Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering*

Our data demonstrate that increased intracellular expression of thymosin β4(Tβ4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tβ4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tβ4(AcSDKPT/4A), Tβ4(KLKKTET/7A), and Tβ4(K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tβ4. Overexpression of Tβ4, Tβ4(AcSDKPT/4A), and Tβ4(K16A) affected intracellular formation of actin filaments. As expected, Tβ4(K16A) uptake by nuclei was impaired. On the other hand, overexpression of Tβ4(KLKKTET/7A) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tβ4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tβ4 and Tβ4(AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants Tβ4(KLKKTET/7A) and Tβ4(K16A) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tβ4. Ku80 and Tβ4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tβ4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tβ4 and mediates its intracellular activity.

T␤4 is considered to be a major actin sequestering molecule, which specifically binds monomeric actin (G-actin) forming a 1:1 complex, or by additionally including profilin, forming a ternery complex (23). Thus, the mechanism by which T␤4 influences cell proliferation, migration, and differentiation is generally believed to be linked with maintaining a dynamic equilibrium between G-actin and F-actin, critical for the rapid reorganization of the cytoskeleton. Under some conditions T␤4 can be found in large amounts in extracellular fluids and shows a broad range of biological activities. For example, the T␤4 level in blood may amount to micromolar concentrations, particularly in prethrombotic states characterized by the enhanced reactivity of blood platelets (24).
Recent observations indicate that T␤4 can express its activity toward different cells via receptor-mediated mechanisms. Thus, T␤4 externally added to endothelial cells: (a) induces expression and release of plasminogen activator inhibitor type 1 (PAI-1) by the mechanism involving activation of the mitogen-activated protein kinase cascade leading to enhanced c-Fos/c-Jun binding to the AP-1-like element present in the PAI-1 promoter (25,26); (b) binds to PINCH and integrinlinked kinase, resulting in activation of the survival kinase Akt, and thus promotes myocardial and endothelial cell migration in the embryonic heart (27) and (c) promotes skin and corneal wound healing through its effects on cell migration, angiogenesis, and possibly cell survival (4,6,11).
However, the precise molecular mechanism(s) through which it functions remains unknown. It is not clear whether T␤4 effects are mediated by (a) extracellular or (b) intracellular receptors, or if (c) T␤4 is taken up by cells and its activity is manifested after interaction with G-actin and modulation of the actin filament system. Therefore, in our present studies we attempt to describe intracellular mechanisms by which T␤4 may activate endothelial cells to up-regulate PAI-1 expression. Particularly, we searched for proteins that may interact in endothelial cells with T␤4 and thus mediate its activating effects. Based on our affinity binding experiments, co-immunoprecipitation, and proteomic analyses we identified the Ku80 subunit of ATP-dependent DNA helicase II to specifically interact with T␤4. Specific down-regulation of its expression by short interference RNA (siRNA) abolished the stimulatory activity of T␤4 toward PAI-1 expression suggesting its contribution in controlling PAI-1 gene activity.

EXPERIMENTAL PROCEDURES
Reagents-All standard tissue culture reagents including Dulbecco's modified Eagle's medium, fetal bovine serum, and Lipofectamine 2000 reagent were from Invitrogen (Eggenstein, Germany). Wizard Miniprep and Maxiprep kits for isolation of plasmid DNA were purchased from Promega Corp. (Madison, WI). Protein A/G-agarose, Enhanced Chemiluminescence (ECL) Western blotting substrate, and BCA Protein Assay Kit were obtained from Pierce. Anti-PAI-1 rabbit polyclonal antibody was from American Diagnostica (Pfungstadt, Germany). Anti-Ku80 rabbit polyclonal antibody and horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibody were from Chemicon (Temecula, CA). Horseradish peroxidase-conjugated goat anti-mouse antibody was purchased from Jackson ImmunoResearch (West Grove, PA). pCMV-Myc and pCMVhemagglutinin (HA) Vector Set with anti-HA tag and anti-Myc tag antibodies were from Clontech. All synthetic short interference RNA duplexes were from Dharmacon (Lafayette, CO). All other reagents, except where noted, were from Sigma.
Plasmids containing the EGFP-T␤4 or EGFP-T␤4 mutants were transfected into EA.hy 926 cells with Lipofectamine. Briefly, 5.0 g of plasmid DNA and 5 l of Lipofectamine solution were incubated for 45 min in 200 l of Opti-MEM (Invitrogen), and then diluted with 800 l of Opti-MEM. This solution was added to growing EA.hy 926 cells in 20% fetal bovine serum medium. Expression of the fusion construct was evaluated by confocal microscopy after 24 and 48 h.
T␤4-binding Proteins-Recombinant T␤4 was expressed in E. coli (BL21(DE3)pLysS) transformed with pRSETa-T␤4. Harvested cells were centrifuged, resuspended in binding buffer (5 mM imidazole, 150 mM NaCl, 20 mM Tris-HCl, pH 7.9), and sonicated on ice. Bacterial lysate was clarified by ultracentrifugation for an hour at 100,000 ϫ g at 4°C, filtered through a 0.45-m syringe filter, and loaded onto a HisTrap column (5 ml, Amersham Biosciences) connected to an FPLC system (Amersham Biosciences). Proteins were eluted with a 5-500 mM imidazole gradient. The purity of recombinant T␤4 was confirmed by reversed phase ultra performance liquid chromatography and mass spectrometry.
Recombinant T␤4 containing a 12-residue His tag (MRGS-HHHHHHGS) at its C terminus was biotinylated with freshly prepared p-diazobenzoyl biocytin according to the manufacturer's protocol (Pierce). To search for T␤4-binding proteins in endothelial cells, total cell extract, nuclear proteins, and the cellular membrane fraction were isolated from EA.hy 926 cells. To obtain the cell lysate, EA.hy 926 cells were extracted with lysis buffer (20 mM Tris-HCl, pH 8.3, containing 5% glycerol, 200 mM NaCl, 1% Triton X-100, 1 mM EDTA and protease inhibitor mixture) and cleared by centrifugation for 30 min at 12,000 ϫ g at 4°C. Crude nuclear extracts of EA.hy 926 cells were prepared essentially as described by Dignam et al. (32), except that modified buffer C was used during nuclear extraction (33). Membranes of EA.hy 926 cells were isolated as described by Thepparit and Smith (34) and solubilized in 20 mM Tris-HCl, pH 8.0, containing 100 mM NaCl, 2 mM MgCl 2 , 1 mM EDTA, 1% Triton X-100 and protease inhibitors.
In typical experiments, the cell lysate, the extract of nuclear proteins or solubilized membranes were divided into two parts: into the first one biotinylated T␤4 was added and incubated overnight at 4°C and the second was used as a control without T␤4, kept under the same conditions. Then, streptavidin-Sepharose beads were added to both samples and incubated for an hour at 4°C. The beads were then washed exhaustively with PBS, resuspended in SDS-PAGE loading buffer (0.1 M Tris-HCl, pH 6.8, 10% glycerol, 1% SDS, 5% ␤-mercaptoethanol, 0.5% Coomassie Brilliant Blue R-250), boiled for 5 min, and separated by SDS-PAGE. Protein bands were visualized by staining with silver according to the method compatible with the analysis of proteins by mass spectrometry (35) and the selected bands subjected to in-gel digestion with trypsin.
Mass Spectrometry, Data Base Search, and Data Processing-Proteins in each gel slice were subjected to reduction with 10 mM dithiothreitol, alkylation with 50 mM iodoacetamide, and tryptic digestion with modified trypsin (10 g/ml; Promega) at 37°C for 14 h. After in-gel digestion, the product peptides were extracted stepwise with three portions of 60 l of 0.1% trifluoroacetic acid in 2% acetonitrile and loaded on an RP-18 precolumn (LC Packings). Peptides were eluted to a nano-HPLC RP18 column (75 m ϫ 15 cm capillary; LC Packings) by acetonitrile gradient in the presence of formic acid and then directly applied into an electrospray (ISI-Q-TOF-Micromass) spectrometer. The spectrometer was working in the regime of data-dependent MS to MS/MS switch giving peptide sequencing data in addition to mass fingerprint data. The NCBI nonredundant protein data base was searched with the Mascot program (Matrix Science). The list of top 20 candidates for each sample was verified by inspection of the quality of sequencing data. We examined their automatic ordering manually in terms of their reliability scores and MS spectrum profiles to pick up only highly reliable peptide data (sorted data).
Likewise, synthetic DNA coding T␤4 sequence was amplified using the oligonucleotide primers pair: 5Ј-CGCCCGCGAAT-TCGGATGTCTGACAAACCC-3Ј and 5Ј-CTCAATAGCG-GCCGCTCATTACGATTCGCCTGC-3Ј. Subsequently, the resulting amplicon was inserted into EcoRI and NotI sites of pCMV-HA vector expressing the protein with HA epitope tag at the N terminus.
Co-immunoprecipitation Experiments-EA.hy 926 cells were transiently cotransfected with pCMV-HA-T␤4 (4 g) and either pCMV-Myc-Ku80 or one of the mutants (pCMV-Myc-Ku80-(1-249), pCMV-Myc-Ku80-(1-460), pCMV-Myc-Ku80-(1-580), pCMV-Myc-Ku80-(368 -372)) (4 g) using Lipofectamine and collected after 48 h in lysis buffer (PBS, 1 mM EDTA, pH 8.0, 0.5% Triton X-100). Cell lysates were then incubated with 2.5 g of rabbit polyclonal antibody to Myc tag on a rotator overnight at 4°C. Subsequently, 100 l of Protein A/Gagarose bead slurry was added to each cell extract and the incubation continued for another 3 h. The beads were washed, suspended in SDS-PAGE loading buffer, and boiled for 5 min. Proteins released from the resin were separated by electrophoresis by 7% SDS-PAGE, electrotransfered onto nitrocellulose membranes, and immunodetected by HA tag mouse monoclonal antibody. Immunoprecipitation of the Ku80⅐T␤4 complex from endothelial cell extracts was performed as described previously (26). siRNA silencing the human glyceraldehyde-3-phosphate dehydrogenase gene served as a positive control, with the following sense strand sequence: 5Ј-UGGUUUACAUGUUCCAAUAUU-3Ј. As negative control the functional non-targeting siRNA was used, containing 4 mismatches for any human, mouse, and rat gene. The sequence of its sense strand was 5Ј-UAGCGACUA-AACACAUCAAUU-3Ј. Synthetic siRNAs (200 pM) were transfected to 50 -80% confluent EA.hy 926 cells with Lipofectamine (10 l) according to the manufacturer's instructions. After 24 h of incubation synthetic T␤4 at 160 nM or tumor necrosis factor-␣ at 10 ng/ml was added to particular cell cultures with the silenced Ku80 gene and concurrently to nontransfected cells. The cells were incubated for another 24 h and then harvested with TriPure reagent for total RNA and protein isolation.
Western Immunoblotting-Protein isolated from EA.hy 926 cells transfected with vectors expressing T␤4 and its mutants, Ku80 or siRNA, were extracted with TriPure reagent according to the manufacturer's protocol. After extraction, pellets were dissolved in 1% SDS aqueous solution and the protein concentrations were measured with BCA Protein Assay Kit and equalized between samples with 1% SDS. Protein extracts were subjected to SDS-PAGE and electroblotted onto nitrocellulose filters. The filters were blocked with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween 20 then immunodetected by anti-PAI-1 and anti-Ku80 or anti-Myc tag mouse monoclonal antibody. Immunodetection was accomplished using the enhanced chemiluminescence kit and Kodak BioMax Light Film (Eastman Kodak). Developed films were scanned and protein bands quantitated by Gel Doc 2000 Gel Documentation System (Bio-Rad).
Confocal Microscopy-For microscopic examination of EA.hy 926 cells transfected with pEGFP-N1-T␤4 or its mutants, cells (5 ϫ 10 4 cells/ml) were plated on Permanox Coverslips in 8-well tissue culture chamber slides (NUNC) with detachable chambered upper structures. After 24 h incubation, they were fixed with ice-cold 3% formaldehyde in PBS for 20 min, washed 3 times with PBS, and incubated with blocking buffer (PBS containing 3% bovine serum albumin). They were also counterstained with TRITC-phalloidin or Hoechst 33258 (Molecular Probes) to visualize actin filaments or nuclei, respectively. Endogenous T␤4 was detected in the untransfected cells with rabbit antibodies specific to T␤4, followed by staining with secondary antibodies conjugated with fluorescein. The cells were then visualized using a helium/neon ion laser (543 nm excitation) and analyzed with MultiScan version 8.08 software. For intracellular probe visualization the confocal laser microscope Leica TCS SP2 system in the Laboratory of Confocal Microscopy in Nencki Institute of Experimental Biology was used. The series of the single 0.2-m optical sections were collected. The image has been scanned at high resolution (ϫ63 oil objective, 1.32 NA).

High Content Forster Resonance Energy Transfer Screening
Analysis-Distribution of pEGFP-N1-T␤4, pEGFP-N1-T␤4 (KLKKTET/7A) , and pEGFP-N1-T␤4 (K16A) in membranes and nuclei of the transfected EA.hy 926 cells was evaluated by high content screening analysis using Cellomics ArrayScan V TI HCS Reader (Thermo Fisher Scientific, Pittsburgh, PA). ArrayScan consists of a high resolution optical system, multiple bandpass emission filter with matched single band excitation filters (XF57 or XF100, Omega Optical), a CCD camera with frame grabber, and proprietary applications software. In this assay, an excitation filter wheel and multiple bandpass emission filters are used to enable multichannel imaging of fluorescence from several fluorophores in the same cells. This system automatically locates, focuses, and exposes fields of cells within blacksided 96-well microtiter plates in a user-defined manner (36,37). Briefly, 0.2 g of plasmid DNA and 0.5 l of Lipofectamine solution were incubated for 45 min in 50 l of Opti-MEM (Invitrogen). This solution was added to EA.hy 926 cells growing on a 96-well plate in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. After 6 h the medium was changed and the cells were further incubated for 24 h. Then, the cells were washed twice with PBS and the staining of plasma membrane and nuclei was performed with the Image-LIVE Plasma Membrane and Nuclear Labeling Kit (Molecular Probes). The plasma membranes were stained with red fluorescent Alexa Fluor 594 wheat germ agglutinin at 5.0 g/ml and the nuclei, with the blue fluorescent Hoechst 33342 at 1 M. Finally, 100 l of PBS was added into each well and the fluorescent images of the cells for three fluorophores were taken at the following wavelengths (excitation/emission; nm) of 591/618, 350/461, and 485/535 for Alexa Fluor 594 wheat germ agglutinin, Hoechst 33342, or GFP, respectively. Triple emission images were acquired for 30 fields in each well of the plate using the ϫ20 objective. The cells were analyzed using Cytoplasm to Cell Membrane Translocation BioApplication and Compartmental Analysis BioApplication software according to the manufacturer's instructions. Statistical analysis of the obtained data were performed using t test for independent samples.
Statistical Analysis-All values are expressed as mean Ϯ S.E. compared with controls and among separate experiments. Paired and unpaired Student's t tests were employed to determine the significance of changes. A p value Ͻ0.05 was considered statistically significant.

Subcellular Localization of T␤4 in Endothelial
Cells-In the course of our studies on the mechanism by which T␤4 influences angiogenesis we examined its intracellular activity in endothelial cells. In preliminary experiments the intracellular distribution of T␤4 and its mutants fused with GFP was evaluated by confocal microscopy. To produce a functional T␤4-GFP fusion protein, GFP was attached to the C-terminal residue of the peptide. The rationale for this fusion strategy was that the C-terminal region does not specify any known function of T␤4. This design avoided modifying the N terminus of T␤4, which is important for interaction with potential receptors assumed to exist, based on the biological activity of its N-termi-
Endogenous T␤4 identified by staining with anti-T␤4 antibodies, followed by second antibodies conjugated with fluorescein, was found in whole cytoplasm, showing a strong punctate staining together with a diffuse cytoplasmic localization. It was focally enriched in regions located subjacent to the plasma membrane and nucleus (Fig. 1A, a, green). This staining was specific because nonimmune IgG was negative in control cells, additionally stained with TRITCphalloidin and Hoechst 33258 (Fig.  1A, b, red and blue). Cells transfected with the GFP-tagged T␤4 showed much higher expression of the fluorescently labeled intact T␤4. Its distribution matched that of the endogenous T␤4, showed much higher accumulation in cytoplasm and was close to the membranes (Fig. 1A, c, green). Endogenous T␤4 similar to GFP-tagged T␤4 colocalized with actin at the edges of cells (Fig. 1B, c, yellow). There were heavy accumulations of GFP-tagged T␤4 and GFP-tagged T␤4 (SDKP/4A) close to the membranes, particularly at the cell-cell contact sites (Fig. 1B, a  and c). Actin filament staining in the same cells was perhaps even more concentrated at the cell-cell contact sites (Fig. 1B, b), and overlapping staining of T␤4 and actin seen as yellow was particularly evident at these sites. Besides the accumulations visualized within the cytoplasm, the data obtained also showed in many cases a punctate nuclear staining, indicating accumulation of intact T␤4 within the cell nucleus. Within the nuclei there was a homogenous distribution of T␤4 with the exception of regions of presumed nucleoli that were void of T␤4. Similar to the wild-type T␤4, overexpression of T␤4 (AcSDKPT/4A) affected intracellular formation of actin filaments that could be found predominantly in the verges of cells, directly under the cell membrane (Fig. 1B,  d, e, and f).
Mutants T␤4 (KLKKTET/7A) and T␤4 (K16A) differ significantly in their cytoplasmic distribution from that of the wild-type T␤4 (Fig. 1B, g, h, i and k, l, m, respectively): their fluorescence is more punctuated and they do not accumulate in the submembrane regions. In contrast to wild-type T␤4 and T␤4 (AcSDKPT/4A) , cells transfected with T␤4 (KLKKTET/7A) showed a well developed system of the actin filament network typical of adhering and spread cells. This indicates that the mutant lacked the actin binding site and thus did not interfere with actin polymerization. It is noteworthy that localization of T␤4 and its mutants in endothelial cells shown in Figs. 1 and 2 was observed in a number of cells (from 47 to 95) analyzed by confocal microscopy during five separate experiments. These data indicate that attachment of EGFP to the polypeptide chain of T␤4 did not influence its primary function, which is to interact with G-actin. To examine whether the point mutation within the nuclear localization sequence K16A reduced accumulation of this mutant within the cell nucleus, optical sections of cells transfected with pEGFP-N1-T␤4 or pEGFP-N1-T␤4 (K15A) were taken by confocal microscopy starting from apical to basal surfaces.
After scanning, the relative brightness of the images compared with an average brightness of the images of the particular nucleus was estimated and plotted ( Fig. 2A). They reflect the concentration of the EGFP-T␤4 or its EGFP-T␤4 (K16A) mutant within the nuclei. These data clearly demonstrate significantly reduced amounts of pEGFP-N1-T␤4 (K16A) in the analyzed cell nuclei when compared with those detected for pEGFP-N1-T␤4. To further support nuclear location of T␤4 and T␤4 (K16A) , crude nuclear extracts were isolated from human endothelial cells transfected with either pEGFP-N1-T␤4 or pEGFP-N1-T␤4 (K16A) , solubilized in 0.02 M HEPES buffer containing 0.14 M NaCl and 1% Triton X-100, pH 7.5, and analyzed spectrofluorimetrically. Their concentration, evaluated by analysis of the emission at 507 nm after excitation of EGFP at 488 nm, clearly show the presence of both proteins in nuclear extracts and much lower concentrations of GFP-tagged T␤4 (K16A) when compared with wild-type T␤4 (Fig. 2B). Finally, to evaluate subcellular distribution of T␤4 and its mutants in living endothelial cells we employed high content Forster resonance energy trans-fer screening analysis using a Cellomics ArrayScan V TI HCS Reader. Due to specific staining of membranes and nuclei with Alexa Fluor 594 wheat germ agglutinin and Hoechst 33342, respectively, this system automatically locates, focuses, and exposes labeled fields of cells. Then, location, translocation, and quantification of T␤4 and its mutants containing yellow GFP showing the optimized spectral overlap with both dyes are accomplished by Forster resonance energy transfer. Fig. 2, C and D, show that T␤4 and its mutants are equally localized close to the membrane but their nuclear accumulation is significantly different. Both, T␤4 (KLKKTET/7A) and T␤4 (K16A) occur in much lower concentrations in nuclei than intact T␤4 indicating that their nuclear uptake is impaired.
Intracellular Expression of T␤4 and PAI-1-In the next experiments we tested whether increased intracellular levels of T␤4 or its functionally modified mutants affect the mechanisms by which expression of the PAI-1 gene is controlled. For this purpose, PAI-1 expression was evaluated at the level of both mRNA and protein synthesis in EA.hy 926 cells transfected with T␤4 and its mutants. Because transfection efficiency differed from one construct to another, expression of PAI-1 mRNA was normalized and the plotted values correspond to those that would be obtained after transfection of all cells. Fig. 3A shows that transfection with pEGFP-N1-T␤4 and pEGFP-N1-T␤4 (AcSDKPT/4A) resulted in inducing the PAI-1 gene almost to the same extent as is demonstrated by real-time PCR analysis of PAI-1 mRNA. Thus, depletion of the N-terminal tetrapeptide AcSDKP did not influence the ability of T␤4 to up-regulate PAI-1 expression. Interestingly, elimination of the actin binding sequence KLKKTET or damage of the nuclear localization sequence by a point mutation (K16A) only partially reduced the ability of such T␤4 mutants to up-regulate PAI-1 expression. When tested at the protein synthesis level, as shown by immunoblotting with anti-PAI-1 antibodies followed by scanning, increased intracellular concentrations of T␤4 and all its mutants also induced PAI-1 expression in transfected cells (Fig. 3B).
T␤4-binding Proteins in Endothelial Cells-To investigate the potential mechanisms through which T␤4 might be influencing endothelial cell migration and survival events, we then searched for T␤4 interacting proteins within the cells. For this purpose, the recombinant T␤4 was biotinylated with p-diazobenzoyl biocytin reagent in such a way that exclusively His res-  n ϭ 15). The image brightness of each section is presented as a % of the average nuclear brightness and reflects the concentration of the EGFP-T␤4 or its mutant within the nucleus. B, the presence of T␤4 and T␤4 (K15A) in whole cell lysate, solubilized membranes, and crude nuclear extracts of human endothelial cells transfected with either pEGFP-N1-T␤4 or pEGFP-N1-T␤4 (K16A) , respectively. Data, obtained during three separate experiments, were collected by analysis of the emission at 507 nm after excitation of EGFP at 488 nm. In each fraction, fluorescence of pEGFP-N1-T␤4 was taken as a control. Distribution of T␤4, T␤4 (KLKKTET/7A) , and T␤4 (K16A) in membranes (C) and nuclei (D) of endothelial cells was also analyzed by high content Forster resonance energy transfer screening. A 36-well plate was seeded with EA.hy 926, transfected with pEGFP-N1-T␤4, pEGFP-N1-T␤4 (KLKKTET/7A) , or pEGFP-N1-T␤4 (K16A) , and imaged for 90 min with ϳ600 fields of view (ϫ20 objective). The wells were then stained with Alexa Fluor 594 wheat germ agglutinin and Hoechst 33342 to label membrane and nuclei, respectively. Data are expressed as mean Ϯ S.D. and were collected during three separate experiments. *, p Ͻ 0.02, and **, p Ͻ 0.001 were determined using t test for independent samples. JANUARY 18, 2008 • VOLUME 283 • NUMBER 3

JOURNAL OF BIOLOGICAL CHEMISTRY 1539
idues of the His tag sequence attached to the N terminus of T␤4 were modified. Thus, the entire sequence of the polypeptide was left unchanged and available for interacting with proteins. To narrow down their location in the cells, extracts of subcellular fractions, including isolated nuclei, membranes, or cytoplasm, were preincubated with the biotinylated T␤4, and then aliquots of streptavidin-Sepharose suspension were added. After incubation, the resin was washed and T␤4-interacting proteins were dissociated with the SDS sample buffer and analyzed by SDS-PAGE. This electrophoretic separation of complexes with biotinylated T␤4 showed the presence of several proteins. Among them, a protein with molecular mass of 80 kDa was consistently found to be bound to T␤4 regardless of the starting material used, namely the total cell lysate, solubilized cell membranes, or the extract of nuclear proteins. This protein was absent in the control samples missing the biotinylated T␤4 (Fig. 4, A-C). It was identified with confidence, by peptide sequencing and peptide mass fingerprinting using an electrospray (ISI-Q-TOF-Micromass) spectrometer, to be ATPdependent DNA helicase II, specifically its Ku80 subunit (Table  1). In addition to some other cellular proteins, large amounts of actin were also detected to be associated with both types of resin, i.e. the control resin missing T␤4 and the one containing immobilized T␤4. We then investigated whether T␤4 can interact in vivo with Ku80 in endothelial cells. To identify Ku80⅐T␤4 complexes consisting of endogenous proteins, we performed co-immunoprecipitation experiments using extracts of untransfected endothelial cells. Proteins were precipitated with specific rabbit polyclonal antibodies to T␤4, and immunoprecipitates were washed and separated by SDS-PAGE. Ku80 bound to T␤4 was identified by Western immunoblotting using antibodies specific to Ku80 or nonimmune rabbit sera (Fig. 4D).
To narrow down localization of the Ku80 binding site for T␤4 and prove the specificity of this interaction, the Ku80 deletion mutants were constructed and expressed in pCMV-Myc vector (Fig. 5A). As detected by immunoblotting with specific antibodies to Myc tag, EA.hy 926 cells after transfection with these constructs showed almost the same expression of Ku80 fragments, namely Ku80(1-249), Ku80-(1-460), Ku80-(1-580), Ku80-(368 -732), or intact molecule (Fig. 5B). EA.hy 926 cells were then cotransfected with pCMV-HA-T␤4 and pCMV-Myc-Ku80 followed by immunoprecipitation performed using rabbit anti-HA antibodies. Fig. 5C shows the presence of Ku80 in the co-precipitating proteins as evidenced by immunoblotting with anti-Myc monoclonal antibody, thus confirming that Ku80 can form the complex with T␤4 in endothelial cells. Fig. 5D shows that in addition to intact Ku80, only Ku80-(1-580) and Ku80-(368 -732) form a complex with T␤4 , and then aliquots of streptavidin-Sepharose suspension were added. After incubation, the resin was washed, and T␤4-interacting proteins were dissociated with the SDS sample buffer and analyzed by SDS-PAGE. In parallel, samples of starting material (SM) and proteins eluted from streptavidin-Sepharose in the absence of biotinylated T␤4 were also analyzed. D, the presence of Ku80 was detected by Western immunoblotting in immunoprecipitates obtained from endothelial cells with antibodies specific to T␤4. In parallel, samples of cell extract were incubated with control normal IgG. Then, proteins precipitated with anti-T␤4, normal IgG, and those present in starting cell extracts were separated by SDS-PAGE and blotted with anti-Ku80 antibodies. In these experiments, total cellular RNA was extracted from cells 48 h after transfection with T␤4 and its mutants, and PAI-1 mRNA was quantified by real-time PCR. This experiment was repeated three times with similar results. Because transfection efficiency differed from one construct to another, expression of PAI-1 mRNA was normalized to 100% of transfection efficiency. B, PAI-1 protein in extracts of the same cells evaluated by Western immunoblotting (inset). Immunodetection of PAI-1 was accomplished using an enhanced chemiluminescence kit, and films were scanned and protein bands quantitated using the Gel Doc 2000 gel documentation system (Bio-Rad). To quantify the densitometric scans, the background was subtracted and the area for each protein peak was determined. Data were obtained from three separate experiments and normalized to 100% of transfection efficiency.
Down-regulation of Ku80 by siRNA and PAI-1 Expression-To explore the impact of Ku80 on PAI-1 expression in T␤4-activated endothelial cells, we employed specific siRNA as a means of depleting this protein in EA.hy 926 cells. For this purpose, siRNAs complementary to four regions of Ku80 were generated, and their efficacy in down-regulating Ku80 in endothelial cells was analyzed. Fig. 6, A and B, show control experiments demonstrating that all four siRNAs to Ku80 mRNA and their equimolar mixture reduced expression of the targeted protein  (368 -732)). In these experiments, total cellular RNA was extracted from cells 48 h after transfection, and PAI-1 mRNA was quantified by real-time PCR. This experiment was repeated three times with similar results. Because transfection efficiency differed from one construct to another, expression of PAI-1 mRNA was normalized to 100% of transfection efficiency. F shows PAI-1 protein in extracts of the same cells evaluated by Western immunoblotting (inset figure). Immunodetection of PAI-1 was accomplished as described in the legend to Fig. 3B. Data were obtained from three separate experiments and normalized to 100% of transfection efficiency.

TABLE 1 T␤4-interacting proteins identified in subcellular fractions of endothelial cells
The subcellular fractions were isolated from HUVECs as described under "Experimental Procedures." The biotinylated T␤4 was added to the total cell lysate, the cell membrane lysate, or nuclear protein fraction. After incubation, the biotinylated T␤4 and bound proteins were separated using streptavidin immobilized on Sepharose. Samples of proteins bound were separated by SDS-PAGE followed by sequencing of protein bands selectively bound to T␤4 (Fig. 4). Protein bands were identified after silver staining and analyzed by sequencing as described under "Experimental Procedures."  (Fig. 6, C and D). In cells treated with control siRNA, T␤4 alone increased PAI-1 to the level comparable with those observed in the untreated cells (not shown). Expression of von Willebrand factor and T␤4 analyzed in the same samples was not affected when analyzed using both RT-PCR (panel E) or Western blotting (panel F). Thus, these data show that inhibition of Ku80 expression resulted in specific abolishment of the PAI-1 signal determined at the level of its mRNA and PAI-1 antigen synthesis. Furthermore, cells depleted of Ku80 with siRNA failed to elicit a robust increase in PAI-1 typical for the untreated cells.

Cell fraction
Taken together these data suggest that Ku80, after formation of complexes with T␤4, is involved in regulation of PAI-1 expression in endothelial cells induced by T␤4.

DISCUSSION
A number of studies showed that T␤4 exhibits biological functions that are important for angiogenesis, wound healing, and the regeneration and remodeling of injured tissues (for review, see Ref. 38). Up to now two bioactive fragments of T␤4 have been identified, namely the N-terminal tetrapeptides AcSDKP (39) and LKKTET, derived from its central actin-binding domain (40). Although there are observations revealing that T␤4 and its fragments affect cellular processes by activation of several signaling pathways and induction of changes in cell properties necessary for both migration and survival, not much is known about the mechanisms by which they express their functions.
The data presented here demonstrate that increased intracellular expression of T␤4 is necessary and sufficient to initiate PAI-1 gene expression in T␤4-activated endothelial cells. This conclusion is based on the constitutively enhanced expression of T␤4 in EA.hy 926 cells prior to the induction of the PAI-1 gene by extracellularly added T␤4. Interestingly, the mechanism of this intracellularly active T␤4 only partially depends on its ability to bind G-actin or the presence of other bioactive regions in the T␤4 molecule such as the N-terminal tetrapeptide and the nuclear localization sequence. This conclusion is based on the following observations: (a) substitution of the N-terminal tetrapeptide with four alanine residues did not have any influence on stimulation of PAI-1 expression in the transfected cells. (b) Mutation of bioactive motifs responsible for binding G-actin and entering the nucleus selectively damaged their functions. Neither mutation abolished the ability of such mutants to up-regulate PAI-1 expression in the transfected cells although they showed an ϳ40% decrease in activation of the PAI-1 gene. (c) T␤4 and T␤4 (AcSDKPT/4A) , showed the same levels of overexpression and a similar distribution in the transfected cells as evaluated by confocal microscopy. They were densely accumulated close to the membranes, particularly at cell-cell contact sites.
One of the notable findings of this study is the identification of previously unknown T␤4-interacting proteins in endothelial cells, especially the Ku80 subunit of ATP-dependent DNA helicase II, which was consistently associated with the biotinylated T␤4. Ku80 specifically interacted with T␤4 when total cell lysate, isolated nuclear proteins, or plasma membranes were taken as a starting material. Moreover, Ku80 and T␤4 con- sistently immunoprecipitated in a common complex from extracts of both control endothelial cells and the cells that were cotransfected with Ku80 and T␤4 fused with peptide tags. Furthermore, co-immunoprecipitation experiments with Ku80 deletion mutants showed that T␤4 directly interacts with the C-terminal arm domain of Ku80 but to stimulate PAI-1 expression it requires both the C-terminal arm and the ␣/␤ domains to be in close proximity. Interestingly, down-regulation of Ku80 expression with siRNA in endothelial cells resulted in a dramatic reduction of PAI-1 synthesis, indicating that Ku80 is involved in signaling initiated by T␤4 that leads to activation of PAI-1 gene expression by a still unknown mechanism.
The Ku proteins were originally identified as autoantigens, recognized by the sera of patients with autoimmune diseases such as systemic lupus erythematosus and scleroderma (41). The two Ku proteins, Ku70 and Ku80, have been well demonstrated to dimerize and function in repair of DNA double strand breaks, DNA telomere length maintenance, transcription regulation, and V(D)J recombination (42)(43)(44)(45). Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival (for review, see Ref. 46). Observations through various experimental models indicate that Ku may act as either a tumor suppressor or an oncoprotein. Although they are predominantly nuclear proteins and primarily can be found in the transcriptionally active regions of the nucleus, recent studies showed that Ku proteins are also expressed in the cytoplasm, on the cell surface, and in the extracellular matrix (47,48). Ku is a component of the DNA-PK complex in membrane rafts of mammalian cells (49) and its membrane expression can be induced at hypoxia. Interestingly, it mediates adhesion of cells to fibronectin (48,50,51), which indicates its role as an adhesion receptor (52). Recently, Ku80 was also identified to be a coreceptor for human parvovirus B19 infection (53). It appears that both Ku80 and signal transduction are coupled (49,54). Furthermore, Ku can interact with matrix metalloproteinase 9 at the cell surface of highly invasive hematopoietic cells of normal and tumor cell origin, and Ku80/ metalloproteinase-9 interaction at the cell membrane may result in contributing to invasion by tumor cells through regulation of extracellular matrix remodeling (48). Ku proteins can also function as transcription factors and bind in a sequencespecific manner to promoter elements (55). For example, Ku86 binds to the promoter and regulates the genes of the heat shock proteins, glucose-regulated peptide 78, grp94 (56), and S100A9 gene expression (57).
At present the mechanism by which Ku80 contributes to T␤4-induced PAI-1 gene regulation is unclear. Ku proteins have been shown to regulate numerous intracellular functions, suggesting that they might be active at multiple locations within the cell. Thus, Ku80 may function at different steps of signal transduction induced by T␤4 and leading to up-regulation of PAI-1 expression. Because it is found in membrane rafts it may play a role of T␤4 coreceptor and coactivator. Ku80 has a nuclear localization sequence and, consistent with data presented in Fig. 3D, may facilitate transport of T␤4 into the nucleus. Finally, Ku80 can be a cofactor that binds to the PAI-1 promotor and potentiates its T␤4-induced activation.
To sum up, in these studies we have provided evidence that increased intracellular expression of T␤4 leads to induction of the PAI-1 gene in T␤4-activated endothelial cells. This effect involves complex formation with Ku80, which functions as a novel receptor for T␤4 and provides an alternative mechanism of the intracellular activity of T␤4 different from sequestering of G-actin.