiluminescence films (Hyperfilm, Amersham Pharmacia).
ACKNOWLEDGMENTS We thank John Moore (Cancer Research UK, Oxford) for support and advice; Toby Hunt, Rosemary Jeffery, and Jan Longcroft (Cancer Research UK, London) for performing in situ analysis; Michael O’Hare (University College, London) for HMME2 cells; and Zsuzsanna Nagy (Department of Neuropathology, University of Oxford) for brain tissue. This work was supported by the Imperial Cancer Research Fund (Cancer Research UK). RECEIVED FOR PUBLICATION OCTOBER 19, 2001; ACCEPTED FEBRUARY 1, 2002.
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GENOMICS Vol. 79, Number 4, April 2002 Copyright © 2002 Elsevier Science (USA). All rights reserved.