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Published as doi: 10.1096/fj.08-107524.
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(The FASEB Journal. 2008;22:4168-4178.)
© 2008 FASEB

Guanylate binding protein-1 inhibits spreading and migration of endothelial cells through induction of integrin {alpha}4 expression

Kristina Weinländer*, Elisabeth Naschberger*, Michael H. Lehmann*, Philipp Tripal*, Wolfgang Paster{dagger}, Hannes Stockinger{dagger}, Christine Hohenadl{ddagger} and Michael Stürzl*,1

* Division of Molecular and Experimental Surgery, Department of Surgery, University of Erlangen-Nuremberg, Erlangen, Germany;

{dagger} Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria; and

{ddagger} Department of Pathobiology, Institute of Virology, University of Veterinary Medicine, Vienna, Austria

1 Correspondence: Division of Molecular and Experimental Surgery, Department of Surgery, University of Erlangen-Nuremberg, Schwabachanlage 10, 91054 Erlangen, Germany. E-mail: michael.stuerzl{at}uk-erlangen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human guanylate binding protein-1 (GBP-1) is a large GTPase that is induced by inflammatory cytokines and acts antiangiogenically through the inhibition of endothelial cell proliferation and migration. In this study, we detected that GBP-1-expressing cells show a significantly reduced spreading and migration on fibronectin matrices. Investigating possible mechanisms of these effects, we found that integrin {alpha}4 (ITGA4) was consistently up-regulated at both the RNA and protein level in GBP-1-expressing cell cultures. Inhibition of cell spreading and migration by GBP-1 was dependent on the binding of ITGA4 to fibronectin. The inflammatory cytokines IL-1β and TNF-{alpha} induced ITGA4 expression in HUVECs and inhibited spreading and migration. Knockdown of GBP-1 by shRNA abrogated inflammatory cytokine induced ITGA4 expression and restored spreading and migration capabilities of the cells. These results show that inhibition of endothelial cell spreading and migration by inflammatory cytokines is mediated by GBP-1 through induction of ITGA4 expression. Endothelial cell migration is a key process during angiogenesis. Therefore, ITGA4 may be a novel molecular target to modulate angiogenesis in human disease.—Weinländer, K., Naschberger, E., Lehmann, M. H., Tripal, P., Paster, W., Stockinger, H., Hohenadl, C., Stürzl, M. Guanylate binding protein-1 inhibits spreading and migration of endothelial cells through induction of integrin {alpha}4 expression.


Key Words: angiogenesis • inflammation • integrins • large GTPase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE FORMATION OF NEW BLOOD vessels is termed angiogenesis. It is associated with proliferation, migration, and invasion of endothelial cells (ECs). Angiogenesis is important in embryonal development, tissue repair, and pathological processes such as tumor growth and metastasis (1 2 3) . Angiogenesis depends on molecular interactions between ECs and components of the surrounding extracellular matrix (ECM) and is tightly controlled by a balance between proangiogenic and antiangiogenic molecules. Interactions of ECs with ECM are regulated predominantly by integrins. Integrins comprise two noncovalently linked subunits termed {alpha} and β that form heterodimeric transmembrane proteins and bind to ECM proteins (4 5 6 7) . The major proangiogenic molecules are vascular endothelial growth factor (VEGF) (8 , 9) and basic fibroblast growth factor (bFGF) (10) . Factors exerting antiangiogenic activities are thrombospondin (11) , endostatin (12) , angiostatin (13) , and inflammatory cytokines (ICs), such as interferon (IFN) -{gamma}, interleukin (IL) -1β, and tumor necrosis factor (TNF) -{alpha}. ICs inhibit EC proliferation and invasiveness (14 15 16 17 18 19) . We have previously shown that the antiangiogenic effects of ICs are mediated by guanylate binding protein (GBP) -1 (17 , 18 , 20) .

GBPs were originally identified as the most abundant proteins induced by IFN-{gamma} (21) . Seven different GBPs with a relative molecular mass of 65–71 kDa have been detected in humans, and 10 GBPs have been discovered in mice (22 23 24) . The GBPs belong to a subfamily within the protein family of large GTPases that includes atlastins (25) , dynamins (26) , and Mx proteins (27) . GBP-1 is the best characterized member of the human GBP family (28 , 29) . Expression of GBP-1 is induced by IFN-{gamma}, IL-1β, and TNF-{alpha} in many different cell types in vitro (17 , 20 , 30 , 31) . In vivo, GBP-1 expression is highly associated with blood vessel ECs and rarely observed in other cells (20) , indicating that GBP-1 may exert specific functions in ECs. Indeed, it has been shown that GBP-1 inhibits EC proliferation (17) and mediates the inhibition of EC invasiveness via down-regulation of matrix metalloproteinase (MMP) -1 expression (18) .

Here, we show that GBP-1 also inhibits spreading and migration of ECs and that these effects are mediated via induction of ITGA4 expression by GBP-1. Our results further support the role of GBP-1 as an antiangiogenic regulatory molecule in inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures
Primary human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (designated as HUVEC1s) (Heidelberg, Germany) and Cambrex Bio Science (designated as HUVEC2s) (Verviers, Belgium) and were maintained in the corresponding endothelial cell basal medium [ECBM (PromoCell) or EBM (Cambrex)] supplemented with 2% fetal calf serum (FCS) at 37°C in a humidified atmosphere at 5% CO2. HUVEC1s were routinely cultivated in uncoated culture flasks (Nunc, Wiesbaden, Germany), whereas HUVEC2s were cultured in flasks coated for at least 2 h with 1.5% bovine skin gelatin, type B (Sigma-Aldrich, Taufkirchen, Germany) in phosphate buffered saline (PBS) (Biochrom AG, Berlin, Germany). For routine cultivation, confluent cells were washed once with PBS, detached by using 1 x 0.5 g/L trypsin and 0.2 g/L ethylene-diamine-tetra-acetic acid in HBSS (trypsin/EDTA) (PAA, Colbe, Germany) for 2–3 min, and passaged in a 1:4 ratio (one passage). All experiments were carried out between passages 5 and 11.

For stimulation with recombinant proteins, cells were seeded on gelatin-coated flasks, incubated overnight in ECBM supplemented with 0.5% FCS (ECBM/0.5% FCS), and subsequently treated with inflammatory cytokines (200 U/ml IL-1β, 300 U/ml TNF-{alpha}, 100 U/ml IFN-{gamma}) in the same medium for the indicated time spans. Recombinant human IL-1β, TNF-{alpha}, and IFN-{gamma} were purchased from Roche (Mannheim, Germany). All cytokines were diluted in PBS containing 0.1% bovine serum albumin (BSA) (Sigma-Aldrich).

Retroviral transduction of HUVECs
Control (CR), GBP-1, and green fluorescent protein (GFP) -HUVECs were obtained by infection of HUVEC cultures with the retroviral vectors pBABEpuro, pBABEpuro-GBP-1, and pBABEpuro-GFP generated from PG13 packaging cells (32) . For generation of HUVECs constitutively expressing shRNAs targeting GBP-1 or GFP, the respective shRNAs were cloned into the retroviral pBABEpuro vector (32) . GBP-1-shRNA: 5'-GGTTGAGGATTCAGCTGACttcaagagaGTCAGCTGAATCCTCAACCTTTTTG-3' (capital letters=sense and antisense strand, small letters=loop sequence), GFP-shRNA used as a control: 5'-GCAAGCTGACCCTGAAGTTCttcaagagaGAACTTCAGGGTCAGCTTGCTTTTTG-3'. Recombinant virus particles were generated in human embryonic kidney (HEK) 293T cells (24) . Retroviral transduction of HUVECs was carried out as described previously (17) .

Western blot analysis
Western blotting from cell lysates was performed as described previously (20 , 33) , with the exception that membrane blocking was carried out in PBS containing 5% skim milk overnight at 4°C with gentle shaking. The following primary antibodies diluted in PBS/2.5% skim milk were incubated for 1 h at room temperature (RT): monoclonal rat anti-human GBP-1 antibody (clone 1B1; 1:500; hybridoma supernatant), and monoclonal mouse anti-human GAPDH antibody (1:70,000) (Chemicon/Millipore, Schwalbach, Germany). Detection of the primary antibodies was performed using goat anti-rat and sheep anti-mouse immunoglobulin G (IgG) coupled to horseradish peroxidase (HRP) (1:5000) (GE Healthcare, Munich, Germany) for 45 min. HRP enzyme reaction was performed using enhanced chemiluminescence (ECL) reagents (GE Healthcare).

Cell spreading assay and quantification of cell surface area
Cell spreading assay with HUVECs was performed in 96-well plates (Nunc) that were coated either with 5 µg/cm2 fibronectin (FN) (BD Biosciences, San Jose, CA, USA) or with a 120 kDa {alpha}-chymotryptic fragment of FN (FN-120) (Chemicon/Millipore, Temecula, CA, USA) for 1 h at 37°C. Nonspecific binding sites were blocked with PBS containing 1% BSA overnight at 4°C. Subsequently, the cells were washed once with PBS and detached by Accutase treatment (PAA). Cells (5x103) were seeded into the coated wells after resuspension in serum-free ECBM (SFM). Cells were allowed to adhere at 37°C and 5% CO2 for 20 min. Unattached cells were removed by gentle washing with PBS containing 0.1% BSA. Attached cells were fixed with 3.7% neutral buffered formalin (Sigma-Aldrich) for 15 min, stained with 0.5% crystal violet in 2% ethanol for 10 min, and briefly washed with water. Images were acquired using an Olympus digital camera (Olympus, Munich, Germany) mounted on an Axiovert 25 microscope (Zeiss, Jena, Germany). The surface area of 100 cells per cell type was measured using the OPTIMAS 6.0 software package (Optimas Corporation, Washington, DC, USA), and the cells were grouped according to their size into 15 increments with increasing surface area (e.g., increment 1: 0–400 µm2; increment 2: 400–800 µm2; ... ; increment 15: 5600–6000 µm2). Cells were classified as "spread" based on size cutoffs of either 1200 or 1600 µm2. Mean values ± SD from at least 3 independent experiments were calculated and are depicted in the respective graphs.

Wound healing assay
CR-, GBP-1-, and shRNA-HUVECs were grown on FN- or FN-120-coated (5 µg/cm2) 35-mm culture dishes (Nunc) until confluence and were starved in ECBM/0.5% FCS overnight. Confluent cell monolayers of CR- and GBP-1-HUVECs were scratched using a sterile 1-ml pipette tip. The monolayers were washed twice with SFM and cultured for 10 h in SFM. During this time span, the wounded area was repopulated with cells, resulting in reduced scratch width. shRNA-expressing HUVECs were stimulated with ICs for 48 h, as described above, before scratching. The cells were wounded and washed twice with ECBM/0.5% FCS after incubation with ICs. shRNA-expressing cells were cultivated for 10 h in ECBM/0.5% FCS because of an increased cell death when cultivated in SFM. Images of the same areas were taken immediately after scratching (time point 0) and every 2 h using an Olympus digital camera mounted on an Axiovert 25 microscope (Zeiss). The widths of the wounding scratches at different time points were measured and expressed as a percentage of the initial distance at time point 0, which was set to 100%. The results shown are depicted as means ± SD from at least 3 independent experiments.

RNA isolation and microarray analysis
Total RNA of CR- and GBP-1-HUVECs was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Residual traces of genomic DNA were removed with DNase I (Qiagen). RNA concentration and purity were determined photometrically (GeneQuant, Amersham Biosciences, Freiburg, Germany), and RNA integrity was controlled by nondenaturing agarose gel electrophoresis. Preparation of cRNA targets (5 to 10 µg total RNA), fragmentation, hybridization of HG-U133 plus 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), washing, staining, and scanning were performed according to manufacturer’s protocols (Affymetrix) with a commercial partner (Dr. Klein-Hitpass, Institute for Cell Biology, University of Essen, Essen, Germany). Signal intensities and detection calls were determined using Affymetrix microarray suite, version 5.0. Comparison files were further filtered to detect differentially expressed genes. Filter criteria included a change of I, a signal log ratio of >1, a value of P < 0.001, and an overall signal intensity of >300; or a change of D, a signal log ratio of <–1, a value of P > 0.999, and an overall signal intensity of >300.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Reverse transcription of total RNA and amplification of GBP-1, ITGA4, and GAPDH cDNA were carried out as described previously (24) . GBP-1 was amplified using the primers 5'-ATGGCATCAGAGATCCACAT-3' (sense) and 5'-TTCGCTTATGGTACATGCCTTTC-3' (antisense) and ITGA4 using the primers 5'-TTCGGAGCCAGCATACTACC-3' (sense) and 5'-GCAGAATCAGACCGAAAAGC-3' (antisense). For amplification of GAPDH, the primers 5'-AGCCACATCGCTCAGAACAC-3' (sense) and 5'-GAGGCATTGCTGATGATCTTG-3' (antisense) were used, as previously described (34) . Gel pictures were acquired by the Gel-Doc system (Bio-Rad). Signal intensities of each PCR product were quantified with the AIDA software package (Raytest, Straubenhardt, Germany).

Ribonuclease protection assay (RPA)
In vitro transcription of the BD RiboQuant Multi probe human integrin template set (BD Biosciences) and RPA were carried out as described previously (35) . Protected RNAs and the biotin-labeled multiple integrin probes were separated on a 6% tris-borate EDTA (TBE) urea gel (Anamed, Darmstadt, Germany) and blotted onto a positively charged nylon membrane (Bio-Rad, Hercules, CA, USA). Detection of RNA fragments was performed with the Chemiluminescent Nucleic Acid Detection Kit (Pierce, Rockford, IL, USA), and signal intensities were quantified with the AIDA software package.

FACS analysis
FACS analysis was performed as described previously (36) . The following primary antibodies were incubated with the cells for 45 min on ice: monoclonal mouse anti-human ITGA4 (CD49d) antibody (clone HP2/1; 1:400) (Chemicon/Millipore) and monoclonal mouse anti-human ITGB1 (CD29) antibody (1:8000). Mouse IgG1 (R&D Systems, Minneapolis, MN, USA) was used as an isotype control in the corresponding concentrations. Primary antibodies were generally detected with an Alexa 488-conjugated goat anti-mouse secondary antibody (1:350) (Invitrogen). An R-phycoerythrin (PE) -conjugated goat anti-mouse secondary antibody (1:300) (Jackson ImmunoResearch, West Grove, PA, USA) was used for the detection of primary antibodies when GFP-expressing cells were stained. The use of a red fluorescing secondary antibody allowed differentiating ITGA4 and ITGB1 signals from GFP signals.

Statistical analysis
Student’s t test was performed using the SPSS 14.0 for Microsoft Windows software (SPSS Inc., Chicago, IL, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ectopic expression of GBP-1 inhibits spreading and migration of ECs on FN matrices
FN is one of the most common proteins in the ECM and has been shown to be involved in the regulation of angiogenesis (37) . In preliminary experiments, we realized that after seeding on FN matrices, spreading of GBP-1-expressing ECs was retarded as compared to GBP-1-nonexpressing cells (data not shown). To analyze the effect of GBP-1 on spreading in more detail, HUVECs were transduced with a retroviral vector encoding GBP-1 (GBP-1-HUVECs) or with the respective control vector (CR-HUVECs). GBP-1 expression was confirmed by Western blot analysis (Fig. 1A ). GBP-1- and CR-HUVECs were plated on FN matrices and were allowed to attach and to spread in SFM. Most of the CR-HUVECs were spread after 20 min on FN (Fig. 1B ; CR), whereas the GBP-1-expressing cells still retained a small, round shape after this time (Fig. 1B ; GBP-1). Quantification of the surface area of GBP-1- and CR-HUVECs was performed by grouping them into 15 increments according to their size (increment 1: 0–400 µm2; increment 2: 400–800 µm2; ... ; increment 15: 5600–6000 µm2). By this means, a clear difference in cell size was recognized between GBP-1- and CR-HUVECs (Fig. 1C ). The percentage of GBP-1-expressing cells (27.0±5.6%) showing a surface area greater than 1200 µm2 (increments 4 to 15) was decreased as compared to CR-HUVECs (87.8±3.6%) (Fig. 1D ; ≥1200 µm2). When using more stringent criteria of cell spreading (increments 5 to 15 or ≥1600 µm2), the difference in numbers of spread CR-HUVECs (74.3±7.1%) compared to those of GBP-1-expressing cells (13.3±3.5%) was still highly significant (P≤0.001) (Fig. 1D ; ≥1600 µm2).


Figure 1
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  		Figure 1. Expression of GBP-1 reduces spreading and migration of HUVECs on FN matrices. A) Western blot analysis of total cell lysates obtained from HUVEC1s transduced with the retroviral vector pBABEpuro (CR) or with pBABEpuro-GBP-1 (GBP-1). Immunochemical detection of GAPDH demonstrates that equal concentrations of proteins were blotted onto the membranes. B) CR- and GBP-1-HUVEC1s were plated on FN-coated dishes. Adherent cells were fixed with 3.7% neutral buffered formalin after 20 min and stained with 0.5% crystal violet. C) Surface area of 100 individual cells from each cell type, stained according to B, was determined as described in Materials and Methods. One increment corresponds to 400 µm2. Representative size distribution of cell surface area as determined in one experiment is shown. D) Relative number of spread CR- and GBP-1-HUVECs is given as the percentage of cells with surface area ≥1200 µm2 (left bars; increments 3–15) or ≥1600 µm2 (right bars; increments 4–15) and is depicted in the diagram. Values are means ± SD from 4 independent experiments; ***P ≤ 0.001. E) Migration activity of CR- and GBP-1-HUVEC1s on FN-coated surfaces was investigated using a wound healing assay. In vitro scratch wounds were generated by scraping confluent cell monolayers with a sterile pipette tip. Pictures from the same area acquired immediately after scratching (0 h) and after 10 h of culture (10 h) are shown. F) Width of the scratch wounds at different time points was determined relative to the initial width at time point 0 and is indicated as a percentage. Results are means ± SD from 3 independent experiments; *P ≤ 0.05.

An important aspect of cell migration is membrane ruffling and protrusion of the leading edge (38 , 39) . Cell spreading mimics these events, which occur at the leading edge of a migrating cell. Migration of CR- and GBP-1-HUVECs was examined by performing in vitro wound healing assays. GBP-1-expressing cells migrated at a significantly slower rate into the wounded area in comparison to control cells (Fig. 1E ). After 10 h, the scratch width of GBP-1-expressing cells was 81.2 ± 2.0% of the initial width, whereas CR-HUVECs reduced the scratch width to 45.6 ± 13.5% of the initial width (Fig. 1F ). These results demonstrate that ectopically expressed GBP-1 inhibits spreading and migration of ECs on FN-coated surfaces.

GBP-1 up-regulates ITGA4 expression in ECs
To elucidate the molecular mechanisms of the inhibitory activities of GBP-1 on cell spreading and migration, we investigated GBP-1-regulated gene expression in ECs. Two different HUVEC cultures (HUVEC1 and HUVEC2) were subjected to the study, in order to exclude cell culture-specific variations of primary cells. In addition to the culture shown in Fig. 1 , a second culture was transduced with a retroviral vector encoding GBP-1 or with the respective control vector (CR). GBP-1 expression in this culture was also confirmed by Western blot analysis (Fig. 2A ). mRNA of both cultures was isolated, and the gene expression profiles of GBP-1-HUVECs were compared with CR-HUVECs by applying oligonucleotide-based microarray technology from Affymetrix (HG-U133 plus 2.0). Of the 38,500 genes analyzed, 6 were highly significantly (P≤0.001) regulated by GBP-1 in both HUVEC cultures tested (Table 1 ).


Figure 2
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  		Figure 2. GBP-1 induces ITGA4 expression at RNA and protein level in HUVECs. A) Western blot analysis of total cell lysates obtained from HUVEC2s transduced with the retroviral vector pBABEpuro (CR) or with pBABEpuro-GBP-1 (GBP-1). Immunochemical detection of GAPDH demonstrates that equal concentrations of proteins were blotted onto the membranes. B) RT-PCR for analysis of GBP-1, ITGA4, and GAPDH expression in CR- and GBP-1-HUVECs (HUVEC1 and HUVEC2). Decreasing amounts of cDNA [undiluted (1), 1:10, 1:100, and 1:1000] were subjected to PCR after reverse transcription of isolated mRNA. Bands obtained with nonsaturated levels of ITGA4 (1:100) were densitometrically quantified and normalized to the corresponding GAPDH signal (1:100). Relative increase of ITGA4 expression in GBP-1-expressing cells as compared to CR-HUVECs is depicted in the diagram (right). C) Total mRNA of CR- and GBP-1-HUVECs was analyzed by Multiprobe RNase protection assay using a set of multiple biotinylated human integrin templates as probes. ITGA4 signal intensities were densitometrically quantified and normalized to the L32 signal. L32 is a housekeeping gene and encodes an essential protein for the 60S ribosomal subunit. Relative increase of ITGA4 expression in GBP-1-expressing cells as compared to CR-HUVECs is depicted in the diagram (right). D) FACS analysis of CR- (black), GBP-1- (red), and GFP-HUVECs (red) stained with a monoclonal antibody against ITGA4 (top panel) and ITGB1 (bottom panel), or an isotype-matched control antibody (gray). Fluorescence intensities from ITGA4- or ITGB1-stained cells were normalized to the isotype control. Normalized signals of ITGA4 and ITGB1 cell surface expression of GBP-1- or GFP-HUVECs relative to CR-HUVECs are given as means ± SD from at least 3 independent experiments.


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  		Table 1. GBP-1-regulated genes in stably transduced HUVEC cultures

ITGA4 was the strongest up-regulated gene in both GBP-1-expressing cultures (Table 1) . It has been reported that ITGA4 specifically dimerizes with the integrin β1- and β7-subunits. Of note, {alpha}4β1 expression has been shown to reduce cell spreading (40 41 42 43) and to inhibit cell migration and invasion on FN (44 45 46 47) . In contrast, other integrins, such as {alpha}5β1 and {alpha}vβ3, promoted those processes.

RT-PCR analysis (Fig. 2B ) and RPA (Fig. 2C ) confirmed the high induction of ITGA4 expression by GBP-1 in both HUVEC cultures. RPA additionally revealed that the expression of other integrin transcripts was either not consistently altered in both cultures (itgav), unchanged (itga2, itga3, itga5, itga6), or not detectable (itga1, itga7, itga8, and itga9) (Fig. 2C ; left).

Subsequently, the presence of ITGA4 on the surface of CR- and GBP-1-HUVECs was analyzed by FACS. In agreement with the RNA expression analyses, the ITGA4 protein level was increased on the cell surface of GBP-1-expressing cells (2.8±0.7-fold HUVEC1; 2.2±0.2- fold HUVEC2), as compared to control cells (Fig. 2D ; ITGA4; GBP-1). ITGB1 forms heterodimers with ITGA4 and belongs to the same integrin family (4 , 5 , 48) . Therefore, ITGB1 was investigated as a control. ITGB1 was highly expressed in HUVECs, but its expression was not altered by ectopically expressed GBP-1 (Fig. 2D ; ITGB1; GBP-1). Expression of GFP as a heterologous control protein, which was detectable in 98% of the cells (data not shown) affected neither ITGA4 nor ITGB1 expression (Fig. 2D ; ITGA4 and ITGB1; GFP). Together, these data demonstrate that ectopically expressed GBP-1 is sufficient to induce ITGA4 expression in ECs at both the RNA and protein level.

The GBP-1-mediated inhibition of cell spreading and migration depends on the presence of the ITGA4 binding site in FN
ITGA4 expression correlated positively with the inhibition of cell spreading and migration on FN matrices. ITGA4 selectively binds to a distinct site near the C terminus of FN (type III repeat 14-IIICS) containing the CS-1 and CS-5 domains (Fig. 3A ; FN) (49 , 50) . All other integrins, such as {alpha}vβ3 and {alpha}5β1, bind to FN within the central integrin binding site (type III repeats 9–10) (Fig. 3A ; FN) (51 , 52) . In case that ITGA4 causes the inhibition of cell spreading, both effects should be abrogated by plating the cells on a 120-kDa fragment of FN (FN-120). FN-120 lacks the C-terminal ITGA4 binding site but still harbors the central integrin recognition site (Fig. 3A ; FN-120). Indeed, spreading of CR- and GBP-1-HUVECs on FN-120 was identical (Fig. 3B, C ). Consistently, migration of GBP-1- and CR-HUVECs was also found to be similar on the FN-120 matrix (Fig. 3D, E ). These results suggest that the GBP-1-mediated inhibition of cell spreading and migration depends on the binding of ITGA4 to its specific binding site in FN.


Figure 3
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  		Figure 3. GBP-1 inhibition of cell spreading and migration depends on the presence of the ITGA4 binding site in FN. A) Schematic illustration of FN and FN-120. FN consists of four types of polypeptide modules, termed type I, II, and III repeats and type III connecting segment (IIICS). Besides a fibrin/heparin and a collagen binding site at the N terminus, FN posseses two integrin binding motives: 1) a central recognition site for various integrins (type III repeat 9–10) and 2) a C-terminal binding site recognized only by integrin {alpha}4β1 and {alpha}4β7 (type III repeat 14-IIICS). FN-120 is a chymotryptic digested fragment of FN and encompasses type III repeats 3 through 11. It contains the central integrin binding motif, but not the C-terminal binding region for integrin {alpha}4β1 and {alpha}4β7. B) CR- and GBP-1-HUVEC1s were plated on FN-120-coated surfaces. Adherent cells were fixed with 3.7% neutral buffered formalin after 20 min and stained with 0.5% crystal violet. C) Surface area of 100 individual cells from each cell type, stained according to B, was determined as described in Materials and Methods. Relative number of spread CR- and GBP-1-HUVECs is given as the percentage of cells with surface area ≥1200 µm2 (left bars) or ≥1600 µm2 (right bars) and is depicted in the diagram. Values are means ± SD from 3 independent experiments. D) Migration activity of CR- and GBP-1-HUVEC1s on FN-120-coated surfaces was investigated using a wound healing assay. In vitro scratch wounds were generated by scraping confluent cell monolayers with a sterile pipette tip. Pictures from the same area acquired immediately after scratching (0 h) and after 10 h of culture (10 h) are shown. E) Width of the scratch wounds at different time points was determined relative to the initial width at time point 0 and is indicated as a percentage. Results are means ± SD from 4 independent experiments.

IL-1β and TNF-{alpha} induce ITGA4 surface expression and provoke an inhibition in cell spreading on FN matrices
Since IL-1β, TNF-{alpha}, and IFN-{gamma} are known inducers of endogenous GBP-1 expression (17 , 20 , 31 , 53) , their effects on ITGA4 cell surface expression in ECs was investigated. To this aim, each of the ICs was applied in concentrations previously shown to induce GBP-1 expression (Fig. 4A ) and a clear biological response in HUVECs (20) . ITGA4 cell surface expression was enhanced in IL-1β- (2.8±0.4-fold) and TNF-{alpha}-stimulated cells (2.0±0.4-fold) as compared to unstimulated cells (Fig. 4B ; ITGA4; IL-1β and TNF-{alpha}). Interestingly, ITGA4 surface expression was down-regulated in cells treated with IFN-{gamma} (0.4±0.0-fold) in comparison to untreated cells (Fig. 4B ; ITGA4; IFN-{gamma}). ITGB1 surface expression was neither affected by IL-1β, TNF-{alpha}, nor by IFN-{gamma} (Fig. 4B ; ITGB1).


Figure 4
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  		Figure 4. IL-1β and TNF-{alpha} induce ITGA4 surface expression and cause ITGA4-dependent inhibition of cell spreading. HUVECs were either left unstimulated (unstim.) or were stimulated for 48 h with IL-1β (200 U/ml), TNF-{alpha} (300 U/ml) or IFN-{gamma} (100 U/ml). A) Cells were analyzed by Western blot analysis to detect GBP-1 protein expression. Immunochemical detection of GAPDH demonstrates that equal concentrations of proteins were blotted onto the membranes. B) FACS analysis of unstimulated (black) and stimulated (red; IL-1β, TNF-{alpha}, or IFN-{gamma}) HUVECs with a monoclonal antibody against ITGA4 (top panel) and ITGB1 (bottom panel) or an isotype-matched control antibody (gray). Fluorescence intensities from ITGA4- or ITGB1-stained cells were normalized to the isotype control. Normalized signals for surface expression of ITGA4 and ITGB1 obtained from stimulated HUVECs relative to unstimulated cells were calculated; values are means ± SD from at least 3 independent experiments. C) Unstimulated and stimulated HUVECs (48 h) were plated on surfaces coated with FN (top panel) or FN-120 (bottom panel) and were allowed to spread for 20 min. Cells were fixed with 3.7% neutral buffered formalin and stained with 0.5% crystal violet. D) Surface area of 100 individual cells from each cell type, stained according to C, was determined as described in Materials and Methods. Relative number of spread unstimulated and stimulated HUVECs is given as percentage of cells with surface area ≥1200 µm2 (left bars) or ≥1600 µm2 (right bars) and is depicted in the diagram. Values are means ± SD from 3 independent experiments; **P ≤ 0.01; ***P ≤ 0.001.

Subsequently, the effect of ICs on the spreading of HUVECs on FN and FN-120 was examined (Fig. 4C ). Quantification of the cell surface area under the different conditions showed that the relative numbers of IL-1β- (26.0±1.7%), TNF-{alpha}- (21.7±2.5%), and IFN-{gamma}-treated cells (20.3±0.6%) with surface area ≥1600 µm2 were significantly decreased, as compared to untreated cells (82.3±3.8%) on FN matrices (Fig. 4D ; FN; ≥1600 µm2). On FN-120-coated surfaces, however, the spreading of IL-1β- and TNF-{alpha}-treated cells was not inhibited and was identical to that of unstimulated cells (Fig. 4C, D ; FN-120; IL-1β and TNF-{alpha}). In contrast, spreading of IFN-{gamma}-treated cells was still inhibited on FN-120 matrices (Fig. 4C, D ; FN-120; IFN-{gamma}).

These findings demonstrate that IL-1β and TNF-{alpha} induce ITGA4 surface expression and reduce HUVEC spreading dependent on ITGA4 binding to FN. IFN-{gamma} also inhibited cell spreading, but in an ITGA4-independent manner. Therefore, in the following experiments, we focused on the effects of ITGA4 on spreading and migration of IL-1β- and TNF-{alpha}-treated cells.

GBP-1 mediates the IL-1β- and TNF-{alpha}-induced inhibition of cell spreading and migration via up-regulation of ITGA4 expression
GBP-1 knockdown experiments were carried out in order to demonstrate that GBP-1 is necessary to mediate the inhibitory effects of IL-1β and TNF-{alpha} on EC spreading. HUVECs were transduced with retroviral vectors encoding an shRNA targeting either GBP-1 (GBP-1-shRNA-HUVECs) or GFP as a control (ctrl-shRNA-HUVECs). GBP-1 expression was analyzed by Western blot analysis after treatment of transduced cells with IL-1β and TNF-{alpha} for 48 h (Fig. 5A ). Expression of GBP-1 in GBP-1-shRNA-expressing cells was decreased to 10% (IL-1β) and 7% (TNF-{alpha}) as compared to the expression level in ctrl-shRNA-HUVECs (Fig. 5A ). In addition, ITGA4 mRNA levels (Supplemental Fig. 1), as well as cell surface expression of ITGA4 (Fig. 5B ; ITGA4), were significantly lower in IL-1β- and TNF-{alpha}-treated cells that expressed GBP-1-shRNA as compared to the respective ctrl-shRNA-expressing cells. The expression of ITGB1 was not altered by the presence of either GBP-1-shRNA or ctrl-shRNA (Fig. 5B ; ITGB1).


Figure 5
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  		Figure 5. GBP-1 mediates the IL-1β- and TNF-{alpha}-induced inhibition of cell spreading and migration. HUVECs stably expressing shRNA targeting GFP (ctrl-shRNA) or GBP-1 (GBP-1-shRNA) were either left unstimulated (unstim.) or were stimulated for 48 h with IL-1β (200 U/ml) or TNF-{alpha} (300 U/ml). A) GBP-1 expression was analyzed by Western blotting of protein extracts obtained from total cell lysates. Immunochemical detection of GAPDH demonstrates that equal concentrations of proteins were blotted onto the membranes. Corresponding signal intensities were densitometrically quantified and normalized to GAPDH in each lane. Numbers indicate the relative amount of GBP-1 in GBP-1-shRNA-HUVECs as compared to ctrl-shRNA-HUVECs. B) Cell surface expression of ITGA4 (top panel) and ITGB1 (bottom panel) on ctrl-shRNA- (black) and GBP-1-shRNA-HUVECs (red) was determined by FACS analysis. Gray indicates isotype-matched control antibody staining. Fluorescence intensities from ITGA4- or ITGB1-stained cells were normalized to the isotype control. Normalized signals of GBP-1-shRNA-HUVECs relative to ctrl-shRNA-HUVECs are given as means ± SD from at least 3 independent experiments. C) Cells were fixed and stained with 0.5% crystal violet 20 min after adhesion to FN-coated surfaces. D) Surface area of 100 individual cells from each cell type, stained according to C, was determined as described in Materials and Methods. Relative number of spread unstimulated and stimulated ctrl-shRNA- or GBP-1-shRNA-HUVECs is given as percentage of cells with surface area ≥1200 µm2 (left bars) or ≥1600 µm2 (right bars) and is depicted in the diagram. Values are means ± SD from 4 independent experiments; ***P ≤ 0.001. E) Migration activity of ctrl-shRNA- and GBP-1-shRNA-expressing HUVECs after stimulation with IL-1β or TNF-{alpha} on FN-coated surfaces was investigated using a wound healing assay. In vitro scratch wounds were generated by scraping confluent cell monolayers with a sterile pipette tip. Width of the scratch wounds at different time points was determined as a percentage of the initial width at time point 0. Diagram shows relative width of wounds of stimulated ctrl-shRNA- and GBP-1-shRNA-HUVECs vs. the respective unstimulated cells (set to 1) at 2, 6, and 10 h. Values are means ± SD from 4 independent experiments; *P ≤ 0.05; **P ≤ 0.01.

Subsequently, the spreading capability of these cells was assessed. Spreading of unstimulated ctrl-shRNA-HUVECs on FN within 20 min was greater as compared to ctrl-shRNA-HUVECs that had been stimulated for 48 h with IL-1β or TNF-{alpha} (Fig. 5C ; ctrl-shRNA). Quantification of these effects indicated that the number of IL-1β- (27.3±5.6%) and TNF-{alpha}-treated (33.0±2.5%) ctrl-shRNA-HUVECs showing surface area ≥1600 µm2 was highly significantly (P≤0.001) decreased in comparison to unstimulated cells (95.5±2.4%) (Fig. 5D ; ctrl-shRNA; ≥1600 µm2). These findings are in agreement with the data obtained for nontransduced HUVECs treated with IL-1β or TNF-{alpha} (compare Fig. 4C, D ). This indicates that transduction of cells with retroviral vectors encoding a nonspecific ctrl-shRNA did not alter their spreading capability. In contrast, the specific knockdown of GBP-1 by introducing a GBP-1-specific shRNA abrogated the inhibitory effect of IL-1β and TNF-{alpha} on cell spreading (Fig. 5C, D ; GBP-1-shRNA). IL-1β- and TNF-{alpha}-treated GBP-1-shRNA-HUVECs exhibited a similar spreading capability on FN-coated surfaces as unstimulated GBP-1-shRNA-HUVECs (Fig. 5C, D ; GBP-1-shRNA).

In a final approach, we investigated the effect of GBP-1 knockdown on migration of IL-1β- and TNF-{alpha}-stimulated HUVECs on FN matrices. Stimulated shRNA-expressing cells showed an increased cell death rate in SFM. Therefore, wounding assays with these cells were carried out in the presence of 0.5% FCS. Treatment of ctrl-shRNA-HUVECs with IL-1β and TNF-{alpha} significantly (P≤0.05) reduced the capability of these cells to migrate into the wounded area as compared to unstimulated cells (Fig. 5E ; ctrl-shRNA). After 10 h the scratch widths in the IL-1β- and TNF-{alpha}-treated ctrl-shRNA-HUVEC cultures were larger (2.5±0.4- and 2.4±0.3-fold) as compared to that obtained in the unstimulated ctrl-shRNA-HUVEC culture (Fig. 5E ; ctrl-shRNA; 10 h). Knockdown of GBP-1 restored migration capabilities of IL-1β- and TNF-{alpha}-treated cells (Fig. 5E ; GBP-1-shRNA). Altogether, these results show that GBP-1 is both necessary and sufficient to mediate inhibition of spreading and migration of IL-1β- and TNF-{alpha}-treated cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
GBP-1 is an antiangiogenic molecule. It is strongly expressed in inflammatory tissues, selectively in blood vessel ECs of tissue areas with high concentrations of ICs, and is absent in areas with high concentrations of angiogenic growth factors (17 , 18 , 20) . ICs induce GBP-1 expression in ECs, and GBP-1 mediates the inhibition of cell proliferation and invasion, which are characteristic direct effects of ICs on ECs (17 , 18) . Here, we described a further antiangiogenic activity of GBP-1. We showed that GBP-1 inhibits spreading and migration of ECs on FN matrices. These inhibitory effects appear to be mediated by the up-regulation of ITGA4 expression in response to GBP-1.

The specific up-regulation of ITGA4 expression by GBP-1 was supported by several lines of experimental evidence. First, two different HUVEC cultures were used for comparative transcriptome analyses in order to compensate for the heterogeneity of EC primary cultures. Expression of the itga4 gene was highly significantly increased by ectopically expressed GBP-1 in both HUVEC cultures. The expression of several other integrin {alpha}-subunits was either unchanged (itga2, itga3, itga5, itga6) or not consistently affected (itgav) by GBP-1. GFP, expressed as a heterologous control protein, did not induce ITGA4 expression. Second, ITGA4 up-regulation was confirmed by semiquantitative RT-PCR and RPA on the RNA level and by FACS analysis on the protein level. Interestingly, ITGA4 expression was not only up-regulated in response to ectopic expression of GBP-1, but also in response to treatment of the cells with IL-1β and TNF-{alpha}. Although each cytokine is known to exert different activities on ECs (54 , 55) , both induced expression of GBP-1 and ITGA4. In agreement with our findings, an increase of ITGA4 cell surface expression in ECs treated with TNF-{alpha} was reported previously (56) . Finally, a specific knockdown of GBP-1 expression using shRNA abrogated the induction of ITGA4 mRNA transcription, as well as cell surface expression in the presence of IL-1β and TNF-{alpha}. These results congruently indicate that GBP-1 is necessary and sufficient to mediate the induction of ITGA4 expression in response to IL-1β and TNF-{alpha}.

In preliminary experiments with HUVECs expressing mutant GBP-1 proteins with a defective GTPase activity (57) , we were able to show that ITGA4 induction was below 36% as compared to wild-type GBP-1-expressing cells (data not shown). This indicates that the GTPase activity is involved in the up-regulation of ITGA4 expression but may cooperate with additional structures/regions of GBP-1, which will need to be determined in future studies.

The capability of cytoplasmic GTPases to regulate gene expression has been generally accepted for the small GTPases Rac, Rho, and Ras (58 59 60 61 62) . Expression of the itga4 gene is regulated through several different transcription factors that can either stimulate [Ets family (63) , Pax-6 (64) , the Wilms tumor suppressor Wt1 (65) , the glial cell missing factor GCMa (66) , c-Myb (67) ] or inhibit [ZEB (67) ] itga4 promoter activity. It will need to be determined in future studies whether GBP-1 regulates itga4 expression via one of these factors or through post-transcriptional regulation of ITGA4 mRNA stability.

Interactions of ECs with components of the ECM, such as FN, are crucial for the regulation of angiogenesis. FN is present in provisional vascular matrices of angiogenic vessels, but not in the basal membrane of mature vessels, underscoring its important function in angiogenesis (68 , 69) . Numerous reports describe interactions of ECs with FN mainly mediated by the integrins {alpha}5β1 and {alpha}vβ3. These interactions promote EC proliferation, spreading, and motility during the growth of new blood vessels (70 71 72 73) . ITGA4 is another FN binding integrin with distinct biochemical and biological functions from the others. ITGA4 binds to FN via a C-terminal binding site (type III repeat 14-IIICS) that is different from the central binding site used by {alpha}5β1 and {alpha}vβ3. ITGA4 binding to full-length FN results in decreased cell spreading (40 41 42 43) and migration (44 45 46 47) . Studies using integrin chimeras have demonstrated that these functional properties are conferred by the cytoplasmic domain of ITGA4 (40 , 42) , which directly and specifically binds to paxillin, a signaling adaptor protein, leading to reduced cell spreading (43) . In agreement with these results, we demonstrated that ITGA4 up-regulation by ectopically expressed GBP-1 is sufficient to mediate the inhibition of EC spreading and migration on FN. These effects were abrogated on a 120-kDa fragment of FN (FN-120) lacking the C-terminal ITGA4 binding site. This demonstrates that ITGA4 binding to the type III repeat 14-IIICS motif is required for its inhibitory activity.

In addition, we were able to show that EC spreading and migration on FN matrices was significantly inhibited in response to IL-1β and TNF-{alpha} through GBP-1-mediated induction of ITGA4 expression. Cell spreading on FN matrices was also impaired when IFN-{gamma} was applied, but in an ITGA4-independent manner. It is known that IL-1β, TNF-{alpha}, and IFN-{gamma} exert differential activities on ECs. For example, IL-1β and TNF-{alpha} induce a prothrombotic and proinflammatory phenotype, whereas IFN-{gamma}-induced activations are predominantly related to host defense against infections (54 , 55) . It is also clear that IL-1β and TNF-{alpha} in comparison to IFN-{gamma} activate different signal transduction pathways. This is also evident in the regulation of GBP-1 expression. In the GBP-1 promoter, a promoter module of an IFN-{alpha} response element (ISRE) and a cRel motif have been described (31) . In response to IL-1β and TNF-{alpha}, cooperative activation of both elements is observed and also required for the induction of GBP-1 expression. In contrast, in IFN-{gamma}-treated cells exclusively the ISRE element is activated, and this is sufficient for the induction of GBP-1 expression. The different activities of IL-1β and TNF-{alpha}, as compared to IFN-{gamma} on cellular activation and on the induction of GBP-1 expression may explain the different effects of GBP-1 on ITGA4 expression in IL-1β/TNF-{alpha}- vs. IFN-{gamma}-stimulated ECs. It can be speculated that GBP-1 inhibits spreading and migration under inflammatory conditions, which are regulated by IL-1β and TNF-{alpha}, and may be engaged in different functions in infectious conditions regulated by IFN-{gamma}. In this framework, it is interesting that GBPs have been shown to exert antiviral activity (74 , 75) and have been found associated with antibacterial responses in mammalian cells (22) .

Our work provides further insight into the function of GBP-1 in inflammation, showing that GBP-1 is necessary and sufficient to mediate the inhibitory effects of IL-1β and TNF-{alpha} on EC spreading and migration on FN matrices. Both of these important angiogenic processes can be inhibited by GBP-1 via induction of ITGA4 expression. These results indicate that ITGA4 may provide an antagonistic signal to ECM interactions of {alpha}5β1 and {alpha}vβ3 during inflammation. This may be a novel mechanism for termination of physiological and pathophysiological angiogenesis and thus may open new strategies for the modulation of angiogenesis in human diseases.


   ACKNOWLEDGMENTS
 
We thank Melanie Nurtsch, Gertrud Hoffmann, and Mahimaidos Manoharan for excellent technical assistance, Susanne Reed and Matthew Miller for reading the manuscript (Division of Molecular and Experimental Surgery, University of Erlangen), and Ludger Klein-Hitpass (Institute for Cell Biology, University of Essen) for Affymetrix GeneChip hybridization and partial analysis. We also thank Dr. Christoph Garlichs (Department of Medicine II, Cardiology and Angiology, University of Erlangen) for kindly providing the FACSCalibur. We are very grateful to Werner Hohenberger (Director of the Department of Surgery, University of Erlangen) for his generous support. This work was supported by grants from the Interdisciplinary Center for Clinical Research (IZKF) of the University of Erlangen-Nuremberg and the Deutsche Forschungsgemeinschaft (STU 317/2-1, DFG-GK 1071, DFG SPP 1130) to M.S. The authors have no conflicting financial interests.

Received for publication February 14, 2008. Accepted for publication July 17, 2008.


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