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Cell and Tumor Biology |
Departments of 1 Cancer Research, 2 Structural Biology, and 3 Molecular Biology, Abbott Laboratories, Abbott Park, Illinois
Requests for reprints: Don Davidson, Department of Cancer Research, Abbott Laboratories, Department 48R, Building AP9, 100 Abbott Park Road, Abbott Park, IL 60064. Phone: 847-937-8547; Fax: 847-937-4150; E-mail: don.j.davidson{at}abbott.com.
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Introduction |
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Several endogenous angiogenesis inhibitors are protein fragments derived from extracellular matrix (13) or hemostatic system proteins (14). Plasminogen is a blood protein that is proteolysed into potent angiogenesis inhibitors, such as angiostatin (kringles 1-4) and kringle 5 (K5; refs. 15–17). Kringle domains contain 
80 amino acids and three similarly linked disulfide bonds (18). Recombinant K5 (rK5) displays the most potent inhibitory activity to endothelial cell proliferation and migration (19–21) of known naturally occurring angiogenesis inhibitors. A recombinant K5 has also been shown to induce apoptosis in proliferating endothelial cells (19). Recently, reports show that rK5 prevents the development and arrests the progression of ischemia-induced retinal neovascularization in a rat model (21) by the down-regulation of VEGF and up-regulation of pigment epithelial-derived factor.
The tertiary structure of K5 has been studied in detail and contains a weak lysine-binding pocket. By contrast, the endothelial-binding site through which the antiangiogenic effects of K5 are mediated has not been defined (22). Here, we report that the antiangiogenic and proapoptotic activity of rK5 depends on a high-affinity binding interaction with glucose-regulated protein 78 (GRP78) exposed on the surface of stimulated endothelial cells and on many hypoxic and cytotoxic stressed tumor cells.
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Materials and Methods |
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Cell culture. Human microvascular endothelial cells (HMVEC), human umbilical arterial vascular endothelial cells, human umbilical endothelial cells, dermal fibroblasts, and neutrophils were obtained from Clonetics Corp. (San Diego, CA). D54 human glioma tumor cells were obtained from University of Texas-Southwestern Medical Center (Houston, TX). All other cell lines were obtained from American Type Culture Collection (Rockville, MD).
Peptide synthesis. All peptides were synthesized using a Symphony (Protein Technology, Inc., Woburn, MA) automated peptide synthesizer. Peptide purification was done using a Gilson high-performance liquid chromatography system equipped with automated liquid handler. The Fmoc-protected amino acids and resins were purchased either from Calbiochem-Novabiochem Corp. (San Diego, CA) or from Bachem, Inc. (Torrance, CA). Mass spectra were recorded using either a Finnigan SSQ7000 (ESI) or JEOL JMS-SX102A-Hybrid (FAB) mass spectrometers.
Cell proliferation assay. The effect of rK5 and rK5 peptides on endothelial cells was assessed using a proliferation assay with 1% bovine serum albumin (BSA) and 3 ng/mL bFGF in serum-free medium. Relative cell numbers in each well of a 96-well microplate after incubation for 72 hours in the absence or presence of inhibitors were determined by using the AQueous Cell Proliferation Assay (Promega, Madison, WI). For all other cell lines tested for proliferation, minimal growth medium was used (16). Results are presented as the percent inhibition of control cell (bFGF-induced) proliferation.
Expression and purification of recombinant kringle 5. K5 fragment was PCR amplified from a human plasminogen cDNA template (American Type Culture Collection) with the following two primers: 5'-CTGCTTCCAGATAGAGA-3' (forward primer for residues 450-457) and 5'-TTATTAGGCCGCACACTGAGGGA-3' (reverse primer for residues 538-543). The PCR fragment was ligated into the pET32a vector (Novagen, San Diego, CA) that had been digested with NcoI and XhoI. The NcoI and XhoI cleavage sites of the pET32a had been filled in to form blunt ends with plaque-forming unit DNA polymerase (2.5 units/µL, Stratagene, La Jolla, CA). XL2-Blue ultracompetent cells (Stratagene) were transformed with the ligation mixture as per the manufacturer's instructions. After sequence confirmation, the pET32a/K5 vector was retransformed into Escherichia coli BL21 cells (DE3, Novagen) for expression as per the manufacturer's instructions. The recombinant protein was recovered from the cell paste by cell lysis in lysis buffer [50 mmol/L Tris, 300 mmol/L NaCl, 1 mmol/L MgCl2 (pH 7.8)] using a French press. The His-tagged protein was purified over a Probond nickel resin (Invitrogen). The His tag was removed from the rK5 molecule by enterokinase (Invitrogen) and the rK5 was repurified over a second Probond nickel column to remove the His tag. Finally, endotoxin contamination was removed by size filtration (5 kDa) chromatography.
Yeast rK5 was expressed as described previously (22). Briefly, the human K5 gene was expressed in the methylotrophic yeast Pichia pastoris (Invitrogen). Genetic transcription of rK5 was under the control of the alcohol oxidase promoter (AOX1). The AOX1 promoter permits high-level expression of heterologous proteins in Pichia. The K5 expression construct also includes a secretion signal sequence to direct transport of the protein to the medium. The plasmid construct was a hybrid of commercially available plasmid sequences from Invitrogen, designated pHIL-S1 and pHIL-D2. The expressed rK5 was purified by octyl-Sepharose and size exclusion chromatography.
Radiolabeled recombinant kringle 5. rK5 was tritiated (3H) by a method published previously (23) Briefly, a carefully controlled particle beam composed of T3+ and T2+ ions and fast T2 molecules were accelerated into rK5 within a vacuum chamber. The 3HK5 was found to be active in the endothelial cell migration assay with an IC50 of 0.2 nmol/L and a specific activity of 8.74 mCi/mg.
Human rK5 potency is highly dependent on the extent of iodination because the molecule contains a readily iodinated tyrosine in its binding sequence. Mutations of this tyrosine to phenylalanine resulted in incorrect protein folding. However, dog rK5 has a phenylalanine in this position naturally and is folded and active in our assays. We therefore relied on 125IrK5 (dog) as a reagent. The radioiodination of rK5 (dog) was done following the procedure published by Markwell (24). The Iodobead reagent (Pierce, Rockford, IL) was used for the radioiodination and the labeling reaction as per protocol. A total of two beads were used with 25 µg rK5 for the reaction. The separation of labeled rK5 from free iodine was accomplished using an iodine trap and a desalting spin filter (Pierce). 125IK5 (dog) was found to be activity in the migration assay.
Endothelial cell migration assays. The effect of rK5 on endothelial cell migration was determined by two different methods. The first assay was done in a 96-well plate with a cellulose membrane between the upper and the lower chambers. HMVECs were starved of growth factors overnight, labeled with fluorescent calcein AM (50-100 nmol/L), plated into a 96-well migration chamber (2.9 x 104 per well, Neuroprobe), and stimulated to migrate with VEGF (5 ng/mL). After 4 hours, migrated cells were measured by fluorescence (25). In a second assay for cellular migration, a standard Boyden chamber was used (26). HMVEC cells were starved overnight in DMEM containing 0.1% BSA and harvested by scraping and resuspended in DMEM with 0.1% BSA at 1.5 x 106 cells/mL. Cells were added to the bottom of a 48-well Boyden chamber. The chamber was assembled and inverted, and cells were allowed to attach for 2 hours at 37°C to polycarbonate chemotaxis membranes (5 µm pore size) that had been soaked in 0.1% gelatin overnight and dried. The chamber was reinverted, test substances, including activators, were added to the wells of the upper chamber, and the apparatus was incubated for 4 hours at 37°C. Growth factors were used, where indicated, at concentrations determined in preliminary experiments to give equivalent migration responses of 100 cells migrated per high-power field (x400). Growth factors and concentrations used were acidic FGF (aFGF; 50 ng/mL), bFGF (15 ng/mL), interleukin-8 (IL-8; 40 ng/mL), transforming growth factor-ß (TGF-ß; 1 pg/mL), VEGF (100 pg/mL), hepatocyte growth factor (HGF; 40 ng/mL), and platelet-derived growth factor (PDGF; 250 pg/mL). Membranes were recovered, fixed, and stained and the number of cells that had migrated to the upper chamber per 10 high-power fields was counted. Background migration to DMEM plus 0.1% BSA was subtracted and the data were reported as the number of cells migrated per 10 high-power fields (x400) or, when results from multiple experiments were combined, as the percent inhibition of migration compared with the positive growth factor control (26).
Assessment of cellular apoptosis. The effects of rK5 and rK5 peptide-induced apoptosis were determined with a histone ELISA apoptosis assay (Roche, Indianapolis, IN; ref. 27); 5,000 cells per well were grown in 96-well plates. rK5 and/or an antibody to GRP78 were added to plates and incubated overnight. Apoptosis was determined from triplicate samples and the apoptotic index was determined by dividing the absorbance from the treated cells by the absorbance from the untreated cells.
Binding of human recombinant kringle 5 to endothelial cells. Tritium-labeled rK5 was added to monolayers of 50,000 HMVEC cells that were either starved or stimulated for 16 hours by 15 ng/mL bFGF and 5 ng/mL VEGF in 96-well plates. The number of counts remaining bound to the cells after extensive washing determined the total amount of rK5 bound (28).
Binding of 125I kringle 5 (dog) to endothelial cells and recombinant kringle 5 to recombinant glucose-regulated protein 78. The same methods as described above for the expression and purification of human K5 were used to express dog rK5 in E. coli. 125IK5 (dog) was added to the wells and incubated at room temperature for 1 hour. After 1 hour, the cells were washed and lysed with M-Per (Pierce) and the amount of 125IK5 (dog) bound was counted. Scatchard plot analysis was done using Prism software (GraphPad Software, Inc., San Diego, CA). Competition binding against 5 nmol/L 125IK5 (dog) was tested using a monoclonal antibody against GRP78 (N-20) or a monoclonal antibody against K5. The antibodies were added to HMVECs at various concentrations for 1 hour at room temperature. Cells were washed and the amount of bound 125IK5 (dog) bound was counted. Competition binding was also tested against 2 nmol/L 3HK5. K5 peptides or a COOH-terminal antibody to GRP78 (A-129, Santa Cruz) were added to HMVECs at various concentrations for 1 hour at room temperature. Cells were washed and lysed and the amount of 3HK5 bound was estimated by scintillation counting.
Immunohistochemical analysis of glucose-regulated protein 78 on human microvascular endothelial cells. Cells were starved overnight with medium alone. Complete medium, containing 10% fetal bovine serum plus 15 ng/mL bFGF and 5 ng/mL VEGF, was added at different times to the cells. GRP78 bound antibody was visualized with horseradish peroxidase (HRP)–reactive substrate visualized by a brown color.
Binding and pull-down of recombinant kringle 5 binding proteins. Binding of rK5 to endothelial cells was measured as described (29). Briefly, HMVEC cells (50,000 per well) were cultured in 96-well microtiter plates for 2 hours, washed, and then incubated with PBS and increasing concentrations of 3HK5 or 125IK5 (dog) for another 2 hours at 4°C. After washing, cells were lysed and bound labeled rK5 was counted.
Cell surface rK5 binding proteins were isolated by two methods. The first method used NH2-terminal biotinylated PRKLYDY active site rK5 peptide with 5 x 107 endothelial or tumor cell lysates. Cell lysate was passed over an agarose-avidin-biotin-PRKLYDY column. The column was washed with two column volumes of 100 nmol/L of the NH2-terminal rK5 peptide. Bound proteins were eluted with excess unlabeled rK5. Mass spectrometry analysis was used to determine the bound proteins.
The second method used to identify cell surface rK5 binding proteins was described previously (29). Surface proteins on 4 x 106 EaHy cells were labeled with NHS-biotin. The cells were washed and lysed (M-Per, Pierce). Cell lysates were mixed with S-tag K5 for 1 hour at room temperature. S-tag K5 bound proteins were precipitated with S-protein agarose (Pierce). Bound proteins were eluted with excess rK5 or excess PRKLYDY peptide. Eluted proteins were visualized with avidin-HRP and a chemiluminescent substrate. Mass spectroscopic analysis was used to identify the major protein bands.
Binding of rK5 to rGRP78 was measured using equilibrium dialysis (30). In the top well of a 96-well equilibrium dialyzer (molecular weight cutoff 50 kDa, Harvard Apparatus, Holliston, MA), 150 µL of 10 nmol/L rGRP78 were added. In the reciprocal (bottom) chamber, 150 µL of increasing concentrations from 0.1 to 50 nmol/L 3HK5 were added. The chambers were shaken at room temperature for 72 hours. The total number of counts from both chambers after dialysis was compared with the number of counts remaining in the 3HK5 chamber.
RNA interference. RNA interference of GRP78 expression was induced with small interfering RNA (siRNA) directed against the GRP78 mRNA. Three different nucleotide siRNA primers were made that targeted human GRP78 mRNA sequence. The siRNAs started at position 139 (AAC GGC CGC GUG GAG AUC AUC), position 1,175 (AAG CUG UAG CGU AUG GUG CUG), and position 1,567 (AAG AUC ACA AUC ACC AAU GAC). A scrambled siRNA from position 1,567 was used as negative control (AAA UCA UAG CGU AUG GUG CUG). All oligonucleotides were from Dharmacon Research (Dharmacon RNA Technologies, Lafayette, CO).
EaHy or HT1080 cells were seeded at a density of 20,000 cells/cm2 the day before transfection and were 40% confluent when they were transfected with 50 nmol/L positive or scramble oligonucleotides in LipofectAMINE 2000 (Invitrogen) and Opti-MEM (Life Technologies, Gaithersburg, MD) without serum or BSA. Before transfection, the cells were washed once with Opti-MEM. Transfection medium was maintained on cells for 3 hours and was then removed and substituted with complete medium. The reduction in GRP78 protein, 48 hours after transfection, was estimated by Western blot analysis (31).
In selected studies, GRP78 siRNA-transfected or scrambled siRNA-transfected EaHy cells were grown in 96-well plates. The medium was changed and 3HK5 in PBS was added to the cells at various concentrations. The cells were incubated at room temperature for 2 hours and then washed thoroughly. Cell counts were measured for bound 3HK5 as described above. Data points were calculated from the average of triplicate samples.
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50 nmol/L (15). Here, we expand on those results using a yeast expressed rK5 (no detectable endotoxin) as well as synthetic K5 peptides to examine their effects on stimulated human endothelial cell proliferation, migration, and apoptosis assays. In these assays, rK5 inhibits stimulated human endothelial cell migration in a dose-dependent manner with an IC50 value (Table 1) of 0.20 nmol/L. rK5 inhibited endothelial cell migration induced by a wide variety of inducers of angiogenesis, including aFGF, bFGF, IL-8, PDGF, TGF-ß, and VEGF (Fig. 1A). It was selective for endothelial cells because it failed to inhibit the migration of neutrophils and fibroblasts even when tested at concentrations up to 1,000-fold higher than that at which it inhibited endothelial cell migration (data not shown). rK5 also showed selectivity for inhibition of proliferation of stimulated endothelial cells and did not cause inhibition of tumor or primary cell proliferation at concentrations as high as 100 µmol/L (Table 2). Apoptosis of stimulated endothelial cells was induced by rK5 (Fig. 1B) in a dose-dependent manner, indicating that the binding of rK5 initiates a cell signaling cascade leading to cell death.
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Glucose-regulated protein 78 is an endothelial cell surface recombinant kringle 5 binding protein. To reduce the cell surface nonspecific protein binding that is often observed with proteins, an immobilized NH2-terminally biotinylated PRKLYDY peptide was used to isolate K5 binding proteins from endothelial cell surfaces. Bound proteins were then eluted with excess rK5. Mass spectrometric sequencing of tryptic peptides from the major protein band (80 kDa; Fig. 2A) revealed sequences corresponding to GRP78 (78 kDa). That GRP78 is a cell surface binding protein for rK5 was further confirmed by coprecipitation of biotinylated surface proteins with S-tagged K5. The major bound biotinylated proteins eluted with excess rK5 from immobilized S-tagged K5 (Fig. 2B) were identified by mass spectrometric analysis as GRP78 and GRP94.
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vß3, had no effect, except at very high concentrations (Fig. 6B). The same NH2-terminal GRP78 antibody also blocked the rK5-induced inhibition of endothelial cell migration (Fig. 6C) and rK5-induced apoptosis of endothelial cells (Fig. 6D). The expression of GRP78 on endothelial cells was significantly reduced by the transfection of a siRNA for GRP78 but not by the transfection of a scrambled siRNA (Fig. 7A). The binding of rK5 to GRP78 siRNA-transfected HMVEC cells was very low compared with the binding of rK5 to scrambled siRNA-transfected cells (Fig. 7B). These results confirm the requirement for GRP78 expression for rK5 inhibitory activity on endothelial cells.
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0.7 nmol/L (Fig. 8A). This binding could be blocked by the addition of unlabeled rK5 (Fig. 8B). However, the rK5(K82A) mutant in concentrations up to 25-fold excess of GRP78 concentration did not block binding, confirming that GRP78 binds with high affinity and specificity to rK5.
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90% decrease in GRP78 expression in transfected HT1080 (GRP78–) cells (data not shown), which was very similar to the siRNA knockdown of GRP78 expression we observed in HMVECs (Fig. 7A). This decrease in GRP78 protein expression significantly eliminated the proapoptotic activity of rK5 on hypoxic HT1080 (GRP78–) cells (Fig. 9C). However, rK5 significantly induced apoptosis of the hypoxic HT1080 cells transfected with scrambled siRNA and control nontransfected cells. These data show the necessary GRP78 expression for rK5 induction of apoptosis on tumor cells.
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Discussion |
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90% of its ability to inhibit in vitro migration, although the kringle was still correctly folded as determined by nuclear magnetic resonance analysis (22). This mutant K5(K82A) did not compete with 3HK5 binding to endothelial cells or with direct binding of 3HK5 to recombinant GRP78 (Kd = 0.25 nmol/L). To further elucidate this binding, immunohistochemical analysis of cells with anti-GRP78 were examined with and without K5 or K5(K82A) mutant. Wild-type rK5 reduced the binding of the GRP78 antibody to cells, but the K5(K82A) mutant had no effect on GRP78 antibody binding. This specificity of binding on cell surfaces was also examined using the siRNA for GRP78 on endothelial cells. When siRNA transfection reduced the GRP78 protein concentration by 90% on EaHy and HT1080 cells, there was greatly reduced binding of 3HK5 to cell surfaces. These observations show that the effects of rK5 on proliferating endothelial cells require direct, specific, high-affinity binding to GRP78. This conclusion is not excluded by the results of a previous study reporting that the K5 endothelial cell surface binding protein was voltage-dependent anion channel (VDAC; ref. 33). That study used confluent, static monolayers of endothelial cells to examine rK5 binding. However, our findings show that under confluent conditions GRP78 is minimally exposed on the endothelial cell surface; hence, other binding proteins may play a more prominent role for rK5 binding under these static growth conditions. Our studies were carried out under subconfluent conditions. There are also reports that show VDAC is chaperoned to cell surfaces by heat shock or glucose-regulated proteins. It is possible that rK5 binds to GRP78, which is associated with VDAC on cell surfaces (34). VDAC siRNA knockdown experiments are under way to determine if rK5 binding to GRP78 may depend on VDAC expression. However, our results challenge the direct binding of rK5 to VDAC because of the tight binding of rK5 to GRP78 (Kd = 0.25 nmol/L) compared with the reported rK5 binding to VDAC (Kd = 28 nmol/L). A recent study showing that rK5 is more effective on hypoxic endothelial cells supports our data of increased rK5 surface binding to endothelial cells with stress and supports our finding of increased apoptosis by rK5 on hypoxic tumor cells. The GRP78 antibody and GRP78 siRNA provide nearly quantitative inhibition of rK5 binding to endothelial cell surfaces providing additional evidence for GRP78 as the dominant rK5 binding protein.
In addition to identifying and establishing the importance of an uncharacterized endothelial cell binding protein for rK5, our results also show that rK5 directly induces tumor cell apoptosis under stressed conditions. Reports have shown that the induction of GRP78 is critical for tumor cells to maintain viability during stress-like hypoxia and glucose deprivation. In addition, the induction of tumor cell surface–expressed GRP78 by hypoxic conditions has been linked to resistance to cytotoxic or radiation therapy in vitro and in vivo (35). Surface-expressed GRP78 was induced by hypoxia on the surface of 8 of the 10 tumor cell lines tested here. Under these conditions, rK5 had a very potent proapoptotic effect on all eight of the hypoxia-stressed tumor cell lines that induced surface-expressed GRP78. The two tumor lines, PC-3 and Lewis lung, on which rK5 had no proapoptotic effect, did not up-regulate GRP78 under hypoxic conditions. These results show the selectivity of rK5 for most stressed tumor cells and activated endothelial cells but not for normal or quiescent endothelial cells.
The identification of GRP78 as an endothelial and tumor cell–binding site required for the antitumor and antiangiogenic activity of rK5 raises several questions concerning the mechanism by which rK5 induces cellular apoptosis. Although rK5 binds to GRP78, there is not a predicted transmembrane domain in GRP78 to transfer signal to the cytosol. However, published reports indicate that GRP78 may protect cells from apoptosis during stress by blocking procaspase-7 activation (36). We hypothesize that rK5 binds to GRP78 and is endocytosed into the cell where rK5 blocks GRP78 binding to procaspase-7. Evidence supporting this hypothesis is shown by our finding that when rK5 is added to stressed endothelial cells or stressed HT1080 cells caspase-7 activity is increased at least 2-fold within 2 hours. We also show that the cell surface concentration of GRP78 is greatly reduced within a few hours after rK5 binding (Fig. 10B). Expression of recombinant procaspase-7 is under way, which will help to determine if rK5 can inhibit the binding rGRP78 to procaspase-7. Although rK5 could block the protection of procaspase-7 leading to active caspase-7 generation, the appearance of other caspases within the cell necessary to activate caspase-7 argues that additional mechanisms for rK5's antiangiogenic activity exist.
In summary, we have shown that an interaction between rK5 and GRP78 on stimulated endothelial and stressed tumor cells underlies the proapoptotic activity of rK5. To our knowledge, this is the first evidence that an antiangiogenic compound is effective directly against stressed tumor cells. Finally, we predict that a two-stage inhibition of in vivo tumor growth by rK5 could exist. First, rK5 would induce apoptosis in activated tumor endothelial cells. Subsequently, the consequent hypoxia would cause tumor cells to present surface-expressed GRP78, allowing rK5 to enhance tumor cell apoptosis. The second state of rK5 action could go beyond tumor stasis and possibly lead to tumor regression. Further efforts to characterize the biology of GRP78 as well as the binding interaction between GRP78 and rK5 may yield insight into both the regulation of angiogenesis and the survival of hypoxic tumor cells. These findings could lead to the development of novel agents targeted toward pathologic angiogenesis and tumor cell survival.
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Acknowledgments |
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Received 9/21/04. Revised 12/17/04. Accepted 3/22/05.
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