Perlecan Protein Core Interacts with Extracellular Matrix Protein 1 (ECM1), a Glycoprotein Involved in Bone Formation and Angiogenesis*

Maurizio MongiatDagger §, Jian FuDagger §, Rachel OldershawDagger , Robert GreenhalghDagger , Allen M. Gown, and Renato V. IozzoDagger ||**

From the Dagger  Department of Pathology, Anatomy and Cell Biology and the || Cellular Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and  PhenoPath Laboratories, Seattle, Washington 98103

Received for publication, October 15, 2002, and in revised form, February 4, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to discover novel partners for perlecan, a major heparan sulfate proteoglycan of basement membranes, and to examine new interactions through which perlecan may influence cell behavior. We employed the yeast two-hybrid system and used perlecan domain V as bait to screen a human keratinocyte cDNA library. Among the strongest interacting clones, we isolated a ~1.6-kb cDNA insert that encoded extracellular matrix protein 1 (ECM1), a secreted glycoprotein involved in bone formation and angiogenesis. The sequencing of the clone revealed the existence of a novel splice variant that we name ECM1c. The interaction was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells, and the specific binding site within each molecule was identified employing various deletion mutants. The C terminus of ECM1 interacted specifically with the epidermal growth factor-like modules flanking the LG2 subdomain of perlecan domain V. Perlecan and ECM1 were also co-expressed by a variety of normal and transformed cells, and immunohistochemical studies showed a partial expression overlap, particularly around dermal blood vessels and adnexal epithelia. ECM1 has been shown to regulate endochondral bone formation, stimulate the proliferation of endothelial cells, and induce angiogenesis. Similarly, perlecan plays an important role in chondrogenesis and skeletal development, as well as harboring pro- and anti-angiogenic activities. Thus, a physiological interaction could also occur in vivo during development and in pathological events, including tissue remodeling and tumor progression.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Perlecan is a modular heparan sulfate proteoglycan harboring five distinct domains that share homology with laminin, the neural cell adhesion molecule and the low-density lipoprotein receptor (1-6). Perlecan is an intrinsic constituent of basement membranes (7) and it can self-assemble into dimers and oligomers (8). It is expressed quite early during development being deposited at the two-cell embryo stage (9) and during acquisition of attachment competence (10). In adulthood, it is present in all the basement membranes and it is richly deposited along the vessel walls where, together with other extracellular matrix molecules, maintains structural integrity in sites of high pressure (1, 11-15). Perlecan has also been detected at the cell surface (16, 17) where it is likely to bind through a member of the integrin family (18-20), an adhesion modulated by glycosaminoglycans (21). Perlecan null mice are embryonic lethal at day 10.5. Surviving mice die shortly after birth because of vascular and cephalic abnormalities, and display chondrodysplasia with dyssegmental ossification of the spine (22, 23), which resembles the human dyssegmental dysplasia Silverman-Handmaker type (24, 25). Notably, perlecan null animals exhibit a high incidence of malformations of the cardiac outflow tract with complete transposition of the great vessels (26), further stressing a central role for perlecan in vasculogenesis.

Perlecan is involved in controlling cell proliferation, thrombosis, tumorigenesis, and angiogenesis (20, 27-30), but the mechanisms and pathways regulating these effects are not completely understood. Its expression is increased in breast and colon carcinomas (27) and metastatic melanomas (31). The effects exerted by perlecan are thought to be in part indirect, because of its ability to bind several molecules via the heparan sulfate chains (32) and the protein core (33). Perlecan heparan sulfate binds several signaling molecules including various members of the fibroblast growth factor (FGF)1 family and the platelet-derived growth factor (34-36). The binding of FGF2 to perlecan promotes receptor activation and mitogenesis (37), and increases blood vessel formation to a higher extent than that induced by FGF2-heparin complexes (37). Recent studies have shown that perlecan protein core binds to several extracellular matrix and growth-promoting proteins including: fibronectin (38), laminin (39), collagen type IV (17, 36), fibulin-2 (36, 38), alpha -dystroglycan (40), platelet-derived growth factor (41), FGF7 (42), and FGF-binding protein (43). A similar distribution in a number of tissues and some aspects of the functionality are shared by extracellular matrix protein 1 (ECM1), a secreted glycoprotein containing six cysteine doublets, with a CC-(X7-10)-C pattern (44). This cysteine arrangement, which is also found in serum albumin and the sea urchin Endo16 protein (45, 46), leads to the formation of double-loop structures that are involved in protein/protein interactions (45, 47). Two splice variants have been described so far: ECM1a, encoded in humans by a 1.8-kb mRNA, and ECM1b, encoded by a 1.4-kb mRNA lacking exon 7 (48). ECM1a is expressed by the highly vascularized heart and placenta. It is also present in liver, ovary, kidney, lung, pancreas, testis, muscle, and colon, while ECM1b expression is confined to skin and tonsils (48, 49).

ECM1 has been shown to play an important role in endochondral bone formation by inhibiting alkaline phosphatase activity and bone mineralization (50), and to promote blood vessel formation in the chicken chorioallantoic membrane assay (51). ECM1 can be detected in close association with the embryonic endothelial cells and has been shown to trigger the proliferation of endothelial cells in vitro (51). More recently, ECM1 has been associated to the lipoid proteinosis, an autosomal recessive disorder characterized by thickening of the skin, mucosae, and certain viscera (52). Loss of functionality of this ECM1, induced by point mutations, leads to thickening of the basement membrane at the dermal-epidermal junction, around blood vessels and adnexal epithelia.

In this paper we discovered a specific binding of the perlecan C terminus to the corresponding C terminus of ECM1, we identified a novel splice variant (ECM1c), and demonstrated co-expression and partial co-distribution of these two molecules in both cells and tissues. We propose that perlecan/ECM1 interactions could be physiological and could potentially influence endochondral bone formation, angiogenesis, and tumorigenesis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Library Screening-- To lessen the number of false positives we employed the MATCHMAKER GAL4 Two-hybrid system 3 (Clontech, Palo Alto, CA), which adopts three independent reporter genes for the selection, His, Ade, and alpha - or beta -galactosidase. A keratinocyte library (Clontech) constructed in the pACT2 vector was amplified and the plasmids were extracted employing the NucleoBond AX Giga plasmid purification kit (Clontech). Perlecan domain V was subcloned into the pGBKT7 vector from a previous construct available in our laboratory (42) using EcoRI and BamHI restriction sites and the construct was used as a bait to screen the keratinocyte library (complexity ~0.5 × 106). The transfected yeast cells were plated onto quadruple minus plates lacking Trp and Leu to select for the presence of both plasmids, and His and Ade that are selective for the putative interactions. The yeast cell clones growing in selective medium were re-plated in quadruple minus plates containing X-alpha -gal. The plasmids from the blue colonies were extracted using the Yeast Plasmid Isolation Kit (Bio 101, Carlsbad, CA) and employed to transfect DH5alpha cells. The pACT2 clones only would grow on ampicillin LB plates. The plasmids were extracted and the inserts were analyzed on agarose gels after restriction digestion with BglII. The inserts were identified by automatic sequencing as described before (43). For the alpha -galactosidase assays, 100-µl aliquots of a X-alpha -gal solution (20 mg/ml) in N,N-dimethylformamide were spread in 10-cm quadruple minus plates. The yeast clones transfected with putative interactive molecules would turn blue (the alpha -galactosidase is a secreted enzyme unlike the beta -galactosidase). For beta -galactosidase assays, the grown yeast colonies were transferred onto Whatman 3MM paper filters, soaked in Z buffer/X-gal solution (0.1 M Na2HPO4, 45 mM NaH2PO4, 10 mM KCl, 2 mM MgSO4, 0.3% beta -mercaptoethanol, and 3.3 mg/ml 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside), frozen in liquid nitrogen for 10 s and incubated at 30 °C for ~8 h to visualize the blue colonies.

Analysis of the ECM1c Splice Variant by RT-PCR and Real Time PCR-- To verify that the ECM1c variant was actually being expressed, RNA from HT1080 and A431 cancer cell lines was extracted employing the TRI-Reagent (Sigma). The RNA was treated with RNase-free DNase for 10 min at 37 °C and reverse transcribed using the avian myeloblastosis virus reverse transcriptase (Promega) and random primers. The following oligonucleotides were used in the RT-PCR: forward 5'-CTCTGAGGGAGGCTTCACG-3' and reverse 5'-CGGGATCCATAGTTGGGGTAAGGAGCC-3'. For the nested PCR the reverse oligonucleotide 5'-ATCTCCTCCTGATATGGAAGAGGT-3' specific for the splice variant was used. The PCR products were then analyzed on agarose gels. Real time PCR was performed on the RNA samples as well. Ten serial 1:1 dilutions of the extracted RNAs were carried out and the samples were reverse transcribed as before. The following oligonucleotides were synthesized: forward 5'-ACCAAAGAGTTCTCACCAAGCA-3' and reverse 5'-GCAGTCATGTTGTGGATCAGC-3', common to all three variants, and forward 5'-GCACCCCAATGAACAGAAGGA-3' and reverse 5'-CGGCATGGAATGGATGATTATC-3', specific of the ECM1c variant. beta -Actin or glyceraldehyde-3-phosphate dehydrogenase were used as a control. The experiments were performed using the QuantiTectTM SYBR Green PCR kit (Qiagen) and the PCR was performed using an ABI PRISMTM 7700 Sequence Detector (Applied Biosystems, Forster City, CA). The real time PCRs were run in 2% agarose gels to confirm the specificity of the fluorescent detection system. The relative amounts of ECM1c were identified using the comparative method (ABI PRISM 7700 sequence bulletin 2) employing the formula 2 - Delta Delta CT, where the Delta CT was determined by subtracting the average beta -actin CT (threshold cycle) value from the average ECM1c CT value. The Delta Delta CT value was finally calculated by subtracting the Delta CT calibration value (Delta CT of all the ECM1 splice variants). A validation experiment was performed prior to the calculations and the absolute value of the slope of the log input amounts of RNA used versus Delta CT values was found to be <0.1. For the comparative expression of ECM1 and perlecan in different cell lines the following oligonucleotides were used: forward 5'-GGATACCCTTGACAAATACTGTG-3' and reverse 5'-ATTCTTCCTTGGGCTCAGA-3', forward 5'-GCGGGTGAATGGTGGAC-3' and reverse 5'-AGGGAAGGCGAGGAAGC-3', respectively. RNA was extracted from the different cell lines and reverse transcribed as described previously. The amplified products were separated onto 1% agarose gels.

Yeast Two-hybrid Constructs-- Seven deletions of perlecan domain V were cloned into the pGBKT7 vector. For deletions Delta 1, Delta 2, and Delta 3, the following forward primer was used: 5'-GGAATTCGAGATCAAGATCACCTTC-3'. The reverse oligonucleotides for these deletions were: Delta 1 5'-CGGGATCCATGTCCAGAGCCTTGTTG-3', Delta 2 5'-CGGGATCCTGTCACACCTTCCTCACA-3', and Delta 3 5'-CGGGATCCGGTGGGGCAGTGGGAGAT-3'. The forward oligonucleotide for deletion Delta 4 was 5'-GGAATTCTGTCGGGACCGGCCCTGC-3' and the reverse primer corresponded to the one used for deletion Delta 2. For deletion Delta 5 the forward oligonucleotide was 5'-GGAATTCGTGACCACCCCCTCGCTG-3' and the reverse primer corresponded to the one used for deletion Delta 1. The reverse primer for deletions Delta 6 and Delta 7 was 5'-CGGGATCCCGAGGGGCAGGGGCGTGT-3', and the normal nucleotides were 5'-GGAATTCTGTGAGCGCCAGCCTTGC-3' and 5'-GGAATTCGGCATAGCAGAGTCCGAC-3', respectively. In all the forward nucleotides an EcoRI site was introduced and a BamHI site introduced in the reverse primers. The ECM1 construct was subcloned into the pGADT7 and pGBKT7 vectors by PCR using the pACT2 construct as a template. The forward primer was 5'-GGAATTCCCCGAAGCCTCCCCATGGAT-3', which introduced an EcoRI site, and the reverse primer was 5'-CGGGATCCCCAAATCCAAGAGGTGTTTAGG-3', which introduced a BamHI site. The forward primers used to amplify the deletion fragments of ECM1 were 5'-GGAATTCGGAACGCCAGCTCCATTT-3', 5'-GGAATTCTGGGAGGAAGCAATGAGC-3', 5'-GGAATTCTGGGAGGATACCCTTGACAAA-3', 5'-GGAATTCGACCGGGACATCTTGACCAT-3' for deletions Delta E1, Delta E2, Delta E3, Delta E4, respectively. For deletion Delta E5 the same oligonucleotide used to amplify the entire ECM1 fragment was employed and the reverse primer was 5'-CGGGATCCATAGTTGGGGTAAGGAGCC-3'. For all the PCR amplifications the GC-Rich Taq polymerase (Roche Molecular Diagnostics GmbH) was used.

Co-immunoprecipitation and Western Blotting-- To corroborate the interaction between ECM1 and perlecan domain V we performed co-immunoprecipitation studies. The two proteins were in vitro transcribed and translated in the presence of [35S]methionine (ICN Pharmaceuticals Inc., Costa Mesa, CA) employing the TNTTM reticulocyte lysate system (Promega). One µg of the pGBKT7 or pGADT7 constructs was employed and the reactions were incubated for 90 min at 30 °C and performed according to the manufacturer's instructions in a total volume of 50 µl. An amount of 15 µl of each translated protein was employed for the co-immunoprecipitation, the proteins were co-incubated for 1 h at 30 °C and 1 ml of co-immunoprecipitation buffer was added (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 5 mg/ml aprotinin, 0.5 mM phenylmethylsulfoniyl fluoride, and 0.1% Tween 20 (v/v)). About 2 µg of the anti-hemagglutinin antibody (the pGADT7 vector carries the hemagglutinin tag sequence) or the anti-c-Myc antibody (the pGBKT7 vector carries the c-Myc tag sequence) were added and the samples were incubated for 18 h at 4 °C with constant rocking. The immune complexes were captured with protein A/G-agarose beads (Pierce). The beads were washed three times with HNTG buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 200 µM Na3VO4, 20 mM NaF), and an EDTA-free protease inhibitor mixture (Roche Molecular Diagnostics GmbH). The bands were separated in polyacrylamide gels and, after fixation, enhanced with AMPLIFYTM (Amersham Biosciences AB). The gels were then exposed to Eastman Kodak films.

To investigate the interaction between ECM1 and perlecan in mammalian cells we stably transfected 293 EBNA cells (human embryonic kidney cells expressing the Epstein-Barr virus nuclear antigen) with 4 µg of pCEP-Pu vector harboring the full-length domain V as described before (53). The EBNA cells allow high episomal replication of the transfected pCEP-Pu vector. Stable clones were obtained by culturing cells in puromycin (1 µg/ml) for at least 4 weeks. Following verification of domain V expression by enzyme-linked immunosorbent assay and microsequencing (53), three domain V-expressing clones were pooled together and transiently transfected with a full-length ECM1-containing pcDNA3.1 plasmid (Invitrogen), harboring the BM-40 signal peptide and a polyhistidine tag at the C terminus, using LipofectAMINE 2000 (Invitrogen). As a further control, we transfected wild type 293-EBNA cells with the ECM1 construct as above. After 48 h of incubation, the serum-free conditioned medium was collected and filtered, and a mixture of protease inhibitors (CompleteTM, Roche Molecular Biochemicals) was added. Rabbit polyclonal anti-ECM1 antiserum (51) was added to 1 ml of conditioned medium (1:200 dilution), and the proteins were immunoprecipitated at 4 °C overnight with continuous rocking. Sepharose G beads (Amersham Biosciences) were added and incubated for another 4 h with rocking, and subsequently washed four times with 20 mM Hepes, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 200 µM Na3VO4, 10 mM NaF. The final pellet was boiled in Laemmli buffer and analyzed by SDS-PAGE. Western immunoblotting was performed using anti-His monoclonal antibodies (Qiagen), followed by horseradish-conjugated secondary antibody and chemiluminescence detection as described before (53).

Immunohistochemical Studies-- Frozen sections of human skin were briefly fixed in acetone, incubated in phosphate-buffered saline containing 0.3% H2O2 and 1% sodium azide for 10 min, blocked in 5% goat serum for 30 min and incubated with the primary antibody, either a rabbit polyclonal anti-ECM1 antiserum (51) or 7B5 mouse monoclonal antibody raised against domain III of human perlecan (12). Incubations were for either 1 or 18 h and dilutions, all in the presence of 5% goat serum, ranged between 1:200 and 1:500 for each antibody. The sections were washed again, incubated with goat anti-rabbit antibodies conjugated with horseradish peroxidase (Envision, DAKO), and finally developed with diaminobenzidine and counterstained with hematoxylin. Paraffin sections of skin were also tested after various antigen retrieval including pretreatment with 1% protease type XXIV, citrate antigen retrieval (S30), 0.1% Pronase (Calbiochem) for 20 min each, or without any pretreatments. Peroxidase blocking, serum blocking, and antibody incubations were the same as described above for the frozen sections. Images were captured with a Pixera digital camera and assembled using Adobe Photoshop version 6.0.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ECM1 Is a Novel Protein Interacting with Perlecan Protein Core-- Only recently the two-hybrid system has been employed to identify interactions among extracellular matrix proteins (43, 54-56). To identify new partners for the perlecan protein core, we employed perlecan domain V as a bait (Fig. 1A) and screened a human keratinocyte cDNA library. This domain is located at the C-terminal end of the protein core and harbors a tandem array of three laminin-G type (LG1-LG3) modules separated by four EGF-like (EG1-EG4) modules, in an arrangement highly conserved across species (1, 57, 58). Initially, domain V was cloned into the pGBK-T7 vector and was tested by itself to ensure that it did not activate the reporter genes HIS3 and ADE. Next, about 0.5 × 106 cDNAs were screened. To minimize the number of false positives, the double transfected yeast cells growing in minimal medium were replated in the same medium containing X-alpha -gal. The screening led to the isolation of ~100 blue colonies. The inserts, ranging between 0.5 and 4 kb, were analyzed either by PCR using specific primers or by restriction digestion. All inserts were sequenced and identified by homology searching with the NCBI BLAST program. More than 10% of the sequenced clones encoded secreted proteins including several extracellular matrix proteins, likely to bind perlecan. The rest of the cDNAs were false positives, being intracellular proteins such as keratins, highly expressed by keratinocytes, and transcription factors, which can independently activate the reporter genes. One of the fastest growing clones contained a ~1.6-kb cDNA insert (Fig. 1B) spanning between nucleotides 260 and 1822 of the ECM1 (Fig. 2). The cDNA isolated with the screening encoded for 487 amino acid residues beginning at residue 53 to the very end of the published sequence (48). ECM1 is a secreted glycoprotein first isolated from an osteogenic mouse cell line in concomitance with studies on bone matrix biology (49). ECM1 contains a 19-amino acid residue signal peptide and it has been divided into four distinct domains (Figs. 1C and 2). The first is a cysteine-free domain followed by the second and third repeat and by the C-terminal domain that contain the CC-(X7-10)-C arrangement of cysteine residues, typically observed in members of the albumin protein family. This pattern generates double-loop structures that are believed to be involved in protein/protein interactions. The ratio of acidic and basic amino acids is balanced; furthermore, the different double loops may have different binding affinities increasing the potential of interaction with a variety of biological ligands (47).


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Fig. 1.   ECM1 is a novel interacting partner for the perlecan protein core. A, schematic representation of the C-terminal perlecan domain V used as bait in the yeast two-hybrid screening. This domain consists of three laminin-G type (LG1-LG3) modules (orange ovals) separated by four EGF-like (EG1-EG4) modules (blue rectangles), in an arrangement highly conserved across species (1, 57, 58). B, agarose gel electrophoresis showing the isolation of the ~1.6-kb insert (ECM1) from the pACT-2 cDNA clone that interacted with perlecan domain V in the yeast two-hybrid screening. The construct was digested with the restriction enzyme BglII and the bands were separated into a 1% agarose gel. The migration of the markers in kb is indicated in the left margin. C, schematic representation of ECM1 and its three splice variants, with ECM1c being the novel splice variants described in this paper (see also Fig. 2). The different domains are color-coded and the key for each domain is provided in the right box. The numbers indicate the amino acid residues. Potential glycosylation sites are indicated.


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Fig. 2.   ECM1c sequence of the pACT-2 clone. Nucleotide and deduced amino acid sequence of human ECM1c (see NCBI accession number NM_004425.2). Vertical blue arrows indicate the beginning and end of the pACT-2 clone interacting with perlecan domain V that was identified during the library screening. The sequence of the novel exon 5a is highlighted in yellow. The horizontal arrows indicate the sequences of the oligonucleotides used in the real time PCR experiments (see Fig. 3). Potential glycosylation sites are underlined.

Identification of the ECM1c Splice Variant and Expression Studies-- Two splice variants of the molecule have been described so far, ECM1a and ECM1b, generating two transcripts of 1.8 and 1.4 kb, respectively. The smaller isoform lacks exon 7 (Fig. 3A). Interestingly, both isoforms are larger in the murine counterpart because of the fact that the sequence of exon 6 was not found in the human homologue. However, this exon appears to be conserved within the 5th intron of the human gene (48). The sequencing of the pACT2 clone of ECM1, isolated from our library screening, revealed the presence of an extra exon, which we named exon 5a (Figs. 2 and 3A). The peptide encoded by the resulting splice variant was highly homologous to the mouse sequence encoded by the murine exon 6 (Fig. 3A). To corroborate the presence of this new splice variant, we extracted RNA from two different cell lines, HT1080 and A431, and amplified an ECM1 fragment by RT-PCR. A nested PCR, employing an internal oligonucleotide was necessary to visualize the ECM1c splice variant, indicating that the expression of this splice form was very low, at least in these two cell lines (Fig. 3B). As a control no cDNA, derived from the first amplification, was added to the reaction. To support these findings and to calculate the relative amounts of this new splice variant, we performed real time PCR experiments. We used glyceraldehyde-3-phosphate dehydrogenase and/or beta -actin as a control. A representative example of an amplification plot is shown in Fig. 3C. The reduced normalized fluorescence values (Delta Rn) are plotted against the cycle value. The threshold cycle (CT) value, defined as the fractional cycle number at which the fluorescence passes the fixed threshold, is indicated in the plot by a bold line. The amplicons were run in 2% agarose gels and showed the bands of the expected size (Fig. 3D), indicating that the fluorescent readings were specific. Comparative analysis of the CT (see "Experimental Procedures") indicated that the amount of ECM1c RNA was ~15% of the total amount of ECM1 RNA in both cell lines. These data confirm the existence of a new ECM1c splice variant, which is expressed, albeit at low levels, in both cell lines. The level of expression between the two cell lines was comparable.


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Fig. 3.   ECM1c is a new splice variant. A, schematic representation of the ECM1 genomic organization. Boxes represent exons, in scale, and thin lines represent introns (not in scale). The new exon, named exon 5a, is in red, whereas exon 7, which is spliced out in ECM1b, is in blue. The 5'- and 3'-untranslated regions are in yellow. The lower rectangle contains the alignment of the two peptides coded by the human exon 5a and the corresponding murine exon 6. Identical and conserved amino acid residues are highlighted in green and cyan, respectively. The amino acid residues present only in the human counterpart are highlighted in yellow. B, agarose gel electrophoresis showing the nested PCR bands amplified using specific ECM1c oligonucleotides for the ECM1c splice variant. An RT-PCR was performed employing specific ECM1 oligonucleotides and RNA from the A431 cell line (lane 2) or the HT1080 cell line (lane 3), and subsequently re-amplified. As a control, the first amplification was not added to the sample (lane 4). Lane 1, molecular markers. C, semi-log amplification plot showing the change in fluorescence of the SYBR Green I dye plotted versus cycle number; the bold line represents the threshold from which the CT values are deducted. The code for each curve is explained in the right margin. D, photograph of a 2% agarose gel showing the amplicons obtained with the real time PCR. Lane 1, molecular markers. The same cell lines were used in this experiment, A431 (lanes 2-4) and HT1080 (lanes 6-8). The total amount of ECM1 RNA (lanes 3 and 7) was compared with the amount of ECM1c (lanes 4 and 8). beta -Actin was used as a positive control in each experiment (lanes 2 and 6). As a negative control, no cDNA was added to the reaction (lanes 5 and 9). E, agarose gel electrophoresis showing the expression of perlecan (upper panel) and ECM1 (lower panel) in different human cells using RT-PCR, including HT1080 fibrosarcoma cells (lane 2), umbilical vein endothelial cells (lane 3), dermal fibroblasts (lane 4), Saos2 osteosarcoma cells (lane 5), and MG63 osteosarcoma cells (lane 6). Molecular weight markers, lane 1; negative control, lane 7. F, agarose gel electrophoresis showing the expression of perlecan (upper panel) and ECM1 (lower panel) in keratinocytes (lane 2), dermal fibroblasts (lane 3), and negative control (lane 4). Molecular weight markers are shown in lane 1. The identity of each amplified band was confirmed by automatic sequencing.

To determine whether perlecan and ECM1 were co-expressed, we tested by RT-PCR a series of normal and malignant cells including fibrosarcoma cells, endothelial cells, dermal fibroblasts, keratinocytes, and two osteosarcoma cells. In every case, with the exception of Saos2 osteosarcoma cells, we found significant co-expression of both genes (Fig. 3, E and F). Thus, cells with a diverse histogenetic origin express ECM1 and perlecan.

Analysis of Perlecan/ECM1c Interactions-- To confirm that ECM1 alone did not trigger the growth of yeast cells, the construct was subcloned into the pGADT7 vector and used with the pGBKT7-53 construct (coding for the p53 molecule) to transfect the AH109 yeast strain. In double minus plates all transfectants grew, indicating that the cells were successfully transfected. On the contrary, only the cells transfected with the positive control were able to grow in minimal media (Fig. 4A), suggesting that the growth observed in the previous experiment was actually induced by the interaction of perlecan domain V with the ECM1 molecule. Next, the interaction was re-tested in this system using the pGADT7-ECM1 and the pGBKT7-domain V constructs in one-to-one interactions. As a further control, the molecules were employed as either a prey or bait. Several clones resulting from the transfection were replated in minimal media containing X-alpha -gal. The growth of the blue colonies and the intensity of the blue color were comparable with the positive control (Fig. 4, B-D).


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Fig. 4.   Specific interaction between perlecan domain V and ECM1. A, ECM1 alone could not trigger the growth of the transfected yeast cells in minimal media. The pGBKT7-Lam construct (containing the lamin c cDNA) was used as a partner for the interaction. All clones were able to grow in double minus media, which select for the presence of both plasmids (boxes 1-3). Box 1, positive control (pGBKT7-53/pGADT7-T); box 2, negative control (pGBKT7-Lam/pGADT7-T); box 3, pGADT7-ECM1/pGBKT7-Lam. The same transfectants were plated on quadruple minus media to check for putative interactions. Growth was observed with the positive control only (box 4); no growth was detected with the negative control (box 5) or with the ECM1 construct (box 6). B, specificity of the interaction was corroborated using the pGADT7 and pGBKT7 plasmids in which the ECM1c cDNA was subcloned. Growth was observed using ECM1 both as a "bait" and "prey" and was comparable with that of the positive control (pGADT7/pGBKT7-53). As a negative control the lamin c construct was used in combination with the ECM1 construct. The name of the respective constructs is indicated in the margins. C and D, beta -galactosidase assays performed on six independent clones using ECM1 as prey or bait, respectively. The pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T constructs were used as a positive (+) and negative (-) control, respectively.

Interaction of ECM1 and Perlecan Domain V in Cell-free Systems and in Mammalian Cells-- To corroborate the results obtained with the yeast two-hybrid system we performed co-immunoprecipitation experiments. The two proteins were in vitro transcribed and translated; as a negative control, we used an empty plasmid. The constructs confer to perlecan domain V a Myc epitope and to ECM1 a hemagglutinin epitope fused to their respective C termini. The translated proteins were analyzed on SDS-PAGE and the molecular weight of the bands corresponded to the predicted sizes of the molecules, i.e. ~81,000 for perlecan domain V and ~61,000 for the ECM1c construct (Fig. 5A, left panel). The anti-Myc monoclonal antibody was capable of co-precipitating both proteins (Fig. 5A, right panel). In contrast, the anti-hemagglutinin monoclonal antibody failed to do so (not shown), suggesting that the antibody might have interfered with the protein/protein interaction.


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Fig. 5.   Corroboration of perlecan domain V/ECM1 interaction. A, autoradiograph of a 10% SDS-PAGE (left panel) showing the in vitro transcribed and translated domain V of perlecan (~81 kDa) and ECM1 (~61 kDa), respectively, as indicated on the top margin. The two molecules were in vitro transcribed and translated using the pGBKT7-DV and pGADT7-ECM1 constructs and [35S]methionine as the labeled precursor. The right panel shows the co-immunoprecipitation of equimolar amounts of domain V and ECM1 using the anti-Myc antibody (the sequence for the Myc epitope is present in the pGBKT7 vector). In contrast no band could be detected when the primary antibody was omitted in the incubation medium (control). B, Western immunoblotting (WB) of media conditioned by 293-EBNA cells transfected with either ECM1 or domain V as indicated. Immunodetection was performed with anti-histidine (alpha His) antibody. The predicted bands of ~61 and 81 kDa for the respective proteins were detected. C, Western immunoblotting of media conditioned by 293-EBNA cells doubly transfected with ECM1- and domain V-containing vectors, following co-immunoprecipitation with the anti-ECM1 antiserum and detection with anti-His monoclonal antibody. The two lanes include media conditioned by the doubly transfected 293-EBNA cells in the absence or presence of fetal bovine serum, respectively. D, same experiment as in C but without co-transfection.

Because both domain V and ECM1 are cysteine-rich glycoproteins with several disulfide bonded residues, a modification that does not occur in yeast, we wanted to confirm the interaction using mammalian cells. We utilized the 293-EBNA cells, human embryonic kidney cells that harbor the Epstein-Barr virus nuclear antigen, that have been previously shown to allow the synthesis and proper glycosylation of a variety of extracellular matrix proteins, including portions of perlecan (53, 58-62). Following transfection with either ECM1 or domain V, the predicted bands of ~61 and 81 kDa for the respective proteins were secreted (Fig. 5B). When the cells were doubly transfected with ECM1- and domain V-containing vectors, the two proteins were co-immunoprecipitated by the anti-ECM1 antiserum and detected by Western immunoblotting with anti-His monoclonal antibody (Fig. 5C). In contrast, untransfected 293-EBNA cells showed no reactive bands (Fig. 5D). Collectively, these data corroborate the results obtained with the yeast two-hybrid system, and indicate that ECM1 is a novel interacting partner for the perlecan protein core.

ECM1 Interacts with the EGF-like Repeats of Perlecan Domain V-- The results of the co-immunoprecipitation studies described above indicated that the interaction identified with the yeast two-hybrid system was specific. Thus, we wanted to analyze in detail the precise site of interaction on the perlecan molecule. To this end, we generated eight deletions of domain V and cloned each one of them into the pGBKT7 vector (Fig. 6A). The plasmids were co-transfected with the ECM1 construct into AH109 cells and the cells were plated on selective media. Four independent clones from each transfection were replated in minimal media containing X-alpha -gal. Only three deletions (Delta 3, Delta 7, and Delta 8) failed to induce growth of the yeast cells (Fig. 6A). The other deletions induced growth and blue color of the yeast cells in a manner comparable with the positive control (Fig. 6B). These results clearly indicate that the sites of interaction reside within the EGF-like repeats flanking the LG2 domain, and further indicate that each individual LG module of perlecan is dispensable for the interaction.


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Fig. 6.   The sites of interaction with the ECM1 molecule reside within the EGF-like repeats of perlecan domain V. A, schematic representation of the full-length perlecan domain V and eight deletions, cloned into the pGBKT7 vector. The full-length pGADT7-ECM1 construct was used as prey. The name of the constructs, the number of the amino acid residues, and the semi-quantitative estimation of growth in selective media are indicated. B, alpha -galactosidase assays conducted on the perlecan domain V constructs. Four independent clones of each transfection were plated on quadruple minus plates containing X-alpha -gal. The pGBKT7- 53/pGADT7-T and the pGBKT7-Lam/pGADT7-T plasmids were used as positive and negative controls, respectively. The results clearly indicate that the ECM1 molecule has affinity for the EGF-like tandem repeats of perlecan domain V.

Perlecan Domain V Binds to the C-terminal Domain of ECM1-- Next, we determined the specific site of perlecan interaction within the ECM1 molecule. We used a strategy similar to that described above. We generated five deletions of ECM1, cloned all of them into the pGADT7 vector and used them to co-transfect cells with the full-length domain V pGBKT7 construct. All the constructs that contained the sequence of the C-terminal fourth domain of ECM1 were able to trigger the growth of the yeast cells and the production of alpha -galactosidase (Fig. 7, A and B), indicating that the interaction with perlecan domain V resides in this region. In fact, with deletion Delta E5, which lacks the terminal 135 amino acid residues (i.e. the fourth domain of ECM1), no growth of the transfected cells was observed (Fig. 7A). Thus, we conclude that the two C termini of perlecan and ECM1 are specifically interacting with each other.


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Fig. 7.   Perlecan domain V binds to the C-terminal domain of ECM1. A, schematic representation of the full-length ECM1 and its five deletion mutants cloned into the pGBKT7 vector. The full-length pGADT7-domain V construct was used as prey. The name of the constructs, the amino acid residue number, and the semiquantitative estimation of growth in selective media are indicated. B, alpha -galactosidase assays conducted on four independent clones for each transfectant. The pGBKT7-53/pGADT7-T and pGBKT7-Lam/pGADT7-T plasmids were used as positive and negative controls, respectively. For this experiment the transfected yeast cells were plated on quadruple minus plates containing X-alpha -gal. No growth or alpha -galactosidase expression was observed employing the ECM1 deletion Delta E5, indicating that the site of interaction resides in the C-terminal ECM1 domain.

Partial Expression Overlap between ECM1 and Perlecan in Skin-- Because both perlecan and ECM1 are widely expressed in human skin we chose to investigate the potential expression overlap of these two proteins in this organ. Frozen sections of human skin were reacted with either a rabbit polyclonal anti-ECM1 antiserum (51) or 7B5 mouse monoclonal antibody raised against domain III of human perlecan (12). The anti-ECM1 antibody has been raised against human recombinant ECM1, and recognizes both ECM1a and ECM1b splice variants. Thus, we can reasonably assume that this antiserum also recognizes ECM1c, because this splice variant harbors both ECM1a and ECM1b epitopes. ECM1 was widely distributed throughout the dermis with minimal expression in the epidermis (Fig. 8A). It was diffusely present around small blood vessels (Fig. 8A, arrows) and hair follicles (Fig. 8B). The reaction was specific because omission of the primary antibody failed to produce any signal (Fig. 8C). Perlecan was prominently expressed within the dermal basement membrane, at the dermal-epidermal junction and dermal blood vessels (Fig. 8D); however, perlecan epitopes were also observed as delicate punctuate deposits throughout the upper dermis. Perlecan expression was also prominent around hair follicle (Fig. 8E) and, as in the case for the ECM1 antiserum, omission of the primary antibody failed to generate any detectable signal (Fig. 8F). No significant signal was obtained with paraffin-embedded tissue, even after several attempts of antigen retrieval. Collectively, the immunolocalization studies confirm the secreted, extracellular nature of ECM1 and further indicate that perlecan protein core could potentially interact in vivo with ECM1 within restricted areas of the skin, including perivascular and perifollicular regions of the dermis, and diffusely within collagen-rich regions of the upper dermis.


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Fig. 8.   Partial expression overlap between perlecan and ECM1. Frozen sections of human skin were reacted with either a rabbit polyclonal anti-ECM1 antiserum (51) (A and B) or 7B5 mouse monoclonal antibody raised against domain III of human perlecan (12) (D and E). Notice that ECM1 is widely distributed throughout the dermis with minimal expression in the epidermis, and is present around dermal blood vessels (arrows) (A). ECM1 is also diffusely expressed around hair follicles (B). The reaction is antibody specific because removal of the primary antibody fails to produce any detectable signal (C). Notice that perlecan is expressed at the dermal epidermal junction and within dermal blood vessels (D); however, perlecan is also present as delicate punctuate deposits throughout the upper dermis (D), and more intensely around hair follicles (E). Removal of the primary antibody failed to generate any detectable signal (F). Primary antibody dilution was 1:500 for both the anti-ECM1 and anti-perlecan. Scale bars = 160 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The screening of a human keratinocyte library using perlecan domain V as bait led to the identification of a novel molecule interacting with the perlecan protein core. The construct of ECM1 consisted of a ~1.6-kb fragment encompassing nucleotides 260-1822 of the published sequence (48). Interestingly, a sequence within the human intron 5, which shares high homology with murine exon 6, was not spliced out in our cDNA. This sequence coded for an additional 19-amino acid residues thereby generating a novel splice variant, which we termed ECM1c. We performed RT-PCR experiments on different cell lines and found this isoform to be expressed by two transformed cell lines, the fibrosarcoma HT1080 and the squamous cell carcinoma A431. To determine whether perlecan and ECM1 were co-expressed, we tested by RT-PCR a series of normal and malignant cells including fibrosarcoma cells, endothelial cells, dermal fibroblasts, keratinocytes, and two osteosarcoma cells. In all the cases, with the exception of Saos2 osteosarcoma cells, we found significant co-expression of both genes, indicating that ECM1 and perlecan are co-expressed by cells with a diverse histogenetic origin. It should be pointed out, however, that other researchers found ECM1 to be expressed by Saos2 osteosarcoma cells (48).

The interaction between ECM1 and perlecan domain V was validated by co-immunoprecipitation studies, using both cell-free systems and mammalian cells. The latter experiments assure proper disulfide bonding and glycosylation of the secreted glycoproteins. Using a battery of deletion mutants we then demonstrated that the major sites of interaction within perlecan domain V reside in the EGF-like repeats connecting the LG subdomains of the molecule, which did not directly participate in the binding. These results clearly show that both the EGF-like tandem repeats are directly involved in the interaction within ECM1, and further suggest that more than one ECM1 molecule might bind to the perlecan protein core insofar as the binding region is quite large. It is not yet known if the binding to ECM1 can alter or influence the binding of other molecules that interact with this domain such as nidogen-1, fibulin-2, heparin, beta 1-integrin, and alpha -dystroglycan (36).

The binding site on the ECM1 molecule was identified in the C-terminal cysteine-rich repeat. The arrangement of the cysteine residues in this molecule is noteworthy following the CC-(X7-10)-C pattern found also in the serum albumin family of proteins (47, 49). This peculiar arrangement of cysteine residues is responsible for the formation of double-loop domains, which may play important roles in protein/protein interactions (47, 63). ECM1 contains three double-loop domains, one present in each of the two central tandem repeats and one in the C-terminal domain. Notably, only the C-terminal domain of ECM1 was found to interact with the perlecan molecule, indicating that the double-loop domains may have different affinity for different ligands. The three cysteine-rich domains present in this molecule are the most conserved among species, the human being 75% identical to the mouse, whereas the overall identity between the two species reached 67%. This indicates that this unique pattern is fundamental in maintaining the structure of the molecule and probably indispensable for the interaction with other extracellular molecules and in particular with perlecan.

Interestingly, loss of function of ECM1 in humans is responsible for lipoid proteinosis (52), an autosomal recessive disease characterized by hoarse voice in early infancy because of thickening of the vocal cords, followed by chicken pox-like scars and thickening of the skin and mucous membranes. Several point mutations have been described, which disrupt the molecule. Because most of the point mutations have been found in exon 7, leading to a truncated molecule lacking the C-terminal repeat, it is possible that the disrupted interaction with perlecan might be responsible for some of the pathological changes observed in this disorder. Notably, the major changes in lipoid proteinosis occur in the dermis and subcutis, with basement membrane thickening at the dermal-epidermal junction, around blood vessels and adnexal epithelia (52). These changes are in agreement with our immunohistochemical data and further stress a physiologically relevant interaction between ECM1 and perlecan protein core.

ECM1 is also involved in the regulation of endochondral bone formation (50). The studies conducted so far have been performed in vitro employing 15-day-old fetal mouse metatarsal bones consisting of undifferentiated cartilage. The explants undergo sequential steps of endochondral bone formation in a 5-day culture period. ECM1 is expressed in the connective tissues surrounding the developing bones and is thought to affect mineralization by regulating the alkaline phosphatase activity, a marker of chondrocyte differentiation. Similarly, both in vitro (64, 65) and gene targeting studies (22, 23) have shown that perlecan affects chondrogenesis and endochondral ossification. Perlecan null mice develop both exencephaly and chondrodysplasia with severe disorganization of the columnar structures of the chondrocytes and defective endochondral ossification (24, 25). Part of the effects observed in perlecan null mice have been ascribed to extracellular matrix disorganization. Because the disruption of these two molecules leads to similar effects, it is possible that the interaction of perlecan with ECM1 is responsible, at least in part, in coordinating chondrocyte differentiation and bone development.

Notably, ECM1 is associated with blood vessels similarly to perlecan, and its expression overlaps with Flk-1, one of vascular EGF receptors (51). Recombinant ECM1 can stimulate the proliferation of endothelial cells and can induce the formation of vessels in chorioallantoic membrane assays (51). Perlecan is expressed quite early during development in tissues of vasculogenesis (11, 13), although the precise mechanism by which perlecan influences the formation of blood vessels is not completely understood. Some of these effects are thought to be indirect, mediated by the ability of the heparan sulfate side chains to bind several growth factors and cytokines (66-68). Perlecan binds FGF2, thereby promoting receptor activation and mitogenesis (34, 35). Experiments conducted using a rabbit ear model, showed that perlecan-FGF2 complexes induce the formation of blood vessels in much higher levels than heparin-FGF2 complexes (37). We have recently discovered that perlecan domain V is a potent anti-angiogenic factor and have named it endorepellin to signify its anti-vascular function (53). Several proteins or degradation products of the extracellular matrix have been shown to control blood vessel formation in a positive or negative way (69-72). Thus, one possibility is that processed forms of matrix proteins may regulate each others' activity. The interaction of domain V/endorepellin and ECM1 around blood vessels could lead to a fine regulation of blood vessel formation and maintenance. Moreover, the influence on angiogenesis exerted by these two molecules may be important in many pathological events such as wound repair (73), diabetic retinopathy, rheumatoid arthritis, as well as tumor growth and metastasis (74, 75), all of which are largely dependent on the formation of new blood vessels.

    ACKNOWLEDGEMENTS

We thank Dr. J. Ni, Human Genome Sciences, for providing the anti-ECM1 antibody and Shelly Campbell and Marilyn Skelly for excellent technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1 CA47282 and RO1 CA39481 (to R. V. I.), United States Department of the Army Grants DAMD17-00-1-0663 and DAMD17-00-1-0425 (to R. V. I.), and a fellowship from the American-Italian Cancer Foundation, New York (to M. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to the results of this work.

** To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Rm. 249 Jefferson Alumni Hall, Thomas Jefferson University, 1020 Locust St., Philadelphia, PA 19107. E-mail: iozzo@lac.jci.tju.edu.

Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M210529200

    ABBREVIATIONS

The abbreviations used are: FGF, fibroblast growth factor; ECM1, extracellular matrix protein 1; LG, laminin-G like module; EGF, epidermal growth factor; RT, reverse transcriptase; X-alpha -gal, 5-bromo- 4-chloro-3-indolyl-alpha -D-galactopyranoside.

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