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Originally published In Press as doi:10.1074/jbc.M305022200 on June 12, 2003

J. Biol. Chem., Vol. 278, Issue 35, 33232-33238, August 29, 2003
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A Novel Interleukin-17 Receptor-like Protein Identified in Human Umbilical Vein Endothelial Cells Antagonizes Basic Fibroblast Growth Factor-induced Signaling*

Ruey-Bing Yang {ddagger}, Chi Kin Domingos Ng, Scott M. Wasserman, László G. Kömüves, Mary E. Gerritsen and James N. Topper

From the Department of Cardiovascular Research, Millennium Pharmaceuticals, Inc., South San Francisco, California 94080

Received for publication, May 13, 2003 , and in revised form, June 6, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-17 (IL-17) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with fibroblast growth factor (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-17 receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-17 receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Interleukin-17 (IL-17)1 is a recently discovered proinflammatory cytokine that has been implicated in a variety of diseases, such as rheumatoid arthritis (1, 2), allergic skin immune response (3, 4), multiple sclerosis (5), and organ transplant rejection (6–9). Recent work on genomic mining clearly defined an IL-17 family of cytokines (10, 11). These newly identified IL-17-like family members (e.g. IL-17B to IL-17F) share ~20–30% homology with IL-17, have four invariant cysteines, and all contain an amino-terminal putative signal peptide typically required for secretion (12–15). The most recently described family member, IL-17F, is expressed selectively in activated T cells and monocytes, has been shown to be capable of inhibiting angiogenesis in human endothelial cells (EC), and can induce EC to produce IL-2, transforming growth factor-{beta}, and monocyte chemoattractant protein-1 cytokines (16). However, the endothelial receptor for IL-17F has remained elusive.

ECs play a key role in a variety of physiologic and pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and atherosclerosis (17–20). We have previously applied a genome-scale strategy to identify all genes expressed in human EC by a combination of large scale EST sequencing and genome-wide microarray expression profiling in human vascular EC cultured under various stimuli (flow, cytokine, angiogenic, etc.) (21). One full-length cDNA identified by this approach encoded a potential type I transmembrane protein. The deduced reading frame of this novel gene contains a putative signal peptide followed by a distinct extracellular domain, one membrane-spanning region, and a cytoplasmic domain that shares sequence homology with the intracellular domain of members of the IL-17 receptor (11).

In addition to endothelial expression, this novel receptor (termed human SEF (hSEF)) is also found in the ductal epithelial cells of several human tissues by in situ hybridization. This novel receptor is capable of forming a homomeric complex and also interacts with human fibroblast growth factor (FGF) receptor 1 in co-immunoprecipitation assays. Most interestingly, overexpression of this protein resulted in an attenuation of FGF-mediated induction of a FGF-responsive reporter gene in human 293T cells. This inhibitory effect appears to act downstream (or at the level) of MEK and upstream (or at the level) of ERK. Based on sequence and molecular functions, this novel IL-17 receptor-like protein (hSEF) appears to be a human homologue of a recent discovered zebrafish gene, Sef (similar expression of fgf genes) (22, 23).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Reagents—Human basic FGF was from Oncogene Research Products (San Diego, CA). Antibodies against activated-ERK1/2, pan-ERK1/2, activated-MEK1/2, and pan-MEK1/2 were from Cell Signaling Technology (Beverly, MA). Anti-FLAG M2 monoclonal antibody was from Sigma. Anti-Myc 9E10 monoclonal antibody was from Covance (Princeton, NJ). Anti-human FGFR1 monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Full-length Cloning and Expression Plasmids—A full-length clone of human SEF (hSEF) was identified by searching databases (NCBI) for sequences similar to the IL-17 receptor coding region using the BlastX algorithm (24). Based on gene prediction and public information (GenBankTM accession number AF458067 [GenBank] ), the entire open reading frame of hSEF was amplified by PCR via PLATINUM Taq DNA polymerase (BD Biosciences) from a mixture of human umbilical vein endothelial cDNAs and cloned into pGEM-T Easy (Promega, Madison, WI). The sequence of hSEF was confirmed by sequencing. The following constructs were prepared by cloning of the corresponding PCR fragments into the mammalian expression vectors. The FLAG.hSEF-FL constructs with the predicted mature hSEF (amino acids 28–739), FLAG.hSEF-{Delta}ICD (amino acids 28–353), and FLAG.hSEF-{Delta}ECD (amino acids 284–739) were made using the pFLAG-CMV-1 expression vector (Sigma). The hSEF-FL.Myc (amino acids 28–739) was constructed by PCR in a similar manner into pcDNA4-Myc/His (Invitrogen).

Quantitative Real-time PCR (TaqMan) Analysis—hSEF mRNA expression was measured by quantitative real-time PCR on a panel of cDNAs from a variety of human primary cells or normal tissues. Probes were designed by PrimerExpress software (PE Biosystems) based on the sequence of hSEF gene. Each hSEF gene probe was labeled using 6-carboxyfluorescein, and the {beta}2-microglobulin reference probe was labeled with a different fluorescent dye (VICTM, ABI, Foster City, CA). The differential labeling of the target gene and internal reference gene thus enabled measurement in the same well. Normalization was performed using {beta}2-microglobulin mRNA levels as controls in the same reaction. TaqMan experiments were carried out on an ABI PRISM 7700 Sequence Detection System (PE Applied Biosystems). The thermal cycler conditions were as follows: hold for 2 min at 50 °C and 10 min at 95 °C followed by two-step PCR for 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min.

Northern Blot Analysis—The human tissue Northern blot was purchased from Clontech and hybridized with a radiolabeled human SEF cDNA probe (nucleotides 206–633) per the manufacturer's protocol.

Construction of FGF-responsive Reporter Gene—The FGF-inducible response element (FiRE) of the mouse syndecan-1 gene was defined as described previously (25). The FiRE fragment amplified from mouse genomic DNA was confirmed by sequencing and cloned into the luciferase reporter plasmid pGL3 (Promega).

Cell Culture and Transfection—Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded in 6-well plates overnight before transfection. The transfection was performed with FuGENE 6 reagent (Roche Applied Science). We constantly observed greater than 90% of transfection efficiency in 293T cells, accessed by the transfection of green fluorescent protein vector (Clontech) as reporter and examined by flow cytometry. The total amount of DNA was kept constant in all transfections by supplementing empty vector DNA. Human umbilical vein endothelial cells (HUVEC) were cultured as previously described (26).

Luciferase Reporter Assay—293T cells were transfected with the indicated expression plasmids together with 0.5 µg of the luciferase reporter plasmid pGL3-FiRE and 0.05 µg of the Renilla luciferase reporter vector as an internal control (27). After serum-free conditions for 20 h, cells were treated with bFGF (50 ng/ml) for 6 h. Luciferase activity was measured by using reagents from Promega and was expressed as relative luciferase activity by dividing firefly luciferase by that of Renilla luciferase.

Immunoprecipitation and Western Blot Analyses—Transfected cells were washed once with phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin). Lysates were clarified by centrifugation at 4 °C for 15 min at 10,000 x g. Cells lysates were incubated with 1 µg of the indicated antibody and 20 µl of 50% (v/v) protein A-agarose (Pierce) for 2 h with gentle rocking. After three washes with lysis buffer, precipitated complexes were solubilized by boiling in Laemmli sample buffer, fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were blocked with phosphate-buffered saline (pH 7.5) containing 0.1% gelatin and 0.05% Tween 20 and were blotted with the indicated antibodies. After two washes, the blots were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) for 1 h. After washing the membranes, the reactive bands were visualized with the enhanced chemiluminescence system (Amersham Biosciences).

Tissue Samples—Formalin-fixed, paraffin-embedded sections of normal human tissues, breast tumor tissues, and breast carcinoma cell lines were obtained from DAKO Corp. (Carpinteria, CA). The breast cancer tissue samples were either individual samples or were represented in tissue microarray slides. Altogether the various tissue samples analyzed are from more than 50 individuals.

In Situ Hybridization—The details of tissue preparation and in situ hybridization have been described earlier (28–30). Following the manufacturer's protocol, digoxigenin-labeled antisense and sense riboprobes were synthesized from hSEF cDNA templates (nucleotides 206–633) using reagents supplied by Roche Applied Science. Sectioning, pretreatment of the sections, and hybridization of the probes were done under strict RNase-free conditions. All reagents were prepared using diethyl pyrocarbonate-treated distilled water. Sections of 15-µm-thick were collected on positively charged slides and dried at 55 °C overnight. The sections were deparaffinized, rehydrated in Histosolve (ThermoShandon, Pittsburgh, PA) and ethanol, and rinsed in diethyl pyrocarbonate-treated distilled water. The sections were treated at room temperature with 0.2 N HCl (20 min), 1.5% H2O2 (15 min), 0.3% Triton X-100 (15 min) followed by proteinase K treatment at 37 °C (30 min). The sections then were washed with triethanolamine buffer followed by acetylation with acetic anhydride. After prehybridization in 2x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate) containing 50% formamide at 37 °C (1 h), the sections were air-dried at room temperature. Sections were hybridized with the probes diluted in hybridization solution (2x SSC, containing 50% formamide, 10x Denhardt's, 0.001% SDS, 10 mM Tris, pH 7.4, 0.005% sodium pyrophosphate, and 500 µg/ml yeast tRNA) at 55 °C overnight. After hybridization, the sections were washed with 4x SSC (2 x 15 min) and 2x SSC (2 x 15 min). The sections then were treated with RNase A at 37 °C for 30 min followed by washes in 2x SSC at 37 °C (15 min), 0.1x SSC at 42 °C (40 min), and finally in 0.1x SSC at room temperature (2 x 15 min). The sections were washed with maleate buffer (30 min) and then blocked with 10 mM Tris buffer, pH 7.6, containing 500 mM NaCl, 4% bovine serum albumin, 0.5% cold-water fish skin gelatin, and 0.05% Tween 20. The sections were then incubated with anti-digoxigenin antibody conjugated to peroxidase (Roche Applied Science) for 1 h. The signal was amplified using TSA-Plus kit (PerkinElmer Life Sciences), and the signal was detected with Vector Blue substrate (Vector Laboratories, Burlingame, CA). After incubation with substrate, the sections were dehydrated in ethanol and Histosolve and coverslipped. Hybridization with the sense control probe did not result in detectable signal, indicating the specificity of hybridization.

Microscopy—Slides were observed with an Olympus BX50 microscope (Olympus US, Inc., Melville, NY), using differential interference contrast illumination. The microscope was equipped with a NIKON DXM1200 digital camera (Technical Instruments San Francisco, Burlingame, CA). Digitized images (1280 x 1024-pixel resolution) were acquired using ACT-1 software (Nikon USA, Melville, NY). Images were resized, cropped and assembled using Photoshop version 6.0 (Adobe Systems, San Jose, CA). Apart from equalizing the background intensities, no other digital modifications of the original digital images were carried out.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Identification and Full-length Cloning of hSEF—We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in HUVEC (21). One cDNA identified by this approach encodes a protein sequence homologous to the cytoplasmic domain of human IL-17R. To obtain the full-length cDNA of this gene, the original cDNA fragment was mapped to human genomic sequence (www.ensembl.org) and was found to be localized on chromosome 3p14.3, where a human gene was predicted based on its homology to zebrafish and mouse Sef (similar expression of fgf genes) (22, 23). Multiple oligonucleotides, based on the public sequence, were used to amplify the entire open reading frame from a mixture of HUVEC cDNAs. This cDNA contains an open reading frame of 2217 nucleotides and encodes a polypeptide of 739 amino acids (Fig. 1a). Hydropathy (31) and protein family (32) analyses predict one 27-residue amino-terminal signal peptide (SP) followed by a 272-residue extracellular domain (ECD), one 20-amino acid membrane-spanning (TM) domain, and a 420-amino acid cytoplasmic domain (ICD) (Fig. 1b). The ECD contains seven potential sites for N-linked glycosylation and shares apparent homology with mouse and zebrafish (mSef and zSef) (22, 23) but is otherwise distinct to all other proteins in the data base. Interestingly, the ICD is highly homologous to that of mSef and zSef, and the sequence similarity extends to the ICD of other members of the IL-17R family (15–20% identical) as well (11). It is noteworthy that eight cysteine residues are invariant among the ECD of human, mouse, and zebrafish SEF homologues, suggesting that these residues probably play a critical role in maintaining the secondary structure and/or functions of SEF.



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  		FIG. 1.
Primary sequence and domain structure of hSEF. a, primary sequence of the 739-amino acid hSEF as deduced by the full-length cDNA clone. The signal peptide and transmembrane domain are marked, and the IL-17 receptor-like region in the intracellular domain is underlined. Seven potential N-linked glycosylation sites are underlined in bold. The margins of two deletion constructs, {Delta}ICD and {Delta}ECD, are indicated. The estimated mature molecular mass is 79,689 Da. b, hydrophobicity plot and domain structure of hSEF. The plot was generated according to the coefficients proposed by Kyte and Doolittle (31). The lower panel shows the domain structure of SEF protein, in which one transmembrane (TM) region separates the ECD from the intracellular IL-17R-like domain (ICD). The regions marked with thick line indicate the putative signal peptide (SP) and transmembrane domain.

 

Cell Type and Tissue Distribution of hSEF Transcript—To validate the EC origin of this novel receptor, hSEF expression was measured by real-time quantitative PCR (TaqMan) in a cDNA panel derived from a variety of human primary cells and tissues (Fig. 2a). We found that hSEF is highly enriched in cultured HUVEC but found low or no expression in human primary coronary smooth muscle cells, peripheral blood leukocytes, macrophages, CD34+ progenitor cells, megakaryocytes, neutrophils, or erythroids (Fig. 2a, top panel). Interestingly, when a panel of human tissues was assessed for hSEF expression by TaqMan, hSEF mRNA was expressed in a variety of normal human tissues with highest levels in ovary and breast (Fig. 2a, bottom panel). This expression pattern suggests that hSEF may also be expressed in epithelial cells. To further validate this tissue distribution, a Northern blot containing poly(A)-enriched mRNA (1 µg) from a variety of human adult tissue was hybridized with a hSEF cDNA radiolabeled probe. The expression level of the hSEF transcripts was highest in kidney followed by heart, skeletal muscle, colon, and small intestine and barely detectable in brain, spleen, liver, placenta, and lung (Fig. 2b), which is broadly consistent with the TaqMan analysis (Fig. 2a, bottom panel).



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  		FIG. 2.
Cell type and tissue distribution of hSEF. a, quantitative real-time PCR (TaqMan) analysis of hSEF expression. hSEF expression was measured by TaqMan in a panel of cDNAs prepared from a variety of human primary cell types (top) or human tissues (bottom). Normalization was performed using the {beta}2-microglobulin mRNA levels as controls in the same reaction as described under "Experimental Procedures." b, Northern blot analysis of poly(A)+ mRNA from a variety of human tissues for SEF. One microgram of poly(A)-enriched mRNA from various human adult tissues was hybridized with hSEF cDNA radiolabeled probe. The hSEF probe identified one major and a minor mRNA species of 8.5 and 4.4 kilobases, respectively. The lower panel shows the same blot hybridized with {beta}-actin probe as a control. PBL, peripheral blood leukocytes; SMC, smooth muscle cells. Sk., skeletal.

 

Expression of hSEF in several highly vascularized tissues, such as kidney or heart, is consistent with the endothelial origin of hSEF; however, we were unable to confirm the EC expression pattern by in situ hybridization. It is possible that the mRNA expression level in tissue ECs is below the sensitivity limit of in situ hybridization method we used.

High expression levels of hSEF in human breast prompted us to further examine a variety of epithelial-rich normal and tumor tissues. Intriguingly, we observed that hSEF mRNA expression was localized to ductal epithelial cells in human salivary gland, seminal vesicle, and the collecting tubules of the kidney by in situ hybridization (Fig. 3). hSEF message is uniformly expressed in normal epithelial cells in the intercalated ducts of the salivary glands (both in the parotid and submandibular glands) (Fig. 3a) and the collecting ducts in kidney (Fig. 3c). In the seminal vesicle, hSEF is expressed in scattered solitary cells in the connective tissue stroma and in the epithelium (Fig. 3b). The precise identity of those cells is currently unknown.



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  		FIG. 3.
Expression of hSEF mRNA in the ductal epithelial cells of a variety of human tissues determined by in situ hybridization. Localization of hSEF (antisense probe) to the ductal epithelial cells (purple stain) was observed in human salivary gland (a), seminal vesicle (b), and kidney collecting tubules (c). The sense probe yielded no detectable signal (not shown).

 

hSEF Expression in Human Breast Tumors and Breast Carcinoma Cell Lines—We also examined hSEF expression in a number of breast tumor samples with different pathologies by in situ hybridization. As shown in Fig. 4, samples of well differentiated ductal carcinomas of the breast expressed hSEF uniformly in the epithelial cells, whereas the connective tissue stroma was negative in all cases (Fig. 4, a and b). Less differentiated forms of ductal carcinomas expressed hSEF at varying levels (Fig. 4c); several of those cases were negative (data not shown). Variable, non-uniform expression of hSEF was found in other types of breast tumors analyzed, such as fibroadenoma, cribriform carcinoma, and mucus adenocarcinoma (data not shown).



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  		FIG. 4.
Expression and localization of hSEF mRNA in a number of human breast tumors and breast carcinoma cell lines by in situ hybridization. Hybridization of an antisense RNA probe of hSEF showed specific signal in human breast ductal carcinomas (a and b), lobular carcinoma (c), or breast carcinoma cell lines MDA-231 (d), MDA-175 (e), or SKBR-3 (f). The sense probe yielded no detectable signal (not shown).

 

hSEF expression was also studied in three human breast carcinoma cell lines (DAKO HercepTest). These cell lines express the HER-2/neu receptor at various levels scored at 3+ (SKBR-3), 1+ (MDA-175), and 0 (MDA-231) (33). hSEF expression, which showed high cell-to-cell variations, correlates well with HER-2/neu oncogene expression in these breast carcinoma cell lines (Fig. 4, d–f). That is, MDA-231 (grade 0, no expression of HER-2/neu) has the weakest hSEF expression (Fig. 4d), whereas SKBR-3 (grade +3 for high expression of HER-2/neu) has the strongest hSEF expression (Fig. 4f). MDA-175 (grade +1 for intermediate expression of HER-2/neu) displayed moderate hSEF expression as compared with SKBR-3 cells (Fig. 4e).

Taken together these expression studies indicate that hSEF is expressed in certain epithelial cell type in vivo and may play a role in the pathology of certain tumors of epithelial origin. In addition, reproducible evidence of hSEF expression is seen in primary cultured human endothelial cells.

hSEF Is Capable of Forming a Homomeric Complex and Interacts with FGFR1—To elucidate the functions of hSEF, several epitope-tagged expression plasmids were constructed to produce the full-length (FL), ICD-truncated ({Delta}ICD), or ECD-truncated ({Delta}ECD) version of hSEF (Fig. 1b). The FLAG epitope was added at the amino terminus followed by signal peptide cleavage site, whereas the Myc tag was joined at the carboxyl terminus for the detection of the recombinant protein expression. We first examined whether or not hSEF, behaving like a putative receptor, could be expressed and targeted to the cell surface. The FLAG.hSEF-FL, {Delta}ICD, or {Delta}ECD proteins were expressed by means of transient expression in human embryonic kidney 293T cells. Forty hours post-transfection, cells were collected and incubated with anti-FLAG M2 antibody followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody, then analyzed by flow cytometry. We found that expression of all three forms of recombinant hSEF proteins resulted in a shift of a population of fluorescein isothiocyanate-labeled cells by fluorescence-activated cell sorter analysis (data not shown), suggesting that hSEF indeed behaves like a receptor and is targeted to the cell surface.

Because families of cytokine receptors or growth factor receptor tyrosine kinases are often capable of forming dimeric or higher ordered structures (34, 35), and because hSEF is an apparent receptor, we hypothesized that oligomeric forms of hSEF may exist. The differential epitope-tagged versions of hSEF were singly or co-transfected in 293T cells, and we then examined their association by co-immunoprecipitation assays (Fig. 5a). Lysates of these cells were immunoprecipitated with anti-Myc antibody, and the precipitates were analyzed by immunoblotting with anti-FLAG antibody. An immunoreactive band of ~100 kDa recognized by the anti-FLAG antibody was observed in the anti-Myc immunoprecipitates from cells coexpressing hSEF-FL.Myc and FLAG.hSEF-FL proteins but not from cells coexpressing FLAG.hSEF-{Delta}ICD or -{Delta}ECD proteins in these assays (Fig. 5a). We did not observe an association between hSEF and human IL-1R1, suggesting specificity of the homomeric interactions between hSEF proteins (not shown). These results demonstrate that hSEF proteins are capable of forming oligomeric complexes. However, both the ECD and ICD of hSEF are required to maintain the stable homomeric interactions, at least in overexpressing cells.



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  		FIG. 5.
hSEF can form a homomeric complex and interacts with FGFR1 in transfected 293T cells. a, homomeric interaction of SEF proteins. The differential epitope-tagged constructs were singly or co-transfected in 293T cells as indicated. Forty hours post-transfection whole cell lysates were immunoprecipitated (IP) followed by Western blot (WB) using antibodies as indicated. Experiments were performed twice with similar results. b, hSEF binds to FGFR1. FLAG-tagged hSEF FL or deletion constructs ({Delta}ICD or {Delta}ECD) were transiently expressed in 293T cells with wild-type FGFR1. Forty hours after transfection, detergent lysates were immunoprecipitated with anti-FLAG antibody and then immunoblotted with anti-FGFR1 antibody.

 

Because zebrafish SEF was demonstrated to be co-immunoprecipitated with FGFRs from lysates of overexpressing COS cells (23), we next determined whether or not hSEF associates with human FGFR1 by co-transfection in 293T cells. As shown in Fig. 5b, hSEF-FL as well as deletion constructs of SEF, {Delta}ICD or {Delta}ECD, co-immunoprecipitated with human FGFR1. We also found that these associations remained equally efficient either in the absence or presence of FGF stimulation (not shown). These data suggest that the ECD or ICD alone is sufficient to establish the association between hSEF and FGFR1, and this interaction is independent of FGFR1 tyrosine kinase activation.

hSEF Blocks FGF-mediated Induction of a FGF-responsive Reporter Gene—The association between hSEF and FGFR1 prompted us to investigate whether or not hSEF can affect FGFR1-mediated signaling. One of the FGF target genes, the syndecan-1 gene, contains an upstream enhancer harboring an FiRE that has been well characterized and appears to be activated selectively by members of the FGF family but not by other tyrosine kinase receptor-activating growth factors (e.g. epidermal growth factor, platelet-derived growth factor, or insulin-like growth factor) (25). We utilized the FiRE from the syndecan-1 gene to create a luciferase reporter gene that could monitor FGF-activated signaling and gene expression. When transfected into 293T cells, this FiRE reporter manifests an approximate 3-fold increase in expression in the presence of bFGF. This bFGF-mediated stimulation was selectively inhibited by co-expression of hSEF in a dose-dependent manner (Fig. 6). Interestingly, the deletion of the IL-17-like ICD in hSEF-{Delta}ICD markedly reduced this inhibitory effect (Fig. 6). Therefore, overexpressed hSEF appears to suppress FGF-induced signal transduction, and this inhibitory effect is dependent upon on a functional intracellular IL-17R-like domain.



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  		FIG. 6.
Overexpression of hSEF results in the blocking of basic FGF-mediated induction of a FGF-responsive reporter gene in human 293T cells. 293T cells were transfected with a luciferase reporter gene driven by the FiRE of the mouse syndecan-1 gene (25) along with empty vector or 0.5 or 0.75 µg of FLAG.hSEF-FL or 0.75 µg of FLAG.hSEF-{Delta}ICD expression plasmid. After 20 h of serum starvation, transfected cells were treated with 50 ng/ml basic FGF for 6 h and subsequently lysed for the measurement of luciferase activity. Cell lysates were also immunoblotted by FLAG antibody to examine the protein expression level of hSEF-FL or hSEF-{Delta}ICD (data not shown).

 

hSEF Is a Negative Feedback Regulator of the FGF-induced ERK Activation—To further dissect the molecular inhibitory effect of hSEF on FGF-induced ERK signaling, 293T cells were transfected with empty vector or various expression plasmids of hSEF, as shown in Fig. 7. After serum starvation, cells were either left untreated or stimulated with bFGF for 10 min. Activation of ERK1/2 was determined by immunoblotting with a monoclonal antibody that specifically recognizes the activated (diphosphorylated) form of ERK1/2 (p42/p44 MAPK). Expression of hSEF-FL and the ECD truncated hSEF-{Delta}ECD, but not ICD deletion of hSEF ({Delta}ICD), resulted in a reduction in the levels of activated ERK1/2, whereas total ERK1/2 protein remained unaltered (Fig. 7a). This hSEF-mediated attenuation of FGF-induced ERK activation is consistent with the reduction in FGF-induced reporter gene activity seen previously (Fig. 6). Interestingly, immunoblotting with a monoclonal antibody that recognizes the upstream activated (phosphorylated) forms of MEK1/2 showed that hSEF overexpression had no effect on FGF-induced MEK activation (Fig. 7b). These data indicate that the targeting site of hSEF, at least in 293T cells, is likely to be downstream (or at the level) of MEK1/2 and upstream (or at the level of) of ERK1/2 (Fig. 7, a and b). Overall, our findings are broadly consistent with the molecular action of zebrafish SEF as previously reported (22, 23), although the precise molecular mechanism underlying the hSEF-dependent blocking of FGF signaling is currently unknown.



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  		FIG. 7.
hSEF attenuates FGF-induced ERK phosphorylation in the absence of upstream MEK inhibition. a, the intracellular domain of hSEF is critical for its inhibitory effect on FGF-induced ERK activation. 293T cells were transfected with empty vector or hSEF expression constructs as indicated. After serum starvation for 20 h, transfected cells were stimulated with bFGF (50 ng/ml) for 10 min followed by lysis and immunoblotting (WB) for activated phosphorylated ERK1/2 (p-ERK1/2), pan ERK, or anti-FLAG antibodies to determine expression of hSEF (not shown, similar to Fig. 3). b, FGF-induced MEK phosphorylation is not affected by hSEF. 293T cells were transfected and stimulated with FGF as described above, cell lysates were immunoblotted with antibodies to phosphorylated MEK (p-MEK), and pan MEK, respectively.

 

Recently, a family of negative regulators of receptor tyrosine kinase signaling, Sprouty proteins, have been identified in fly and mammals (36–40). Both epidermal growth factor and FGF up-regulate the expression of Sprouty genes (41, 42). Interestingly, we reproducibly observed that hSEF mRNA expression was induced upon epidermal growth factor and FGF stimulation in HUVEC (data not shown). This supports the hypothesis that hSEF is a feedback-induced antagonist of FGFR1/ERK-mediated FGF signaling. It had been previously demonstrated that membrane translocation of Sprouty proteins is necessary for their phosphorylation, which is essential for their inhibitory activity (43, 44). However, unlike Sprouty, hSEF is already membrane-anchored and interacts with FGFR1. In addition, we did not observe tyrosine phosphorylation of hSEF in response to growth factor stimulation (not shown).

Because members of the IL-17 cytokine family are expanding (11) and because hSEF shares sequence homology with IL-17R, we tested whether or not hSEF can bind to members of IL-17 cytokine family in a binding assay. However, incubation of the recombinant ECD of hSEF-IgG fusion protein with individual recombinant IL-17-like cytokines failed to show an association in a protein A-agarose pull-down assay (data not shown). This study does not exclude a role for hSEF in IL-17R-mediated functions; however, together with data that hSEF can inhibit FGF signaling, these studies suggest that the functions of hSEF in vivo are complex.

Is Human SEF Involved in Tumorigenesis?—Given the negative feedback nature of hSEF on growth factor-receptor tyrosine kinase signaling, it is conceivable that a defect (e.g. loss-of-function mutation) in the hSEF gene might lead to unchecked, malignant cell proliferation. Consistent with this hypothesis, the hSEF gene is located on chromosome region 3p14 (www.ensembl.org) where defined translocations have been identified in a number of human malignancies (45, 46). For instance, a tumor suppressor locus for renal cell carcinoma was physically and functionally mapped within human chromosome region 3p14 (47, 48). This raises the possibility that the hSEF gene may be a viable candidate for this tumor suppressor locus.

In summary, we have identified a novel FGFR negative feedback regulator encoded by a type I transmembrane receptor. Based on sequence and functional similarities, this gene appears to be the human homologue of a recent discovered zebrafish SEF and may function as a critical modulator for receptor tyrosine kinase-mediated biology such as cell proliferation, migration, and angiogenesis.


   FOOTNOTES
 
* This work was supported by a NHLBI, National Institutes of Health Award HL-62823 (to J. N. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Cardiovascular Research, Millennium Pharmaceuticals, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 650-246-7344; Fax: 650-244-9270; E-mail: rueybing{at}yahoo.com. Present address: Institute of Biomedical Sciences, Academia Sinica, 128 Academia Road, Sec. 2, Taipei 115, Taiwan, Republic of China.

1 The abbreviations used are: IL-17, interleukin-17; IL-17R, IL-17 receptor; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; EC, endothelial cells; FGF, fibroblast growth factor; bFGF, basic FGF; FGFR, FGF receptor; FiRE, FGF-inducible response element; HUVEC, human umbilical vein endothelial cells; hSEF, human SEF; ECD, extracellular domain; ICD, intracellular domain; FL, full-length. Back


   ACKNOWLEDGMENTS
 
We thank Drs. James E. Tomlinson, Nancy Stagliano, and Nicole Avitahl for advice and help on quantitative real-time PCR analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Lubberts, E., Joosten, L. A., van de Loo, F. A., van den Gersselaar, L. A., and van den Berg, W. B. (2000) Arthritis Rheum. 43, 1300–1306[CrossRef][Medline] [Order article via Infotrieve]
  2. Chabaud, M., Lubberts, E., Joosten, L., van Den Berg, W., and Miossec, P. (2001) Arthritis Res. 3, 168–177[CrossRef][Medline] [Order article via Infotrieve]
  3. Albanesi, C., Scarponi, C., Sebastiani, S., Cavani, A., Federici, M., De Pita, O., Puddu, P., and Girolomoni, G. (2000) J. Immunol. 165, 1395–1402[Abstract/Free Full Text]
  4. Albanesi, C., Scarponi, C., Cavani, A., Federici, M., Nasorri, F., and Girolomoni, G. (2000) J. Invest. Dermatol. 115, 81–87[CrossRef][Medline] [Order article via Infotrieve]
  5. Matusevicius, D., Kivisakk, P., He, B., Kostulas, N., Ozenci, V., Fredrikson, S., and Link, H. (1999) Mult. Scler. 5, 101–104[Abstract/Free Full Text]
  6. Van Kooten, C., Boonstra, J. G., Paape, M. E., Fossiez, F., Banchereau, J., Lebecque, S., Bruijn, J. A., De Fijter, J. W., Van Es, L. A., and Daha, M. R. (1998) J. Am. Soc. Nephrol. 9, 1526–1534[Abstract]
  7. Loong, C. C., Lin, C. Y., and Lui, W. Y. (2000) Transplant. Proc. 32, 1773 (abstr.)[CrossRef][Medline] [Order article via Infotrieve]
  8. Antonysamy, M. A., Fanslow, W. C., Fu, F., Li, W., Qian, S., Troutt, A. B., and Thomson, A. W. (1999) Transplant. Proc. 31, 93 (abstr.)[CrossRef][Medline] [Order article via Infotrieve]
  9. Antonysamy, M. A., Fanslow, W. C., Fu, F., Li, W., Qian, S., Troutt, A. B., and Thomson, A. W. (1999) J. Immunol. 162, 577–584[Abstract/Free Full Text]
  10. Aggarwal, S., and Gurney, A. L. (2002) J. Leukocyte Biol. 71, 1–8[Abstract/Free Full Text]
  11. Moseley, T. A., Haudenschild, D. R., Rose, L., and Reddi, A. H. (2003) Cytokine Growth Factor Rev. 14, 155–174[CrossRef][Medline] [Order article via Infotrieve]
  12. Shi, Y., Ullrich, S. J., Zhang, J., Connolly, K., Grzegorzewski, K. J., Barber, M. C., Wang, W., Wathen, K., Hodge, V., Fisher, C. L., Olsen, H., Ruben, S. M., Knyazev, I., Cho, Y. H., Kao, V., Wilkinson, K. A., Carrell, J. A., and Ebner, R. (2000) J. Biol. Chem. 275, 19167–19176[Abstract/Free Full Text]
  13. Li, H., Chen, J., Huang, A., Stinson, J., Heldens, S., Foster, J., Dowd, P., Gurney, A. L., and Wood, W. I. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 773–778[Abstract/Free Full Text]
  14. Lee, J., Ho, W. H., Maruoka, M., Corpuz, R. T., Baldwin, D. T., Foster, J. S., Goddard, A. D., Yansura, D. G., Vandlen, R. L., Wood, W. I., and Gurney, A. L. (2001) J. Biol. Chem. 276, 1660–1664[Abstract/Free Full Text]
  15. Hymowitz, S. G., Filvaroff, E. H., Yin, J. P., Lee, J., Cai, L., Risser, P., Maruoka, M., Mao, W., Foster, J., Kelley, R. F., Pan, G., Gurney, A. L., de Vos, A. M., and Starovasnik, M. A. (2001) EMBO J. 20, 5332–5341[CrossRef][Medline] [Order article via Infotrieve]
  16. Starnes, T., Robertson, M. J., Sledge, G., Kelich, S., Nakshatri, H., Broxmeyer, H. E., and Hromas, R. (2001) J. Immunol. 167, 4137–4140[Abstract/Free Full Text]
  17. Cines, D. B., Pollak, E. S., Buck, C. A., Loscalzo, J., Zimmerman, G. A., McEver, R. P., Pober, J. S., Wick, T. M., Konkle, B. A., Schwartz, B. S., Barnathan, E. S., McCrae, K. R., Hug, B. A., Schmidt, A. M., and Stern, D. M. (1998) Blood 91, 3527–3561[Free Full Text]
  18. Topper, J. N., and Gimbrone, M. A., Jr. (1999) Mol. Med. Today 5, 40–46[CrossRef][Medline] [Order article via Infotrieve]
  19. Chavakis, E., and Dimmeler, S. (2002) Arterioscler. Thromb. Vasc. Biol. 22, 887–893[Abstract/Free Full Text]
  20. Ross, R. (1999) N. Engl. J. Med. 340, 115–126[Free Full Text]
  21. Yang, R. B., Ng, C. K., Wasserman, S. M., Colman, S. D., Shenoy, S., Mehraban, F., Komuves, L. G., Tomlinson, J. E., and Topper, J. N. (2002) J. Biol. Chem. 277, 46364–46373[Abstract/Free Full Text]
  22. Furthauer, M., Lin, W., Ang, S. L., Thisse, B., and Thisse, C. (2002) Nat. Cell Biol. 4, 170–174[CrossRef][Medline] [Order article via Infotrieve]
  23. Tsang, M., Friesel, R., Kudoh, T., and Dawid, I. B. (2002) Nat. Cell Biol. 4, 165–169[CrossRef][Medline] [Order article via Infotrieve]
  24. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403–410[CrossRef][Medline] [Order article via Infotrieve]
  25. Jaakkola, P., Vihinen, T., Maatta, A., and Jalkanen, M. (1997) Mol. Cell. Biol. 17, 3210–3219[Abstract]
  26. DiChiara, M. R., Kiely, J. M., Gimbrone, M. A., Lee, M. E., Perrella, M. A., and Topper, J. N. (2000) J. Exp. Med. 192, 695–704[Abstract/Free Full Text]
  27. Yang, R. B., Mark, M. R., Gray, A., Huang, A., Xie, M. H., Zhang, M., Goddard, A., Wood, W. I., Gurney, A. L., and Godowski, P. J. (1998) Nature 395, 284–288[CrossRef][Medline] [Order article via Infotrieve]
  28. Komuves, L. G., Hanley, K., Lefebvre, A. M., Man, M. Q., Ng, D. C., Bikle, D. D., Williams, M. L., Elias, P. M., Auwerx, J., and Feingold, K. R. (2000) J. Invest. Dermatol. 115, 353–360[CrossRef][Medline] [Order article via Infotrieve]
  29. Komuves, L. G., Feren, A., Jones, A. L., and Fodor, E. (2000) J. Histochem. Cytochem. 48, 821–830[Abstract/Free Full Text]
  30. Stelnicki, E. J., Komuves, L. G., Kwong, A. O., Holmes, D., Klein, P., Rozenfeld, S., Lawrence, H. J., Adzick, N. S., Harrison, M., and Largman, C. (1998) J. Invest. Dermatol. 110, 110–115[Medline] [Order article via Infotrieve]
  31. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–132[CrossRef][Medline] [Order article via Infotrieve]
  32. Sonnhammer, E. L., Eddy, S. R., and Durbin, R. (1997) Proteins 28, 405–420[CrossRef][Medline] [Order article via Infotrieve]
  33. Brown, R. E. (2002) Ann. Clin. Lab. Sci. 32, 12–21[Abstract/Free Full Text]
  34. Davies, D. R., and Wlodawer, A. (1995) FASEB J. 9, 50–56[Abstract]
  35. Sun, P. D., and Davies, D. R. (1995) Annu. Rev. Biophys. Biomol. Struct. 24, 269–291[CrossRef][Medline] [Order article via Infotrieve]
  36. Kramer, S., Okabe, M., Hacohen, N., Krasnow, M. A., and Hiromi, Y. (1999) Development 126, 2515–2525[Abstract]
  37. Hacohen, N., Kramer, S., Sutherland, D., Hiromi, Y., and Krasnow, M. A. (1998) Cell 92, 253–263[CrossRef][Medline] [Order article via Infotrieve]
  38. Tefft, J. D., Lee, M., Smith, S., Leinwand, M., Zhao, J., Bringas, P., Jr., Crowe, D. L., and Warburton, D. (1999) Curr. Biol. 9, 219–222[CrossRef][Medline] [Order article via Infotrieve]
  39. de Maximy, A. A., Nakatake, Y., Moncada, S., Itoh, N., Thiery, J. P., and Bellusci, S. (1999) Mech. Dev. 81, 213–216[CrossRef][Medline] [Order article via Infotrieve]
  40. Minowada, G., Jarvis, L. A., Chi, C. L., Neubuser, A., Sun, X., Hacohen, N., Krasnow, M. A., and Martin, G. R. (1999) Development 126, 4465–4475[Abstract]
  41. Sasaki, A., Taketomi, T., Wakioka, T., Kato, R., and Yoshimura, A. (2001) J. Biol. Chem. 276, 36804–36808[Abstract/Free Full Text]
  42. Gross, I., Bassit, B., Benezra, M., and Licht, J. D. (2001) J. Biol. Chem. 276, 46460–46468[Abstract/Free Full Text]
  43. Hanafusa, H., Torii, S., Yasunaga, T., and Nishida, E. (2002) Nat. Cell Biol. 4, 850–858[CrossRef][Medline] [Order article via Infotrieve]
  44. Impagnatiello, M. A., Weitzer, S., Gannon, G., Compagni, A., Cotten, M., and Christofori, G. (2001) J. Cell Biol. 152, 1087–1098[Abstract/Free Full Text]
  45. Kovacs, G., Brusa, P., and De Riese, W. (1989) Int. J. Cancer 43, 422–427[Medline] [Order article via Infotrieve]
  46. Pathak, S., Strong, L. C., Ferrell, R. E., and Trindade, A. (1982) Science 217, 939–941[Abstract/Free Full Text]
  47. Sanchez, Y., el-Naggar, A., Pathak, S., and Killary, A. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3383–3387[Abstract/Free Full Text]
  48. Julicher, K., Marquitan, G., Werner, N., Bardenheuer, W., Vieten, L., Brocker, F., Topal, H., Seeber, S., Opalka, B., and Schutte, J. (1999) J. Natl. Cancer Inst. 91, 1563–1568[Abstract/Free Full Text]

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