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First published online 13 June 2007
doi: 10.1242/jcs.008607

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The C-terminus of ephrin-B1 regulates metalloproteinase secretion and invasion of cancer cells

¹ Department of Growth Factor Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Tokyo 104-0045, Japan
² Department of Pharmacology, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan

View larger version (37K): [in this window] [in a new window]   Fig. 1. The ectodomain of ephrin-B1 is secreted into the culture medium of human pancreas cancer cells. (A) Various pancreas cancer cell lines were cultured in medium containing 0.5% FBS. After 6 hours, conditioned medium was collected and subjected to immunoprecipitation (IP) and immunoblotting (IB) with polyclonal antibodies against the extracellular domain of ephrin-B1 (ephrin-B1 ECD). The immunoprecipitated 35 kDa ephrin-B1 fragment is indicated by an arrowhead. The expression of ephrin-B1 in each cell lysate was confirmed by immunoblotting (bottom). (B) A diagram of ephrin-B1 is shown at the top. TM, transmembrane domain; Y, tyrosine phosphorylation sites. Dotted line indicates the cleavage site of ephrin-B1, and the PDZ domain-binding motif is indicated as a gray box at the C-terminus. Four aa residues around the cleavage site were changed to alanine (ephrin-B1 4A) destroying the MMP-8 cleavage site. The conditioned medium of COS-1 cells transfected with wild-type (wt) or mutant ephrin-B1 (4A), or independent PANC-1 clones stably expressing ephrin-B1 were collected and subjected to immunoprecipitation and immunoblotting as described in A. (C) SUIT-4 cells were either treated with EphB2-Fc (4 µg/ml) for 2 hours (+) or left untreated (–). The ephrin-B1 fragment in the medium was detected as in A (left) or the cell lysates were subjected to immunoblotting with anti ephrin-B1 C18 (right panel). Open and filled arrowheads indicate uncleaved ephrin-B1 and its processed fragment (p17), respectively. (D) SUIT-4 cells were transiently transfected with ephrin-B1 tagged with HA at the C-terminus. Transfected SUIT-4 cells were overlayed on a monolayer of parent HEK293T cells or HEK293T cells stably expressing EphB2 for 2 hours. Cell lysates were prepared from co-cultured cells to detect HA-tagged p17 fragment derived from exogenously expressed ephrin-B1 in SUIT-4 cells by immunoprecipitation. # and * indicate the IgG heavy chain and light chain, respectively. Open and filled arrowheads indicate uncleaved ephrin-B1 and its processed fragment (p17), respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Activation of ephrin-B1 cleavage requires its C-terminus. (A) Wild-type and various mutants of ephrin-B1 tagged with Flag at the N-terminus were expressed in Capan-1 cells by retrovirus-mediated gene transfer. The cells were treated with EphB2-Fc (2 µg/ml) for 1.5 hours (+) or left untreated (–) and conditioned medium was assayed for the presence of ephrin-B1 fragments by immunoprecipitation (IP) and immunoblotting (IB) with anti-flag antibody. The filled arrowhead indicates the ephrin-B1 ectodomain fragment. (Bottom) Expression of wild-type or mutated ephrin-B1 in cell lysates after treatment with EphB2-Fc. The open arrowhead indicates ephrin-B1 C19. (B) SUIT-4 cells were treated with EphB2-Fc and PP2 or control PP3or left untreated, as indicated. Conditioned medium was collected after 2 hours and subjected to immunoprecipitation and immunoblotting. In the bottom panel, suppression of tyrosine phosphorylation of ephrin-B1 by PP2 treatment is shown. # and * indicate the IgG heavy chain and light chain, respectively.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Screening of a protease that cleaves the extracellular domain of ephrin-B1. (A) Panc-1 cells transiently expressing Flag-tagged ephrin-B1 were incubated with various protease inhibitors for 4 hours in medium containing 0.5% FBS: leupeptin and E64d (Loxistatin), a cystein protease inhibitor; GM6001, a pan-MMP inhibitor; PD150606, a calpain inhibitor; pepstatin A, an aspartic protease inhibitor; DCI (3,4-dichloroisocoumarin), a serine protease inhibitor; TPCK (N-tosyl-phe chloromethyl ketone), a chymotrypsin inhibitor. The cleavage of ephrin-B1 ectodomain was examined as described in Fig. 2A. (B) Left: SUIT-4 cells were incubated with EphB2-Fc (2 µg/ml) together with or without TIMPs (100 nM for each) as indicated for 2 hours. The processed ephrin-B1 fragment was detected in the culture medium. Right: THP-1 cells were treated with PMA (10 ng/ml) together with or without TIMP-3 (100 nM) for 6 hours. Open arrowhead indicates the processed fragment of TNF- in the medium detected by immunoprecipitation with anti TNF- antibody. (C) SUIT-4 cells were treated with various MMP inhibitors as indicated (1 µM for MMP-8 inhibitor and 5 µM for others) for 4 hours. Ephrin-B1 fragment was detected in the medium. (D) Purified ephrin-B1-Fc protein was incubated with activated MMP in vitro for 1 hour at 37°C, separated by SDS-PAGE, and immunoblotted with anti-Fc mouse IgG or anti-ephrin-B1. Bottom: a schematic representation of ephrin-B1-Fc with the MMP-8 cleavage site indicated by a dotted line. Open and filled arrowheads indicate uncleaved ephrin-B1-Fc and the processed fragments, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 4. MMP-8 is the key protease of ephrin-B1 cleavage. (A) Expression of MMP-8 in cell lysates. Left: The indicated cells were seeded on plates not to reach confluence. Cell lysates were prepared on the day after plating. Right: cell lysates were prepared 4 hours (4h) or 3 days (d3) after being plated on dishes. The cells were confluent on day 3 after plating. (B) SUIT-4 cells treated with either MMP-8 siRNA or control scrambled siRNA (control), or left untreated. The cells were detached 48 hours later, replated on new plates and further incubated for 24 hours. Left: Cellular levels of MMP-8 were analyzed 72 hours after treatment of SUIT-4 cells with siRNAs. Right: the culture medium was replaced with one fresh medium or medium containing EphB2-Fc (2 g/ml) and incubated for 2 hours to detect ephrin-B1 ectodomain in the medium. (C) SUIT-4 cells were transiently transfected with the indicated volume of a plasmid encoding Flag-tagged activated MMP-8 cDNA. After 48 hours of transfection, the medium was replaced and the cells were further incubated for 6 hours to detect processed ephrin-B1 ectodomain in the medium. The expression of transfected MMP-8 in each cell lysate was confirmed by immunoblotting with anti-Flag antibody (bottom). (D) The lysate of L ephrin-B1 cells was immunoprecipitated with anti-MMP-8 polyclonal antibody or control rabbit IgG1, and subjected to immunoblotting with anti-ephrin-B1 C18 antibody. HRP-conjugated anti-rabbit IgG (TrueBlot) was used as the secondary antibody for immunoblotting to avoid cross reaction with denatured rabbit IgG heavy chain of the antibody used for immunoprecipitation. The arrowhead indicates coprecipitated ephrin-B1. The asterisk indicates the IgG light chain.

View larger version (53K): [in this window] [in a new window]   Fig. 5. Stimulation of ephrin-B1 with EphB2 increases MMP-8 secretion. (A) SUIT-4 cells were left untreated or were treated with EphB2-Fc or co-cultured with either parent or EphB2-expressing HEK293 cells for 4 hours. Cellular levels of MMP-8 were analyzed by RT-PCR using GAPDH as a control. (B) SUIT-4 cells were incubated with or without EphB2-Fc for 2 hours in the presence or absence of cyclohexamide (CHX, 100 µg/ml) or actinomycin D (ACND, 5 µg/ml) to detect the ephrin-B1 ectodomain in the medium. (C) SUIT-4 cells or PANC-1 cells stably expressing ephrin-B1 were treated with actinomycin D (5 µg/ml) together with or without EphB2-Fc (2 µg/ml) for 4 hours. Cell lysates were prepared and intracellular expression levels of MMP-8 were analyzed by western blotting using -tubulin as a loading control. (D) Conditioned medium of SUIT-4 cells were collected after the cells were treated with EphB2-Fc or left untreated for 4 hours in serum-free medium. Proteins secreted in the medium were precipitated with trichloroacetic acid (10%), resuspended in sample buffer, and subjected to immunoblotting with anti MMP-8 polyclonal antibody. Arrowheads indicate MMP-8 protein (proenzyme and activated). (E) SUIT-4 cells were metabolically labeled with [35S]methionine, then treated with EphB2-Fc or control Fc for 2 hours. The amount of labeled MMP-8 (left panel) or MMP-7 (right panel) in the medium was evaluated through immunoprecipitation from the conditioned medium followed by SDS-PAGE and autoradiography. Arrowhead indicates MMP-8 (proenzyme and activated; left) or MMP-7 (right) in the medium. (F) Wild-type and mutants of ephrin-B1 were expressed in Capan-1 cells as in Fig. 2A, and [35S]methionine-labeled MMP-8 was detected in the medium (left panel). Right: The total amount of MMP-8 in the conditioned medium of Capan-1 cells was evaluated using the trichloroacetic acid (TCA) precipitation procedure, followed by immunoblotting with anti MMP-8 antibody as in D.

View larger version (35K): [in this window] [in a new window]   Fig. 6. Stimulation of ephrin-B1 with EphB2 activates Arf1. (A) Flag-tagged ephrin-B1 was expressed in Capan-1 cells. The cells were treated with or without EphB2-Fc (2 µg/ml) and brefeldin A (10 µg/ml) as indicated for 1.5 hours, and conditioned medium was assayed for the 35 kDa ectodomain of ephrin-B1. (B,C) The activity of Arf1 was analyzed in the indicated cells. (C) Wild-type or mutant ephrin-B1 was expressed in Capan-1 cells as in Fig. 2A. The cells were incubated with EphB2-Fc (4 µg/ml) for 20 minutes before being lysed. Arf1-GTP was pulled down with GST-GGA3 bound to glutathione-Sepharose. As controls, lysates of COS-1 cells transiently transfected with plasmids encoding Arf1 T31N or Arf1 Q71L, HA tagged at the C-terminus were analyzed (B, right panel). Open arrowhead indicates cross-reacted GST-GGA3 used for the pull-down assay. (D) Suppression of Arf1 activation decreased the MMP-8 secretion. Capan-1 cells stably expressing ephrin-B1 were used. In the right lane, Arf1 T31N was also transiently expressed in the cells by retrovirus mediated gene transfer. All cells were treated with EphB2-Fc together with (middle lane) or without brefeldin A for 4 hours, and the conditioned medium was subjected to TCA precipitation to detect MMP-8 through immunoblotting. The filled arrowhead indicates endogenous Arf1, and the open arrowhead indicates HA-tagged Arf1 T31N (bottom).

View larger version (71K): [in this window] [in a new window]   Fig. 7. The C-terminus of ephrin-B1 regulates the invasion of cancer cells. (A) Wild-type (WT) ephrin-B1 or C4 ephrin-B1 mutant was expressed in Capan-1 cells. The cells were seeded onto a Transwell membrane coated with a collagen matrix (25 µg/cm2) in serum-free medium containing control Fc or EphB2-Fc (4 µg/ml) with or without addition of the MMP-8 inhibitor (1 µM). In the lower chamber, medium containing 5% FBS was added as a chemoattractant. After 8 hours incubation, the wells were harvested and cells that had invaded the collagen were counted. Representative fields are shown. (Right) The results from three independent experiments, each in duplicate, are shown as the mean ± s.d. *P<0.01. (B) PANC-1 ephrin-B1 cells or PANC-1 cells transfected with a mock vector (mock) were injected intraperitoneally into nude mice. The representative appearance of intestinal loops 8 weeks after injection is shown. Arrows indicate disseminated tumor nodules in the mesentery. The right panel shows the histology of the tumors in the mesentery (x100). The asterisk indicates a tumor nodule. Microscopic invasion of cancer cells was observed in the mesenteric sheet (blanket). (C) Representative appearance of the tumors of panc1 cells expressing either mock vector, wild-type or C4 ephrin-B1 in the rectouterine region was compared. Yellow and red arrowheads indicate uterine horns and tumor nodules, respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Diagram showing the possible mechanism of ephrin-B1-mediated stimulation of MMP-8 secretion and cell invasion. When ephrin-B1 is stimulated by EphB receptors, Arf1 GTPase is activated through signaling mediated by the C-terminus ephrin-B1, which may stimulate the transport of MMP-8 for extracellular release. The increase of secreted MMP-8 triggers the degradation of extracellular matrix (ECM) and cleavage of ephrin-B1.