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EXTRACELLULAR MATRIX, CELL INTERACTIONS
1Institute of Biomedicine/Anatomy, University of Helsinki, Helsinki, Finland; 2Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo, Japan; 3Laboratory of Electronics Production Technology, Helsinki University of Technology, Espoo, Finland; 4Renal Division, Washington University School of Medicine, St. Louis, Missouri; and 5Institut de Biologie et Chimie des Protéines, Unité Mixte de Recherche 5086, Institut Fédératif de Recherche 128 BioSciences Lyon-Gerland, Lyon, France
Submitted 14 June 2005 ; accepted in final form 11 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of 5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin 5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed Mr 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins 
1 and v3, whereas in the adhesion to laminin-10/11, Lu and integrin 1 were involved. In the cells adhering to the 5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that 5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to 5 laminins in collaboration with integrins 1 and v3. integrin; cycloheximide
Basement membranes (BMs) are specialized sheets of ECM found in intimate contact with endothelia and epithelia, as well as with certain individual cells, such as adipose, muscle, and Schwann cells. In addition to the function of BMs as structural scaffolds for cells and tissues, the components of BMs are ligands for cell surface receptors and have effects on the adhesion, differentiation, migration, proliferation, and survival of the cells. Laminins (Ln), which belong to the main constituents of BMs, are a family of heterotrimeric molecules, each composed of -, -, and 
-chains. To date, five -, three -, and three -chains have been identified, and they form at least 15 laminin isoforms. All laminin chains share structural similarities, but the laminin -chains in particular possess many receptor binding sites, are differently recognized by the cells, and are expressed in a tissue-specific and developmentally regulated manner (6, 38, 47).
Laminin 5-chain is a component of Ln-10 (511), Ln-11 (521), and the recently identified Ln-15 (523) (6, 38). Ln-10 has the broadest expression pattern of laminins and is a constituent of most BMs of fetal and adult tissues (34, 37, 54). Ln-11 has a more restricted distribution and is expressed, for example, in the glomerular BM of kidney, neuromuscular synaptic cleft, and BMs of arterial smooth muscle cells (36). The laminin 5-chain is produced by rodent endothelial cells and found in endothelial BMs (37, 54). Lack of the laminin 5-chain in knockout mice leads to several developmental defects, including defective vascularization of placenta and kidney glomeruli (33, 35), which suggests an important role for this laminin chain in endothelial development and function.
The effects of laminins on cell behavior are mediated by cell surface receptors. Among the integrins, 21 (48), 31 (26, 56), 61 (25, 56), 64 (25, 28), and v3 (18, 51) have been reported to mediate the adhesion of various cell types to 5 laminins. Also, dystroglycan glycoprotein complex binds to laminin 5-chain (28, 67).
Lutheran blood group glycoproteins (Lu) have recently been shown to function as adhesion receptors specific to the laminin 5-chain (24, 43). They include two proteins of 78 and 85 kDa (7, 46), which are products of alternatively spliced RNA transcripts of a single gene (11, 44, 50). The smaller protein is also known as basal cell adhesion molecule (1, 16). The role of Lu as a laminin receptor was recognized in studies of normal and sickle red blood cells (10, 60, 68), Lu-transfected human erythroleukemia cells (10, 43), and Lu-transfected murine fibroblasts (10). Kikkawa et al. (27) recently proposed that Lu alone would be unable to mediate the adhesion of human mesangial cells to Ln-10/11.
Only a few studies have focused on the interaction of human endothelial cells and 5 laminins (9, 15). These studies have evaluated the role of integrins in this interaction, and the role of other potential receptors, such as Lu, has remained elusive. In this study we evaluated the roles of 5 laminins and Lu in the adhesion of human endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Northern blot analysis. Total RNA was isolated from HDME and HPAE cells by acid phenol-guanidinium thiocyanate-chloroform extraction using standard methods (5). Poly(A)+ RNAs were enriched using DynaBeads oligo(dT)25 beads (Dynal, Oslo, Norway) according to the manufacturer's instructions. The RNA was then separated according to size in a denaturing 1.2% agarose gel and transferred onto Hybond membranes (Amersham Biosciences, Uppsala, Sweden) by upward capillary transfer. The Northern hybridization was performed using the DIG High Prime DNA labeling and detection starter kit II (Roche, Mannheim, Germany) according to the manufacturer's instructions. A cDNA expression plasmid containing the full-length human Lu coding region was purchased from Invitrogen (Carlsbad, CA) and modified as previously described (24). A 700-bp probe was created by restriction with SmaI (Promega, Madison, WI). Prehybridization was carried out at 55°C for 30 min, and the hybridization was carried out at 55°C for 24 h. The blots were exposed to Hyperfilm MP (Amersham Biosciences). As size markers, a 0.24- to 0.5-kb RNA ladder (Invitrogen) and a 0.28- to 6.58-kb RNA marker (Promega) were used.
Western blot analysis. For Western blot analysis, samples of HPAE and HDME cells were made by boiling the detached cells in nonreducing sample buffer. SDS-PAGE was performed according to Laemmli's procedure with 8% gels. The proteins were transferred onto nitrocellulose filters and blocked with 5% dry milk in PBS. The filters were exposed to MAb BRIC221 against Lu (Serotec, Oxford, UK). Immunoreactive bands were visualized using peroxidase-coupled goat anti-mouse IgG (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) with nickel intensification and diaminobenzidine as substrate (Sigma, St. Louis, MO) according to the manufacturer's instructions. Sigmamarker with high-molecular-weight range (Sigma) was used as a size marker.
Radioactive metabolic labeling and fluorography.
Endothelial cell cultures were incubated in methionine-deficient culture medium with or without cycloheximide (10 µg/ml; Sigma) for 1 h. Next, 25 µCi/ml S35-labeled methionine (Amersham Biosciences) was added to the culture medium, and the cells were incubated for 2–3 h. The culture medium of the cells was collected, cleared by centrifugation, supplemented with normal mouse serum and 0.5% Triton X-100, preabsorbed with uncoupled GammaBind Plus Sepharose beads, and applied to the GammaBind Plus Sepharose beads precoupled with MAb 4C7 to laminin 5-chain (12, 58) or with MAb 52DH1 to fibronectin (61). The bound proteins were eluted with Laemmli's sample buffer, and SDS-PAGE was performed according to Laemmli's procedure with 5% gels under reducing conditions. Fluorography was performed using Hyperfilm MP (Amersham Biosciences) according to standard methods.
Quantitative cell adhesion assay.
Quantitative cell adhesion assays were performed using a method based on intracellular acid phosphatase (49, 56). Human Ln-10 was purified from the culture medium of PANC-1 pancreatic adenocarcinoma cells by using immunoaffinity chromatography as previously described (56). Human placental Ln-10/11 and mouse Ln-1 from Engelbreth-Holm-Swarm tumor (EHS-Ln) were obtained from Sigma. Human Ln-5 was immunopurified from the culture medium of SCC25 cells as previously described (62). Fibronectin was purified from outdated human plasma (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) by performing gelatin-Sepharose affinity chromatography (Amersham Biosciences) according to the method of Engvall and Ruoslahti (13). The following function-blocking MAbs to integrins were used: PIE6 to integrin 2-subunit (Chemicon, Temecula, CA), PIB5 (Chemicon) and 3G8 (Ref. 25; a kind gift from Prof. K. Sekiguchi, Institute for Protein Research, Osaka University, Osaka, Japan) to integrin 3-subunit, GoH3 to integrin 6-subunit (Chemicon), LM609 to integrin v3-subunit (Chemicon), and 13 to integrin 1-subunit (Ref. 65; kindly provided by Prof. K. M. Yamada, Craniofacial and Developmental Biology and Regeneration Branch, National Institute for Dental and Craniofacial Research, Bethesda, MD). The concentration of the MAbs in the experiments was 2 µg/ml, with the exception of MAb 3G8, which was used at a concentration of 6 µg/ml.
Wells of 96-well plates were coated with laminins or fibronectin (4 µg/ml) at room temperature (RT) for 1 h and washed twice with PBS. Thereafter, part of the wells was exposed to soluble recombinant protein (10 µg/ml in PBS) corresponding to the extracellular domain of Lu (Sol-Lu; Ref. 24) at RT for 1 h, whereas part of the wells was exposed only to PBS. The wells were then washed twice with PBS, subsequently treated with 3% BSA in PBS at RT for 1 h, and washed again twice with PBS. Cycloheximide was added (10 µg/ml) to the culture medium of the cells 1 h before the cells were plated, as well as to the adhesion medium (EGM-2 or EGM-2MV without fetal calf serum). The cells were detached with trypsin and EDTA, exposed to trypsin-neutralizing solution (PromoCell), and washed with the adhesion medium. Thereafter, the function-blocking MAbs were added to the cell suspensions. The cells were plated at 2 x 104 cells/well, and the plates were incubated at 37°C in 5% CO2 for 1 h. The wells were carefully washed to remove nonadherent cells, whereas the control wells showing the amount of the cells originally plated were not washed. The plate was centrifuged with a Hermle Z 400 K centrifuge (Hermle Labortechnik, Wehingen, Germany) at 500 rpm for 5 min to minimize loss of the cells from the control wells, showing the amount of cells plated, and the adhesion media of these wells were carefully removed. Substrate solution (Sigma 104 phosphatase substrate, 6 mg/ml in 50 mM sodium acetate buffer with 0.1% Triton-X100, pH 5) was added to each well, and the plates were incubated at 37°C for 1 h. The reaction was stopped with 1 M NaOH, and the absorbances were measured at 405 nm in an ELISA reader. BSA-coated wells were used as controls showing unspecific adhesion. Experiments were performed in triplicate, and the amount of adhered cells is expressed as a percentage of the cells originally plated (±SD of 3 wells). The difference between two variables was tested with a two-sided, unpaired t-test with a significance level of = 0.05.
Morphological adhesion assays. For visualization of the morphology and adhesion structure formation of the adhering cells, we also performed morphological adhesion assays. For this purpose, cell culture dishes with glass coverslips were coated with Ln-10, Ln-10/11, or Ln-10 combined with fibronectin at RT for 1 h. After two washes with PBS, the dishes were subsequently coated with 3% BSA in PBS at RT for 1 h and washed again twice with PBS. The cells were detached with trypsin and EDTA, exposed to trypsin-neutralizing solution, washed with the adhesion medium (EGM-2 or EGM-2MV without fetal calf serum), plated onto the coverslips in the adhesion medium, and incubated at 37°C in 5% CO2 for 2 h. For certain experiments, cycloheximide (10 µg/ml) was added to the culture medium 1 h before the cells were plated, as well as to the adhesion medium. The nonadherent cells were removed by washing the samples twice carefully with PBS, and the morphology of the cells was visualized with either field emission scanning electron microscopy (FESEM) or indirect immunofluorescence microscopy.
Field emission scanning electron microscopy. For FESEM, the cells cultured on glass coverslips were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, at RT for 45 min. The specimens were coated with 20 nm of chromium with Emitech K575X sputter, and the cells were studied with a Jeol JSM 6335F microscope at a 5-kV operating voltage and 40° inclination.
For comparison of the cell morphology on different adhesion substrata, the adhered cells were counted in three independent visual fields and divided according to their morphology into three groups of nonspread (round), moderately spread (protrusion forming), and well-spread (lamellipodia forming) cells. The results for each group are expressed as percentages of the all adhered cells (±SD of 3 visual fields).
Immunofluorescence microscopy.
For indirect immunofluorescence microscopy, the following antibodies were used: MAb 102DF5 to integrin 1-subunit (66), MAb LM142.69 to integrin v-subunit (Ref. 4; a kind gift from Prof. D. A. Cheresh, Scripps Clinic and Research Foundation, La Jolla, CA), rat MAb BIE5 to integrin 5-subunit (Ref. 64; a kind gift from Prof. Z. Werb, Department of Anatomy, University of California, San Francisco, CA), MAb BRIC221 to Lu (Serotec), MAb TA205 to talin (Serotec), MAb FB11 to vinculin (Biohit, Helsinki, Finland), rabbit antiserum to vinculin (30), MAb 52DH1 to fibronectin (61), and rabbit antiserum to fibronectin (Dako, Glostrup, Denmark).
HPAE and HDME cells cultured on glass coverslips were fixed in methanol at –20°C for 15 min. The specimens were first exposed to MAbs at RT for 30 min, followed by either FITC-coupled goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA) or Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes, Eugene, OR) at RT for 30 min. For double-labeling experiments, the specimens were subsequently exposed to rat MAbs or rabbit polyclonal antisera, followed by tetramethylrhodamine isothiocyanate (TRITC)-coupled goat anti-rat IgG (Jackson Immunoresearch), TRITC-coupled goat anti-rabbit IgG (Jackson Immunoresearch), or Alexa Fluor 594 goat anti-rabbit IgG (Molecular Probes). The specimens were embedded in sodium veronal-glycerol buffer (1:1, pH 8.4) and examined using a Leica Aristoplan microscope equipped with appropriate filters.
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RESULTS |
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In a quantitative cell adhesion assay, 50% of the plated cells adhered to Ln-10 and 35% adhered to Ln-10/11, whereas 70% adhered to fibronectin (Fig. 1A). The morphology of the attached cells was visualized using FESEM. To compare the adhesion-promoting properties of the different substrata, we counted the attached cells in three individual visual fields in each sample and divided them into three groups of nonspread (round), moderately spread (protrusion forming), and well-spread (lamellipodia forming) cells (Fig. 1B). On Ln-10, 15% of the adhered cells did not spread and showed a round morphology and 75% of the cells spread moderately (the cell body was round, and cells formed multiple protrusions), whereas 10% of the cells spread well and showed some lamellipodia (Fig. 1, B and C). On fibronectin, 10% of the adhered cells showed a round morphology and 60% of the cells spread moderately and formed multiple protrusions, whereas 30% of the cells spread well and formed some lamellipodia (Fig. 1, B and D).
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5 (Fig. 2C) in colocalization in fibrillar structures in the center of the cells. On the other hand, within 2 h of adhesion to Ln-10, the cells showed diffuse cytoplasmic immunoreactivity for vinculin (Fig. 2D). Immunoreactivities for talin (Fig. 2E) and fibronectin (Fig. 2F) were colocalized in fibrillar structures in the cells.
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220,000 (Fig. 3A), corresponding to the size of fibronectin (61), and immunoprecipitation with MAb 4C7 showed that the cells synthesized two polypeptides of Mr 380,000 and 390,000 (Fig. 3B), corresponding to laminin 5-chain (2). Exposure of the cells to cycloheximide prevented the synthesis of the proteins (Fig. 3, A and B).
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5 laminins, endothelial cells express integrins 21, 31, 61, and v3 (8, 9). Garin-Chesa et al. (16) suggested that human umbilical vein endothelial cells express a Lu isoform of Mr 90,000. Before evaluating the role of these receptors in the adhesion of endothelial cells to Ln-10, we studied whether HPAE and HDME cells express Lu.
Northern blot analysis of HPAE (Fig. 6A) and HDME (not shown) cells showed two Lu transcripts of 2.5 and 4.0 kb, of which the smaller one was more pronounced than the larger one. Western blot analysis of endothelial cell lysates with MAb BRIC 221 to Lu showed two polypeptides of Mr 78,000 and 85,000 (Fig. 6B), of which the larger one was more prominent than the smaller one. In immunohistochemistry of overnight cultures of HPAE (Fig. 6C) and HDME cells (Fig. 6D), Lu showed a uniform punctate immunoreactivity on the cells.
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5 chain (27). The specificity of the inhibitory effect of Sol-Lu in the assays was tested using fibronectin, Ln-5 (332), and EHS-Ln (111) as adhesion substrates. The cells adhered to these proteins, but the adhesion was not inhibited substantially with the preincubation of the proteins with Sol-Lu (Fig. 7, A–D).
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1, the adhesion diminished to 15%, whereas with the combination of Sol-Lu and MAb to integrin 1, the adhesion was <10%. With the combination of Sol-Lu and MAb to integrin v3, the adhesion was <5%. On the other hand, in the adhesion of HDME cells (Fig. 7D), 30% of the plated cells adhered to Ln-10. With Sol-Lu, the adhesion decreased to 10%. With MAb to integrin 1, the adhesion was diminished to 5%, whereas with the combination of Sol-Lu and MAb to integrin 1, as well as with Sol-Lu and MAb to integrin v3, the adhesion was even less. The inhibitory effects of MAbs to integrins 2 (not shown), 3, and 6 (Fig. 7, C and D) were negligible as single agents, as well as in various combinations (not shown).
Because of the limited availability of native human Ln-10, a mixture of Ln-10 and -11, produced by pepsin digestion from human placenta, has been used as adhesion substrate in many cell adhesion studies. For comparison, we also performed the quantitative cell adhesion experiments with the mixture Ln-10/11. In the adhesion of HPAE cells, 45% of the plated cells adhered to this substrate (Fig. 8A). With Sol-Lu, the adhesion diminished to 35%. With MAb to integrin 1, the adhesion diminished to 30%, and with the combination of Sol-Lu and MAb to integrin 1, the adhesion was <20%. On the other hand, 35% of the plated HDME cells adhered to Ln-10/11 (Fig. 8B). With Sol-Lu, the adhesion diminished to 25%. With MAb to integrin 1, the adhesion was 10%. When combined with Sol-Lu, the inhibitory effect of MAb to integrin 1 did not increase substantially. MAbs to integrins 2 (not shown), 3, 6, and v3 (Fig. 8, A and B) had smaller inhibitory effects on the adhesion as single agents, as well as in various combinations (not shown).
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31 (9, 15). We repeated the experiments, shown above with the MAb PIB5 to integrin 3, using the function-blocking MAb 3G8 to integrin 3, but it did not inhibit the cell adhesion to Ln-10 or Ln-10/11 alone or in combination with Sol-Lu (not shown). Therefore, using quantitative cell adhesion assay, we also studied whether the endothelial cells have a functional 3-integrin in their adhesion to native human Ln-5. Of the plated HDME cells, 20% adhered to this laminin. With the MAb PIB5 to integrin 3, the adhesion diminished to 10%, and MAb to integrin 1 prevented the adhesion totally (Fig. 8C).
The distributions of Lu and integrins v and 1 in the endothelial cells adhering to Ln-10 were studied using immunofluorescence microscopy. In the absence of cycloheximide, immunoreactivity for integrin v was uniformly distributed on most of the cells, but in some cells it was located to tiny fibrillar structures (Fig. 9A). Immunoreactivity for integrin 1 was located to small fibrillar structures, which often appeared in circular formation near the periphery of the cells (Fig. 9B). Immunoreactivity for Lu had a uniform punctate distribution on the endothelial cells (Fig. 9C). In the presence of cycloheximide, immunoreactivities for integrins v (Fig. 9D) and 1 (Fig. 9E) were diffusely distributed on the cells, and immunoreactivity for Lu showed a uniform punctate distribution on the cells (Fig. 9F).
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DISCUSSION |
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5 laminins in the adhesion of endothelial cells, we first studied the morphology and adhesion structure formation of these cells in their adhesion to Ln-10. The results show that in overnight cultures, endothelial cells formed typical nail-like focal adhesions immunoreactive for vinculin, as well as fibrillar adhesions immunoreactive for fibronectin and integrin 5. In their 2-h adhesion to Ln-10, on the other hand, the cells spread and formed tiny fibrillar adhesions (also known as ECM adhesions) immunoreactive for talin and fibronectin, which are related to the reorganization of ECM fibronectin into fibronectin fibrils (17), but no typical focal adhesions, which are considered to be devoid of fibronectin (3).
The fibronectin immunoreactivity of the adhesion structures formed in the adhesion to Ln-10 suggested to us that the cells produced endogenous proteins that they could use as adhesion substrates during the adhesion assay. The immunoprecipitation results supported these findings by showing that the cells secreted within 2–3 h were ECM proteins such as fibronectin and laminin 5-chain, and the production of these proteins could be prevented with cycloheximide. To avoid the effects of these endogenous proteins on the results of adhesion assays, we performed the assays in the presence of cycloheximide. Under these conditions, the cells spread on Ln-10 but formed neither fibrillar nor focal adhesions.
By definition (17, 40), cell adhesion in the absence of cell spreading and focal adhesion formation indicates weak adhesion, a transient phase of attachment failing to support cell survival, whereas adhesion and cell spreading without focal adhesion formation indicates intermediate adhesion considered to support cell survival and motility. Firm adhesion correlates with formation of focal adhesions and actin stress fibers and is crucial for survival, growth, and maintenance of the differentiated phenotype of anchorage-dependent cells. Our findings show that Ln-10 stimulates endothelial cell spreading without focal adhesion formation, suggesting that instead of anchoring the cells firmly to the substratum, this laminin retains the motile phenotype of the cells.
Some immunohistochemical studies have suggested that Lu is found in blood vessel walls (45, 52), and Moulson et al. (39) showed that it is located around the smooth muscle cells of blood vessels. Garin-Chesa et al. (16) proposed that human umbilical vein endothelial cells produce a Mr 90,000 isoform of Lu. In the present study Northern and Western blot analysis showed that the endothelial cells produced two RNA transcripts of 2.5 and 4.0 kb and two protein isoforms of Mr 78,000 and 85,000, which correspond to the Lu isoforms detected in human red blood cells, tumor cells, and many human tissues (7, 46, 50). The long-tail isoform of Lu, which is encoded by the smaller, 2.5-kb transcript, was predominantly expressed by the cells. Immunofluorescence microscopy showed that Lu presented a punctate distribution on the endothelial cells, which did not resemble the distribution of any of the known adhesion structures.
Because function-blocking antibodies to Lu, suitable for our experimental setup, were unavailable, the role of Lu in endothelial cell adhesion was studied by inhibition of Lu function by saturation of the Lu-binding sites of laminin 5-chain with recombinant protein Sol-Lu as described previously (27). Although Sol-Lu clearly inhibited endothelial cell adhesion to Ln-10, it did not inhibit adhesion to fibronectin, EHS-Ln, or Ln-5, suggesting that the inhibitory effect of Sol-Lu in the adhesion assay was specific for the laminin 5-chain.
Compared with the adhesion without any function-blocking MAbs or Sol-Lu, Sol-Lu alone inhibited the endothelial cell adhesion to Ln-10 by 60–70%. MAb to integrin 1 inhibited the adhesion by 65–85%, whereas with the combination of Sol-Lu and MAb to integrin 1, the adhesion was nearly abolished. These findings suggest that endothelial cell adhesion to Ln-10 requires both Lu and integrin 1. Interestingly, MAb to integrin v3 alone had no inhibitory effect on the adhesion, but in combination with Sol-Lu, it prevented the cell adhesion to Ln-10 almost completely. The inability of MAb to integrin v3 alone to inhibit the adhesion suggests that this integrin is not crucial for endothelial cell adhesion to Ln-10. It could, for example, partially replace the function of primary adhesion receptors Lu and integrin 1, if the function of either one of them is prevented. Another explanation for this phenomenon could be the trans-dominant inhibition of other adhesion receptors, such as integrin 1 via integrin v3 as previously suggested by Hynes (20).
Fujiwara et al. (15) recently suggested that human microvascular endothelial cells form focal adhesions on Ln-10/11. The likely reason for the discrepancy with our results is the production of endogenous proteins during the adhesion assays, which they did not take into consideration. We showed that the effects of these endogenous proteins on the results of the adhesion assays could be prevented with the use of cycloheximide. However, this raises the question of whether cycloheximide prevents the formation of focal adhesions. Although some previous studies about the effects of cycloheximide on the focal adhesion formation have remained controversial (22, 57), our results show that in the presence of cycloheximide, the addition of fibronectin to Ln-10 coat induced typical focal adhesions, indicating that cycloheximide did not prevent the formation of these adhesion structures.
Furthermore, Fujiwara et al. (15) showed that the spreading of human microvascular endothelial cells on Ln-10/11 was completely inhibited with function-blocking MAb to integrin 1. Of the function-blocking MAbs to -subunits, MAb 3G8 to integrin 3 had the strongest effect by inhibiting the cell spreading by 40%. Doi et al. (9) studied the adhesion of human saphenous vein endothelial cells and immortalized mouse brain capillary endothelial cells to recombinant human Ln-10. Of the function-blocking MAbs, MAb PIB5 to integrin 3 and MAb to integrin 1 had the best, but only partial, inhibitory effects on the adhesion. Although in neither of these studies did MAbs to integrin 3 inhibit the adhesion completely, the investigators concluded that the adhesion of human endothelial cells to 5 laminins was most probably mediated by integrin 31.
Our results regarding the role of integrin 1 are in agreement with the aforementioned studies (9, 15); however, although we used the same two MAbs to integrin 3 (PIB5 and 3G8), our results did not support the suggested primary role for integrin 3 in the adhesion of the endothelial cells to Ln-10. Although integrin 31 appears to be the primary receptor for Ln-10 in some cell types (26, 56), the binding specificity of certain integrins in endothelial cells differs from their specificity in other cell types (29). The binding specificity of an integrin depends on the amount of its expression, activation state, and interaction with other proteins such as CD151 (19, 21, 41). Therefore, we studied whether the 31-integrin is functional in our cells. Ln-5 is one of the high-affinity ligands for integrin 31 (59), and some studies have proposed that this laminin is located to BMs of some capillaries (32, 63), suggesting that human microvascular endothelial cells could interact with Ln-5 via this receptor. The results show that the adhesion of HDME cells to human Ln-5 is clearly reduced with MAb PIB5 to integrin 3, indicating that these cells have a functional 3-integrin.
Because the endothelial cells of capillaries live in a microenvironment different from that of the endothelial cells of larger vessels, we performed the adhesion experiments with both HDME and HPAE cells. The MAb to integrin 1 clearly inhibited the adhesion of HDME cells better than that of HPAE cells, and the inhibitory effect of Sol-Lu was more pronounced with HPAE cells than with HDME cells, suggesting that even the microvascular and pulmonary artery endothelial cells differ to some extent in their adhesion characteristics.
The MAbs to both integrin 1 and Sol-Lu had more pronounced effects on the cell adhesion to native Ln-10 than to the Ln-10/11 preparation. Furthermore, the combination of Sol-Lu and MAb to integrin v3 did not inhibit the cell adhesion to Ln-10/11, as it inhibited the adhesion to native Ln-10, suggesting that experiments performed with the mixture of Ln-10/11 are not fully comparable to the experiments performed with native Ln-10. The commercially available Ln-10/11 is produced by pepsin digestion and contains partially degraded Ln-10 and -11 (14, 25, 53). The proteolysis can expose masked receptor binding sites and can therefore lead to erroneous results.
In conclusion, the findings show that Ln-10 is an adhesion substrate of endothelial cells but does not stimulate focal adhesion formation. The adhesion to Ln-10 is mediated by Lu together with integrins 1 and v3. Lu has previously been shown to mediate the adhesion of normal and sickle red blood cells, Lu-transfected human erythroleukemia cells, and Lu-transfected murine fibroblasts (10, 43, 60, 68). On the basis of a study of human mesangial cells, Kikkawa et al. (27) recently suggested that Lu alone would be unable to mediate the adhesion of adherent human cells. Our findings show that Sol-Lu alone markedly inhibited the adhesion of human endothelial cells to Ln-10, suggesting a role for Lu in the adhesion of adherent human cells. Moreover, the findings suggest interplay between Lu- and integrin-mediated adhesion processes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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