ECSM2, An Endothelial Specific Filamin A Binding Protein That Mediates Chemotaxis
Objective— We aimed to characterize the expression and function of a novel transcript that bioinformatics analysis predicted to be endothelial specific, called endothelial-specific molecule-2 (ECSM2).
Methods and Results— A full-length cDNA was isolated and predicted ECSM2 to be a putative 205–amino acid transmembrane protein that bears no homology to any known protein. Quantitative polymerase chain reaction analysis in vitro and in situ hybridization analysis in vivo confirmed ECSM2 expression to be exclusively endothelial, and localization to the plasma membrane was shown. Knockdown of ECSM2 expression in human umbilical vein endothelial cells using siRNA resulted in both reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane, both processes involved in angiogenesis. A yeast 2 hybrid analysis using the ECSM2 intracellular domain identified filamin A as an interacting protein. This interaction was confirmed by precipitation of filamin-A from endothelial cell lysates by a GST-tagged intracellular domain of ECSM2.
Conclusion— This study is the first to characterize a novel cell surface protein ECSM2 that regulates endothelial chemotaxis and tube formation, and interacts with filamin A. These studies implicate a role for ECSM2 in angiogenesis via modulation of the actin cytoskeleton.
Endothelial cells perform a wide variety of physiological functions and also play a central role in the pathogenesis of many diseases including atherosclerosis and cancer. As such, many laboratories have sought to identify novel endothelial genes using a variety of techniques.1–8 We previously described a bioinformatics approach9 to identify novel endothelial genes such as Robo410,11 and endothelial cell–specific molecule-2 (ECSM2) now described here. The ECSM2 EST encoded a conceptual protein of 205 amino acids that contained a putative transmembrane region but no other functional domains and had no homology to any known protein. Pelossi et al subsequently showed that ECSM2 is a marker of primary hemangioblasts and endothelial progenitors and not other hematopoietic progenitor cells,12 but little else has been published on the protein.
We now report the isolation of the ECSM2 cDNA, the identification of putative orthologs, and analysis of the ECSM2 protein. The bioinformatics prediction of endothelial specificity9 was confirmed both in vitro and in vivo by ECSM2 expression analysis. Predicted plasma membrane expression was confirmed by cell surface expression of an ECSM2-GFP fusion protein in endothelial cells. To characterize the function of ECSM2, its expression was knocked down using siRNA. This resulted in reduced chemotaxis and impaired tube formation on matrigel, a solubilized basement membrane extract. These data suggest a role for ECSM2 in angiogenesis, a complex process involving the migration, proliferation, and lumen formation by endothelial cells. A yeast 2 hybrid screen was performed and identified filamin A as a binding partner for the ECSM2 intracellular domain. Filamin A anchors transmembrane proteins to the actin cytoskeleton acting as a scaffold for various signaling proteins13 and is known to interact with and regulate the function of several endothelial transmembrane molecules.14–16
Detailed information on methods are given in the online Methods, available at http://atvb.ahajournals.org. The nucleotide sequence reported in this paper has been submitted to GenBank/EBI Data Bank with accession number DQ462572.
Identification of Full-Length cDNA Sequence and Cloning of ECSM2 cDNA
5′ and 3′ rapid amplification of cDNA ends (RACE) was used to determine the full sequence of the ECSM2 transcript, using the SMART RACE cDNA Amplification kit (BD Biosciences). The ECSM2 transcript was amplified by PCR on total human umbilical vein endothelial cell (HUVEC) cDNA using the upstream primer 5′- tactcgagatggacagagcctccactga-3′ designed to include the XhoI site and the downstream primer 5′- taccgcggcacctcatcactttccttgc-3′ designed to include the SacII site and inserted into the pBluescript vector (Stratagene).
cDNA was prepared using total RNA and the random priming High-Capacity cDNA Archive kit (Applied Biosystems). For the larger cell line screen, standard curve analysis was performed to obtain relative expression levels for ECSM2 and the housekeeping gene β-2-microglobulin to which ECSM2 expression was normalized. To perform the primary cell type screen, the housekeeper genes flotillin-2, ubiquitin C, and β-actin were chosen using the method described by Vandesompele et al with the software geNorm.17 Data were analyzed using a method described by Pfaffl.18
In Situ Hybridization
In situ hybridization analysis was performed using radioactively labeled probes as described by Poulsom et al.19 In some cases slides were also stained with CD34 antibody using a method described by Jeffery et al.20 The ECSM2 transcript specific probe (nucleotides 15 to 980) was used to generate the in situ hybridization probe.
Subcellular Localization Studies
ECSM2 was tagged at the c terminus with either myc (pCDNA3.1 Myc-His) or GFP (pEGFP-N1). HUVEC were transfected with these or pEGFP-N1 using Transpass D2 transfection reagent (NEB) for HUVEC according to the manufacturers instructions. Two days after transfection, cells were either viewed live in PBS, or cells were fixed before analysis. Fixed cells were stained with either phalloidin (A12380 Alexa Fluor 568, Molecular probes) to visualize actin, or when transfected with ECSM2-myc were stained with antimyc (9E10) and antimouse fluorescein isothiocyanate (FITC). HUVECs transfected with ECSM2-myc were viewed on a Zeiss Axioplan microscope (Zeiss) using Smartcapture 2 software. HUVECs expressing ECSM2-GFP were viewed using an Zeiss LSM 510 confocal microscope using Zeiss LSM Image Browser image analysis software.
Identification of Interacting Proteins
A yeast 2 hybrid screen using the ECSM2 intracellular domain against a placental cDNA library was performed using the BD Matchmaker Library Construction and Screening kit (Clontech, BD Biosciences). The filamin A interaction was confirmed by a pull down assay using a bacterially expressed ECSM2 intracellular domain-GST fusion protein bound to glutathione agarose beads and HUVEC lysate. To detect precipitation of filamin A, samples were separated using SDS-PAGE and either Coomassie stained for visualization of the GST fusion proteins or immunoblotted using antifilamin A monoclonal antibody (Chemicon).
Transfection With siRNA and Functional Assays
106 HUVECs were seeded into 10 cm plates the day before transfection. The duplexes used were Negative control duplex (Eurogentech), D1 (ACAATGACCCAGACCTCTA) and D2 (TCAGAGGCTAACAGGCCAA). Transfection was performed using duplexes at 50 nmol/L and 0.3% lipofectamine RNAiMax (Invitrogen) in optiMEM (Invitrogen). The transfection mix was incubated with the cells for 4 hours. Cells were used for assays at 48 hours after transfection and knockdown was assessed by quantitative PCR as described above. Knockdown of protein expression was assessed by making cell lysates and Western blotting with polyclonal rabbit antisera raised to a GST fusion of the ECSM2 intracellular domain, as a control samples were blotted with a monoclonal antitubulin antibody (Sigma). Matrigel tube forming assays were carried out using standard methods,21 at a density of 1.4×105 cells per well of a 12-well plate coated with mouse natural BD Matrigel (VWR). Images were recorded after 12 hours, and nodes with 1, 2, 3, or ≥4 branches were counted and the mean and standard errors from 6 fields of view calculated. Chemotaxis was assayed using a 48-well modified Boyden chamber with 8-μm pore size polycarbonate nucleopore filters (Neuro Probe). Filters were coated with 0.1% gelatin and placed over a lower chamber containing 10% fetal calf serum and endothelial cell growth supplements (TCS) as the chemoattractant factors. Cells were rested in serum-free media for 30 minutes before the assay, and upper chambers were seeded at a density of 2×104 cells per well in media containing 1% fetal calf serum. After 5 hours incubation at 37°C with 5% CO2, the filters were removed, fixed in methanol, and stained with 0.5% Crystal Violet. Migrated cells in one quarter the area of the well were counted. The mean and standard error were calculated from the counts from 9 wells per condition of 3 independent experiments.
Identification, Cloning, and Sequence Analysis of ECSM2
To obtain the full length ECSM2 transcript sequence, the sequence of an ECSM2 EST (BI823114) was used to design primers for 5′ and 3′ rapid amplification of cDNA ends (RACE). RACE was performed using HUVEC RNA. Sequencing of RACE experimental products revealed the ECSM2 transcript to be 1030 bp in length. Subsequent Northern blot analysis on human breast tissue RNA confirmed the RACE data with expression of a major transcript at approximately 1 kb (data not shown). Primers were subsequently designed to the 5′ and 3′ ends of the full length ECSM2 sequence, which enabled PCR cloning of the transcript from HUVEC cDNA. Translation of the entire cDNA sequence revealed ECSM2 to contain an open reading frame (ORF) of 618 bp with a 67 bp 5′ untranslated region (UTR) and 345 bp 3′ UTR. The complete ECSM2 nucleotide sequence did not match any database sequences other than ESTs (GenBank/EBI). Database searches and fluorescence in situ hybridization analysis (data not shown) revealed that the ECSM2 gene mapped to human chromosome 5 at position 5q31, and spans 10.3 kb.
The conceptual ECSM2 protein comprised 205 amino acids, and extensive database searches revealed that ECSM2 had no known homologues and contained no functional domains. Analysis of the predicted amino acid sequence revealed that ECSM2 contained a putative 24–amino acid signal sequence and 27 amino acid sequence transmembrane domain (residues 120 to 147). ECSM2 was predicted to contain multiple O-linked glycosylation sites and a single N-linked glycosylation site at residue 96 by the presence of the universal acceptor sequence Asn-X-(Ser/Thr). Antisera was raised to the intracellular domain of ECSM2, and Western blotting using this revealed that endogenous protein comprised at least 3 bands ranging in size from 40 to 60 kDa (supplemental Figure I). The identification of the bands was confirmed by the observation that they were greatly reduced as a result of ECSM2 specific siRNA mediated knockdown. The heterogeneous size of ECSM2, which is at least 20 to 40 kDa bigger than the size predicted from the amino acid sequence, strongly suggests that ECSM2 is highly glycosylated. ESTs from several other species showed homology to ECSM2 (Figure 1). Sequence alignment of the hypothetical proteins (Figure 1) showed extensive conservation of the predicted intracellular domain of the ECSM2 protein, but much less in the extracellular domain. The figure shows that while the transmembrane and intracellular domains showed 75% similarity between zebrafish and man, conservation of the extracellular domain was poor, even within mammals.
Quantitative Analysis of ECSM2 Expression
Quantitative PCR analysis was performed on a selection of human cells to examine the pattern of ECSM2 expression. ECSM2 was expressed in all 4 endothelial cell types investigated but absent in nonendothelial cell lines or primary isolates (Figure 2). After endothelial cells, the highest expression was seen in aortic smooth muscle cells but was only 4% of that in HUVECs. There are 2 vascular smooth muscle cDNA libraries in the public databases, the HCASM2 and Sugana coronary artery smooth muscle cell libraries which between them encode 16 254 ESTs. A blast search of the ECSM2 nucleotide sequence (NM_001077693) against the 2 libraries identified no hits, and it is possible that the 4% signal detected in the real-time PCR analysis of the vascular smooth muscle cells could be attributable to trace contamination of the isolate with endothelium.
In Situ Hybridization Analysis of ECSM2 Expression
In situ hybridization studies of ECSM2 expression were performed to determine expression of ECSM2 in human tissues in vivo. Endothelial restricted expression of ECSM2 was observed in human breast carcinoma (Figure 3A through 3B), human ganglioglioma (Figure 3C through 3D), the skin in a psoriasis patient biopsy (Figure 3E through 3F), placenta (Figure 3G through 3H), and fetal tissue (Figure 3I through 3J). To confirm that expression was endothelial, human breast carcinoma sections were also immunostained for the endothelial specific marker CD34. Colocalization of the signals for ECSM2 and CD34 confirmed endothelial specific expression of ECSM2 in vivo (Figure 3K through 3L).
Subcellular Localization of ECSM2
To examine the cellular localization of ECSM2, the full-length protein was expressed with either myc or GFP fused at its C terminus and transfected into HUVECs. HUVECs were transfected with ECSM2-myc and immunofluorescence performed using antimyc antibodies. This showed that ECSM2 was localized to the membrane, and counterstaining with the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI) confirmed that the expression seen was at the plasma membrane and not the nuclear membrane. Examination of live cells expressing ECSM2-GFP showed localization at the cell surface and particularly on cellular protrusions such as filopodia (Figure 4B). ECSM2-GFP–expressing HUVECs were also fixed and stained with phalloidin to visualize F-actin, which is known to associate around the plasma membrane. Membrane expression of ECSM2 mostly colocalized with F-actin expression, particularly where many filopodia were present (Figure 4C).
Functional Studies With siRNA
ECSM2 expression was reduced to 20% of normal levels in HUVECs using 2 independent siRNA duplexes D1 and D2, compared to mock transfected or negative control duplex transfected cells (Figure 5A). Reduction of ECSM2 protein was confirmed by Western blotting (supplemental Figure I). Cells were then assayed in a scratch wound, chemotaxis and matrigel assays. Cells expressing reduced ECSM2 showed no change in their ability to move in a scratch wound assay (data not shown) but showed a severe defect in their ability to undergo chemotaxis in response to a gradient of fetal calf serum and endothelial growth supplements (Figure 5B), and an impairment in their ability to form tubes when plated on matrigel (Figure 5C and 5D). In the tube forming assay it was found that the cells with reduced ECSM2 formed a less stable and connected network. The matrigel assays were quantitated by counting the numbers of nodes containing 1, 2, 3, or 4 or more branch points (1 branch point indicating an unconnected end). In cells with knockdown of ECSM2, there was an increase in the nodes containing 1 or 2 branch points and a reduction in the nodes containing three or more branch points. This reduced complexity was more evident at 12 hours compared with 6 hours, suggesting that these cells are less able to form a stable network resulting in retraction of some of the connections and an increase in unconnected branch points. Introduction of siRNA into cells can give artifactual results attributable to induction of interferon (INF) secretion from the transfected cell (the so-called INF response). Real-time PCR quantitation of the INF sensitive genes (ISG20 and OAS1) showed no difference between transfectants and controls (data not shown) and confirmed that these duplexes do not induce the INF response at 50 nmol/L 48 hours after transfection. Finally, the duplexes are too small to bind to Toll3 and produce off target effects.22
Identification of Filamin A as an ECSM2 Binding Protein
To determine the mechanism by which ECSM2 was acting, proteins interacting with the intracellular domain of ECSM2 were identified by yeast 2 hybrid. The intracellular domain of ECSM2 (residues 147 to 205) was used as bait in a yeast 2 hybrid analysis of a human placental library. Three clones isolated encoded the C-terminal region of filamin A. The 3 clones encoded amino acids 1658 to 1846 and 2092 to 2310, which are in the 15 to 16 and 19 to 21 beta repeat sheets regions respectively. To confirm this interaction, the intracellular domain of ECSM2 fused to GST was used to pull down endogenous filamin A from a HUVEC lysate revealing a filamin A band at 280 kDa which was absent in the GST-fusion control (Figure 6).
Endothelial cells are one of the most transcriptionally active cell types,23 as they are involved in many physiological processes and play a role in the development of diseases such as atherosclerosis and cancer. As a result, many laboratories seek to identify novel endothelial specific genes. There is a particular interest in the identification of membrane spanning proteins that respond to environmental signals at the cell surface and transduce that information to the cytoskeleton. In this study, we describe the identification and characterization of the novel transmembrane protein ECSM2, a previously uncharacterized filamin A binding protein involved in endothelial chemotaxis and tube formation in vitro.
We originally identified ECSM2 (Genbank DQ462572) by bioinformatics,9 and it was named Endothelial Cell Specific Gene-2 (ECSM2) because of its putative endothelial restricted expression but has otherwise remained a hypothetical protein. This work has confirmed the ECSM2 mRNA as a 1030-bp transcript and validated its highly restricted endothelial expression experimentally. Quantitative PCR analysis of primary cell isolates and cell lines cultured in vitro showed that ECSM2 was expressed only in endothelial cells (Figure 2). In situ hybridization analysis of ECSM2 expression in a range of human tissues showed that the ECSM2 transcript displayed highly restricted endothelial expression, with little or no expression observed in other cell types (Figure 3). Endothelial expression of ECSM2 was confirmed by immunostaining of adjacent sections for the endothelial marker CD34. Our findings are supported by the work of Ho et al, who reported that an EST now known to belong to ECSM2 was the most endothelial specific of 64 endothelial genes identified in a combined bioinformatics and microarray screen.3 Thus, ECSM2 shows potential as an endothelial marker, and the ECSM2 transcript has been used as a measure of endothelial cell contamination24 and differentiation.12
The ECSM2 gene is expressed from a 10.3-kb locus on human chromosome 5q31; it encodes a 205 amino acid protein, which sequence analysis predicted to contain a signal sequence and transmembrane region. Extensive database searches revealed the ECSM2 to be unique in that it bore no homology to any known protein and contained no functional domains. Putative ECSM2 orthologs were identified in other vertebrate species (Figure 1). Sequence alignment of the orthologs revealed that they all contained a highly conserved intracellular domain and a highly variable extracellular domain. The domain specific conservation of ECSM2 may prove to be significant in understanding the function of the protein. The conservation may indicate that there is a strong selective pressure to maintain the integrity of the intracellular domain sequence, or alternatively, interaction with another factor (eg, a virus) may have driven rapid evolution of the extracellular domain. The size of the ECSM2 protein is more than twice that predicted from the amino acid structure, and it is heterogeneous in nature. This experimental evidence, combined with a large number of serine and threonine residues predicted to be sites of O-linked glycosylation in the extracellular domain, which are conserved across species, strongly suggests that ECSM2 is heavily O-glycosylated. A single N-linked glycosylation site is not conserved beyond primates and unlikely to be functionally critical.
Cell surface expression of ECSM2 was confirmed by using an ECSM2-GFP fusion protein in HUVECs (Figure 4). ECSM2 expression was uniform across the entire membrane in 293 embryonic kidney cells (data not shown). By contrast, in HUVECs expression was concentrated at certain points such as the filopodia, which may be a feature of ECSM2 expression in the presence of its interacting proteins.
A mechanism for controlling the localization of ECSM2 within the plasma membrane is through the intracellular interactions of ECSM2 with other proteins. To investigate ECSM2 protein interactions, yeast 2 hybrid analysis was performed using the intracellular domain of ECSM2 as bait to screen a human placental cDNA library. The screen identified a putative interaction between filamin A and ECSM2, and this was subsequently confirmed by using an ECSM2 fusion protein to precipitate endogenous filamin A from HUVEC lysate (Figure 6). Filamin A belongs to the filamin family of actin binding proteins that link actin filaments at the cell membrane and help maintain cell structure. Filamins are considered to be key players in mammalian cell locomotion.25
Mammalian filamins exhibit marked promiscuity in their protein interactions and have been shown to bind to more than 30 different proteins.26 Despite the diversity of the interacting proteins the regions of the filamin that they bind to are principally specified by their function. Thus, a few smaller proteins that participate in signaling processes recognize repeats 1 to 15. In contrast, receptor proteins that make up the largest group of interactors all recognize repeats 16 to 24, and it is this interaction that mediates cross-talk between the extracellular environment and the actin matrix. The interaction of the intracellular domain of ECSM2 with repeats 19 to 21 (and to a lesser extent 15 and 16) are consistent with ECSM2 bearing such a role in endothelial cells that are known to express filamin A.27
An effect of ECSM2 knockdown on endothelial movement in a chemotaxis (Boyden chamber) but not a chemokinetic (scratch wound) assay is in accord with the cells sensing a ligand concentration gradient via ECSM2. The media used as chemoattractant in the Boyden chamber was complex (serum, endothelial cell growth supplements), and at this point it is not clear what an ECSM2 ligand could be. A second possibility is that ECSM2 may act to modulate another, for example, vascular endothelial growth factor, chemokinetic signal. It is also not known what function the extensive O-glycosylation of ECSM2 performs, but in other proteins it has been shown to confer resistance to proteases and modulate the intracellular trafficking of proteins among others. Further work, including in vivo studies, is needed to define the exact role of ECSM2 in angiogenesis and vascular biology.
We thank Rosemary Jeffery and Toby Hunt for help with the in situ analysis, and Holger Gerhardt for the use of and help with the confocal microscope.
Sources of Funding
This work was funded by Cancer Research UK Programme Grant number A6766 and European Union FP6 LSHC-CT-2003-503233 grant ‘STROMA’.
L.-J.A. and V.L.H. contributed equally to this study.
Original received February 19, 2007; final version accepted June 2, 2008.
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