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Originally published In Press as doi:10.1074/jbc.M412653200 on March 23, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20833-20841, May 27, 2005
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Metastasis-associated Protein S100A4 Induces Angiogenesis through Interaction with Annexin II and Accelerated Plasmin Formation*

Alexandre Semov{ddagger}, Maria J. Moreno§, Anatoli Onichtchenko{ddagger}, Abedelnasser Abulrob§, Marguerite Ball§, Irena Ekiel¶, Grzegorz Pietrzynski{ddagger}, Danica Stanimirovic§, and Valery Alakhov{ddagger}||

From the {ddagger}Supratek Pharma Inc., Montreal, Quebec H9S 1A9, Canada, §Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada, and Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada

Received for publication, November 9, 2004 , and in revised form, March 8, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many advanced tumors overexpress and secrete the S100A4 protein that is known to promote angiogenesis and metastasis development. The mechanisms of this effect and the endothelial receptor for S100A4 are both still unknown. Here we report that extracellular S100A4 interacts with annexin II, an endothelial plasminogen co-receptor. Co-localization and direct binding of S100A4 and annexin II were demonstrated, and the binding site was identified in the N-terminal region of annexin II. S100A4 alone or in a complex with annexin II accelerated tissue plasminogen activator-mediated plasminogen activation in solution and on the endothelial cell surface through interaction of the S100A4 C-terminal lysines with the lysine-binding domains of plasminogen. A synthetic peptide corresponding to the N terminus of annexin II prevented S100A4-induced plasmin formation in the endothelial cell culture. Local plasmin formation induced by circulating S100A4 could contribute to tumor-induced angiogenesis and metastasis formation that makes this protein an attractive target for new anti-cancer and anti-angiogenic therapies.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
S100A4 protein, also known as Mts-1 or metastasin, belongs to the S100 family of Ca2+-binding proteins that are implicated in a variety of cellular events, including growth, signaling, differentiation, and motility (1–3). S100A4 and several other S100 proteins, such as S100A2, S100A6, and S100B, have attracted significant attention because of their established significance in the progression of metastatic tumors (4). S100A4 is a well established marker of tumor progression, invasion, and metastasis formation, as well as poor survival prognosis (1, 2). Strong overexpression of S100A4 was found in different types of tumors including breast (5), esophageal squamous (6) and colon carcinomas (7), invasive pancreatic carcinomas (8), non-small lung cancers (9), primary gastric cancers (10), bladder cancer (11), and some others. Recently, a strong up-regulation of S100A4 expression has been also reported for several non-cancer pathologies associated with activated endothelium, such as ocular neovascularization (12), artheriopathy (13), inflammation (14), and fibrosis (15).

S100A4 has been originally described as an intracellular protein that interacts with multiple protein targets and interferes with their phosphorylation. For example, S100A4 binds to p53 tumor suppressor protein and inhibits its phosphorylation by protein kinase C (PKC)1 that modulates the expression of p53-regulated genes, such as p21 and bax (16). It has been suggested that S100A4 cooperates with wild-type p53 to stimulate apoptosis and selection of more aggressive cancer cell populations (16). The binding of S100A4 to liprin {beta}-1 and heavy chain IIA non-muscle myosin results in the inhibition of their phosphorylation by PKC and casein kinase II (CKII) in vitro (17, 18) and subsequent disruption of myosin self-assembly (18). Other binding partners of S100A4 include non-muscle tropomyosin (19), another component of the cytoskeleton, methionine aminopeptidase 2, a well known target for angiogenesis inhibitors fumagillin and ovalicin (20), and nephroblastoma-overexpressed gene (also known as CCN3 or IGFBP9) (21).

S100A4 is secreted by cancer cells and is detectable in the serum of cancer patients (22). Endothelium (23, 24), neurons (25), and astrocytes (26) are primary targets of extracellular S100A4. The exogenous addition of S100A4 stimulates endothelial cell motility in vitro (24) as well as induces corneal neovascularization (22) and metastasis formation in vivo (27). Although angiogenic effects of extracellular S100A4 on endothelial cells have been clearly documented, its putative cell membrane receptors are still unknown. The identification of S100A4 endothelial receptor could facilitate the development of anti-angiogenic agents and metastasis inhibitors.

This report provides evidence that angiogenic effects of S100A4 are induced through its interaction with annexin II (ANXA2) on the surface of endothelial cells. Similarly to S100A10 (also known as p11 or small subunit of annexin II heterotetramer) (28), S100A4, either alone or in a complex with annexin II, accelerates t-PA-mediated conversion of plasminogen to plasmin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Primary human cerebromicrovascular endothelial cells (HCEC) were obtained from small intracortical microvessels and capillaries (20–112 µm) harvested from surgically excised human temporal cortex as previously described (29). HCEC growth conditions and phenotypic properties were described in detail elsewhere (29).

Recombinant Proteins—Reverse transcription-PCR products produced from human placenta cDNA and corresponding to full-size human S100A4, S100A10, and annexin II were cloned in pGEX-4T or pGEX-6P vectors (Amersham Biosciences). N-terminal truncated forms of annexin II with its first 15 or 26 amino acids deleted, A2-del15 and A2-del26, respectively, and C-terminal mutant of S100A4 with its two last lysines substituted with leucines, S100A4-KK/LL, were also cloned as reverse transcription-PCR products in pGEX-4T vector. Glutathione S-transferase fusion proteins were purified using glutathione-Sepharose 4 beads and cleaved with thrombin or PreScission protease to remove glutathione S-transferase tag according to the manufacturer's instructions (Amersham Biosciences). The quality of cloned proteins was assessed by Western blot.

Western Blot Analysis—S100A4 was detected using rabbit polyclonal antibody (NeoMarkers). This antibody did not cross-react with recombinant S100A10 (data not shown). S100A10 and annexin II were detected with mouse monoclonal antibodies from BD Biosciences. Secondary antibodies were sheep anti-mouse (Amersham Biosciences) and goat anti-rabbit (BD Biosciences) antibodies conjugated to horseradish peroxidase.

Capillary-like Tube Formation—In vitro angiogenesis was assessed by capillary-like tube formation by HCEC grown in a basement membrane matrix, MatrigelTM (BD Biosciences), as previously described (30, 31). 24-well plates were coated with 300 µl of unpolymerized growth factor-reduced Matrigel (5–7 mg protein/ml) and allowed to polymerize for 30 min at 37 °C. HCEC (40,000 cells) were suspended in 500 µl of DMEM alone or DMEM containing different concentrations of S100A4 (10–100 nM). In some experiments, HCEC were preincubated with 10 mM 6-aminocaproic acid (6-ACA) for 10 min prior to the addition of S100A4. Capillary-like tube formation was analyzed after 24 h using an Olympus 1X50 microscope.

Immunofluorescence Detection—Co-localization of S100A4 with annexin II in HCEC was studied by double immunocytochemistry. The cells were cultured on fibronectin-coated glass coverslips until 80% confluence. The cells were then exposed to 1 µM S100A4 in serum-free DMEM for 1 h at 37 °C, washed five times, and then fixed with 4% paraformaldehyde for 10 min. Following fixation, the cells were permeabilized with 0.1% Triton X-100 for 10 min and subsequently blocked for nonspecific sites using 4% goat serum for 1 h. The cells were then incubated with anti-S100A4 antibody (1:100 dilution, 1 h, room temperature), washed, and exposed to fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (Invitrogen, 1:500, 1 h). After blocking overnight with 4% goat serum, the cells were incubated with the anti-annexin II antibody (1:100; 1 h, room temperature), washed, and incubated with the secondary Alexa 568-labeled anti-mouse IgG antibody (Invitrogen, 1:500, 1 h). Omission of primary antibodies resulted in no staining. No cross-reactivity between the primary and non-corresponding secondary antibodies was detected. Imaging of the slides was performed using a confocal laser microscope Zeiss LSM 410 (Carl Zeiss).

Immunoprecipitation—S100A4/annexin II complex formation was performed at equimolar concentrations of S100A4 and annexin II in 20 µl of Tris-buffered saline containing 2 mM CaCl2 or 2 mM EGTA at 37 °C. The resulting complexes were analyzed by immunoprecipitation. The reaction mixture was diluted 2-fold with Tris-buffered saline and incubated for 1 h with 10 µg/ml anti-S100A4 or anti-annexin II antibody and then precipitated with protein G-Sepharose beads (Amersham Biosciences) for 1 h at room temperature. The beads were washed with Tris-buffered saline and used for Western blot analysis with anti-annexin II or anti-S100A4 antibody, respectively.

For the detection of S100A4/annexin II complex in HCEC culture, cells were washed three times with Hanks' balanced salt solution and treated with 1 µM S100A4 for 1 h at 37 °C in serum-free medium or left untreated as controls. After the addition of EGTA in a final concentration of 10 mM, the extraction of membrane-bound proteins was performed at room temperature for 20 min. Protease inhibitor mixture (Roche Applied Science) was added to extracts, and they were centrifuged to remove cell debris. Supernatants were concentrated on YM-3 Centriplus (Amicon) devices, and 1 µg of total protein was used for immunoprecipitation with anti-annexin II antibody followed by Western blot analysis with anti-S100A4 antibody as described above.

Peptide Synthesis—The peptide, ANXA2-N13, corresponding to the first 13 amino acids of human annexin II, STVHEILSKLSLE-amide, was synthesized by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy and purified by reverse phase high pressure liquid chromatography. The structure of the peptide was confirmed by mass spectrometry analysis.

NMR Studies—NMR studies of the interactions between the ANXA2-N13 peptide and S100A4 protein were performed using 15N-enriched protein that was produced in Escherichia coli using M9 medium containing 15N ammonium sulfate as a sole nitrogen source. The protein product was purified as described above for the non-labeled S100A4. 1H-15N HSQC NMR spectra (32) were acquired at 303K using a Bruker Avance 500 spectrometer. The sample containing 0.3 mM S100A4 in 20 mM Bis-Tris, pH 6.5, 150 mM NaCl, 2 mM CaCl2 and 10 mM dithiothreitol was measured before and after the addition of the peptide to the final concentration of 0.6 mM. The data processing and analysis were performed using the Bruker XWINNMR software.

Plasminogen Activation Assay—The kinetics of t-PA-mediated plasminogen activation was determined by measuring the amidolytic activity of plasmin formed during the activation of plasminogen as previously described (28, 33) with some modifications. All of the experiments were done in a 100-µl volume in 96-well plates in triplicates. Gluplasminogen (Calbiochem) at the concentration of 100 or 50 nM was preincubated at 25 °C for 1 h in the presence or absence of 1 µM recombinant S100A4, S100A10, or annexin II in a buffer consisting of 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, and 5 mM CaCl2 or 5 mM EGTA. In some experiments, 10 mM 6-ACA was preincubated together with proteins. The reaction was initiated by the addition of 1.6 or 5 nM t-PA (Calbiochem) and 125 µM chromogenic plasmin substrate, Chromozym PL (Roche Applied Science). The reactions were monitored at 405 nm for 1.5 h in a SPECTRAmax PLUS384 microplate spectrophotometer (Molecular Devices) under the control of the SOFTmax PRO kinetic program (Molecular Devices). The initial rates of plasmin generation during the first 10–15 min were calculated using linear regression analysis of plots of A405 nm versus time2 as described before (28, 33). The initial rates of plasmin generation were reported in units (K) of A405 nm/min2 x 103. In the absence of t-PA, plasmin generation did not take place regardless of the presence of S100A4, S100A10, or annexin II (data not shown). The titration data were analyzed with the four-parameter logistic equation, K = (A - D)/(1 + (x/C)B)D.If A (asymptotic minimum) = 0, this equation is converted to the Michaelis-Menten equation, K = D/(1 + (C/[S])B), where [S] is substrate concentration, D = Vmax, C = Km, and B is the Hill coefficient.

Plasminogen Activation on the Surface of Endothelial Cells— HCEC (105 cells/well) were seeded in 24-well plates and allowed to grow until confluent. The cells were washed twice in DMEM and then exposed to S100A4 (25–200 nM), S100A4 (100 nM) preincubated with ANXA2-N13 peptide (10 µM) for 1 h, placental growth factor (20–100 nM; R&D Systems), insulin-like growth factor I (75–200 nM; R&D Systems), or basic fibroblast growth factor (1–200 nM; R&D Systems) for 15 min at 37 °C. The control cells were incubated in DMEM. Each well was then supplemented with t-PA (10 nM) and incubated for 1 h. The cells were washed twice with phenol red-free DMEM and plasminogen (100 nM), and 25 µM plasmin fluorogenic substrate (D-Ala-Leu-Lys-7-amido-4-methylcoumarin, Sigma) were added. Plasmin formation was monitored at 360-nm excitation/460-nm emission setting in a cytofluorimeter plate reader (Bio-Tek FL600) at 5-min intervals for 6 h at 37 °C. The basal fluorescence obtained at 0 min was subtracted from each time point.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
S100A4 Induces Capillary-like Tube Formation in Primary Human Cerebromicrovascular Endothelial Cells—Angiogenic properties of S100A4 were demonstrated in vitro using HCEC grown in a mixture of basement membrane components, Matrigel. HCEC used in this study have been previously shown to form capillary-like tube networks when stimulated with a variety of angiogenic factors (30, 31). HCEC incubated in DMEM exhibited a typical spindle-shaped morphology (Fig. 1a). HCEC exposed to S100A4 formed an intricate capillary-like tube network (Fig. 1b). This effect was concentration-dependent with a minimal effective S100A4 concentration of 10 nM (data not shown). The S100A4-induced capillary-like tube formation was prevented by incubating HCEC with 6-ACA prior to the addition of S100A4 (Fig. 1b). The anti-angiogenic effect of 6-ACA has been previously described (34) to be related to its ability to suppress plasmin formation catalyzed by t-PA upon plasminogen and t-PA binding to annexin II or annexin II heterotetramer on the endothelial surface (33, 35). Because 6-ACA suppressed the angiogenic response of HCEC to S100A4, we have hypothesized that the membrane annexin II may be directly involved in S100A4-induced angiogenesis.

S100A4 Induces Translocation of Annexin II and Co-localizes with It on the Surface of HCEC—Cellular localization of annexin II and the exogenously added S100A4 in HCEC was analyzed by double immunocytochemistry staining using monoclonal anti-annexin II and polyclonal anti-S100A4 antibodies. Among the primary endothelial cells cultures derived from various tissues, cerebromicrovascular cells showed the highest level of annexin II expression (36). In control HCEC, both endogenous annexin II (Fig. 2A) and S100A4 (Fig. 2B) exhibited diffuse cytoplasmic and nuclear distribution, which is consistent with previously published results (37, 38) The merged image of endogenous S100A4 and annexin II staining (Fig. 2C) showed similar nuclear and cytoplasmic co-localization of both proteins. In HCEC exposed to the exogenously added S100A4 for 1 h, annexin II redistribution from the cytoplasm to the plasma membrane (Fig. 2D) coincided with HCEC morphological conversion into "angiogenic" phenotype. Moreover, annexin II (Fig. 2E) and S100A4 (Fig. 2F) immunostaining co-localized on the HCEC plasma membrane (Fig. 2F), suggesting interaction of exogenous S100A4 with annexin II.



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  		FIG. 1.
S100A4 induces capillary-like tube formation in primary human cerebromicrovascular endothelial cells. a, HCEC (4 x 104 cells/well) were seeded into Matrigel-precoated wells and cultured in serum-free DMEM. b, formation of a complex capillary tube network by HCEC exposed to S100A4 (10 nM). c, pretreatment with 10 mM 6-ACA suppresses the angiogenic activity induced by S100A4 (10 nM) in HCEC. The experiments were repeated three times with similar results.

 
Binding of Recombinant S100A4 to Annexin II and Identification of the Binding Region in the Annexin II Molecule—To further confirm that S100A4 binds with annexin II, we studied the interaction between these two proteins in vitro. Both annexin II and S100A4 were cloned in bacterial vectors as glutathione S-transferase fusions and purified as individual proteins by using glutathione-Sepharose and thrombin proteolysis for elution. The interaction of S100A4 and annexin II was evaluated by immune co-precipitation assay. The results of these experiments shown in Fig. 3 demonstrate that S100A4 and annexin II form a complex that can be precipitated with either anti-S100A4 or anti-annexin II antibodies and detected by Western blot with anti-annexin II (Fig. 3A) or anti-S100A4 (Fig. 3B) antibodies, respectively. Surprisingly, the strongest interaction of the two proteins was observed in the absence of Ca2+ (EGTA buffer), whereas the addition of Ca2+ reduced the complex formation (Fig. 3, A and B, lanes 3 and 4).



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  		FIG. 2.
Co-localization of S100A4 and annexin II in HCEC. Immunostaining for endogenous S100A4 (green fluorescence) (A) and annexin II (red fluorescence) (B) in control HCEC demonstrates nuclear and diffuse cytoplasmic staining. C, the merged image of endogenous S100A4 and annexin II staining shows some nuclear and cytoplasmic co-localization. Immunostaining for S100A4 (green fluorescence) (D) and annexin II (red fluorescence) (E) in HCEC exposed to 1 µM S100A4 for 1 h and then washed demonstrates substantial alterations in localization of both proteins. F, the merged image of S100A4 and annexin II staining shows co-localization on the cell surface (yellow). The images are representative of at least five separate experiments yielding similar results.

 
The N-terminal region of the annexin II molecule is responsible for its interactions with binding partners, such as S100A10 and t-PA (39, 40) and contains sites for Tyr and Ser phosphorylation (41). To examine whether the N-terminal region is involved in the annexin II binding to S100A4, we have produced two truncated forms of annexin II: 1) A2-del15, which was lacking the first 15 amino acids in the N terminus including t-PA and S100A10-binding sites, and 2) A2-del26 with an additional deletion of 11 amino acids including Tyr23 and Ser25 that are known to be phosphorylation sites for protein tyrosine kinase pp60srk (srk oncogene) and PKC, respectively (41). The immune co-precipitation assay showed reduced binding of the A2-del15 mutant to S100A4 and no binding in the case of the A2-del26 mutant (Fig. 3C), indicating that the N-terminal part of annexin II contains the S100A4-binding site. On the other hand, S100A10 did not bind either mutant form of annexin II, which is consistent with the previous report that the S100A10-binding site is localized within the first 13 amino acids (39). Therefore, the results of this experiment indicate that S100A4 and S100A10 interact with the similar region of annexin II. However, it appears that the S100A4-binding region is localized distal to the S100A10-binding site and overlaps with the phosphorylation sites. It has been previously reported that the S100A4-binding sequences of several S100A4 targets, including p53 (16), liprin {beta}-1 (17), or the heavy chain of non-muscle myosin (18), contain sites for PKC or CKII phosphorylation. Because annexin II is one of the major targets for PKC phosphorylation (41), S100A4 binding to its phosphorylation site might represent a regulatory pathway for annexin II functions.

To compare the annexin II-binding potencies of S100A4 and S100A10, equimolar mixtures of S100A4 and S100A10 with annexin II were incubated and the complex formation was analyzed. Whereas S100A4 and S100A10 were each separately capable of forming complexes with annexin II (Fig. 3D, lanes 3 and 4), only S100A4/annexin II complex was detected when the mixture of S100A4 and S100A10 was incubated with annexin II (Fig. 3D, lane 5), suggesting that S100A4 had higher affinity for annexin II than S100A10. These results provided additional experimental evidence to the assumption that S100A4 interacts with the S100A10-binding region of annexin II.



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  		FIG. 3.
Interaction of recombinant S100A4 and annexin II proteins. A, immunoprecipitation (IP) of annexin II/S100A4 complex by anti-S100A4 antibody and Western blot analysis with anti-annexin II antibody. Lanes 1 and 2, control proteins without precipitation; lanes 3 and 4, IP by anti-S100A4 antibody. IB, immunoblotting. B, immunoprecipitation of annexin II/S100A4 complex by anti-annexin II antibody and Western blot analysis with anti-S100A4 antibody. Lanes 1 and 2, control proteins without precipitation; lanes 3 and 4, IP by anti-annexin II antibody. C, binding of truncated annexin II mutants, A2-del15 and A2-del26, to S100A4 (upper row) or S100A10 (lower row). Complexes were immunoprecipitated by anti-S100A4 or anti-S100A10 antibody, respectively, followed by Western blot analysis with anti-annexin II antibody. Lanes 1, 3, and 5, control proteins without precipitation; lanes 2, 4, and 6, IP by anti-S100A4 or anti-S100A10 antibody. Full-size annexin II was used in lanes 1 and 2, A2-del26 was used in lanes 3 and 4, and A2-del15 was used in lanes 5 and 6. D, competition of S100A4 and S100A10 for annexin II complex formation in the presence of EGTA. Immunoprecipitation by anti-annexin II antibody and Western blot analysis with anti-S100A4 or anti-S100A10 antibody. Lanes 1 and 2, control proteins without precipitation. Lanes 3, 4, and 5, IP by anti-annexin II antibody. E, immunoprecipitation of S100A4/annexin II complex from HCEC culture treated with 10 mM EGTA. IP was done with anti-annexin II antibody, and Western blot analysis was done with anti-S100A4 antibody. Lane 1, control S100A4 protein without precipitation. Lanes 2 and 3, IP by anti-annexin II antibody from control (lane 2) and S100A4-treated cells (lane 3). Equal intensities of protein G bands reflect equal loading of protein G-Sepharose beads on gel. IB, immunoblotting.

 
Detection of Annexin II/S100A4 Complex in HCEC Culture—To confirm that the interaction of the extracellular S100A4 and membrane annexin II takes place upon S100A4-induced activation of endothelial cells, we have carried out the immune co-precipitation assay using HCEC culture. Consistent with the previously reported data (36, 42), in the untreated endothelial cells, annexin II was well detected in the cell lysates, as well as in the conditioned media of the cells treated with EGTA, which is known to release the membrane form of annexin II in the medium (42). At the same time, no S100A4 was detected in the conditioned medium with or without EGTA treatment, indicating that this protein is not secreted by HCEC. However, when HCEC were treated with 1 µM S100A4, a strong co-precipitation of annexin II with S100A4 was observed in the conditioned medium of the EGTA-treated cells (Fig. 3E), whereas no co-precipitation was observed in the cell lysates (data not shown). These results provide additional evidence that extracellular S100A4 interacts with the membrane form of annexin II during the endothelial cell activation.

Analysis of the Interactions between 15N-S100A4 and N-terminal Fragment of Annexin II by 1H-15N NMR—To further confirm that S100A4 interacts with the N-terminal region of annexin II, we synthesized a peptide, STVHEILSKLSLE-amide, corresponding to the first 13 amino acid sequence of annexin II, which was previously shown to contain the binding site for S100A10 (39, 43). The Cys8 residue of this peptide was replaced with Ser to avoid oxidation prior to complex formation (39). The binding of the synthetic peptide to S100A4 was examined by analyzing chemical shifts on two-dimensional 1H-15N HSQC NMR spectrum (32) of the protein after adding the peptide (44, 45). The shifts were considered essential if they were at least twice larger than the accuracy of the measurement (0.02 ppm for 1H dimension and 0.3 ppm for 15N dimension). As shown in Fig. 4, A and B, the 1H-15N HSQC spectrum of the 15N-labeled S100A4 displays essential shifts of resonance signals upon the addition of excess amounts of the annexin II fragment, indicating the direct binding between S100A4 and annexin II N terminus. Only a subpopulation of the amide signals representing ~25% total S100A4 sequence is affected (marked with asterisk in Fig. 4A), indicating binding specificity. Approximately 10% signals in the S100A4 spectrum, showing particularly large shifts (>0.1 ppm for 1H dimension and/or 1 ppm for 15N dimension), correspond to the amino acids most likely involved in the binding of the peptide (marked *+ in the Fig. 4A).

S100A4 Accelerates t-PA-mediated Conversion of Plasminogen to Plasmin—Having demonstrated that 6-ACA, which is known to inhibit plasminogen activation, suppressed S100A4-induced angiogenesis, we examined whether S100A4 could accelerate t-PA-dependent conversion of plasminogen to plasmin. The kinetics of t-PA-mediated plasminogen processing were analyzed by measuring the amidolytic activity of the produced plasmin. The results of this experiment (Fig. 5A) revealed that the addition of S100A4 produced a significant increase in the plasminogen conversion rate, which was more pronounced in the presence of Ca2+. No activation of plasminogen to plasmin took place in the absence of t-PA (S100A4 plus plasminogen, data not shown), whereas the basal t-PA activity (plasminogen plus t-PA) was Ca2+-independent (data not shown). The stimulation of the plasminogen conversion by S100A4 was concentration-dependent with the half-maximal effect at 2.4 µM (Fig. 5B).



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  		FIG. 4.
Binding of the ANXA2-N13 peptide to S100A4. Two-dimensional 1H-15N HSQC spectra of 15N-labeled S100A4 in the absence (A) and presence (B) of the unlabeled peptide. Approximately 25% of all S100A4 amide signals display significant changes in chemical shifts (at least 0.04 ppm in 1H dimension or/and 0.5 ppm in the 15N dimension), 10% signals display particularly large changes (more than 0.1 ppm for 1H dimension, and/or 1 ppm for 15N dimension). These signals are marked asterisk and *+, respectively, in the spectrum of free S100A4 (A).

 
The stimulatory effects of S100A4 and S100A10 were compared in the next experiment. The kinetic constants of plasmin generation for the two proteins were calculated and are shown in Fig. 5C. These results indicate that S100A4 accelerated the conversion of plasminogen to plasmin by 15-fold in the presence of Ca2+ and by 11-fold in the presence of EGTA. The effect of S100A10 was comparable to that of S100A4 in the presence of Ca2+. However, the activity of S100A10 was Ca2+-independent, which is consistent with the previous report (28). In contrast to all other S100 proteins, S100A10 does not bind Ca2+ because of mutations in both Ca2+-binding domains (46).

The kinetic parameters of t-PA-mediated plasminogen conversion were evaluated in the presence and absence of S100A4 as a function of plasminogen concentrations ranging from 3 nM to 3 µM (Fig. 5D). The titration curves demonstrated that S100A4 significantly reduced Km of the t-PA-mediated plasminogen conversion from 429 to 59 nM but did not substantially affect the Vmax of the reaction, conferring a 7.6-fold increase in the catalytic efficiency (Vmax/Km) of t-PA in the presence of EGTA. The analogous titration curves were constructed for S100A10 (data not shown), and the kinetic parameters for the two proteins are summarized in Fig. 5E. S100A10 did not also change the Vmax of the reaction but decreased the Km down to 47 nM, increasing the catalytic efficiency of t-PA by 9.4-fold.



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  		FIG. 5.
Stimulation of t-PA-dependent plasminogen activation by S100A4. A, plasminogen was preincubated in the presence (2 and 3) or absence (1)of1 µM S100A4 in Ca2+-(2) or EGTA-containing buffer (3) after the addition of t-PA and chromogenic plasmin substrate amidolytic activity of plasmin was monitored at 405 nm. B, concentration dependence of the stimulation of t-PA-dependent plasminogen activation by S100A4 in the presence of Ca2+. The rates of plasmin generation were calculated as depicted under "Materials and Methods." Fitting by four-parameter logistic equation. The data represent the mean ± S.D. (n = 3; R2 = 0.99). C, comparison of plasminogen activation by 1 µM S100A4 or S100A10 in the presence of Ca2+ or EGTA. The data represent the mean ± S.D. (n = 9; *, p < 0.001 by Student's t test). D, titration of plasminogen in the presence of t-PA alone (t-PA) or t-PA and 2.5 µM S100A4 (S100A4) in EGTA-containing buffer. The data were analyzed by fitting a four-parameter logistic equation from which parameters Km and Vmax were estimated. The data represent the mean ± S.D. (n = 3; R2 = 0.98). E, kinetic parameters for S100A4- and S100A10-accelerated t-PA-dependent plasminogen activation in the presence of EGTA.

 
C-terminal Lysine(s) of S100A4 Are Important for Plasminogen Activation—The mechanism of plasminogen conversion involves the binding of C-terminal lysines in plasminogen-binding proteins to lysine-binding sites in the kringle domains of plasminogen (47). Because both S100A4 and S100A10 contain C-terminal lysines, we investigated the role of these residues in plasminogen activation using a C-terminal lysine analogue, 6-ACA. The addition of 6-ACA completely inhibited S100A4-dependent activation of plasminogen both in the presence of Ca2+ (Fig. 6A) and EGTA (data not shown), confirming the involvement of lysine-binding sites. 6-ACA also completely inhibited S100A10-dependent activation of plasminogen in the presence of Ca2+ (Fig. 6B) and EGTA (data not shown).

The role of C-terminal lysine(s) of S100A10 was previously confirmed in mutagenesis experiments. The mutant S100A10 with its two C-terminal lysines deleted and ending with glutamine retained only 15% of its activity compared with the wild-type protein (28). We have produced a mutant of the S100A4 protein with two C-terminal lysines mutated to leucines and found that this mutant completely lost the ability to activate t-PA-mediated plasmin formation (Fig. 6B). These experiments demonstrate that C-terminal lysines of S100A4 are necessary for its effect on plasminogen activation.



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  		FIG. 6.
C-terminal lysine(s) of S100A4 are important for plasminogen activation. A, plasminogen activation in Ca2+ -containing buffer in the absence (1) or presence of S100A4 (2) or S100A4 and 6-ACA (3). B, 6-ACA inhibits plasminogen activation accelerated by both S100A4 and S100A10. S100A4 mutant (S100A4-KK/LL) with two C-terminal lysines mutated to leucines completely lost the activity. The data represent the mean ± S.D. (n = 6; **, p < 0.001 by Student's t test).

 
S100A4 Cooperates with Annexin II in Plasminogen Activation—Since S100A4 was found to form a complex with annexin II, we next investigated the combined effect of S100A4 and annexin II on plasminogen conversion. Preincubation of S100A4 with annexin II further increased the reaction rate compared with that observed in the case of either S100A4 or annexin II alone. This effect was well pronounced only in the presence of EGTA (Fig. 7A), conditions that are favorable for the formation of the S100A4/annexin II complex (see Fig. 3, A and B). At these conditions, the effect of S100A4/annexin II complex on plasmin formation (K = 0.32 ± 0.06) was rather synergistic than additive compared with that of the individual proteins (K = 0.09 ± 0.01 and 0.15 ± 0.01 for S100A4 and annexin II, respectively). These data demonstrate that S100A4/annexin II complex is more potent in acceleration of plasmin generation than S100A4 or annexin II alone. Similar results were previously reported for S100A10/annexin II tetramer, showing that the complex is more active in plasminogen activation than the individual proteins (28). In the presence of Ca2+, the addition of annexin II did not influence the activity of S100A4, which is consistent with the decreased efficiency of complex formation in this environment.

The titration of plasminogen in the presence of S100A4/annexin II complex (EGTA conditions) revealed further increase in the t-PA activity (11.3-fold compared with 7.6-fold observed in the case of S100A4 alone). The titration of plasminogen in the presence of S100A10/annexin II complex also demonstrated an increase in the t-PA activity (10.7-fold compared with 9.4-fold observed for S100A10 alone), but it was less pronounced than in the case of S100A4/annexin II complex. The kinetic parameters of these reactions are summarized in Fig. 7B.

S100A4 Accelerates t-PA-mediated Plasmin Formation on the Surface of HCEC—Based on the ability of S100A4 to induce angiogenesis in HCEC cultures and accelerate t-PA-dependent plasminogen activation in solution, we next investigated the ability of S100A4 to promote plasminogen activation on the surface of HCEC. Extracellular S100A4 induced a concentration-dependent acceleration of plasmin production by HCEC with a minimum effective concentration of 50 nM (Fig. 8A). The plasmin production by HCEC was not induced by other angiogenic growth factors, such as placental growth factor (20–100 nM), insulin-like growth factor I (75–200 nM), or basic fibroblast growth factor (1–200 nM) (data not shown).



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  		FIG. 7.
S100A4 cooperates with annexin II in plasminogen activation. A, plasminogen was preincubated either alone or in the presence of S100A4 or annexin II or both in Ca2+- or EGTA-containing buffer. After the addition of t-PA and chromogenic substrate, the amidolytic activity of plasmin was monitored at 405 nm. The rates of plasmin generation were calculated as depicted under "Materials and Methods." The data represent the mean ± S.D. (n = 6). B, kinetic parameters of t-PA-dependent plasminogen activation by S100A4/annexin II and S100A10/annexin II complexes. Km and Vmax values were evaluated from plasminogen titration curves in the presence of EGTA.

 
The S100A4-induced plasmin production in HCEC cultures was completely inhibited by the annexin II N-terminal (1–13) peptide (Fig. 8B), confirming that it was S100A4 binding to the cell surface annexin II that induced plasminogen activation concomitantly with endothelial angiogenic shift.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A high value of S100A4 as a prognostic marker in oncology (1, 2, 5–11) and some other diseases associated with activated endothelium (12–14) has been well demonstrated in several clinical studies. The pathogenic role of this protein in oncology has been also confirmed (1–3, 12, 27). However, S100A4 has not been broadly considered as a target for drug discovery, mainly due to the lack of mechanistic understanding of its interactions with endothelial tissues. In this work, we present evidence that annexin II is the endothelial receptor for S100A4 and that their interaction triggers the functional activity directly related to pathological properties of S100A4.

Interactions between some S100 protein family members and annexins have been previously reported including heterotetramer formation between annexin I and S100A11 (48), annexin II and S100A10 (49), annexin II and S100A6 (50), annexin VI and S100A1 or S100B (51), annexin VI and S100A6 (50), and annexin XI and S100A6 (52). However, the possibility of direct interaction of S100A4 with one of the annexins and specifically with annexin II has not been previously considered.

Annexins are intracellular proteins located on the cytosolic face of the plasma membrane. Among annexins, annexin I in neutrophils and activated endothelial cells, annexin V in the activated platelets and endothelial cells, and annexin II in activated endothelial and some tumor cells are exposed on the extracellular membrane surface (41). Annexin II alone or in a heterotetrameric complex with S100A10 is the only annexin known to directly control endothelial functions via t-PA-mediated plasmin formation (33, 35). S100A10 binding to plasminogen and its activation is mediated by two C-terminal lysine residues of S100A10 (28, 53). Among S100 proteins, according to our analysis, only S100A4, S100A13, and S100A10 contain two C-terminal lysines (S100A5, S100Z, and S100P contain one C-terminal lysine). Furthermore, the C-terminal sequences of these three proteins are highly homologous (KQPRKK-COOH in S100A4, LKIRKK-COOH in S100A13, and KQKGKK-COOH in S100A10), suggesting that they may share similar functional ability to activate t-PA-mediated plasminogen processing. Based on these considerations, we have hypothesized that, similarly to S100A10, S100A4 interacts with annexin II and plasminogen.

The immune co-precipitations in solution and in conditioned medium, cellular co-localization, and NMR studies provided strong evidence for the S100A4 and annexin II binding. The observations that the S100A4-induced angiogenesis was inhibited by C-terminal lysine analogue, 6-ACA, and the fact that the N-terminal fragment of annexin II inhibited the S100A4-induced plasmin formation in HCEC and complete disappearance of this activity upon mutation of the C-terminal lysine residues in S100A4 confirmed the relevance of S100A4 and annexin II binding to the endothelial functions. Furthermore, the experiments with the truncated annexin II fragments and NMR analysis of interaction of S100A4 with the N-terminal (1–13) synthetic fragment of annexin II have suggested that both S100A4 and S100A10 interact with the similar binding site of annexin II. Combined with the kinetic analysis of the S100A4- and S100A10-accelerated plasminogen activation, these results confirmed the functional similarity between these two proteins.



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  		FIG. 8.
Effect of S100A4 on t-PA-mediated plasminogen activation on the surface of HCEC.. A, plasmin formation by HCEC in control culture (t-PA) or treated with increasing concentrations (50–200 nM) of S100A4. B, inhibition of S100A4-induced plasmin formation by 70 µM ANXA2-N13 peptide. The data represent the mean ± S.D. (n = 3).

 



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  		FIG. 9.
Scheme for S100A4-induced angiogenesis. S100A4 secreted by tumor cells induces translocation of annexin II on the cell surface of endothelial cells where S100A4/annexin II complex locally increases the t-PA-mediated activation of plasminogen to plasmin. Active plasmin then activates pro-MMPs, and active MMPs and plasmin induce extracellular matrix remodeling, facilitating angiogenesis. EC, endothelial cell; A2, annexin II; Plg, plasminogen; Pl, plasmin; ECM, extracellular matrix.

 
The previously reported co-crystal analysis (39) and mutagenesis studies on the interaction between the N-terminal region of annexin II and S100A10 protein (43) have revealed that the binding interface of the annexin II/S100A10 complex is highly hydrophobic and involves side chains of Val3, Ile6, Leu7, Leu10 in the N-terminal region of annexin II. The analysis of the x-ray structure of the complex has shown that the above amino acids interact with the hydrophobic and acidic residues of S100A10 in the binding pocket formed by residues located in the loop L2 and helices 3 and 4 of one S100A10 chain and helix 1 of the other chain in the S100A10 homodimer, including Glu5, Glu9, Phe38, Phe41, Leu78, Tyr85, and Met90. The amino acids in equivalent positions in S100A4 have a similar character (Glu6, Asp10, Leu42, Phe45, Ile82, Phe89, and Pro94) that further supports the similarity between S100A10 and S100A4 interactions with annexin II.

The data reported here identify for the first time annexin II as the endothelial receptor of S100A4 and link the interaction between the two proteins to the t-PA-mediated plasminogen activation. Plasmin formed during this reaction is the major component of tissue fibrinolysis process that is closely linked to the known pathogenic roles of S100A4, including angiogenesis, metastasis, and inflammation. The ability of S100A4, secreted by circulating cancer cells or activated fibroblasts or some other cells, to mimic S100A10 in annexin II/S100A10 plasminogen co-receptor on the endothelial surface provides mechanistic insight in the pathological events induced by S100A4.

The invasive properties of cancer cells strongly correlate with overexpression of matrix metalloproteinases (MMPs) and S100A4 (54). Down-regulation of S100A4 with ribozyme reduced the activity of several MMPs, including MMP2, as well as cell motility and invasiveness (55, 56). On the other hand, treatment of tumor or endothelial cells with S100A4 increased their proteolytic activity, in particular, the activity of MMP13, and invasive growth (24, 27). The role of plasmin in the activation of several MMPs, including MMP2 and MMP13, is well known (57, 58). From the collective evidence presented in this work, the following mechanistic cascade (Fig. 9) has been proposed. S100A4 is secreted by tumor cells and induces translocation of annexin II on the cell surface of endothelial cells where formation of S100A4/annexin II complex takes place. Endothelial cells constitutively secrete t-PA, and S100A4/annexin II complex on the surface of endothelial cells locally increases the t-PA-mediated plasmin production from plasminogen. Active plasmin then activates pro-MMPs, and active MMPs and plasmin induce extracellular matrix remodeling facilitating angiogenesis and tumor invasion. The role of S100A4 in plasmin activation thus explains the importance of S100A4 in tumor-related angiogenesis. The currently unresolved question relates to the described plasmin conversion to angiostatins upon S–S bond reduction and autoproteolysis or cleavage by several MMPs. This process probably serves as a regulatory feedback loop that controls the production of plasmin and prevents uncontrolled tissue proteolysis.

In conclusion, S100A4/annexin II interaction on the surface of activated endothelium represents a new and important target for the discovery of novel anti-cancer and anti-inflammatory therapies. Given that high circulatory levels of S100A4 serve as a poor prognosis indicator in cancer patients (22), if such drug candidates become available, circulatory S100A4 could be used as a surrogate marker in clinical studies to enable monitoring of the treatment efficacy and robust patient selection criteria.


   FOOTNOTES
 
* 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

|| To whom correspondence should be addressed: Supratek Pharma Inc., 215 Blvd. Bouchard, Suite 1315, Dorval, Quebec H9S 1A9, Canada. Tel.: 514-422-9191; Fax: 514-422-9410; E-mail: Valery.Alakhov{at}Supratek.com.

1 The abbreviations used are: PKC, protein kinase C; HCEC, human cerebromicrovascular endothelial cells; 6-ACA, 6-aminocaproic acid; HSQC, heteronuclear single quantum correlation; ANXA2, annexin II; NMR, nuclear magnetic resonance; MMPs, matrix metalloproteinases; t-PA, tissue-type plasminogen activator; DMEM, Dulbecco's modified Eagle's medium. Back



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