Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M703342200 on September 11, 2007

J. Biol. Chem., Vol. 282, Issue 45, 32811-32820, November 9, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/45/32811    most recent
M703342200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gonzalez-Gronow, M.
Right arrow Articles by Pizzo, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gonzalez-Gronow, M.
Right arrow Articles by Pizzo, S. V.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Plasminogen Structural Domains Exhibit Different Functions When Associated with Cell Surface GRP78 or the Voltage-dependent Anion Channel*

Mario Gonzalez-Gronow1, Steven J. Kaczowka, Sturgis Payne, Fang Wang, Govind Gawdi, and Salvatore V. Pizzo

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, April 20, 2007 , and in revised form, September 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Both the voltage-dependent anion channel and the glucose-regulated protein 78 have been identified as plasminogen kringle 5 receptors on endothelial cells. In this study, we demonstrate that kringle 5 binds to a region localized in the N-terminal domain of the glucose-regulated protein 78, whereas microplasminogen does so through the C-terminal domain of the glucose-regulated protein 78. Both plasminogen fragments induce Ca2+ signaling cascades; however, kringle 5 acts through voltage-dependent anion channel and microplasminogen does so via the glucose-regulated protein 78. Because trafficking of voltage-dependent anion channel to the cell surface is associated with heat shock proteins, we investigated a possible association between voltage-dependent anion channel and glucose-regulated protein 78 on the surface of 1-LN human prostate tumor cells. We demonstrate that these proteins co-localize, and changes in the expression of the glucoseregulated protein 78 affect the expression of voltage-dependent anion channel. To differentiate the functions of these receptor proteins, either when acting singly or as a complex, we employed human hexokinase I as a specific ligand for voltage-dependent anion channel, in addition to kringle 5. We show that kringle 5 inhibits 1-LN cell proliferation and promotes caspase-7 activity by a mechanism that requires binding to cell surface voltage-dependent anion channel and is inhibited by human hexokinase I.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasminogen (Pg)2 is a single-chain 92-kDa glycoprotein containing 791 amino acid residues (1, 2). There are seven distinct domains in the Pg molecule: an N-terminal peptide (NTP), five kringle domains, and a serine proteinase domain (3). The kringle domains, each comprising ~80 amino acids, are double-looped disulfide-bonded structures that contain the lysine-binding sites (LBS) responsible for Pg binding to extracellular matrix molecules (4) and cell receptors (5, 6). In addition to the LBS, Pg also has three benzamidine binding sites (BBS), one in K5, a second in the active site of the proteinase domain, and a third unknown site in the Pg proteinase domain (7).

Pg is the precursor of angiostatins, a group of anti-angiogenic molecules, consisting of kringles 1-3 (K1-3), 1-4, and 1-5, as well as the single kringles 1 (K1), 2, 3, and 5, but not K4 (8-10). We have extensively studied the functions of K1-3 and K5 on both endothelial and prostate tumor cell lines (11, 12). We reported that K5 binding to the voltage-dependent anion channel (VDAC) on the surface of human umbilical vein endothelial cells (HUVEC) interfered with mechanisms controlling the regulation of intracellular Ca2+ producing a decrease in intracellular pH (11). A recent study also suggests, that K5 binds with high affinity to surface-expressed glucose-regulated protein 78 (GRP78) (13). Both VDAC and GRP78 are expressed on the cell surface in lipid rafts (14, 15). Because trafficking of VDAC to the cell membrane is associated with the heat shock proteins GRP75 and HSP70 (16, 17), we hypothesized a possible association between VDAC and GRP78 in regulating the functions of VDAC on the cell surface.

The purpose of these studies was to differentiate functions of these proteins, either when acting singly or as a complex. We used human hexokinase I (HK-I) as a specific ligand for VDAC. In the search of a specific ligand for GRP78, we identified microplasminogen as a candidate, and found a putative sequence in this Pg domain containing the third BBS of the Pg molecule, with affinity for a domain in the C-terminal domain of GRP78. We demonstrate that a delicate balance exists between the interaction of HK-I and K5 with VDAC, which may be physiologically relevant to protect cells from apoptosis. We also determined that K5 induces a Ca2+ signaling cascade only through VDAC, whereas microplasminogen does so via GRP78.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Culture media were from Invitrogen. Porcine pancreatic elastase, gastric mucosa pepsin, and trypsin inhibitor were from Sigma. Endothelial cell growth supplement was from Collaborative Research Inc. (Waltham, MA). Na125I was obtained from Perkin-Elmer Life Sciences (Boston, MA). The 20-amino acid peptide SWGLGCARPNKPGVYVRVSR (Ser759-Arg778) of human Pg was obtained from Genemed Synthesis, Inc. (San Francisco, CA). Fura-2/AM was purchased from Molecular Probes, Inc. (Eugene, OR). Protein A-Sepharose was purchased from Sigma.

Proteins—Human Pg purified by affinity chromatography on l-Lysine-Sepharose (18) was digested with elastase and fractionated by gel and affinity chromatography to obtain essentially pure mini-Pg, K1-3, K4, and K5 (3, 7, 19, 20). A functionally active human microplasminogen without kringle structures was produced by incubation of Pg with urokinasefree plasmin at an alkaline pH (21). Human tissue plasminogen activator (t-PA) was purchased from American Diagnostica, Inc. (Stamford, CT). Human brain hexokinase I (clone pET-24b-HK-I), a kind gift of Dr. Herbert J. Fromm, Iowa State University, Ames, IA, was produced in Escherichia coli and purified as previously described (22). Recombinant murine GRP78, a kind gift of Dr. Sylvie Y. Blond, College of Pharmacy, University of Illinois, Chicago was purified as previously described (23). Radioiodination of proteins was carried out by the method of Markwell (24). Incorporation of 125I label was 2 x 107 cpm/nmol of protein. Radioactivity was measured in a Pharmacia-LKB Biotechnology 1272 {gamma}-radiation counter.

Antibodies—Anti-GRP78 IgG from sheep serum was purified by affinity chromatography on protein A-Sepharose (25), followed by immunoadsorption to GRP78 coupled to Sepharose 4B. Goat IgG antibodies against an N-terminal region (N-20), a central domain (H-129) or a C-terminal region of GRP78 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A rabbit IgG antibody against a region including the amino acids 185NDGTEFGGSIYQK197 of VDAC1 was obtained from Affinity Bioreagents (Golden, CO). IRDye 800 DX-conjugated affinity-purified anti-sheep or anti-rabbit IgGs were purchased from Rockland Immunochemicals, Inc. (Gilbertsville, PA). Alexa Fluor 488-conjugated donkey anti-sheep IgG and Alexa Fluor 568-conjugated goat anti-rabbit IgG were obtained from Molecular Probes, Inc. (Eugene, OR).

Cell Cultures—All tumor cell lines were grown in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/ml penicillin G, and 100 µg/ml streptomycin as described previously (26). HUVEC were obtained from Clonetics (San Diego, CA) and grown in Dulbecco's modified Eagle's medium supplemented with 2% bovine calf serum, 100 units/ml penicillin/streptomycin, 2.5 µg/ml amphotericin B, 2 mM glutamine, 5 units/ml sodium heparin, and 200 µg/ml endothelial cell growth supplement (ECGS) (Collaborative Research). For experiments under hypoxic conditions (2% O2, 5% CO2, N2 balanced), the cells were incubated for 24 h in a C-chamber® connected to a ProOx® controller, both from Biospherix (Redfield, NY).

Immunofluorescence Staining and Laser Scanning Confocal Microscopy of Non-permeabilized Cells—1-LN cells were grown on glass coverslips in RPMI 1640 containing 5% FBS. The cells were washed with Hanks' balanced salt solution (HBSS) containing 1 mM CaCl2 and MgCl2, and fixed for 30 min at room temperature in 7% fresh formaldehyde in PBS. The coverslips were then incubated in 5% bovine serum albumin (BSA) in PBS for 90 min at room temperature. Excess serum was drained from the coverslips, and cells were incubated with the primary sheep anti-GRP78 or rabbit anti-VDAC1 IgGs in PBS containing 1% BSA, and 5 mM EDTA for 90 min at 4 °C. The cells were then rinsed three times in PBS and incubated with a secondary antibody containing either a 1:400 dilution of an Alexa Fluor 488-conjugated donkey anti-sheep IgG, an Alexa Fluor 568-conjugated goat anti-rabbit IgG or a mixture of both for 90 min at 4 °C in the dark. As controls, cells were incubated with the secondary IgG alone. Finally, the cells were washed three times with PBS, and the coverslips were placed face down on 1.0-mm thick microscopy slides using mounting medium consisting of 90% glycerol, 0.1 M n-propylgalate, and 0.01% sodium azide. The coverslips were then sealed with nail polish and stored at 4 °C in a light tight box. Confocal microscopy was performed using a Zeiss LSM 410 laser scanning confocal microscope with a krypton/argon laser and 40X/1.30 oil immersion lens (Carl Zeiss). Alexa Fluor 488 and Alexa Fluor 568 fluorophores were detected with 510-560 band pass and 590 long pass filters, respectively. All images are single confocal sections prepared with Adobe Photoshop 5.0 software.

Measurement of Intracellular Calcium Levels—The intracellular calcium concentration ([Ca2+]i) was measured using the fluorescent indicator Fura-2/AM (27) in 1-LN cell monolayers plated on sterile coverslips on 35-mm tissue culture dishes. Fura-2/AM (2 mM) was added to the plates and incubated at 37 °C for 30 min, followed by rinsing with Hank's balanced salt solution. Glass coverslips, containing the cell monolayers in serum-free RPMI 1640 cell culture medium, were then placed on the inverted microscope and [Ca2+]i was measured using a digital imaging microscopy system employing dual excitation ratio imaging techniques. The overall digital imaging microscopy system consists of an IC-300 imaging work station (Inovision Corp., Research Triangle Park, NC), a Zeiss IM35 inverted microscope with a 75 watt xenon excitation lamp and a x40 UVF 1.3 N, a Nikon objective, a low-light level ISIT 66 camera (Dage-MTI, Michigan City, IN), computer-controlled excitation and neutral density filter wheels, and a temperature controller maintaining temperature at 37 °C using controlled heated air circulation and a heated cell chamber. After collecting baseline data, the ligands are added to the cell monolayers to determine their effect on [Ca2+]i. Digitized video images are obtained by averaging up to 260 frames with the following filter combination: Fura-2 excitation, 240 and 380 nm; and emission >450 nm. Routinely, excitation intensity was attenuated 100-1000-fold before reaching the cell, to obtain background images. [Ca2+]i was measured by subtracting the background from images on a pixel basis. To obtain [Ca2+]i for an individual cell, the mean value of the pixel ratio for the cell was compared with values obtained with the same equipment using Fura-2-containing Ca2+-EGTA buffers (27).

On Cell Westerns—Tumor cell monolayers were grown in 48-well culture plates until confluent and rinsed in HBSS. All binding assays were done at 4 °C. Ice-cold 2% paraformaldehyde was added to each well, and the plate was incubated at room temperature for 20 min. Cells were washed three times with 400 µl of PBS and incubated with a blocking solution containing 3% BSA and 5% nonimmune goat serum for 90 min at room temperature with gentle rocking. Blocking buffer was removed, and cells were covered with 200 µl of a solution containing sheep anti-GRP78 or rabbit anti-VDAC1 IgGs (50 ng/ml) in blocking buffer. Cells were then incubated overnight at 4 °C with gentle rocking. The next day, wells were rinsed three times in PBS and incubated with 200 µl of a solution containing a 1:800 dilution of an IRDye 800 DX-conjugated affinity-purified anti-sheep or anti-rabbit IgGs in blocking buffer. Plates were kept under low light conditions following addition of IR-conjugated antibodies. Following a 60-min incubation at room temperature with gentle rocking, plates were again washed three times with PBS. They were then dried and imaged using the LI-COR Odyssey System. Images were analyzed using Excel (Microsoft Corp., Redmond, WA) and Prism (GraphPad Software, San Diego, CA) software.

Ligand Binding Analysis—The cells were grown in 96-well strip tissue culture plates until the monolayers were confluent. The cells were then rinsed in HBSS. All binding assays were performed at 4 °C in serum-free RPMI 1640 containing 2% BSA. Increasing concentrations of 125I-labeled ligand were incubated with cells for 60 min. Free and bound ligand were separated by aspirating the incubation mixture and washing the cell monolayers rapidly three times with RPMI 1640 containing 2% BSA. The cells were stripped from the plates and radioactivity determined. The bound K5 and microplasminogen were calculated after subtraction of nonspecific binding measured in the presence of 50 mM p-aminobenzamidine. The bound hexokinase I (HKI) was calculated after subtraction of nonspecific binding measured in the presence of 50 µM nonlabeled HKI. The Kd and Bmax were determined after Scatchard Plot analyses using the statistical program GraphPad Prism® 4 from Graph-Pad Software, Inc. (San Diego, CA).

Cell Proliferation Assays—Cells suspended in RPMI 1640 containing 5% FBS at a density of 1 x 105/ml were plated in 96-well culture plates (0.1 ml/well) containing increasing concentrations of the tested ligands at a final volume of 0.2 ml/well. Cell proliferation was determined at 72 h using a BrdUrd labeling and colorimetric immunoassay detection method (Roche Applied Science, Indianapolis, IN). Results were expressed as absorbance at 372 nm (reference wavelength: 492 nm). Control cell proliferation was determined in the absence of any ligand.

RNA Interference—RNA interference of GRP78 expression was induced with siRNA directed against the GRP78 mRNA as previously described (28). The chemical synthesis of dsRNA to the target GRP78 370KIQQLVK376 mRNA, and a Silencer® Negative control siRNA (control 1) was performed by Ambion (Austin, TX). The sense 5'-AAU ACA GCA AUU AGU AAA GTT-3' and the antisense 5'-CUU UAC UAA UUG CUG UAU UTT-3' oligonucleotides were annealed according to the manufacturer's instructions. Cells were seeded at a density of 1 x 105 cells/well in 12-well plates the day before the transfection with 40 nM positive or control oligonucleotides in Lipofectamine® 2000 from Invitrogen (Gaithersburg, MD). Transfection medium was maintained on cells for 5 h and was then removed and substituted with complete medium. The reduction in GRP78 expression on the cell surface, 48 h after transfection, was estimated by on cell Western and PCR analyses.

Measurement of GRP78 and VDAC mRNA Levels by Reverse Transcription—Total RNA from HUVEC or 1-LN cells treated with Lipofectamine or GRP78 dsRNA-Lipofectamine was extracted by a single step method using a RNeasy® Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Total RNA was reverse-transcribed with 1 µg of RNA in a 20-µl reaction mixture, using M-MLV (Moloney murine leukemia virus) reverse transcriptase (200 units) and oligo(dT) as the primer for 1 h at 42 °C. The resulting cDNA (5 µl) was used as a template, and a 276-bp segment of the GRP78 cDNA was amplified using a 21-mer upstream primer (5'-AGAAATGAGTTGGAAAGCTAT-3') identical to positions corresponding to amino acids Arg562-Tyr568 and a 21-mer downstream primer (5'-CTAACAACTCATCTTTTTCTGCTG-3') complementary to positions Ala649-Leu654 of the amino acids encoded in the GRP78 mRNA. Similarly, a 237-bp segment of the VDAC1 cDNA was amplified using a 21-mer upstream primer (5'-GGCCTGACGTTTACAGAGAAA-3') identical to positions corresponding to amino acids Gly68-Lys74 and a 21-mer downstream primer (5'-CTCGTAACCTAGCACCAGAGC-3') complementary to positions Ala141-Glu147 of the amino acids encoded in the VDAC1 mRNA. A 302-bp segment of mouse beta-actin (constitutive internal control) cDNA was co-amplified using a set of PCR primers provided in a R&D Systems kit (Minneapolis, MN). Amplification was carried out in a Whatman-Biometra T3 thermal cycler (Whatman Inc., Florham Park, NJ) for 35 cycles (one cycle: 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 45 s). PCR products were analyzed on a 1.2% (w/v) agarose-ethidium bromide gel. The gels were photographed, and the intensity of the individual GRP78 or VDAC1 and beta-actin mRNA bands were evaluated and quantified as GRP78/beta-actin or VDAC1/beta-actin ratios.

Caspase-7 Activity Assay—Caspase-7 activity was assayed with the chromogenic substrate Ac-DEVD-pNA (29) from Alexis Biochemicals (San Diego, CA). For preparation of lysates, 1-LN cells grown in 24-well plates (4 x 106 cells/well) were incubated as required, washed twice with ice-cold PBS, and resuspended in lysis buffer containing 50 mM Hepes (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, and 10% glycerol. The cells were frozen and thawed three times in dry ice, and then centrifuged at 100,000 x g for 30 min at 4 °C. The protein concentration in the supernatant fractions (the lysate) was determined by the Bradford assay from Bio-Rad. Assay mixture was prepared in a 96-well plate in a 0.2-ml reaction mixture containing 20 µg of lysate protein and the substrate Ac-DEVD-pNA (0.25 mM) in lysis buffer and incubated at 37 °C for 90 min. Substrate hydrolysis was quantified using a molar extinction coefficient of 8800 M-1 x cm-1 for pNA at 405 (30). All experiments were done in triplicate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Binding of K5 and Microplasminogen to HUVEC and 1-LN Human Prostate Cancer Cells under Normoxic and Hypoxic Conditions—Both K5 and microplasminogen bind to a large number of sites on HUVEC and 1-LN cells in a dose-dependent manner with high affinity. We had previously assessed only one binding site of K5 to HUVEC (11); however, here, we studied the binding of K5 at lower concentrations and assessed a second high affinity binding site of this ligand to either HUVEC or 1-LN cells under normoxic and hypoxic conditions. The binding isotherm of K5 to HUVEC under normoxic conditions (Fig. 1A), and a Scatchard plot analysis, demonstrate both the lower affinity (Kd of 24 ± 4 nM) and the higher affinity (Kd of 0.4 ± 0.2 nM) binding sites (Fig. 1A, inset). Similarly, 1-LN cells possess both binding sites (Table 1). We previously identified the lower affinity binding site in HUVEC and 1-LN cells as VDAC (11). The higher affinity binding site for K5 was identified as GRP78 and is localized in a region, which is blocked with an antibody (N-20) specific for the GRP78 N-terminal domain (13). When increasing concentrations of K5 were added to HUVEC in the presence of the antibody N-20 (5 µM), K5 still binds to the cells; however, it binds only to the lower affinity binding site (Fig. 1B and inset). A similar experiment in the presence of an anti-VDAC IgG (5 µM) masks only the lower affinity binding site, clearly demonstrating the higher affinity K5 binding site (Fig. 1C and inset). These experiments demonstrate that GRP78 is the higher affinity binding site for K5, whereas VDAC is its lower affinity counterpart. The total number of K5 binding sites on the surface of these cells is large (24 ± 6 x 105 and 15 ± 3 x 105 binding sites for 1-LN and HUVEC cells, respectively) (Table 3).


View this table:
[in this window]
[in a new window]

  		TABLE 1
Binding of plasminogen fragments and HK1 to 1-LN and HUVEC cells under normal oxygen conditions

 


View this table:
[in this window]
[in a new window]

  		TABLE 3
Binding of plasminogen fragments and HK1 to 1-LN and HUVEC cells under normal and hypoxia conditions

 


Figure 1
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 1.
Binding of K5 and microplasminogen to HUVEC under normoxic conditions. All experiments were performed on cells grown in 96 strip-well tissue culture plates (2 x 104 cells/well). A, increasing concentrations of 125I-K5 were added to HUVEC cell monolayers. B, increasing concentrations of 125I-K5 were added to HUVEC cell monolayers in the presence of anti-GRP78 (N-20) IgG (5 µM). C, increasing concentrations of 125I-K5 were added to HUVEC cell monolayers in the presence of anti-VDAC IgG (5 µM). Bound ligand was calculated after subtraction of nonspecific binding measured in the presence of 50 mM p-amino-benzamidine. Insets, Scatchard plot analyses of the binding isotherms. The data are the means ± S.D. from experiments performed in triplicate.

 
Next, we studied the kinetics of binding of K5 to HUVEC and 1-LN cells under hypoxic conditions. The results show changes in affinity of K5 for both the lower and the higher affinity binding sites (Table 2); whereas, the number of binding sites increases nearly 2-fold in both cells types (53 ± 7 x 105 and 35 ± 5 x 105 binding sites for 1-LN and HUVEC cells, respectively) (Table 3), thereby suggesting that hypoxia induces changes in the affinities and number of binding sites of both GRP78 and VDAC for K5.


View this table:
[in this window]
[in a new window]

  		TABLE 2
Binding of plasminogen fragments and HK1 to 1-LN and HUVEC cells under hypoxic conditions

 
Binding of Microplasminogen to Immobilized GRP78, HUVEC, and 1-LN Human Prostate Cancer Cells—Pg has three benzamidine binding sites, K5, the active site of the proteinase domain and an unknown site in the proteinase domain (7). Microplasminogen, the segment of Pg including amino acids Lys530-Asn790, contains an as yet unidentified binding site for benzamidine. Because K5 binds to GRP78, and the binding is mediated by its benzamidine binding site (data not shown), we assessed the capacity of microplasminogen to bind to GRP78. Both 125I-labeled K5 and microplasminogen bind in a dose-dependent manner (Fig. 2A); however, K5 shows a higher affinity for GRP78 than microplasminogen (Kd of 5 ± 2 and 28 ± 3 nM for K5 and microplasminogen, respectively). The binding of K5 to GRP78 is inhibited by Pg fragments containing K1-3, K4 and miniplasminogen (a K5 containing Pg fragment), but not by microplasminogen (Fig. 2B). Microplasminogen binds to HUVEC and 1-LN cells in a dose-dependent manner to a single class of binding sites. The binding isotherm and a Scatchard plot of microplasminogen binding to HUVEC under normal oxygen conditions are shown in Fig. 2C. Binding of 125I-labeled microplasminogen to GRP78 is inhibited only by non-labeled miniplasminogen or microplasminogen, but not by K5 (Fig. 2D). Taken together, these experiments suggest that K5 and microplasminogen bind to different regions of GRP78.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 2.
Binding of K5 to GRP78 and microplasminogen to HUVEC and immobilized GRP78. A, increasing concentrations of both 125I-labeled K5 (•) or microplasminogen ({circ}) were added to GRP78 immobilized on 96 strip-well tissue culture plates. B, inhibition of binding of 125I-K5 (100 nM) to immobilized GRP78 by increasing concentrations of nonlabeled K4 (•), K1-3 ({blacksquare}), miniplasminogen ({blacktriangleup}), or microplasminogen ({diamondsuit}). C, binding of increasing concentrations of 125I-labeled microplasminogen were added to HUVEC cell monolayers. Inset, Scatchard plot analysis of the binding isotherm. D, inhibition of binding of 125I-labeled microplasminogen to immobilized GRP78 by increasing concentrations of nonlabeled K5 (•), miniplasminogen ({blacktriangleup}), or microplasminogen ({blacksquare}). The data are the means ± S.D. from experiments performed in triplicate.

 


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 3.
Binding of HK-I to HUVEC and 1-LN cells. Increasing concentrations of 125I-labeled HK-I were added to HUVEC cell monolayers (2 x 104 cells/well) (A) or 1-LN cells (B) grown on 96 strip-well tissue culture plates. Bound ligand was calculated after subtraction of nonspecific binding measured in the presence of 5 µM nonlabeled HK-I. Insets, Scatchard plot analyses of the binding isotherms. The data are the means ± S.D. from experiments performed in triplicate.

 
The binding data (Table 1) shows that microplasminogen binds to both cell types with similar affinities, under normoxic conditions (Kd of 28 ± 4 and 22 ± 2 nM for 1-LN and HUVEC cells, respectively) and to a large number of binding sites (36 ± 8 x 105 and 18 ± 1 x 105 binding sites for 1-LN and HUVEC cells, respectively). Binding of microplasminogen to cells under hypoxia was also assessed. Under these conditions, the affinity for microplasminogen increases (Kd 4 ± 1 and 12 ± 0.6 nM for 1-LN and HUVEC cells, respectively). As observed above for K5 the number of binding sites for microplasminogen also increases for both HUVEC and 1-LN cells under hypoxia (Table 3).

Binding of HK-I to HUVEC and 1-LN Human Prostate Cancer Cells—HK-I binds to mitochondrial VDAC via a hydrophobic 15-amino acid sequence in its N-terminal region (31, 32). In our experiments we employed HK-I as specific ligand for VDAC expressed on the surface of HUVEC and 1-LN cells. HK-I binds to both cell types in a dose-dependent manner (Fig. 3, A and B) with similar affinity (Table 1) to a single site (Fig. 3, A and B, insets). Experiments conducted under hypoxic conditions show a decrease in affinity of HK-I, whereas its number of binding sites is increased in both cell types (Tables 2 and 3).

Effect of K5 and Microplasminogen Binding on 1-LN cells [Ca2+]i—Because both K5 and microplasminogen bind to GRP78, we investigated the effects induced by these Pg fragments on 1-LN cells. First, we assessed the changes in [Ca2+]i induced by K5 in the presence of antibodies against GRP78 and VDAC. The results (Fig. 4A) show that K5 (100 nM) added to 1-LN cells induces a transient rise in [Ca2+]i lasting about 200 s before returning to base line. Addition of the N-20 anti-GRP78 against the N-terminal domain, which blocks binding of K5 to GRP78 (13) or the C-20 antibody against the C-terminal domain of GRP78 did not influence the changes in [Ca2+]i induced by K5; however, an antibody against a region including the amino acids 185NDGTEFGGSIYQK197 of VDAC1 inhibited the response induced by K5 on 1-LN cells (Fig. 4A). Addition of BAPTA, a cytosol permeable Ca2+ chelator, did not produce any changes in the [Ca2+]i response induced by K5; however, both EDTA, an extracellular Ca2+ chelator, or ruthenium red, a hexavalent polysaccharide stain, which induces VDAC closure (33, 34) inhibited the response induced by K5 (Fig. 4B). Incubation of 1-LN cells with HK-I, induces a rise in [Ca2+]i lasting about 150 s. This incubation with HK-I completely blocks the [Ca2+]i induced by K5 (Fig. 4C). When the cells were incubated with HK-I in the presence of glucose 6-phosphate (1 mM), an antagonist of HK-I binding to VDAC (34), addition of K5 induced a [Ca2+]i response similar to that induced in the absence of HK-I (Fig. 4C). These experiments clearly show that only antibodies blocking binding to VDAC are able to inhibit the [Ca2+]i response initiated by K5 on 1-LN cells. Furthermore, HK-I, a specific VDAC agonist, is able to inhibit the action of K5, thereby suggesting that VDAC alone is the mediator of K5 induced [Ca2+]i responses. These experiments also suggest that the VDAC region including the sequence 185NDGTEFGGSIYQK197 is the binding region for HK-I. Interestingly, this region is structurally localized in the vicinity of the sequence Lys234-Lys255, which we previously identified as the K5 binding region of VDAC (11).


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 4.
Changes in [Ca2+]i in 1-LN cells exposed to K5. The cells were preloaded with Fura-2/AM (4 µM), and the changes in [Ca2+]i were measured after addition of K5 (100 nM). The arrows indicate the time of addition of the ligands. A, effect of K5 alone (•) or in cells preincubated (30 min) with 100 nM anti-GRP78 N-20 IgG ({square}), 100 nM anti-GRP78 C-20 IgG ({triangleup}), or 100 nM anti-VDAC IgG ({blacktriangleup}). B, effect of K5 alone (•) or in the presence of 1 mM EDTA({square}), 50 µM ruthenium red ({triangleup}), and 5 µM BAPTA ({blacktriangleup}). C, effect of HK-I (100 nM) for 3 min followed by K5 (•), or HK-I (100 nM) in the presence of glucose 6-phosphate (200 nM) for 3 min, followed by addition of K5 ({circ}).

 


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 5.
Changes in [Ca2+]i in 1-LN cells exposed to microplasminogen. The cells were preloaded with Fura-2/AM (4 µM), and the changes in [Ca2+]i were measured after addition of microplasminogen or the 20-amino acid Ser759-Arg778 peptide of human Pg (100 nM). The arrows indicate the times of addition of each ligand. A, effect of microplasminogen (•), the Ser759-Arg778 peptide ({square}) or the Ser759-Arg778 peptide in the presence of 1 mM p-aminobenzamidine ({triangleup}). B, effect of the Ser759-Arg778 peptide alone (•), or in the presence of 50 µM ruthenium red ({triangleup}), 1 mM EDTA ({square}), and 5 µM BAPTA ({blacktriangleup}). C, effect of the Ser759-Arg778 peptide alone ({circ}), or in the presence of 100 nM anti-GRP78 N-20 IgG (•), 100 nM anti-GRP78 C-20 IgG ({blacktriangleup}), 100 nM anti-GRP78 H-129 IgG ({triangleup}), and 100 nM anti-VDAC IgG ({blacksquare}).

 


Figure 6
View larger version (37K):
[in this window]
[in a new window]

  		FIGURE 6.
Immunofluorescence microscopy of GRP78 and VDAC on 1-LN cell surface. Cells were incubated with sheep anti-GRP78 or rabbit anti-VDAC IgGs as described under "Experimental Procedures." A, immunofluorescent staining with Alexa 488-conjugated anti-sheep IgG alone. B, immunofluorescent staining with Alexa 568 anti-rabbit IgG alone. C, same field of 1-LN cells under visible light. D, immunofluorescent staining with anti-GRP78 IgG followed by the Alexa 488-conjugated anti-sheep IgG. E, immunofluorescent staining with anti-VDAC IgG followed by the Alexa 568-conjugated anti-rabbit IgG. F, confocal fluorescent stain obtained after merging in a single picture the two previous images.

 
GRP78 is a receptor for Dengue virus serotype 2 (35). Dengue patients develop high antiviral antibody titers cross-reactive with Pg (36, 37). A Pg region containing amino acids Ser759-Phe778 is homologous to the amino acid sequence Gly100-Phe119, contained in the Dengue type 2 envelope glycoprotein (E) (36). Therefore, we hypothesized that the peptide stretch Ser759-Phe778 could contain the binding region of microplasminogen to GRP78. Incubation of 1-LN cells with microplasminogen (100 nM) produced changes in [Ca2+]i, which lasted for 250 s (Fig. 5A), an effect which was mimicked by the Ser759-Phe778 peptide (100 nM) and inhibited when 1-LN cells were incubated with the peptide in the presence of p-aminobenzamidine (1 mM). Inhibition of binding of the Ser759-Phe778 peptide by p-aminobenzamidine suggests this region is the third benzamidine binding region of the Pg molecule (7). Neither ruthenium red (20 µM) or EDTA (1 mM) were able to inhibit the [Ca2+]i response induced by the Ser759-Phe778 peptide (100 nM); however, BAPTA (5 µM) was able to suppress the response (Fig. 5B). 1-LN cells incubated with the anti-GRP78 antibodies N-20, H-129 or anti-VDAC before addition of the Ser759-Phe778 peptide (100 nM) had a normal [Ca2+]i response; however, when the cells were incubated with the anti-GRP78 C-20 antibody against the C-terminal domain of GRP78, the [Ca2+]i response was almost completely inhibited (Fig. 5C).

Taken together these results suggest that K5 binds to both VDAC and GRP78 on the cell surface, but only VDAC is able to mediate a Ca2+ signaling cascade. Conversely, GRP78 mediates aCa2+ signaling cascade when it binds microplasminogen. Furthermore, K5 binds to a region on the N-terminal domain of GRP78, whereas microplasminogen binds to the C-terminal domain of GRP78. The [Ca2+]i response induced by K5 involves an influx of Ca2+ from the extracellular environment toward the cytosol, whereas microplasminogen induces a [Ca2+]i response involving release of Ca2+ from intracellular stores.

Immunofluorescent Microscopy of GRP78 and VDAC1 on the Surface of 1-LN Human Prostate Cancer Cells—1-LN cells express both these proteins on their surface, as demonstrated by their high reactivity with specific antibodies anti-GRP78 or anti-VDAC in non-permeabilized cells (Fig. 6, D and E). Control experiments performed with secondary antibodies alone (Fig. 6, A and B) show minimal reactivity. These two proteins appear to be co-localized (Fig. 6F) and show a plasma membrane staining pattern concentrated around areas of contact between cells. Because both GRP78 and VDAC are expressed on hepatocyte cell surfaces accumulated in lipid rafts (14, 15), a similar association between these two proteins may also exist in 1-LN cells.

Silencing the GRP78 Gene and Expression of GRP78 and VDAC on the Surface of 1-LN Cells—Because both GRP78 and VDAC are colocalized on the surface of 1-LN cells, and heat shock proteins are known to be involved in membrane-associated trafficking of VDAC and modulation of channel properties (16, 17), studies were performed to demonstrate that silencing of the GRP78 gene with dsiRNA was effective in suppressing not only expression of GRP78, but possibly VDAC as well. 1-LN cells were seeded on 12-well plates (1 x 105cells/well) and treated with a dsiRNA to GRP78 or a negative control dsRNA and incubated as described under "Experimental Procedures." Expression of target proteins on the cell surface was analyzed by on-cell Western analyses (Fig. 7, panels B1, B2, and B3, for GRP78 and Fig. 7, panels B4, B5, and B6 for VDAC, respectively). Analyses of cells treated with a negative control dsRNA show very little change on the expression of these proteins on the cell surface; however, cells incubated with dsiRNA targeted to the GRP78 gene show a significant decrease on the expression of both GRP78 and VDAC on the cell surface. Analyses of mRNA levels by PCR amplification show a decrease of both GRP78 and VDAC mRNA levels on cells treated with dsRNA targeted to GRP78 (Fig. 7, panels C3 and C6 for GRP78 and VDAC, respectively) when compared with cells treated with a negative control dsRNA(Fig. 7, panels C2 and C5 for GRP78 and VDAC, respectively) or untreated cells (Fig. 7, panels C1 and C4 for GRP78 and VDAC, respectively). This experiment suggests that changes in the expression of GRP78 affect the expression of VDAC on the cell surface.

Effect of Pg Fragments on 1-LN Cell Proliferation—1-LN cell proliferation is inhibited by K5 (100 nM), but remains unaffected by K4 alone (100 nM) (Fig. 8A). Incubation of the cells with K5 (100 nM) in the presence of K4 (500 nM) does not alter the change induced by K5; however, cells incubated with K5 (100 nM) in the presence of HK-I show a proliferation rate similar to that of untreated cells (Fig. 8B), thereby suggesting that HK-I is able to reverse the effect of K5 via interaction with VDAC.


Figure 7
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 7.
Expression of GRP78 and VDAC on 1-LN cells after silencing the GRP78 gene with dsiRNA. A, phase contrast images of 1-LN cell monolayers (1 x 105 cells/well). A1, A2, A3, images of untreated control cells, cells transfected with a negative control dsRNA or cells transfected with a GRP78 siRNA, respectively, followed by staining with anti-GRP78 IgG. A4, A5, A6, images of untreated control cells, cells transfected with a negative control dsRNA or cells transfected with a GRP78 siRNA, respectively, followed by staining with anti-VDAC IgG. B, on-cell Western immunocytochemical assay of GRP78 and VDAC on 1-LN cell surfaces performed as described under "Experimental Procedures." B1, B2, B3 images of untreated control cells, cells transfected with a negative control dsRNA or cells transfected with a GRP78 siRNA, respectively, stained with anti-GRP78 IgG. B4, B5, B6, images of untreated control cells, cells transfected with a negative control dsRNA or cells transfected with a GRP78 siRNA, respectively, stained with anti-VDAC IgG. C1, C2, C3, measurement of GRP78 mRNA levels by reverse transcription of untreated cells, cells transfected with a negative control dsRNA, and cells transfected with the GRP78 siRNA, respectively. C4, C5, C6, VDAC mRNA levels of untreated cells, cells transfected with a negative control dsRNA, and cells transfected with the GRP78 siRNA, respectively.

 
Effect of K5 on Procaspase-7 Activation—Published reports indicate that opening of VDAC precedes caspase activation leading to cell apoptosis (38), and also that GRP78 may protect cells from apoptosis during stress by blocking procaspase-7 activation (39). K5 binds to both receptors, but only its interaction with VDAC appears to induce a Ca2+ signaling cascade or affect 1-LN cell proliferation. When 1-LN cells were incubated with K5 (100 nM) there was an augmentation in caspase-7 activity, at levels larger than those of cells treated with a similar concentration of the pro-apoptotic TNF-{alpha} (Fig. 9A), whereas cells incubated with a similar concentration of HK-I or microplasminogen show caspase-7 activity levels similar to those of untreated cells. A similar experiment performed under hypoxic conditions shows a moderate increase in caspase-7 activity in cells incubated with K5 (Fig. 9A). When 1-LN cells were treated with a single concentration of K5 (100 nM) and increasing concentrations of HK-I, the caspase-7 activity levels decreased to levels close to those of untreated cells, both under normal oxygen or hypoxic conditions (Fig. 9B). These experiments suggest again that apoptosis induced by K5 is mediated by VDAC on 1-LN cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Pg contains three BBS, one in K5, a second in the active site, and a third in the protease binding domain (7, 40, 41). Of all these BBS, K5 binds with the highest affinity to intact fibrin increasing the rate of Pg activation (42, 43). Then, it is reasonable to speculate a similar role for K5 when Pg binds to cell surface receptors. Activation of Pg on the cell surface enhances cell proliferation and allows for extracellular matrix degradation leading to increased cell migration, tumor invasion, and metastasis (43). These effects ultimately result in a poor prognosis for patients (44, 45). K5 alone, however, is a potent suppressor of angiogenesis (19) and also possesses an anti-proliferative effect on the tumor via a proinflammatory pathway that stimulates recruitment of tumor-associated neutrophils and NKT lymphocytes (47). It would therefore appear that counteracting forces may be acting simultaneously on tumor cells with Pg activation enhancing tumor progression and K5 alone acting to suppress growth. We therefore sought to identify potential mechanisms by which tumor cells evade the growth suppressing effects of K5. Because K5 binds to both VDAC and GRP78 receptors on the cell surface (11, 13), we assessed binding of K5 to both these receptors in HUVEC and human prostate 1-LN tumor cells under normoxic and hypoxic conditions.


Figure 8
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 8.
Effect of K4, K5, and HK-I on 1-LN cell proliferation. 1-LN cells in 96-well culture plates (1 x 104 cells/well) were incubated for 72 h in RPMI 1640 medium containing 5% FBS in the absence or presence of plasminogen kringles. A1, cell proliferation in culture medium alone. A2, cells incubated in culture medium in the presence of K4 (100 nM). A3, cells incubated in culture medium in the presence of K5 (100 nM). B1, cell proliferation in culture medium alone. B2, cells incubated in culture medium in the presence of K5(100 nM). B3, cells incubated in culture medium with a combination of K5 (100 nM) and K4 (100 nM). B4, cells incubated in culture medium with a combination of K5 (100 nM) and HK-I (1000 nM).

 
Tumor hypoxia is an innate environmental factor encountered during the development of many types of human tumors, including malignant prostate tumors (48). Strong evidence suggests hypoxia is a condition necessary for the tumor progression, angiogenesis, metastasis, and therapy resistance (49). Several reports demonstrate that enhanced expression of VDAC under hypoxia may affect Ca2+ homeostasis of the vascular cell (50). Intracellular Ca2+ concentration controls a variety of cell functions including contraction, growth, and gene expression (50). In melanoma cells, expressing GRP78 in large numbers on their cell surface (51), the hypoxic response also stimulates expression of VDAC (52). Our observations suggest that a situation similar to that observed in melanoma cells may also exist in human prostate 1-LN cells. Our experiments show that hypoxia enhances significantly the number of both GRP78 and VDAC receptors on the surface of 1-LN cells, as reflected by an increase in the number of bound K5, microplasminogen, or HK-I molecules. We also demonstrate changes in the affinities of these molecules for their receptors. Similar changes in the number of cell surface receptors or their ligand affinities have been previously observed in endothelial cells exposed to hypoxia, which are attributable to changes in the plasma membrane fluidity resulting from changes in the phospholipid contents of cell plasma membranes (53, 54).


Figure 9
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 9.
Caspase-7 activity assays in 1-LN cell lysates. 1-LN cells in 24-well culture plates (4 x 106 cells/well) were incubated with a single concentration (100 nM) of TNF-{alpha} (positive control), HK-I, microplasminogen, or K5 for 24 h in RPMI 1640 containing 5% FBS under normal oxygen ({blacksquare}) or hypoxic conditions ({square}). Cell lysates, protein concentrations, and caspase-7 activity assays with the colorimetric substrate Ac-DEVD-pNA were measured as described under "Experimental Procedures." A1, cells alone. A2, cells incubated with TNF-{alpha}. A3, cells incubated with HK-I. A4, cells incubated with microplasminogen. A5, cells incubated with K5. B, caspase-7 activity in 1-LN cell lysates incubated with a single concentration of K5 (100 nM) and increasing concentrations of HK-I under normal oxygen (•) or hypoxic conditions ({circ}).

 
Pg also binds to heat shock proteins 27, 60, and 70 through its LBS (55) and the three of them interfere with Pg binding to actin. Heat shock protein 27 interacts predominantly with the Pg domain containing K1-3, whereas GRP78 interacts predominantly with Pg domains through benzamidine binding sites (K5 and microplasminogen). In this fashion, the physiology of Pg bound to the cell surface may be controlled through mechanisms involving several heat shock proteins.

In this report we have demonstrated that both K5 and microplasminogen induce a Ca2+ signaling cascade on HUVEC or 1-LN cells. Unlike K5, which binds to the N-terminal region of GRP78, microplasminogen binds to the C-terminal region of GRP78 and induces a [Ca2+]i response via opening of intracellular Ca2+ stores. Our evidence suggests that VDAC is the signaling receptor for K5. Furthermore, HK-I, a known agonist of VDAC is able to block the effect of K5 on 1-LN at the Ca2+ signaling or cell proliferation levels. HK-I promotes VDAC closure and prevents apoptotic cell death (34), whereas K5 induced opening of VDAC would promote apoptosis. Both HK-I and HK-II bind to VDAC and their surface expression is a strong sign for a negative prognosis in cells from thyroid (56), esophageal (57), or lung cancers (58). Because opening of VDAC precedes procaspase activation (38), the inhibition of caspase-7 activity by HK-I suggests an additional mechanism used by the tumor cell to protect itself from injury triggered by K5, thereby stimulating tumor cell proliferation. Furthermore, in addition to the multiple anti-apoptotic mechanisms already provided by GRP78 (46, 51, 59), this molecule may also protect the cell from apoptosis through binding to K5. Because both GRP78 and VDAC appear to be co-localized on the surface of human prostate 1-LN tumor cells and their trafficking to the cell surface are linked, our experiments suggest that their interaction is critical for protection of the cell from apoptosis.


   FOOTNOTES
 
* This work was supported by Grant HL-24066 from the NHLBI, National Institutes of Health and Grant CA-86344 from the NCI, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-8879; Fax: 919-684-8689; E-mail: gonza002{at}mc.duke.edu.

2 The abbreviations used are: Pg, plasminogen; K5, plasminogen kringle 5; VDAC, voltage-dependent anion channel; GRP78, 78-kDa glucose-regulated protein; HK-1, hexokinase 1; HSP, heat shock proteins; t-PA, tissue plasminogen activator; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FBS, fetal bovine serum; LBS, lysine-binding sites; siRNA, small interfering RNA; HUVEC, human umbilical vein endothelial cells; BBS, benzamidine binding sites. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wallen, P., and Wiman, B. (1970) Biochim. Biophys. Acta 221, 20-30[Medline] [Order article via Infotrieve]
  2. Forsgren, M., Råden, B., Israelsson, M., Larson, K., and Heden, L. O. (1987) FEBS Lett. 213, 254-260[CrossRef][Medline] [Order article via Infotrieve]
  3. Sottrup-Jensen, L., Claeys, H., Zajdel, M., Petersen, T. E., and Magnusson, S. (1978) Prog. Chem. Fibrinol. Thromb. 3, 191-209
  4. Knudsen, B. S., Silverstein, R. L., Leung, L. L., Harpel, P. C., and Nachman, R. L. (1986) J. Biol. Chem. 261, 10765-10771[Abstract/Free Full Text]
  5. Silverstein, R. L., Friedlander, R. J., Nicholas, R. L., and Nachman, R. L. (1988) J. Clin. Investig. 82, 1948-1955[Medline] [Order article via Infotrieve]
  6. Miles, L. A., Dahlberg, C. M., Plescia, J., Felez, J., Kato, K., and Plow, E. F. (1991) Biochemistry 30, 1982-1991
  7. Varady, A., and Pathy, L. (1981) Biochem. Biophys. Res. Commun. 103, 91-102
  8. Cao, R., Wu, H. L., Veitonmaki, N., Linden, P., Farnebo, J., Shi, G., and Cao, Y. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5728-5733[Abstract/Free Full Text]
  9. Cao, Y., Ji, R. W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, S. G., O'Reilly, M. S., Llinas, M., and Folkman, J. (1996) J. Biol. Chem. 271, 29461-29467[Abstract/Free Full Text]
  10. O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328[CrossRef][Medline] [Order article via Infotrieve]
  11. Gonzalez-Gronow, M., Kalfa, T., Johnson, C. E., Gawdi, G., and Pizzo, S. V. (2003) J. Biol. Chem. 278, 27312-27318[Abstract/Free Full Text]
  12. Gonzalez-Gronow, M., Grenett, H. E., Gawdi, G., and Pizzo, S. V. (2005) Exp. Cell Res. 303, 22-31[CrossRef][Medline] [Order article via Infotrieve]
  13. Davidson, D. J., Haskell, C., Majest, S., Kherzai, A., Egan, D. A., Walter, K. A., Scheneider, A., Gubbins, E. F., Solomon, L., Chen, Z., Lesniewski, R., and Henkin, J. (2005) Cancer Res. 65, 4663-4672[Abstract/Free Full Text]
  14. Triantafilou, K., and Triantafilou, M. (2003) Virology 317, 128-135[CrossRef][Medline] [Order article via Infotrieve]
  15. Bae, T. J., Kim, M. S., Kim, J. W., Kim, B. W., Choo, H. J., Lee, J. W., Kim, K. B., Lee, C. S., Kim, J. H., Chang, S. Y., Kang, C. Y., Lee, S. W., and Ko, Y. G. (2004) Proteomics 4, 3536-3548[CrossRef][Medline] [Order article via Infotrieve]
  16. Schwarzer, C., Barnikol-Watanabe, S., Thinnes, F. P., and Hilschmann, N. (2002) Int. J. Biochem. Cell Biol. 34, 1059-1070[CrossRef][Medline] [Order article via Infotrieve]
  17. Szabadkai, G., Bianchi, K., Varnai, P., De Stefani, D., Wiechowski, M. R., Cavagna, D., Nagy, A. I., Balla, T., and Rizzuto, R. (2006) J. Cell Biol. 175, 901-911[Abstract/Free Full Text]
  18. Deustch, D., and Mertz, B. T. (1970) Science 170, 1095-1096[Abstract/Free Full Text]
  19. Cao, Y., Chen, A., An, S. S. A., Ji, R. W., Davidson, D., and Llinas, M. (1997) J. Biol. Chem. 272, 22924-22928[Abstract/Free Full Text]
  20. Thewes, T., Ramesh, V., Simplaceanu, E. L., and Llinas, M. (1987) Biochim. Biophys. Acta 912, 254-269[CrossRef][Medline] [Order article via Infotrieve]
  21. Shi, G.-Y., and Wu, H.-L. (1988) J. Biol. Chem. 263, 17071-17075[Abstract/Free Full Text]
  22. Skaff, D. A., Kim, C. S., Tsai, H. J., Honzatko, R. B., and Fromm, H. J. (2005) J. Biol. Chem. 280, 38403-38409[Abstract/Free Full Text]
  23. King, L. S., Berg, M., Chevalier, M., Carey, A., Elguindi, E. C., and Blond, S. Y. (2001) Prot. Expr. Purif. 22, 148-158[CrossRef][Medline] [Order article via Infotrieve]
  24. Markwell, M. A. (1982) Anal. Biochem. 125, 427-432[CrossRef][Medline] [Order article via Infotrieve]
  25. Goding, J. W. (1978) J. Immunol. Methods 20, 241-253[CrossRef][Medline] [Order article via Infotrieve]
  26. Gonzalez-Gronow, M., Grenett, H., Gawdi, G., and Pizzo, S. V. (2001) Biochem. J. 355, 397-407[CrossRef][Medline] [Order article via Infotrieve]
  27. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450[Abstract/Free Full Text]
  28. Misra, U. K., Gonzalez-Gronow, M., Gawdi, G., Wang, F., and Pizzo, S. V. (2004) Cell Signal. 16, 929-938[CrossRef][Medline] [Order article via Infotrieve]
  29. Lee, A. T. C., Azimahtol, H. L. P., and Tan, A. N. (2003) Cancer Cell Int. 3, 16-23[CrossRef][Medline] [Order article via Infotrieve]
  30. Erlanger, B. R., Kokowsky, N., and Cohen, W. (1961) Arch. Biochem. Biophys. 95, 271-278[CrossRef][Medline] [Order article via Infotrieve]
  31. Xie, G., and Wilson, J. E. (1988) Arch. Biochem. Biophys. 267, 803-810[CrossRef][Medline] [Order article via Infotrieve]
  32. Linden, M., Gellefors, P., and Nelson, B. D. (1982) FEBS Lett. 141, 189-192[CrossRef][Medline] [Order article via Infotrieve]
  33. Gincel, D., Zaid, H., and Shoshan-Barmatz, V. (2001) Biochem. J. 358, 147-155[CrossRef][Medline] [Order article via Infotrieve]
  34. Azoulay-Zohar, H., Israelson, A., Abu-Hamad, S., and Shoshan-Barmatz, V. (2004) Biochem. J. 377, 347-351[CrossRef][Medline] [Order article via Infotrieve]
  35. Jindadamrongwech, S., Thepparit, C., and Smith, D. R. (2004) Arch. Virol. 149, 915-927[CrossRef][Medline] [Order article via Infotrieve]
  36. Markoff, L. J., Innis, B. L., Houghten, R., and Henchal, L. S. (1991) J. Infect. Dis. 164, 294-301[Medline] [Order article via Infotrieve]
  37. Chungue, E., Poli, L., Roche, C., Gestas, P., Glaziou, P., and Markoff, L.J. (1994) J. Infect. Dis. 170, 1304-1307[Medline] [Order article via Infotrieve]
  38. Elinder, F., Akanda, N., Tofighi, R., Shimizu, S., Tsujimoto, Y., Orrenius, S., and Ceccatelli, S. (2005) Cell Death Differ. 12, 1134-1140[CrossRef][Medline] [Order article via Infotrieve]
  39. Marshall, J. M., Brown, A. J., and Ponting, C. P. (1994) Biochemistry 33, 3599-3606[CrossRef][Medline] [Order article via Infotrieve]
  40. Cockell, C. S., Marshall, J. M., Dawson, K. M., Cederholm-Williams, S. A., and Ponting, C. P. (1998) Biochem. J. 333, 99-105[Medline] [Order article via Infotrieve]
  41. Thorsen, S., Clemmensen, I., Sottrup-Jensen, L., and Magnusson, S. (1981) Biochim. Biophys. Acta 668, 377-387[Medline] [Order article via Infotrieve]
  42. Wu, H., Chang, B. I., and Wu, D. H. (1990) J. Biol. Chem. 265, 19658-19664[Abstract/Free Full Text]
  43. Miles, L. A., Hawley, S. B., Baik, N., Andronicos, N. M., Castellino, F. J., and Parmer, R. J. (2005) Front. Biosci. 10, 1754-1762[Medline] [Order article via Infotrieve]
  44. Schmitt, M., Harbeck, N., Thomssen, C., Wilhelm, O., Magdolen, V., Reuning, U., Ulm, K., Höfler, H., Jänicke, F., and Graeff, H. (1997) Thromb. Haemost. 78, 285-296[Medline] [Order article via Infotrieve]
  45. Reuning, U., Magdolen, V., Wilhelm, O., Fischer, K., Lutz, V., Graeff, H., and Schmitt, M. (1998) Int. J. Oncol. 13, 893-906[Medline] [Order article via Infotrieve]
  46. Reddy, R. K., Mao, C., Baumeister, P., Austin, R. C., Kaufman, R. J., and Lee, A. S. (2003) J. Biol. Chem. 278, 20915-20921[Abstract/Free Full Text]
  47. Perri, S. R., Martineau, D., François, M., Lejeune, L., Bisson, L., Durocher, Y., and Galipeau, J. (2007) Mol. Cancer Ther. 6, 441-449[Abstract/Free Full Text]
  48. Anastasiadis, A. G., Stisser, B. C., Ghafar, M. A., Burchardt, M., and Buttyan, R. (2002) Curr. Urol Rep. 3, 222-228[CrossRef][Medline] [Order article via Infotrieve]
  49. Wouters, B. G., Koritzinsky, M., Chiu, R. K., Theys, J., Buijsen, J., and Lambin, P. (2003) Semin. Radiat. Oncol. 13, 31-41[CrossRef][Medline] [Order article via Infotrieve]
  50. Shimoda, L. A., Wang, J., and Sylvester, J. T. (2006) Microcirculation 13, 657-670[CrossRef][Medline] [Order article via Infotrieve]
  51. Gonzalez-Gronow, M., Cuchacovich, M., Llanos, C., Urzua, C., Gawdi, G., and Pizzo, S. V. (2006) Cancer Res. 66, 11424-11431[Abstract/Free Full Text]
  52. Stockwin, L. H., Blonder, J., Bumke, M. A., Lucas, D. A., Chan, K. C., Conrads, T. P., Issaq, H. J., Veenstra, T. D., Newton, D. L., and Rybak, S. M. (2006) J. Proteome Res. 5, 2996-3007[CrossRef][Medline] [Order article via Infotrieve]
  53. Chen, G. D., and Zhao, H. B. (2007) Hear. Res. 226, 14-21[CrossRef][Medline] [Order article via Infotrieve]
  54. Botto, L., Beretta, E., Daffara, R., Miserocchi, G., and Palestini, P. (2006) Respir. Res. 7, 7-17[CrossRef][Medline] [Order article via Infotrieve]
  55. Dudani, A. K., Mehic, J., and Martyres, A. (2007) Mol. Cell. Biochem. 300, 197-205[CrossRef][Medline] [Order article via Infotrieve]
  56. Hooft, L., Van der Veldt, A. A. M., Van Diest, P. J., Hoekstra, O. S., Berkhof, J., Teule, G. J. J., and Molthoff, C. F. M. (2005) J. Clin. Endocrinol. Metab. 90, 328-334[Abstract/Free Full Text]
  57. Tohma, T., Okazumi, S., Makino, H., Cho A., Mochiduki, R., Shuto, K., Kudo, H., Matsubara, K., Gunji, H., and Ochiai, T. (2005) Hepatogastroenterology 52, 486-490[Medline] [Order article via Infotrieve]
  58. Mamede, M., Higashi, T., Kitaichi, M., Ishizu, K., Ishimori, T., Nakamoto, Y., Yanagihara, K., Li, M., Tanaka, F., Wada, H., Manabe, T., and Saga, T. (2005) Neoplasia 7, 369-379[CrossRef][Medline] [Order article via Infotrieve]
  59. Misra, U. K., and Pizzo, S. V. (2005) J. Leukoc. Biol. 78, 187-194[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Infect. Immun.Home page
C. Attali, C. Durmort, T. Vernet, and A. M. Di Guilmi
The Interaction of Streptococcus pneumoniae with Plasmin Mediates Transmigration across Endothelial and Epithelial Monolayers by Intercellular Junction Cleavage
Infect. Immun., November 1, 2008; 76(11): 5350 - 5356.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/45/32811    most recent
M703342200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gonzalez-Gronow, M.
Right arrow Articles by Pizzo, S. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gonzalez-Gronow, M.
Right arrow Articles by Pizzo, S. V.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement