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Home , Matrix metalloproteinase, MMP2, MMP9

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7069-7074, July 1996 Cell Biology

Matrix 2 releases active soluble ectodomain of fibroblast growth factor 1 (fibroblast growth factor receptor cleavage/fibroblast growth factor receptor crosslinking/ A) EHUD LEVI*t, RAFAEL FRIDMANtt, HUA-QUAN MIAO*, YONG-SHENG MA§, AVNER YAYON§, AND ISRAEL VLODAVSKY*¶ *Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, Israel; tDepartment of Pathology, Wayne State University School of Medicine, Detroit, MI 48201; and §Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel Communicated by Leo Sachs, Weizmann Institute of Science, Rehovot, Israel, April 5, 1996 (received for review February 25, 1996)

ABSTRACT Recent studies have demonstrated the exis- The 72-kDa [ metalloproteinase type 2 (MMP2)] and tence ofa soluble fibroblast growth factor (FGF) receptor type the 92-kDa (MMP9) are two members of a large 1 (FGFR1) extracellular domain in the circulation and in family of -dependent , the matrix metal- vascular basement membranes. However, the process of loproteinases (MMP), known to cleave ECM in FGFR1 ectodomain release from the plasma membrane is not normal and pathological conditions (12-16). Like other mem- known. Here we report that the 72-kDa (matrix bers of the MMP family, the gelatinases are secreted in a latent metalloproteinase type 2, MMP2) can hydrolyze the Val368- form, requiring activation for proteolytic activity, and are Met369 peptide bond of the FGFR1 ectodomain, eight amino inhibited by the tissue inhibitors of acids upstream of the transmembrane domain, thus releasing (TIMPs; refs. 12-14). Recent studies demonstrated that latent the entire extracellular domain. Similar results were obtained proMMP2 is activated by a novel subgroup of MMPs, the regardless of whether FGF was first bound to the receptor or membrane-type MMPs, which are bound to the plasma mem- not. The action of MMP2 abolished binding of FGF to an brane of normal and tumor cells by a unique transmembrane immobilized recombinant FGFR1 ectodomain fusion domain (17, 18). The localization of active MMP2 and mem- and to Chinese hamster ovary cells overexpressing FGFR1. brane-type MMPs on the cell surface may play a role in the The released recombinant FGFR1 ectodomain was able to degradation of ECM components in areas of cell-matrix bind FGF after MMP2 cleavage, suggesting that the cleaved interactions. It is conceivable that nonmatrix cell surface soluble receptor maintained its FGF binding capacity. The proteins, including cell surface receptors, may also be the activity of MMP2 could not be reproduced by the 92-kDa target of membrane-bound MMPs. It has been recently shown (MMP9) and was inhibited by tissue inhibitor of that MMP2 can hydrolyze ,B-amyloid isolated from brains of metalloproteinase type 2. These studies demonstrate that Alzheimer disease patients (19, 20), and galectin-3, a cell FGFR1 may be a specific target for MMP2 on the cell surface, surface lectin involved in cell-cell and cell-matrix interactions yielding a soluble FGF receptor that may modulate the in tumor cell (21). mitogenic and angiogenic activities of FGF. The biological activity of FGF may be regulated by several mechanisms, including binding to high and low affinity recep- The fibroblast growth factors (FGFs) constitute a family of tors on the cell surface (1-3), release of FGF from ECM by nine structurally related polypeptides characterized by high heparanase (22, 23), and as recently proposed, release of the affinity to heparin. The FGFs participate in a wide array of entire ectodomain of the FGFR into the circulation and ECM biological activities, including the induction of cellular prolif- (24, 25). A soluble truncated FGFR1 was recently detected in eration, tissue regeneration, neurite outgrowth, , the of retinal vascular endothelial cells by and embryonic mesoderm induction (1-3). This family use of an antibody raised against the extracellular domain of includes the prototypes acidic FGF (aFGF) and basic FGF FGFR1 (25). The mechanism by which FGFR may be released (bFGF) that, unlike most other polypeptide growth factors, are from the cell surface was not elucidated, but it may involve the primarily cell associated proteins, consistent with a lack of a action of a acting on the cell surface and cleaving the conventional signal sequence for secretion (1-3). FGFs elicit extracellular domain of the high affinity receptor. In our study, their biological responses by binding to cell surface tyrosine we examined the ability of the gelatinases to hydrolyze murine kinase receptors. Four distinct but highly related FGF recep- FGFR1 and FGFR2 ectodomains. Here we demonstrate that tors (FGFRs) have been identified (4-6). The prototype human recombinant MMP2, but not MMP9, cleaves a recom- FGFR contains three IgG-like domains in its extracellular binant FGFR1 fusion protein, releasing the entire ectodomain. portion, a single transmembrane domain, and a tyrosine kinase MMP2 hydrolysis was achieved with both free and FGF- domain that is split into two segments by a short interkinase occupied receptor. The MMP2-hydrolyzed ectodomain re- region (5). In addition to the four distinct FGFRs, an alter- We MMP2 native splicing mechanism gives rise to multiple forms of tained its ability to bind FGF. also show that FGFR type 1 (FGR1), FGFR2, and FGFR3 (4-9). In addition treatment of cells overexpressing FGFR1 markedly reduces to interacting with their high affinity receptors, FGFs also interact with lower affinity binding sites identified as heparan Abbreviations: FGF, fibroblast growth factor; FGFR, FGF receptor; aFGF, acidic FGF; bFGF, basic FGF; FGFR1, FGF receptor type 1; sulfate proteoglycans and found on the surface of cells and in ECM, ; MMP, ; MMP2, the extracellular matrix (ECM; refs. 1-3). Cells that naturally MMP type 2; TIMP2, tissue inhibitor of metalloproteinase type 2; do not express heparan sulfate proteoglycans but overexpress proMMP, latent MMP; AP, alkaline phosphatase; APMA, p- high affinity receptors for FGFs fail to respond mitogenically aminophenylmercuric acetate. FRAP, FGFR1 ectodomain-AP fusion to aFGF or bFGF, unless heparin is added (10, 11). protein; BAP, mouse Bek ectodomain-AP fusion protein; DSS, disuccinimidylsuberate; CHO, Chinese hamster ovary. tThe first two authors contributed equally to this work. The publication costs of this article were defrayed in part by page charge ITo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Oncology, Hadassah Hospital, P.O. Box 12000, Jerusalem 91120, accordance with 18 U.S.C. §1734 solely to indicate this fact. Israel.

7069 Downloaded by guest on September 24, 2021 7070 Cell Biology: Levi et al. Proc. Natl. Acad. Sci. USA 93 (1996) binding of FGF. The results suggest a novel mechanism of FGF Treatment of Immobilized FGFR-AP Fusion Protein with regulation modulated by the action of MMP2. MMPs Followed by 125I-bFGF Binding and Crosslinking. Ninety-six-well plates. FGFR-AP fusion protein [FRAP or mouse Bek ectodomain-AP fusion protein (BAP)] immobi- MATERIALS AND METHODS lized onto 96-well plates was incubated (37°C, 5 h, except when Materials. Sodium heparin from porcine intestinal mucosa stated otherwise) with recombinant proMMP2 or proMMP9 was obtained from Hepar (Franklin, OH). Recombinant hu- (3 ,ug/ml, except when stated otherwise) in the presence or man bFGF, kindly provided by Takeda (Osaka), and aFGF (a absence of 1 mM APMA, 10 mM EDTA, or recombinant gift of G. Neufeld, Technion, Haifa, Israel) were iodinated by TIMP2 (1:1 molar ratio of MMP/TIMP2). The wells were then chloramine T as described (26). The specific activity was washed three times and incubated (2 h, 22°C) with 5 ng/ml -1.7 x 105 cpm/ng of 1251-bFGF and -0.4 x 105 cpm/ng of iodinated aFGF or bFGF in the absence and presence of 0.1 125I-aFGF, and the labeled preparations were kept for up to 3 ,tg/ml or 0.02 Ag/ml heparin, respectively. The wells were weeks at -70°C. 1251-Na was purchased from Amersham. washed once with binding medium followed by one wash with Mouse anti-human placental alkaline phosphatase (AP) mAbs Hepes buffer, pH 7.4, containing 0.5 M NaCl (aFGF) or 2 M coupled to agarose were purchased from Sigma. Rabbit anti- NaCl (bFGF), and a final wash with binding buffer. The bound Bek polyclonal antiserum was generated by injecting glutathi- 1251-FGF was released with 2 M NaCl in 20 mM sodium one S- fused with the ectodomain region of mouse acetate, pH 4.5, and counted in a y counter. Bek (9). Sepharose beads. The immobilized FGFR-AP (FRAP or Expression and Purification of Recombinant MMPs and BAP) was equilibrated with MMP reaction buffer (50 mM TIMPs. Human recombinant proMMP2, proMMP9, and Tris-HCl, pH 7.2/5 mM CaCl2/0.02% sodium azide/1 mM TIMP type 2 (TIMP2) were all expressed in HeLa cells using APMA) and incubated (5 h, 37°C) with 3 p,g/ml recombinant a recombinant vaccinia virus expression system (Vac/T7) as proMMP2 in a final volume of 80 ,lI. The beads were washed described (27, 28). proMMP2 and proMMP9 were purified by and suspended in 80 Al of MMP reaction buffer. EDTA (10 -affinity chromatography from the media (Opti-MEM mM) was then added to both the supernatant and the pellet to I, GIBCO/BRL) of HeLa cells coinfected with the appropri- inhibit the activity before bFGF binding and crosslink- ate recombinant viruses as described (27, 28). TIMP2 was ing. The supernatant containing the released FGFR1 ectodo- purified by affinity chromatography using an mAb (CA-101) main and the pellet containing the FRAP fragment remaining against human TIMP2, as described (27, 28). The concentra- bound to the beads were diluted (4x) with Hepes-buffered tions of the purified and TIMP2 were determined by saline, pH 7.5. 125I-bFGF (150 ng/ml) was then incubated (2 analysis (29). h, 40C) with both the supernatant and the pellet in the presence Enzyme Activation. The gelatinases, diluted in 5 mM of heparin (0.2 ,ug/ml) and subjected to crosslinking (30 min, Tris-HCl, 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35 220C) with 0.15 mM disuccinimidylsuberate (DSS; dissolved in buffer, were activated (30 min, 37°C) with a final concentration dimethyl sulfoxide and diluted 1:100 in PBS). The crosslinking of 1 mM p-aminophenylmercuric acetate (APMA). The pres- reaction was then quenched with 10 mM ethanolamine-HCl, ence of active forms and the actual activity of the recombinant pH 8.0, for 30 min at 24°C. Samples were diluted with Sx enzymes were determined by zymography and gelatinase SDS/PAGE sample buffer and subjected to SDS/PAGE assays (7.5% gel) under reducing conditions. The gels were dried, and using [3H]gelatin (27, 28), respectively. the 125I-bFGF was visualized by autoradiography on Kodak Electrophoresis and Immunoblotting. SDS/PAGE was per- XAR film. formed under reducing conditions, using a 7.5% polyacryl- In some experiments, treatment with MMPs was performed amide separating gel and a 3.5% stacking gel. The gels were after the crosslinking of 125I-bFGF to FRAP beads. For this stained with Coomassie blue and/or subjected to autoradiog- purpose, 125I-bFGF (150 ng/ml) was incubated (2 h, 22°C) with raphy. For immunoblot analysis, the separated proteins were the FRAP beads in the presence of heparin (0.2 Ag/ml) in 30 transferred to a nitrocellulose membrane, followed by blocking Al of binding medium. The beads were then washed once with (3% BSA and 3% nonfat dry milk) and incubation with the 2 M NaCl in 25 mM Hepes, pH 7.4, and once with PBS. The corresponding primary antibodies diluted in 50 mM Tris HCl pellet was suspended in 150 ,lI of DSS (0.15 mM final (pH 7.5), 150 mM NaCl, and 0.1% Tween 20. The immuno- concentration, 30 min, 24°C). The crosslinked 125I-bFGF- detection of the antigen was performed using the appropriate FRAP beads were washed four times with PBS, centrifuged, antibodies and an enhanced chemiluminescence kit (Amer- and equilibrated with MMP reaction buffer. Recombinant sham) for development. proMMP2 or proMMP9 (0.2-3 ,ug/ml) was then added to the Preparation of Immobilized FGFR1 Ectodomain-AP beads in the presence of 1 mM APMA, and the reaction (FRAP) Fusion Protein. A soluble FGF receptor ectodomain mixture (-80 ,ul final volume) was incubated for 5 h at 37°C. was constructed by cloning the extracellular domain of murine At the end of the incubation, the beads were centrifuged, and FGFR1 into the APtag expression vector (11, 30). The FRAP the pellets and supernatants were each dissolved in Sx SDS/ fusion protein was immobilized onto either Sepharose beads or PAGE sample buffer and subjected to SDS/PAGE analysis 96-well plates with use of antibodies to AP. Briefly, protein and autoradiography as described above. A-Sepharose beads (30 ,l; RepliGen) were incubated (2 h, High Affinity Binding of FGF to Chinese Hamster Ovary 22°C) with 10 gl of affinity-purified rabbit anti-human pla- (CHO) Cells. proMMP2 was activated with APMA, dialyzed cental AP antiserum. The beads were washed twice and to remove the APMA, and incubated (3 ,ug/ml, 3 h, 370C) with incubated (2 h, 22°C) with FRAP fusion protein (12 ml of pgsA-745-flg CHO cells (2.5 x 105 cells per 16-mm well) in conditioned medium; -0.5 ,ug of FRAP per ml) followed by PBS containing Ca+2 and Mg+2. The culture was washed twice, three washes with 0.1% BSA in DMEM containing 25 mM and receptor binding was performed as described (31-33). Hepes, pH 7.5 (binding buffer). Wells of 96-well plates (F96 Microsequence Analysis. FRAP beads were treated (5 h, Maxisorp, Nunc-immuno plate) were incubated (18 h, 4°C) 37°C) with MMP2 at an enzyme/ ratio of 1:5 (wt/wt). with monoclonal anti-human placental AP antibodies (Sigma) The beads were washed and boiled in sample buffer, and the diluted (1:1000) in 0.1 M bicarbonate buffer, pH 8.0 (75 ,ul per solubilized proteins were separated by SDS/PAGE under well). The wells were then washed two times with binding reducing conditions and transferred to an Immobilon [poly- buffer (250 ,ul per well), incubated (2 h, 220C) with FRAP (vinylidene difluoride)] membrane (Millipore). The trans- conditioned medium (-0.5 ,ug of FRAP per ml, 100 ,ul per ferred proteins were stained with 1% -amido black in 20% well), and washed once with binding buffer. isopropanol and 10% acetic acid, and the major -70-kDa band Downloaded by guest on September 24, 2021 Cell Biology: Levi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7071 was cut and subjected to NH2-terminal sequencing (19 cycles) that binding of either aFGF or bFGF to this receptor was not on an Applied Biosystems 475A gas-phase protein sequencer. abrogated by pretreatment with MMP2 or MMP9 (not shown). Identification of the MMP2 Cleavage Site. To determine whether MMP2 was cleaving within the FGFR1 ectodomain or RESULTS within AP, FRAP attached to Sepharose beads was treated Effect of MMP2 on Binding of aFGF and bFGF to FGFR1. with MMP2, and proteins remaining bound to the beads were The ability of MMP2 and MMP9 to cleave the FGFR was solubilized and subjected to immunoblot analysis using an determined using a FRAP construct attached to anti-AP- antibody to AP. The results demonstrated the presence of a coated wells of a 96-well plate. The functionality of the FRAP 70-kDa protein, possibly representing the full-length AP. fusion protein was demonstrated by the binding of 125I-aFGF Untreated FRAP or FRAP exposed to MMP2 in the presence or 125I-bFGF that was enhanced in the presence of heparin of EDTA showed a molecular mass of 155 kDa (not shown), (Fig. 1), in agreement with previous results (11, 33). Treatment consistent with the size of the full-length FRAP fusion protein. (5 h, 37°C) of the immobilized FRAP with 3 ,ug/ml MMP2 These studies suggested that the MMP2 cleavage must have abolished the binding of 125I-aFGF (Fig. 1) or 125I-bFGF (not occurred within the FGFR1 ectodomain or within AP residues shown) to the FRAP construct, regardless of heparin presence. adjacent to FGFR1. Since antibodies to FGFR1 ectodomain About 50% inhibition of FGF receptor binding was obtained were not available, we analyzed the supernatant fraction, when the immobilized FRAP was treated with 0.3 ,ug/ml containing the MMP2 cleavage product of FRAP, by SDS/ MMP2 (not shown). Addition of TIMP2 or EDTA, two PAGE and Coomassie blue staining. A degradation product inhibitors of MMPs, to the reaction mixture abrogated the (-85 kDa) was identified, consistent with the molecular mass inhibitory effect of MMP2 on FGF-receptor binding. MMP9 of the recombinant ectodomain of FGFR1. Similar studies or proMMP2 had no effect on FGF binding to the FRAP performed with the BAP fusion protein (9), using an antibody construct (Fig. 1). In addition, purified aFGF and bFGF were to FGFR2 (anti-Bek), confirmed the resistance of BAP to not susceptible to cleavage by the MMPs. These results suggest MMP2 cleavage since only a 155-kDa protein was detected in that MMP2 but not MMP9 can hydrolyze the FRAP fusion association with the beads (Fig. 2, pellet). These experiments protein, abrogating its capacity to bind FGF. Similar experi- indicate that of the two FGF receptor ectodomains, only ments performed with a fusion protein formed by AP and FGFR1 may be a substrate susceptible to MMP2 cleavage. FGFR2 (mouse Bek) ectodomain (designated BAP) showed To determine the precise cleavage site of MMP2 in the FRAP fusion protein, the pellet containing the 70-kDa MMP2 35 - degradation product was separated by SDS/PAGE, blotted onto poly(vinylidene difluoride) membrane, and subjected to 30 - N-terminal sequencing. A sequence of 18 amino acids was obtained, identical to the known FRAP sequence around the O 25 - fusion point between FGFR1 ectodomain and AP (Fig. 3). The P- N terminus of the 70-kDa protein was found to be within the 20 - 0X receptor sequence, beginning from Met369, eight residues o 4) (MTSPLYLE) upstream of the transmembrane domain that 15 - starts at Arg377 of the murine FGFR1 sequence (5). Thus, PQ3 MMP2 hydrolyzes the Val368-Met369 bond of the FGFR1 10 - ectodomain. Beyond the eight amino acids of the receptor, the C: g other 10 adjacent amino acids (RSSGIIPVEE) that were 5-! sequenced were identical to those of AP, starting at position _-4 377. Thus, AP, together with eight amino acids of the FGFR1 I I I I I ~I I I ectodomain, was the immobilized FRAP fragment remaining after MMP2 cleavage. FGF Binding to FGFR1 Ectodomain Released by MMP2. * + + + + + + The above results demonstrate that MMP2 cleaved the FGF

IMMP-2 - - 4 + s P s P kOa ++ + + + 1981- 116- + 66

FIG. 1. Effect of MMP2 on binding of aFGF to FGFR1 (FRAP). FRAP immobilized onto an anti-AP-conjugated 96-well plate was incubated (5 h, 37°C) with or without 3 ,g/ml recombinant proMMP2 1 2 3 4 or proMMP9 in the presence of 1 mM APMA in MMP reaction buffer (110 ,ul per well). Some of the wells were incubated in the presence of FIG. 2. Western blot analysis of BAP fusion protein treated with EDTA (10 mM) or recombinant TIMP2 (1:1 molar ratio of MMP/ MMP2. BAP immobilized onto anti-AP conjugated Sepharose beads TIMP2) or in the absence of APMA (i.e., proMMP2) as indicated. The was treated (5 h, 37°C) with MMP2 (3 jig/ml) in MMP reaction buffer. wells were washed three times and further incubated (2 h, 22°C) with The beads were centrifuged, and both the supernatant (S) and the 5 ng/ml 125I-aFGF in the absence and presence of 0.1 ,tg/ml heparin washed pellet (P) were boiled in SDS/PAGE sample buffer. Solubi- in binding medium. The wells were washed with binding medium and lized proteins were subjected to SDS/PAGE (7.5% gel) followed by salt solutions, and the bound 1251-aFGF was released and counted as electroblotting and detection with anti-Bek antibodies. Lane 1, super- described in the text. Similar results were obtained with 1251-bFGF. natant; lane 2, pellet of immobilized BAP that was not exposed to Each point represents the mean ± SD from triplicate wells. All MMP2; lane 3, supernatant; lane 4, pellet of immobilized BAP that was experiments were performed at least three times, and the variation treated with MMP2. Molecular weight standards are marked on the between different experiments did not exceed ± 10%. left. Downloaded by guest on September 24, 2021 7072 Cell Biology: Levi et al. Proc. Natl. Acad. Sci. USA 93 (1996)

FGFR-1 ectodomain A N P Is I P Is

kDa EA LEER PAVMTS PLY LER*SS GIIPV EE 195-[ t 112- EALEERPAVMTS B '11 iW "I 84

M, i 1 2 3 4 FIG. 4. Binding of 125I-bFGF to MMP2 cleavage products of FRAP. FRAP immobilized onto anti-AP-conjugated Sepharose beads was incubated (5 h, 37°C) with MMP2 (3 g/g/ml) in MMP reaction buffer. The beads were centrifuged, and both the pellet and the supernatant were incubated (2 h, 4°C) with 125I-bFGF (150 ng/ml) in the presence of 0.2 /Lg/ml heparin. Binding was followed by crosslink- FIG. 3. Schematic presentation of the MMP2 cleavage site in the ing with DSS, boiling in SDS/PAGE sample buffer, electrophoresis, FGFR1 ectodomain. The FGFR ectodomain-AP fusion protein (A) is and autoradiography as described in text. Lanes 1 and 2, 125I-bFGF cleaved at the Val368-Met369 peptide bond (arrow) of the ectodomain. binding to the pellet (P) and supernatant (S) of control immobilized The Arg residue with the asterisk indicates the start of the AP in the FRAP that was not exposed to MMP2; lanes 3 and 4: 125I-bFGF fusion protein. For comparison, the natural murine FGFR1 is shown binding to the pellet and supernatant of immobilized FRAP that was in B. III, immunoglobulin domain III; TM, transmembrane domain; k, first exposed to MMP2. Molecular weight standards are marked on the kinase domain. left. receptor ectodomain immediately next to the transmembrane domain, maintaining the FGF binding domain intact. There- Effect of MMP2 on FGF Binding to CHO Cells. To test the fore, we examined whether the MMP2-cleaved FGFR1 effect of MMP2 treatment on the ability of cells to bind FGF, ectodomain can bind FGF after being released from the FRAP we exposed a CHO cell line (pgsA-745-flg) overexpressing the beads. Immobilized FRAP was first subjected to MMP2 murine FGFR1 (31-33) to recombinant MMP2. The pgsA- cleavage in the absence and presence of EDTA. Then, both the 745-flg CHO cells were also defective in their metabolism of beads (pellet) and the released material (supernatant) were heparan sulfate, making them dependent on exogenous hep- crosslinked to 125I-bFGF in the presence of heparin. The complex was then analyzed by SDS/PAGE followed by auto- Pellet I Sup radiography. As demonstrated in Fig. 4 (lane 4), the superna- P-2 : 4- tant showed a 105-kDa the '25I-bFGF EDTR : + - - + product representing kDa crosslinked to the solubilized FGFR1 ectodomain. In contrast, ,il." ",i~ii the FRAP fragment remaining bound to the beads almost did not bind 125I-bFGF (lane 3), consistent with the release of the 175- ectodomain into the supernatant by MMP2. A lack of 1251- bFGF crosslinking was also observed in the supernatant of FRAP beads treated with MMP2 in the presence of EDTA (not shown). These results demonstrate that following hydro- lysis of the FGFR1 ectodomain by MMP2, the soluble ectodo- main is still capable of binding bFGF. Effect of MMP2 on FRAP-bFGF Complex. Since the cleav- 1 2 3 4 5 6 age site of MMP2 was found to be immediately upstream of the FIG. 5. Cleavage of '25I-bFGF-FRAP complexes by MMP2. 125I_ transmembrane domain, we examined whether a FRAP fusion bFGF (150 ng/ml) was incubated (2 h, 22°C) with FRAP immobilized protein bound to FGF was still susceptible to MMP2 cleavage. onto anti-AP conjugated Sepharose beads in the presence of 0.2 glg/ml To this end, immobilized FRAP was crosslinked to 125I-bFGF heparin in binding medium. The beads were washed with PBS (pH in the presence of heparin, as described in Materials and 7.4), subjected to crosslinking with DSS, washed three times with PBS, Methods. The FRAP-125I-bFGF was then treated with and equilibrated with MMP reaction buffer as described in the text. complex The beads were then incubated with MMP2 in and both the beads and the were (5 h, 37°C) (3 ALg/ml) MMP2, supernatant subjected the absence or presence of 10 mM EDTA, followed by centrifugation to SDS/PAGE analysis followed by autoradiography. As and boiling of both the pellet and the supernatant (Sup) fractions in shown in Fig. 5 (lane 1), the untreated FRAP-125I-bFGF SDS/PAGE sample buffer. Solubilized proteins were subjected to complex showed a molecular mass of 175 kDa. Treatment with SDS/PAGE and autoradiography. Lanes 1 and 4, pellet and super- MMP2 released into the supernatant a 105-kDa protein (lane natant of control immobilized 125I-bFGF-FRAP complexes that were 5) representing the crosslinked 125I-bFGF/FGFR1 ectodo- not exposed to MMP2, respectively; lanes 2 and 5, pellet and super- main complex. In the presence of EDTA, all the radioactivity natant of immobilized bFGF-FRAP complexes that were exposed to remained associated with the beads in the form of the 175-kDa MMP2, respectively; lanes 3 and 6, pellet and supernatant of immo- bilized bFGF-FRAP complexes that were exposed to MMP2 in the complex (Fig. 5, lane 3). Taken together, the above studies presence of 10 mM EDTA, respectively. The molecular masses of demonstrate that an occupied FGFR1 ectodomain is still untreated (175 kDa, pellet) and MMP2-treated (105 kDa, superna- susceptible to hydrolysis by MMP2. tant) 125I-bFGF-FRAP are marked on the left. Downloaded by guest on September 24, 2021 Cell Biology: Levi et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7073 arin for binding of FGF (31, 34). The CHO cells were treated even after a long exposure to MMP2, indicating that the (2 h, 37°C) with MMP2 (3 ,ug/ml), washed with PBS, and then cleavage of the Val368-Met369 peptide bond was unique. In incubated with 125I-aFGF at 4°C for 2 h in the presence of contrast to FGFR1, BAP was not hydrolyzed by either MMP2 heparin. High affinity binding of 125I-aFGF was tested as or MMP9. This difference is most probably due to a lack of described in Materials and Methods. As shown in Fig. 6, MMP2 sequence identity between these two FGF receptors, particu- treatment of the pgsA-745-flg CHO cells caused a marked larly in the area adjacent to the transmembrane domain that reduction in the binding of 125I-aFGF to the cells when is susceptible to MMP2 hydrolysis in FGFR1. Indeed, align- compared with the binding to untreated cells. Moreover, ment of the sequences of FGFR1 and FGFR2 reveals that the FGFR1 ectodomain was detected in the incubation medium of VMTSPLY motif of FGFR1 is replaced by ITASPDY in MMP2 treated CHO cells, and this medium competed with FGFR2 (5, 6). aFGF binding to untreated CHO cells. In other studies, The hydrolysis of the FGFR1 ectodomain by MMP2 was pretreatment of bovine aortic endothelial and smooth muscle observed with both an unoccupied ectodomain, or with an cells with MMP2 resulted in a significant (approximately 40% ectodomain crosslinked to bFGF, indicating that the MMP2 and 30%, respectively) loss of aFGF and bFGF binding to high cleavage site is accessible to MMP2 attack regardless of the affinity cell surface receptor sites, indicating that FGFR1 is a presence of FGF. In addition, FGFR1 ectodomain retained its major receptor for aFGF and bFGF in these cells. These ability to bind FGF after MMP2 hydrolysis. Thus, MMP2 studies indicate that FGF receptors expressed on the cell cleavage of the receptor yields a functional soluble FGFR1 surface are susceptible to MMP2 hydrolysis, which in turn ectodomain capable of FGF binding. The molecular mass of decreases the binding of FGF. the extracellular domain ofFGFR1 cleaved by MMP2 from the fusion protein was approximately 85 kDa, similar to that of the free FGFR1 ectodomain recently identified in human plasma DISCUSSION and in the ECM of retinal vascular endothelial cells (24, 25). The present studies demonstrate that human recombinant It is tempting to speculate, based on our data, that the presence MMP2, but not MMP9, can hydrolyze the Val368-Met369 of the ectodomain of FGFR1 in plasma and ECM results from peptide bond of FGFR1 located eight residues upstream of the the effect of MMP2 on FGFR1. We have shown here that transmembrane domain. This cleavage resulted in release of treatment of heparan sulfate-deficient CHO cells overexpress- the entire FGFR1 ectodomain from an immobilized AP- ing FGFR1 with MMP2 markedly reduced their binding of FGFR1 ectodomain fusion protein and inhibited binding of FGF. A significant loss of FGFR1 was also observed in 1251-FGF to the remaining immobilized fusion protein. The MMP2-treated vascular endothelial and smooth muscle cells. cleavage of the FGFR1 ectodomain was MMP2-dependent Thus, it is likely that a plasma membrane-bound FGFR1 is since presence of either TIMP2, the natural inhibitor of MMP2 susceptible to MMP2 hydrolysis. The released ectodomain (35), or EDTA, a known chelating inhibitor of MMPs (12, 13), binds extracellular FGF, possibly controlling the biological inhibited hydrolysis and subsequent solubilization of the availability and growth promoting activity of FGF. ectodomain. It also required proMMP2 activation since with In a recent study, the ECM produced by bovine endothelial latent enzyme no hydrolysis occurred. Sequencing data re- cells was found to contain proMMP2, free of TIMP2 and susceptible to release by MMP9 and to activation by APMA vealed that the MMP2 cleavage site was within the FGFR1 (36). Once activated, the ECM-resident MMP2 may cleave the ectodomain and not within the AP enzyme. No evidence for FGFR1 of cells that contact the ECM, resulting in ECM further processing of the FGFR1 ectodomain was observed sequestration and/or release of the FGFR1 ectodomain. MMP2 expression has been reported in a variety of endothelial 14 cells (37), and MMP2 can be induced by FGF (38, 39). Also, thrombin was recently reported to elicit activation of MMP2 in 12 vascular endothelial cells (40). This may result in a more efficient cleavage of FGFR1, possibly providing a feedback control mechanism that will down-regulate cellular responses 0+ to bFGF. 8 A cell surface localization of MMP2 can facilitate the access of the protease to membrane-bound proteins susceptible to 6 MMP hydrolysis. Studies of human tumors showed that the o~Z4~~ o10i gelatinases localize on the tumor cells in a pericellular pattern m61~~~~~~~~c (41-44). In addition, the invadopodia of cultured transformed chicken embryo fibroblasts showed the presence of MMP2 (45), and plasma membranes isolated from phorbol ester- LO) treated fibrosarcoma HT1080 cells were found to contain the P-4 0 0 62-kDa active form of MMP2 (unpublished results). It is conceivable that on the cell surface an appropriate local concentration and availability of both MMP2 and FGFR1 can *+ *I be achieved, so that a physiologically efficient cleavage of the cm4 receptor can take place. A similar situation was proposed for FIG.0~6.0feto ~M2o.idn ~ f 2IaG 0-ops-4 H other cell surface (i.e., receptor bound urokinase) (46) and/or ECM-bound molecules (i.e., plasminogen and thrombin) (47, 48) that are protected and/or better situated for interaction with effector molecules when immobilized to a solid support, FIG. . Effect of MMP2 on binding of 125I-aFGF to pgsA-745 CHO as compared with their behavior in a fluid phase (48). The cells. PgsA-745-flg CHO cells transfected with FGFR1 were treated (3 MMP2 on and other cellular such as h, 37°C) with MMP2 (3 ,ug/ml) in PBS containing Ca+2 and Mg+2. effect of FGFR1 proteins Untreated and MMP2-treated cells were then incubated (2 h, 4°C) with galectin-3 (21) and j-amyloid (19, 20) suggests that this 1251-aFGF (5 ng/ml) in the absence and presence of 0.2 ,ug/ml heparin, proteinase may also play a role in other biological process and the amount of radioactivity specifically bound to high affinity cell besides ECM degradation and remodeling. This property is not surface receptor sites was determined. Each point represents the unique to MMP2. MMP1 and MMP3 were-shown to hydrolyze mean ± SD from triplicate wells. insulin growth factor-binding protein type 3 in solution and in Downloaded by guest on September 24, 2021 7074 Cell Biology: Levi et al. Proc. Natl. Acad. Sci. USA 93 (1996) rat pregnancy serum (49) and MMPs are also involved in the 25. Hanneken, A., Maher, P. 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