Original Paper

Oncogene (2004) 23, 5487–5495. doi:10.1038/sj.onc.1207720 Published online 3 May 2004

Extracellular S100A4(mts1) stimulates invasive growth of mouse endothelial cells and modulates MMP-13 matrix metalloproteinase activity

Birgitte Schmidt-Hansen1, Dorte Örnås1, Mariam Grigorian1, Jörg Klingelhöfer1, Eugene Tulchinsky1,2, Eugene Lukanidin1 and Noona Ambartsumian1

  1. 1Department of Molecular Cancer Biology, Institute for Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
  2. 2Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester LE 7 LX, UK

Correspondence: N Ambartsumian, E-mail: na@cancer.dk

Received 4 September 2003; Revised 10 March 2004; Accepted 11 March 2004; Published online 3 May 2004.

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Abstract

S100A4(mts1) protein expression has been strongly associated with metastatic tumor progression. It has been suggested as a prognostic marker for a number of human cancers. It is proposed that extracellular S100A4 accelerates cancer progression by stimulating the motility of endothelial cells, thereby promoting angiogenesis. Here we show that in 3D culture mouse endothelial cells (SVEC 4-10) respond to recombinant S100A4 by stimulating invasive growth of capillary-like structures. The outgrowth is not dependent on the stimulation of cell proliferation, but rather correlates with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. beta-Casein zymography demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis-stimulating activity of S100A4(mts1).

Keywords:

metastasis-promoting, S100A4, extracellular activity, MMP-13 induction, angiogenesis

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Introduction

Understanding the mechanisms of metastatic spread of tumors is one of the major challenges in cancer research. Metastasis is a complex process that involves both cancer cells and cells of the surrounding stroma (Fidler, 2003). A number of molecules are produced by both tumor and stroma cells to determine the outcome of this process.

Degradation and penetration of the extracellular matrix (ECM) is a hallmark of tumor invasion and metastasis (Hanahan and Weinberg, 2000). Concerted action of multiple proteinases and their inhibitors, particularly the matrix metalloproteinases (MMP) and the plasminogen activator (PA) system, are required to accomplish the invasion and spread of tumor cells to their new location (Duffy et al., 2000).

Penetration of ECM is an essential step during the angiogenesis and formation of new blood vessels. Angiogenesis is crucial for cancer progression, because it supplies the proliferating tumor cells with necessary nutrients and oxygen and at the same time provides escape routes for invading tumor cells. Proteinases, such as MMPs and PA, promote angiogenesis both by degradation of ECM and by liberating factors that promote or maintain the angiogenic phenotype (Pepper, 2001).

It has been demonstrated in a number of studies that the S100A4(mts1) gene product is involved in the promotion of metastases. The S100A4 gene was originally isolated as a gene differentially expressed in highly metastatic mouse mammary adenocarcinoma cells (Ebralidze et al., 1989). Introduction of the S100A4 gene into nonmetastatic tumor cell lines and suppression of its activity in metastatic ones proved its involvement in metastasis formation (Grigorian et al., 1996; Maelandsmo et al., 1996; Takenaga et al., 1997; Lloyd et al., 1998; Uozumi et al., 2000). Stimulation of tumor metastasis was demonstrated in two transgenic mouse models with overexpression of the S100A4 gene (Ambartsumian et al., 1996; Davies et al., 1996). In a number of human cancers of different origin, the enhanced expression of S100A4 was associated with poor prognosis (Platt-Higgins et al., 2000; Yonemura et al., 2000; Davies et al., 2002; Rosty et al., 2002).

S100A4 protein belongs to the S100 family of Ca2+-binding proteins. Members of this family are functioning both intra- and extracellularly. Inside a cell they are implicated in a variety of activities such as cell proliferation and differentiation, cytoskeleton dynamics and apoptosis. When released into extracellular space, S100 proteins stimulate neuronal differentiation and astrocyte proliferation, modulate activity of inflammatory cells and stimulate angiogenesis (for a review, see Donato, 2001, 2003; Heizmann et al., 2002). The function of S100A4 in promoting metastases is not completely understood. A number of intracellular interacting partners, such as the heavy chain of nonmuscle myosin (Kriajevska et al., 1994; Ford and Zain, 1995), liprinbeta-1 (Kriajevska et al., 2002), p53 (Grigorian et al., 2001) and recently discovered methionine aminopeptidase (Endo et al., 2002) were described for the S100A4 protein, suggesting its participation in cell motility, adhesion and proliferation. This raised the possibility that S100A4 participates in metastasis by interfering with any of these processes.

When applied extracellularly, S100A4 stimulates neurite outgrowth (Novitskaya et al., 2000) and migration of astrocytic tumor cells by reorganizing the actin cytoskeleton (Belot et al., 2002). Furthermore, as an extracellular cytokine it stimulates angiogenesis, by promoting chemotactic motility of endothelial cells (Ambartsumian et al., 2001). This implies that S100A4 might contribute to tumor progression by stimulating neovascularization of the tumor. An association between S100A4 expression, MMP activity and metastatic potential of human tumor cells has been reported (Andersen et al., 1998; Bjornland et al., 1999).

It was shown that extracellular activity of S100A4 is associated with the oligomeric conformation of the protein (Novitskaya et al., 2000; Ambartsumian et al., 2001). This conformation is detected both in vitro, in the conditioned media from the cells secreting S100A4, and in vivo, in the blood serum.

To clarify the involvement of S100A4 in angiogenesis, we analysed the influence of S100A4 on different stages of angiogenesis with emphasis on its ability to affect the production of matrix degrading enzymes.

Here, we demonstrate that S100A4 stimulates invasive growth and formation of capillary-like structures by mouse microvascular endothelial cells. This feature correlates with the modulation of expression of genes involved in the proteolytic degradation of ECM. Our results indicate that S100A4 stimulates production of MMP-13 from endothelial cells, suggesting a casual relationship between this activity and the angiogenic activity of S100A4.

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Results

Stimulation of capillary-like growth of microvascular endothelial cells

The ability to form capillary-like structures in vitro is a characteristic feature of endothelial cells. The mouse immortalized microvascular endothelial cell line, SVEC 4-10, is capable of forming branching networks on the top of Matrigel (O'Connell and Edidin, 1990). This feature was used for the development of a morphological model of endothelial cell function (Wilasrusmee et al., 2002). To study both the invasive growth and the ability to form capillary-like structures by SVEC 4-10 cells, we modified this model by cultivating the cells in a thick layer of Matrigel in a 3D culture. Preformed aggregates of endothelial cells sealed in a Matrigel form long linear protrusions growing out of the clumps and invading the Matrigel (Figure 1). Invasive capillary-like growth was observed already after 24 h in culture (Figure 1a). Treatment of 3D cultures of SVEC 4-10 cells with the S100A4 oligomeric protein stimulated the formation of protrusions (Figure 1b and e). The addition of the Y75F mutant of S100A4 that is capable of forming dimers only (Novitskaya et al., 2000) did not stimulate growth of the protrusions (Figure 1c and e). The observed capillary-like growth was also stimulated by tumor necrosis factor-alpha (TNF-alpha) and hepatocyte growth factor (HGF), indicating that this assay could be used for analysis of the stimulatory effect of different angiogenic molecules. Anti-S100A4 antibodies inhibited the ability of S100A4 protein to stimulate invasive growth (Figure 1d and e). Invasive growth stimulated by HGF was not blocked by treatment with anti-S100A4 antibodies (Figure 1e). The extent of the outgrowth was quantified by measuring the distance from the edge of the preformed cell clump and the end of the protrusion in 30 locations evenly distributed along the perimeter of the cell aggregate. This enabled us to compare the results of different treatments and made the assay semiquantitative. The outgrowth extent was dose-dependent with a maximum stimulation at a concentration of 0.5 mug/ml (Figure 1e). Taken together, the obtained results indicated that oligomeric conformation of S100A4 is capable of stimulating invasive capillary-like growth of endothelial cells in 3D Matrigel. This outgrowth could be specifically blocked by anti-S100A4 antibodies.

Figure 1.
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Stimulation of invasive capillary-like growth of SVEC 4-10 cells by S100A4. (a) No treatment. (b) Cell clumps treated with 0.5 mug/ml S100A4. (c) Cell clumps treated with 0.5 mug/ml of Y75F mutant of S100A4. (d) Blocking of the S100A4 stimulated outgrowth (0.5 mug/ml) with anti-S100A4 antibodies. (e) Quantification of extent of the outgrowth from clumps treated with different concentrations of S100A4, TGF-alpha, HGF and affinity purified rabbit polyclonal anti-S100A4 antibodies

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S100A4 does not stimulate proliferation of endothelial cells

Enhanced outgrowth in 3D Matrigel culture in response to S100A4 treatment could be due to the increase of the proliferation rate of the cells. We therefore tested the possible influence of extracellular S100A4 on the proliferation of endothelial cells. The relative proliferation rate of SVEC 4-10 cells with and without addition of S100A4 was measured by cell counting of exponentially growing cultures and by MTT assay. The doubling time for SVEC 4-10 cells was not affected by treatment with S100A4. S100A12, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) as well as recombinant myosin fragment used as a negative control did not stimulate proliferation (Figure 2a). The only cytokine that was found to stimulate proliferation of SVEC 4-10 cells under our experimental conditions was HGF. MTT assay confirmed that the proliferation rate was unchanged by treatment of the SVEC 4-10 cells with S100A4 (data not shown).

Figure 2.
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S100A4 has no effect on the proliferation of the SVEC 4-10 cells. (a) Doubling time of the SVEC 4-10 cells in exponentially growing culture treated with S100A4, myosin fragment and different cytokines. (b) The mean rate of 3[H]thymidine incorporation per hour into the SVEC 4-10 cells grown in 3D Matrigel with and without different additives

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Since the Matrigel could affect the proliferation rate of SVEC 4-10 cells in 3D cultures, we also tested the influence of exogenously added S100A4 on the proliferation rate of the cells grown in 3D Matrigel culture. The proliferation rate was determined by measuring the level of 3[H]thymidine incorporation into the cells under 3D culturing conditions. Again, SVEC 4-10 cells proliferated with comparable rates in the presence or in the absence of S100A4 (Figure 2b).

We conclude therefore that S100A4 did not stimulate proliferation of the SVEC 4-10 cells but rather modulated their invasion and motility.

Activation of the NF-kappaB transcription complex in response to S100A4

The direct action of S100A4 on endothelial cells suggests an interaction with the cell surface with consequent activation of signaling pathway, which in turn leads to stimulation of invasion and motility of the cells. A receptor for advanced glycation end products (RAGE), which is expressed in human umbilical vein endothelial cells (HUVEC), was shown as interacting with two members of the S100 protein family, leading to activation of the nuclear factor-kappaB (NF-kappaB) (Hofmann et al., 1999). We tested the possible activation of NF-kappaB in response to S100A4 treatment. Electrophoretic mobility shift assay (EMSA) showed that S100A4 treatment activated the NF-kappaB complex in the SVEC 4-10 cells (Figure 3a). A slight activation was observed after treatment of the cells with S100A12 known to stimulate NF-kappaB complex in HUVEC (Hofmann et al., 1999). NF-kappaB complex in SVEC 4-10 cells was activated after 4 h of S100A4 treatment (Figure 3c). The ubiquitous Sp-1 transcription factor-specific complex was used as an internal control in these experiments (Vallone et al., 1997) (Figure 3a). Supershift assays with anti-NF-kappaB antibodies demonstrated that the induced NF-kappaB complexes were composed of p50 and p65 but not of c-Rel or RelB proteins (Figure 3b). We also detected translocation of the p65 and p50 proteins to the nucleus of S100A4-treated cells (Figure 3d). However, RAGE-specific RNA was not detected in SVEC 4-10 cells (data not shown), raising the possibility that the S100A4-induced signal might be mediated via a receptor other than RAGE.

Figure 3.
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Activation of nuclear factor-kappaB specific complex in SVEC 4-10 cells treated with S100A4. (a) EMSA assay with NF-kappaB-specific oligonucleotides. Sp-1-specific oligonucleotide was used as a loading control. (b) Supershift assay with anti-p50, anti-p65, anti cRel and anti-relB antibodies. (c) Level of the NF-kappaB-specific complex activation (d) Translocation of NF-kappaB transcription complex into the cell nucleus. Immunostaining with anti-p65 and anti-p50 antibodies

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Expression of mRNA of proteases and their inhibitors in cells treated with S100A4

NF-kappaB transcription factor is implicated in the regulation of MMPs (Kim and Koh, 2000; Mengshol et al., 2000; Bond et al., 2001; Vincenti and Brinckerhoff, 2002; Philip and Kundu, 2003). MMP-mediated degradation of ECM is essential for endothelial cells to invade the surrounding tissues.

We therefore studied the transcription of genes involved in the degradation of ECM and that might influence the invasive properties of the SVEC 4-10 cells (Pepper, 2001). The panel of genes we chose for analysis included genes encoding for MMPs and their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMP) as well as the components of plasminogen activator–plasmin system. Northern blot analysis of RNA isolated from SVEC 4-10 cells showed that MMP-2, MMP-9 and MMP-3 transcripts were not detected in the cells, whereas MMP-11, MMP-13 and MMP-14 were expressed together with TIMP-1, TIMP-2 and TIMP-3. Extracellular serine protease, uPA and its endogenous inhibitor PAI-1 mRNA were also transcribed in the SVEC 4-10 cells (Table 1).


When the cells were treated with S100A4, the transcription of MMP-2, MMP-9 and MMP-3 was not initiated (data not shown). Meanwhile, we detected an increase of MMP-11, MMP-13, MMP-14 and uPA mRNA transcription (Figure 4a–d). The most profound effect was observed for MMP-13 where the level of specific transcript was raised 3.5 times, peaking at 5–6 h of treatment (Figure 4b). Expression of TIMP-2 mRNA was not altered, whereas transcription of PAI-1, TIMP-1 and TIMP-3 was downregulated (Figure 4e–h). Except for MMP-13 mRNA the effects observed were rather mild.

Figure 4.
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Expression of mRNA coding for proteases and their inhibitors in the cells treated with S100A4 for different periods of time. Expression profile of MMP-11 (a), MMP-14 (b), MMP-13 (c), uPA (d), TIMP-1 (e), TIMP-2 (f), TIMP-3 (g) and PAI1 (h) obtained by Northern blot hybridization analysis of RNA isolated from the SVEC 4-10 cells treated with S100A4 for different periods of time

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These observations raised the possibility that stimulation of motility and invasion of SVEC 4-10 cells by the S100A4 protein are interposed by modulation of expression of proteins mediating degradation of ECM.

Stimulation of protease activity in the conditioned media of SVEC 4-10 cells

We attempted to determine whether activation of MMP-13 RNA transcription led to the production of secreted MMP-13 in the conditioned media (CM).

CM from cells treated with S100A4 contained substantially more MMP-13 protein than from nontreated cells, as could be seen from the Western blot analysis (Figure 5a). These differences were detected at 24 h of treatment, but not at 6 h (Figure 5a). TNF-alpha, in combination with phorbol myristate acetate (PMA), a stimulator of MMP production, was used as a positive control (Figure 5a). Accumulation of MMP-13 in the media was substantially reduced when anti-S100A4 antibodies were added together with the S100A4 protein (Figure 5b). MMP 13 is secreted from the cells as an inactive proenzyme of 58 kDa that is later processed into an enzymatically active 48 kDa form. The anti-MMP-13 antibodies used for Western blot analysis enabled us to detect only the latent 58 kDa form in the CM.

Figure 5.
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Detection of MMP-13 and changes in proteolytic activity of CM from the cells treated with S100A4. (a) Western blot analysis of CM with anti-MMP-13 antibodies. CM from the cells treated with PMA and PMA+TNFalpha was used as a positive control. (b) Blocking of the MMP-13 protein production by anti-S100A4 antibodies detected by Western blot analysis of CM with anti-MMP-13 antibodies. (c) beta-Casein zymography of the CM. Cells were treated with S100A4 for 24 h. (d) Level of presumable MMP-13 proteolytic activity determined from three independent beta-casein gels. (e) Gelatin zymography of the CM from the cells treated with S100A4 for 6 and 24 h. (f) Extent of the protrusions formed by SVEC 4-10 cells in 3D Matrigel cultures treated with S100A4 and CL-82198

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We also studied the proteolytic activity of the CM from cells treated with S100A4 by zymography. Figure 5c and e shows typical examples of beta-casein and gelatin zymography. beta-Casein zymography revealed enhanced caseinolytic activity determined by the appearance of a cleared zone of 48 kDa (probably MMP-13) in the CM from the cells treated with S100A4 for 24 h (Figure 5c). The intensity of the cleared zone at 48 kDa was 2.2 times stronger in samples treated with S100A4 (Figure 5d). At 6 h of treatment we also detected a proteolytic activity of 70 kDa absent in the CM of non-treated cells. This activity was diminished at 24 h of treatment (data not shown).

To verify whether the cleared bands detected in beta-casein zymography were due to MMP activity, a parallel gel was incubated in a buffer containing 10 mM EDTA. This treatment eliminated the development of cleared areas, indicating that the observed activity indeed belonged to MMP (data not shown).

Gelatin zymography did not reveal substantial differences in the CM from S100A4 treated and nontreated cells. A cleared area corresponding to 55 kDa was detected in CM from both treated and nontreated cells. The size of this band did not correspond to the expected size of active MMP13 (48 kDa).

Based on the size of the band (48 kDa) revealed in beta-casein zymography and its induction in response to the S100A4 treatment, we propose that this activity corresponds to MMP-13.

We can speculate that enhanced proteolytic activity detected in the CM of cells treated with S100A4 is correlated with stimulation of invasiveness of SVEC 4-10 cells. This should be at least in part be dependent on the activity of MMP-13. To further support this hypothesis, we tested the possibility of inhibition of invasive activity of SVEC 4-10 cells stimulated by S100A4 with a specific inhibitor of MMP-13 activity – CL-82198. The formation of capillary-like protrusions of SVEC 4-10 cells in 3D Matrigel stimulated by S100A4 was inhibited by treatment with CL-82198 (Figure 5f). This indicates that proteolytic activity of MMP-13 could be responsible for the S100A4 stimulated induction of the invasive growth of SVEC 4-10 cells.

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Discussion

Metastasis-promoting S100A4 is a typical representative of S100 family of Ca-binding proteins. These proteins are characterized by a dual extra- and intracellular function (Donato, 2003). The mechanism by which S100A4 promotes metastasis remains unclear. Expression of S100A4 in tumor cells leads to changes in their motility and invasion (Heizmann et al., 2002). This offers an explanation about how intracellular S100A4 facilitates metastatic spread of tumor cells.

On the other hand, S100A4 exhibits extracellular functions. When released into extracellular space it causes differentiation of neurons and osteoclasts (Novitskaya et al., 2000; Duarte et al., 2003). Furthermore, S100A4 acts as an angiogenic factor by stimulating motility of endothelial cells (Ambartsumian et al., 2001). Motility of astrocytic tumor cells was also increased in response to S100A4 treatment (Belot et al., 2002). It has been shown that S100A4 could be released both by tumor and normal cells (Ambartsumian et al., 2001; Duarte et al., 2003). One can propose then that when released into the extracellular space, S100A4 could create a microenvironment that facilitates invasion and motility of both endothelial and tumor cells, thus stimulating dissemination of tumor cells in the organism.

Here we show that S100A4 is capable not only of stimulating motility of endothelial cells but also enhancing their ability to invade the surrounding ECM. S100A4 stimulated invasive growth of microvascular endothelial cells in 3D Matrigel.

Degradation and remodeling of ECM is an obligatory step of angiogenesis. Proteolytic activity is required during the formation of the capillary bud in order for endothelial cells to migrate out through the peri-capillary membrane and through the ECM. Capillary elongation, the lumen formation and ECM remodeling all require proteolytic activity (Folkman, 1995).

Endothelial cells themselves produce numerous proteases that degrade ECM and create conditions for them to invade surrounding tissues. A number of angiogenic factors regulate the production of MMPs from endothelial cells. For example, TSP-1 (thrombospondin) stimulates secretion of MMP-9 (Qian et al., 1997), HGF modulates expression of MMPs in human endothelial cells (Wang and Keiser, 2000) and MMP-2 production from endothelial cells is stimulated by the retinoic acid (Braunhut and Moses, 1994).

Invasive growth of endothelial cells in response to S100A4 was accompanied by activation of transcription factor NF-kappaB. This suggested that S100A4 was capable of modulating the expression of proteases with the consecutive release of active proteolytic enzymes to degrade the ECM.

Transcription of MMP-11, MMP-13, MMP-14 and uPA was increased in response to S100A4 treatment. The most pronounced increase was documented for the MMP-13 gene. At the same time, we observed transcriptional downregulation of two out of three genes coding for protease inhibitors – TIMP-1 and TIMP-3, as well as PAI-1. Transcriptional activation of MMP13 was followed by secretion of the protein into CM. Moreover, we detected increased levels of active enzyme, presumably MMP-13, by casein zymography. MMP-13 (collagenase 3) is also reported to possess a gelatinolytic activity (Lemaitre et al., 1997; Peeters-Joris et al., 1998), but we did not detect any proteolytic activity that might correspond to MMP-13 in the gelatin zymography. Gelatin zymography is reportedly a very sensitive assay for gelatinases, but it is much less sensitive for collagenases; collagenase activity is measured therefore by casein zymography (Yu and Woessner, 2001). We suppose that the gelatinolytic activity of MMP-13 is lower that the caseinolytic one; therefore, we were not able to detect it in these experimental conditions.

Stimulation of invasive growth by S100A4 could be reduced by treatment with CL-82198, a specific inhibitor of MMP-13 activity. This observation indirectly points to the possibility that the activity detected in beta-casein zymography is indeed MMP-13. The stimulation of production of proteases proves to be significant for S100A4 stimulation of endothelial cell invasion.

Fibrillar collagens are the most abundant structural components of the connective tissues. It is conceivable that the ability to degrade them is crucial for invasion and metastasis of neoplastic cells. MMP-13 degrades native fibrillar collagens and several other ECM components very efficiently (Freije et al., 1994; Vihinen and Kahari, 2002). The wide proteolytic capacity of MMP-13 suggests its role as a powerful invasion tool. In fact, the expression of MMP-13 has been detected in a number of invasive neoplastic tumors, such as breast carcinomas, squamous cell carcinomas, head and neck carcinomas (Freije et al., 1994; Airola et al., 1997; Johansson et al., 1997; Cazorla et al., 1998; Johansson et al., 1999). MMP-13 expression is increased substantially in the endothelial cells under the conditions stimulating vascular development (Heikkila et al., 2002; Zaragoza et al., 2002a, 2002b; Hattori et al., 2003). Based on these observations, MMP-13 is regarded as one of the key players in remodeling of the specific cell microenvironment.

Treatment of the endothelial cells with S100A4 did not affect the expression of TIMP-2, but downregulated the expression of TIMP-1 and TIMP-3, the inhibitors that are most efficient in interaction with MMP-13 (Stamenkovic, 2000; Yeow et al., 2002). Moreover, latent MMP-13 is a substrate for MMP-14, a membrane-bound metalloproteinase, whose expression was also increased in response to S100A4 treatment.

Taking into consideration the role of S100A4 in tumor progression, the fact that its presence in the extracellular space could stimulate production of matrix-degrading enzymes offers a new mechanistic explanation to its role as a metastasis stimulating protein.

Previous investigations have demonstrated that inhibition of S100A4 production displays reduced in vitro invasive properties and alters the expression levels of MMPs and TIMPs in the tumor cells (Bjornland et al., 1999; Bjornland et al., 2001). Here, we extend the understanding of this process by demonstrating that extracellular S100A4 interacts with endothelial cells and stimulates the production of matrix-degrading enzymes.

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Materials and methods

Materials

Mouse lymphoid microvascular endothelial immortalized cell line, SVEC 4-10 (O'Connell and Edidin, 1990), was obtained from the ATCC. Dulbecco modified Eagle medium (DMEM) and DME-F12 medium were purchased from Gibco BRL.

An active oligomeric fraction of S100A4 was obtained from recombinant His6-tagged protein by gel filtration, as described in Novitskaya et al. (2000).

A recombinant C-terminal peptide of human myosin heavy chain named Hmyo4-3B (1762–1961 amino acids) was isolated and purified as described (Kriajevska et al., 1998). Human recombinant S100A12 was isolated and purified as described (Mikkelsen et al., 2001).

VEGF was purchased from Biosource, bFGF from Boehringer Mannheim, HGF from Sigma. TNF-alpha and PMA were from Calbiochem. Basement membrane matrix (Matrigel) was purchased from Biomedical Technologies. beta-Casein and gelatin were from Sigma. CL-82198 was purchased from Biomol, USA.

Invasive growth in 3D Matrigel culture

A detailed protocol for invasive assay in 3D Matrigel was described elsewhere (Ambartsumian, in press). Briefly, 6 times 105 cells were incubated in a hanging drop of DME medium supplied with 10% FCS and glucose to form a clump, which was then placed on a layer of Matrigel containing DMEM and 10% FCS. The clump was covered with a drop of Matrigel and incubated at 37°C for polymerization. DMEM supplied with 10% FCS and proteins used for the experiments were added to the cells sealed in the Matrigel and incubated for different periods of time. Proteins and anti-S100A4 antibodies were added to the Matrigel at the same time point. The specific inhibitor of MMP-13, CL-82198, was added to the cell clump at a concentration of 10 mug/ml. The extent of the outgrowth was followed in an inverted microscope using times 10 objective. The distance from the edge of the clump to the end of the protrusion was used to quantify the extent of the outgrowth. For this, the images of the cell aggregates were photographed and analysed using Image Gauge software (Fujifilm). The length of the protrusions was measured in 30 locations evenly distributed along the perimeter of the cell clump. This permitted a randomized measure of the extent of the protrusions. The averageplusminuss.d. was taken as a measure of outgrowth for each experiment. Outgrowth observed with the untreated cells was taken as 100%.

Proliferation assays

To determine the rate of cell proliferation, SVEC 4-10 cells were plated in T25 TC flasks. At 1 day after plating, the media were exchanged to a DMEM supplemented with 10% FCS and the proteins to be investigated. Triplicate flasks for each treatment were harvested and the cells were counted at various times. Each experiment was repeated two times. The doubling time was calculated as a function of the increase of the amount of cells at each 24 h. The MTT assay was performed using the MTT cell proliferation kit (Boehringer Mannheim), according to the protocol of the manufacturer. Cells were plated at a concentration of 1 times 103 cell per well in a 96-well plates and treated with different proteins for 24 h prior to assay. All experiments were performed in quadruplicate and repeated at least twice. The averageplusminuss.d. was taken as a measure for all the experiments.

For measuring the rate of 3[H]thymidine incorporation in 3D Matrigel culture, the cells were resuspended in Matrigel supplemented with DMEM and 10% FCS at a concentration 1 times 106 cells/ml. After 24 h of cultivation, 2 muCi/ml of 3[H]Thymidine was supplemented together with the proteins of interest. Cells were further incubated for 3, 6 and 24 h, and washed three times with PBS prewarmed to 37°C. Matrigel-containing cells were then dissolved in ice-cold PBS. The cells were lysed by addition of 1% SDS, and the content was precipitated by 5% TCA. The precipitate was collected on glass fiber filters and the cell-bound radioactivity was determined in a liquid scintillation counter (RacBeta, LKB Wallak). All the experiments were repeated three times with five parallel determinations for each time point. The rate of 3[H]thymidine incorporation was determined from the corresponding curves. To compare the rates from different experimental points, the rate of incorporation of untreated cells was set as 100%. The averageplusminuss.d. was taken as a measure for all the experiments.

Nuclear extract preparation, EMSA and nuclear translocation assays

SVEC 4-40 cells were treated with S100A4 oligomer (0.5 mug/ml) for 2 and 4 h, and nuclear extracts were prepared as described in Andrews and Faller (1991). EMSA was performed as described earlier (Tulchinsky et al., 1997). NF-kappaB- and Sp-1-specific oligonucleotides were used for the assay. The level of NF-kappaB complex activation was measured as a ratio of the amount of radioactivity in the NF-kappaB complex normalized to the amount of radioactivity of the SP-1 complexplusminuss.d. Supershift assay was performed by incubating nuclear extracts with anti-p50, anti-p65, anti-cRel and anti-RelB antibodies (Santa-Cruz Biotechnologies) for 1 h prior to the addition of the radiolabeled oligonucleotide probe. The results were reproduced three times.

For testing the translocation of p50 and p65 to the nucleus of the cells, SVEC 4-10 cells were grown on glass coverslips. The media were shifted to a serum-free DME/F12 medium for 24 h followed by treatment with different proteins for 4 h. Cells were fixed and immunostained with anti-p50 and anti-p65 antibodies, according to the recommendations of the manufacturer (Santa-Cruz Biotechnologies). FITC-labeled anti-rabbit secondary antibodies (Vector laboratories) were used for the visualization of the staining.

RNA analysis

Total RNA from the SVEC 4-10 cells after different treatments was isolated according to Chromoczymski and Saechi (1987); after separation by gel-electrophoresis the RNA was transferred onto the nylon membrane (Hybond-N, Amersham) and hybridized with different RNA probes. The following 32P-labelled probes were used for hybridization: MMP-13, MMP-14 and TIMP-3 were synthesized by RT–PCR using the following primers: MMP-14 sense 5'-ACACCCTTTGATGGTGAAGG-3' antisense 5'-TCGGAGGGATCGTTAGAATG-3', MMP-13: sense 5'-CTATCCTGGCCACCTTCTTCTT-3' antisense 5'-GGGACCATTTGAGTGTTCTAGG-3', TIMP-3: sense 5' GACCACAACAGCTACCATGACT-3' antisense 5'-GCCACAAAGACTTTCAGAGGCT-3'. Probes specific for MMP-2, MMP3, MMP-9 and MMP-12 were kindly provided by Dr Mamen Overejro, CCBR.

Probes specific for mouse MMP-11, TIMP-1, TIMP-2 uPA and PAI1 were kindly provided by Dr D Kramerov, IGB RAN.

The amount of mRNA on the filters was calibrated by hybridization with italic gamma-32P ATP-labeled poly(U) probe (Ambartsumian et al., 1996).

To quantify the intensities of the bands, membranes were scanned using Fujifilm FLA-3000 computing densitometer with Image Gauge software. The amount of specific RNA was normalized to the poly(U) content of the lanes. Each expression profile was determined in two to five independent experiments. Each expression profile included at least five time points of treatment with S100A4.

Analysis of CM

Cells were grown to 90% confluency in the flasks pretreated with a low concentration of Matrigel (1 mg/ml). The medium was exchanged with a serum-free DME/F12 medium containing proteins of interest. The cultures were sustained for required periods of time, harvested media were then filtered through a 0.45 mum membrane filters and concentrated 20–50 times using Vivapore and Vivaspin concentrators (Vivascience Ltd, UK). Subsequently, the cells were trypsinized and counted to equilibrate the quantity of CM used for further analysis.

Concentrated CM was used for Western blot analysis. After electrophoresis in 10% SDS—PAGE, the samples were transferred to Immobilon membrane (Millipore) and immunoprobed with anti-MMP-13 antibodies (Neomarkers, USA). The bands were visualized by incubation with HRP-conjugated rabbit anti-mouse antibodies (DAKO A/S) and the ECL plus chemiluminescent substrate (Amersham) according to the directions of the manufacturer. CM was assayed for protease activity using gelatin or beta-casein zymography. For these experiments the cells were grown in the flasks without pretreatment with Matrigel. Samples were separated in 10% SDS–PAGE containing gelatin (0.5 mg/ml, Sigma) or beta-casein (0.5 mg/ml, Sigma); all the procedures were performed as described (Fernandez-Resa et al., 1995). The cleared zones were quantified using an LAS-1000 analyser with subsequent quantification with Image Gauge computing program (Fujifilm).

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Acknowledgements

We thank Claus Christensen for fruitful discussions of both the experimental design and the content of the manuscript, and Birgitte Kaas and Inge Skibshøy for excellent technical assistance. This work was supported by grants from Danish Cancer Society, Danish Research Council and Dansk Kraeftforsknings Fond.