From the Departments of Pathology and Virology, Haartman Institute, University of Helsinki, and Helsinki University Hospital, FIN-00014 Helsinki, Finland
Received for publication, December 19, 2002 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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LTBPs belong to the LTBP/fibrillin family of extracellular matrix proteins, which includes LTBPs 14 and fibrillins 1 and 2. LTBP-1 and -2 co-localize with extracellular fibrillin microfibrils as well as with fibronectin in cultured cells at certain points of matrix formation (7, 8). LTBP-3 is especially important for bone development (9). LTBP-1 and -3 possess the strongest ability to bind small latent TGF- as assessed by complex formation assays (10). LTBP-4 is also important for regulating TGF-
1 function, since the LTBP-4 gene-targeted mouse suffers from defects in TGF-
targeting (11). Unlike the other LTBPs, LTBP-2 is unable to associate with the small latent TGF-
(10). Human LTBP-2 is expressed mostly in the lung and to a lesser extent in the liver, skeletal muscle, placenta, and heart (12). LTBP-2 deficiency in the mouse causes early embryonic lethality, indicating the importance of the molecule in development, possibly during implantation (13). LTBP-2 contains the common integrin recognition sequence RGD in its N terminus, suggesting a possible role in cell adhesion. Interactions between fibrillin-1 and integrin
V
3 (14) and also between LAP-TGF-
1 and integrins
V
1,
V
6, and
8
1 have been reported (see Refs. 1518, respectively).
Integrins are heterodimeric transmembrane receptors consisting of and
subunits (reviewed in Refs. 19 and 20). They mediate cell-matrix and cell-cell interactions and play important roles in many biological processes such as wound healing, maintenance of tissue integrity, cell growth, and survival. Integrins participate also in various pathological conditions like inflammation and invasion of cancer cells. Integrins are widely distributed on various cell types, and 24
heterodimers are currently known. Their ligands are usually immobilized extracellular matrix proteins, and their intracellular tail is anchored to the actin cytoskeleton through proteins like talin, paxillin, and vinculin (2123). Integrin receptors lack intrinsic enzymatic activity, but the cytoplasmic domains are the site of accumulation of many signaling molecules such as Src-type kinases and Src substrates (21). In addition, there are proteins that interact with the transmembrane part of an integrin-like adaptor protein, caveolin (24, 25). Through these multiple interactions, integrins mediate signals from the cell surroundings into the cell and vice versa.
The 1 subfamily of integrin receptors includes 12 members, which have partially overlapping ligand specificities (26, 27). Fibronectin, collagens, and laminins are common ligands, but there are also receptors for tenascin and VCAM-1 in this family. Melanoma cells are known to express a wide variety of integrin receptors, of which the function of the
V
3 is best characterized in the context of malignant melanoma (2830).
In the current work, we have analyzed the ability of LTBP-2 to act as an adhesion protein for different cultured cells. In general, LTBP-2 did not mediate cell adhesion but acted as an adhesion protein for all melanoma cell lines tested. This feature of LTBP-2 is new and provides new views to the regulation of melanoma cell adhesion and spreading.
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EXPERIMENTAL PROCEDURES |
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Cells and Cell Culture ConditionsCell lines used were human embryonic lung fibroblasts (HEL-299; CCL-137, American Type Culture Collection, Manassas, VA) and human melanoma cell lines Bowes (CRL-9607; ATCC); G361 (CRL-1424; ATTC); WM793, WM852, WM163, WM164, and WM239 (WM, Wistar melanoma; isolated from melanoma metastasis, established at Wistar Institute); and Chinese hamster ovary cell clones expressing recombinant LTBP-2 (31) or its fragments. Cells were cultured in minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS; Invitrogen), 100 IU/ml penicillin, and 50 µg/ml streptomycin. Stably transfected LTBP-2 expressing CHO-L2 clone was grown in MEM containing 0.2% bovine serum albumin, supplemented with FCS and antibiotics as above and 0.4 mg/ml neomycin, G418 (Invitrogen).
Production and Purification of Recombinant LTBP-2 and LTBP-2 FragmentsThe cloning of the expression construct, production, and purification of full-length LTBP-2 have been described elsewhere (31). Briefly, conditioned medium from CHO-L2 clone was precipitated with 30% (w/v) (NH4)2SO4 at +4 °C. The precipitate was dissolved in 50 mM Tris-HCl, pH 7.0, after which it was filtered and applied to MonoQ HR 5/5 column (Amersham Biosciences). The bound proteins were eluted with a 210700 mM NaCl gradient. LTBP-2-containing fractions were combined, and 4 M urea was added to achieve the final concentration of 2 M. The sample was applied to a MonoQ 5/5 column equilibrated in 50 mM Tris-HCl, pH 7.0, containing 2 M urea. Proteins were eluted with a 01 M NaCl gradient. The fractions containing LTBP-2 were fractionated on a Superdex 200 gel filtration column (Amersham Biosciences) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 2 M urea. The buffer of LTBP-2 fractions was then changed to phosphate-buffered saline (PBS) with Fast Desalting column HR 10/10 (Amersham Biosciences). Expression and purification of LTBP-2 fragments as IgG Fc-tail fusion proteins was carried out essentially as described for LTBP-1 (32).
Cell Adhesion AssaysPlates of 96 wells were coated with the indicated proteins diluted in PBS at +37 °C for 2 h. The residual protein binding sites were saturated with 1% heat-denaturated bovine serum albumin (BSA) in PBS (0.14 M NaCl, 10 mM sodium phosphate buffer, pH 7.4) at room temperature for at least 30 min. The wells were washed with PBS before the addition of cells. Semiconfluent cultures of cells were detached with trypsin-EDTA and suspended at a density of 6.0 x 105 cells/ml in serum-free minimal essential medium. Cell suspension (50 µl) was then transferred to each well. In inhibition studies, the cells were incubated with antibodies or other agents of interest for 15 min before transfer to the wells. The antibodies were used at concentrations up to 25 µg/ml, the RGD peptide was used at 100500 µM, and heparin was used at up to 800 IU/ml. Each experimental condition was tested in triplicate. The cells were permitted to attach at +37 °C for 1 h, after which the wells were washed with PBS to remove nonattached cells. Cells were then fixed and stained simultaneously with 0.1% Coomassie Blue in 10% acetic acid and 40% methanol and washed with the same fixative without the dye. Subsequently, the cells were lysed in 100 µl of 1% SDS in PBS, and the absorbance was measured at 620 nm.
Immunofluorescence StainingsGlass coverslips were coated with either LTBP-2 or laminin (10 or 20 µg/ml) at +37 °C for 2 h and then treated with 1% heat-denaturated BSA at room temperature for 1 h to prevent nonspecific binding of cells. BSA-coated coverslips were used as negative controls. Bowes melanoma cells were detached with trypsin-EDTA and suspended at 1 x 105 cells/ml in serum-free MEM. The coated coverslips were placed in six-well plates, and 1 x 105 cells were transferred to each well. The cells were then allowed to attach at +37 °C for 2 h, and nonattached cells were removed by washing with PBS, after which the cells were fixed with 3% paraformaldehyde for 15 min. Permeabilization of cells was performed with 0.5% Nonidet P-40 for 5 min. The cells were then treated with 5% BSA for 30 min, washed with PBS, and incubated with monoclonal antibodies against the integrin 1 subunit (P5D2), paxillin, or vinculin at pretested concentrations for 1 h. Unbound proteins were removed by washing with PBS with subsequent incubation either with biotinylated secondary antibodies or with fluorescein isothiocyanate-conjugated anti-mouse antibody. The coverslips were washed again and Streptavidin-fluorescein isothiocyanate was used as fluorescence conjugate when needed. The coverslips were washed and mounted on glass slides using Vectashield (Vector Laboratories, Burlingame, CA). The fluorescent images were obtained using epifluorescent microscope.
The 3 integrin staining was performed as described above with one exception; the primary antibody, mAb ASC-6, was permitted to bind to living cells on ice for 20 min before fixation. The following steps were carried out as for other stainings.
Cell Migration AssaysMigration assays were performed using modified Boyden chambers (Falcon cell culture inserts with 8-µm pores). The upper and/or lower sides of the membranes of inserts were coated on either with the indicated concentrations of LTBP-2 or fibronectin at +37 °C for 1 h, washed with PBS, and then treated with 1% heat-inactivated BSA for 30 min to prevent nonspecific binding. The inserts were then washed twice with PBS and placed in 24-well cell culture plates. Bowes melanoma cells were detached with trypsin-EDTA, and the cell number was determined by an optical electronic counter. The cells were suspended in MEM containing 0.1% FCS at a density of 1 x 105 cells/ml. Cell suspension (500 µl) was applied to upper chambers, and 750 µl of MEM with 0.1% FCS was added to lower chambers. The cells were allowed to migrate in 5% CO2 at +37 °C for 6 h. The cells were then fixed and stained with 40% methanol, 10% acetic acid, and 0.1% Coomassie Blue for 5 min and washed with the same fixative without the dye. The cells that had not migrated through the pores were removed from the upper surface of the membrane with cotton swabs. The number of migrated cells was counted from three randomly chosen microscope fields of each membrane.
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RESULTS |
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To test the effects of LTBP-2 on cell spreading, we coated glass coverslips with 10 µg/ml LTBP-2. Coverslips coated with 1% denatured BSA were used as controls. Cells were seeded on coated coverslips and allowed to adhere for 90 min, after which they were fixed. The cell morphology was visualized by staining the actin cytoskeleton with phalloidin-TRITC. By microscopic analysis, we found that Bowes cell adhesion to LTBP-2 was associated with cell spreading (Fig. 1C). Actin staining was mostly seen at the cell cortex, which is typical for these cells. Cells seeded on LTBP-2 formed also extensive filopodia, in contrast to cells adhered to BSA, which appeared more round and less spread.
Antibodies to 1 Integrin Totally Prevent Bowes Cell Adhesion to LTBP-2Since LTBP-2 contains an RGD sequence in its N terminus, we assumed that the interaction of Bowes melanoma cells with LTBP-2 could be mediated by one or more integrin receptors. We took the approach of inhibiting cell adhesion by a number of monoclonal anti-integrin antibodies. Cells were incubated with function-blocking antibodies for 20 min at room temperature before seeding onto LTBP-2-coated 96-well plates. Among the antibodies, the anti-
1 antibody 6S6 completely prevented melanoma cell adhesion to LTBP-2 (Fig. 2A). The inhibitory effect was clearly concentration-dependent (Fig. 2C). The antibody against the integrin
3 subunit decreased cell adhesion by 50%, whereas mAbs recognizing the
subunits 1, 2, 4, 5, and V did not have any effect on Bowes cell adhesion to LTBP-2 (Fig. 2A). mAb against the integrin
6 subunit had a small decreasing effect on the adhesion and enhanced the inhibitory effect of the anti-
3 antibody when used in combination (Fig. 2B). Together, the antibodies against
3 and
6 integrins decreased Bowes cell adhesion to LTBP-2 almost by 80%, which was comparable with their effect on Bowes cell adhesion to laminin. However, when used individually, both anti-
3 and anti-
6 antibodies were more effective in preventing Bowes cell adhesion to laminin, as could be expected, since
3
1 and
6
1 are known as laminin receptors (summarized in Ref. 33). The effect of these antibodies on Bowes cell adhesion to collagen I was negligible. Since antibodies against integrin
3 and
6 subunits did not totally prevent Bowes cell adhesion to LTBP-2, some other interactions are evidently involved in the adhesion.
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V
3 integrin has been shown to be an important integrin receptor in melanoma cell adhesion and invasion in various studies (28, 29, 34). Although the antibody against
V subunit did not have any effect on melanoma cell adhesion to LTBP-2, we tested monoclonal antibodies against the
3 subunit and an antibody against the entire
V
3 in inhibition assays. We could not observe any effect on cell adhesion (data not shown), confirming that
V
3 integrin is not involved in adhesion to LTBP-2.
Cells Seeded on LTBP-2 Express the 1 and
3 Integrins on Their Surface and Form Focal AdhesionsThe involvement of the
1 integrin in melanoma cell attachment to LTBP-2 was further verified by immunofluorescence. Cells were allowed to adhere to LTBP-2- or laminin-coated (20 µg/ml) coverslips for 2 h before fixing and staining. The fluorescence signal was localized mostly at the cell membrane, especially in the lamellipodia (Fig. 3, uppermost panel, on the left). Some perinuclear staining was observed as well. In control cells seeded on laminin,
1 integrin was seen in lamellipodia and also inside the cell (Fig. 3). Marginal, more diffuse staining was seen in cells attached to BSA. The
3 integrin staining was similar to
1 localizing mainly to the cell cortex in Bowes cells seeded on either LTBP-2 or laminin (Fig. 3). Both integrins were seen in plaque-like structures on the cell membrane or just beneath it, indicating receptor clustering after ligand recognition.
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Connection to actin cytoskeleton is important for integrin functions. The connection is formed through many adaptor proteins (e.g. vinculin and talin at the ECM contact sites called focal contacts or focal adhesions) (35). We explored next whether Bowes cells form this kind of structure when attached to LTBP-2. We carried out immunofluorescence stainings with antibodies against paxillin and vinculin after Bowes cells were allowed to attach to LTBP-2-coated coverslips. Both paxillin and vinculin were localized to the cell periphery in dot- or plaque-like structures in cells adhered to LTBP-2 or laminin, which indicates that cells are firmly attached and form focal contacts.
Melanoma Cell Attachment to LTBP-2 Is Not Dependent on RGD SequenceSince the 3
1 integrin is known to recognize the RGD sequence in some ligands (36, 37), and the LTBP-2 molecule also contains one at its N terminus, we tested whether an RGD peptide could interfere with Bowes melanoma cell adhesion to LTBP-2. Cells were allowed to attach to LTBP-2-coated wells in the presence of RGD peptide. In our experiments, the RGD peptide decreased Bowes cell adhesion to LTBP-2 by 40% at a concentration of 500 µM. Lower concentrations did not have any effect (data not shown). To control the function of the RGD peptide, we tested its ability to inhibit Bowes cell adhesion to fibronectin and vitronectin. The peptide totally prevented both at a concentration of 500 µM (Fig. 4A).
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Cell surface proteoglycans are important modulators of cell adhesion, and heparan sulfate proteoglycans cooperate with integrins in many cell-matrix interactions (38). This led us to examine whether Bowes cell adhesion to LTBP-2 would be affected by treating cells with heparin, a wide range inhibitor of cell adhesion (38), before seeding cells onto LTBP-2-coated wells. Heparin inhibited Bowes cell adhesion to LTBP-2 in a concentration-dependent manner (Fig. 4B). The effect of heparin was similar to the inhibition of cell adhesion to fibronectin (data not shown). Total inhibition, comparable with adhesion to BSA, was achieved at a concentration of 800 IU/ml. We explored also the effect of heparan sulfate in the same assay but did not observe any effect on Bowes cell adhesion to LTBP-2 (data not shown). This may implicate the involvement of very specialized form of cell surface heparan sulfate proteoglycan.
Bowes Cell Adhesion to LTBP-2 Is Decreased by Polyclonal Antibody against Recombinant LTBP-2 but Only Slightly by the Soluble Form of the ProteinTo confirm that the adherence of Bowes cells to LTBP-2 is not caused by some impurity in the protein preparation, we tested the effect of polyclonal antibody, abL22 (affinity-purified IgG), generated against recombinant LTBP-2, in the adhesion assay. Before seeding the cells into LTBP-2-coated wells, the wells were incubated with the antibody at room temperature for 20 min. After that, the adhesion assay was carried out as described under "Experimental Procedures." Treatment of the LTBP-2-coated wells with abL22 decreased the melanoma cell adhesion by 40% (Fig. 5). We have observed that abL22 does not recognize the N-terminal epitopes of LTBP-2 as well as the C-terminal ones, which may explain the inhibitory effect being only partial.2
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We analyzed next whether soluble LTBP-2 could compete with melanoma cell adhesion to an immobilized form of the protein. To test this, Bowes cells were incubated before plating with soluble LTBP-2 (5 µg/ml) at room temperature for 20 min. We observed that soluble LTBP-2 decreased melanoma cell adhesion by 15%, suggesting that cells cannot efficiently bind soluble LTBP-2 and that the adhesive properties of LTBP-2 are dependent on its deposition to substratum, namely tissue culture plate in this case.
N-terminal Fragment of LTBP-2 Is the Domain Responsible for Bowes Cell AdhesionWe took the recombinant fragment approach to identify the adhesive site in the LTBP-2 protein. To investigate this, we produced several partially overlapping LTBP-2 fragments as Ig fusion proteins covering the whole protein and used the purified fragments in adhesion assays (Fig. 6B). As expected, Bowes cells did not adhere to any of the fragments as efficiently as to full-length LTBP-2. The N-terminal fragments L2-III, L2-V, and L2-VII were the most potent in mediating cell attachment (Fig. 6A). These fragments contain a common proline-rich region. Of these fragments, L2-III contains the RGD sequence, but the shorter fragment L2-V, lacking this RGD, was slightly more potent in mediating Bowes cell adhesion. Fragment L2-II containing the RGD sequence was ineffective, confirming that Bowes cell adhesion to LTBP-2 is independent of the RGD sequence. Some binding was observed to fragment L2-IX, which contains another proline-rich region, and to the pIg control protein. On the other hand, cells did not adhere to purified mouse IgG.
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LTBP-2 Supports Migration of Bowes Melanoma CellsWe explored next whether LTBP-2 supports melanoma cell migration in addition to adhesion. The migration assay measures the ability of cells to migrate through a porous membrane, which can be coated with the test protein either on one side or both. We used purified LTBP-2 for coating and made control experiments with fibronectin, since fibronectin is haptotactic (39) for a variety of cell lines. In the experiment where the LTBP-2 was coated only on the upper surface of the membrane of the cell culture insert, the cells migrated very poorly (Fig. 7). When LTBP-2 was coated on the lower surface of the membrane, the cells migrated toward it. Coating of both sides with LTBP-2 further increased cell migration, the pattern being very similar to Bowes cell migration on fibronectin. These data suggest that LTBP-2 can support melanoma cell migration and that LTBP-2 is sufficient for induction of cell migration by itself in the absence of other attractants.
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DISCUSSION |
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Our earlier observation was that LTBP-2 is unable to bind small latent TGF- (10). Since the LTBP-2 null mice die during the time of implantation, we hypothesized that LTBP-2 could have a cell adhesion-mediating or -modulating function in the ECM. The current study was carried out to explore the effects of LTBP-2 on cell adhesion. We tested several adherent cell lines, both normal diploid and transformed cells, and found that only melanoma cells were able to adhere to immobilized recombinant LTBP-2. We have, in fact, observed that LTBP-2, and especially its N-terminal fragment, L2-V, which mediates melanoma cell adhesion, acts as an antiadhesive molecule for at least fibroblastic cells, leading to decreased actin stress fiber formation.3 Melanoma cell adhesion to LTBP-2 was accompanied by actin stress fiber formation as well as focal contact formation in a manner similar to cells adhering to laminin. This indicates that LTBP-2 acts as a real adhesion-mediating protein, not just as a passive binding partner.
Integrins are the most common cell surface receptors for extracellular matrix proteins. They are found in a number of species from sponges to vertebrates. In fibrillin-1, fibroblasts adhere to its RGD site via V
3 integrin, but in addition, some cell types recognize another, non-RGD binding site within the fibrillin molecule (14). The nature of the second adhesion site is not known. Integrins
3,
V, and
1 are involved in human skin fibroblast and murine L-cell adhesion to fibrillin-2 (44). The motif mediating the adhesion was localized to the 12th module of the fibrillin-D segment, which contains an RGD sequence, and the adhesion could be prevented by RGD peptides. Although RGD sequence is present in LTPB-2, RGD peptides could not compete melanoma cell adhesion to LTBP-2. In addition, melanoma cells did not bind to the smallest RGD-containing fragment but bound to an adjacent short fragment, L2-V.
1 integrin family contains both kind of receptors, namely RGD-binding and RGD-independent (45). Integrin
3
1 recognizes RGD sequence in some ligands, but it also recognizes several ligands lacking a functional RGD sequence (36). For example, the RGD sites in laminins are often cryptic and inaccessible to integrin receptor. Our findings suggest that melanoma cells adhere to LTBP-2 in an RGD-independent manner.
Cell adhesion to ECM via integrins regulates cell behavior and fate in many ways being crucial for tissue morphogenesis and in guiding cell migration during embryonic development. Melanoma cell adhesion via V
3 integrin is widely characterized and appears to be important in melanoma metastasis (30). In our studies, melanoma cells did not adhere to LTBP-2 by
V
3 integrin. Inhibition assays indicated that adhesion to LTBP-2 was mediated by
1 integrin, the
3 subunit being the most obvious partner. The mAb against the
6 subunit had an additive inhibitory effect on melanoma cell adhesion. However, cell adhesion to LTBP-2 does not depend only on the expression of
3
1 or
6
1 integrins, since many of the cell lines we have tested (e.g. HT-1080 fibrosarcoma cells) express either one or both of these receptors (46, 47) and still could not bind to LTBP-2 (Fig. 1A). The ligand binding specificities of integrins are known to vary from cell type to another, and various other cell surface proteins, like urokinase-type plasminogen activator receptor, thrombospondin, or tetraspanins, modulate the integrin functions (48, 49). Thus, this kind of regulation of integrin activity may explain why melanoma cells but none of the other tested cells adhered to LTBP-2. The
3
1 integrin has a role in melanoma cell migration toward laminin, fibronectin, and collagen IV and in invasion through Matrigel (50), and its expression is increased in some metastatic melanomas (51). Our results indicate that melanoma cell adhesion to LTBP-2 could be mediated at least partially by the
3
1 integrin receptor.
In our studies, heparin markedly decreased Bowes melanoma cell adhesion to LTBP-2. This could implicate that cell surface proteoglycans play a role in the adhesion. We were not able to inhibit the adhesion by competing with heparan sulfate, which could imply the involvement of very specialized form of heparan sulfate proteoglycan, since they are very heterogenic. Melanoma cells express cell surface chondroitin sulfate proteoglycans that modulate tumor cell adhesion and motility (reviewed in Ref. 52). chondroitin sulfate proteoglycans bind the 4
1 integrin and are needed for
4
1-mediated melanoma cell adhesion (53). Involvement of chondroitin sulfate proteoglycans in melanoma cell adhesion to LTBP-2 has not been ruled out here.
Soluble LTBP-2 did not efficiently prevent Bowes cell adhesion to the substratum-bound form of the protein. This suggests that in solution LTBP-2 adopts a conformation in which cell adhesion sites are masked and not accessible to cell surface receptors. When coated on a solid surface, the conformation is obviously changed, exposing a plausible cryptic site in LTBP-2, and may act more like in extracellular matrix in vivo.
The polyclonal antibody generated against the full-length LTBP-2, AbL22, inhibited only partially Bowes cell adhesion to LTBP-2. This may be due to possible differences between the protein conformations in solution and in immobilized form, since these antibodies have been generated against the soluble form. Melanoma cells adhered to some extent to an N-terminal fragment of LTBP-2, and the AbL22 does not recognize the N-terminal fragments as well as the C-terminal ones. This may explain the relatively inefficient inhibitory effect of AbL22 in the adhesion assays.
LTBP-2 deficiency in the mouse leads to embryonic lethality during the time of implantation (13), which raises a question of whether cell adhesion to LTBP-2 would play a role during development. Our findings indicate that LTBP-2 can mediate melanoma cell adhesion in an integrin-dependent manner. Bowes cells were also able to migrate on LTBP-2. In general, the ability to adhere to different ECM proteins is beneficial for tumor cells during migration and invasion and when adapting to a new environment after invasion. LTBP-2 is an abundant protein in human lung, and it is also expressed in the liver, where it may serve as an adhesion protein for melanoma cells during cancer spreading.
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FOOTNOTES |
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To whom correspondence should be addressed: Department of Pathology, P.O. Box 63, Biomedicum Helsinki, University of Helsinki, FIN-00014 Helsinki, Finland. Tel.: 358-9-191-25566; Fax: 358-9-191-25573; E-mail: Jorma.Keski-Oja{at}Helsinki.fi.
1 The abbreviations used are: LTBP, latent TGF--binding protein; TGF, transforming growth factor; ECM, extracellular matrix; LAP, latency-associated peptide; BSA, bovine serum albumin; MEM, minimal essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; mAb, monoclonal antibody; TRITC, tetramethylrhodamine isothiocyanate.
2 M. Hyytiäinen, P. Vehviläinen, and J. Keski-Oja, unpublished data.
3 M. Hyytiäinen and J. Keski-Oja, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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