Cell migration—The role of integrin glycosylation

Biochimica et Biophysica Acta 1800 (2010) 545–555

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n

Review

Cell migration—The role of integrin glycosylation Marcelina E. Janik a,⁎, Anna Lityńska a, Pierre Vereecken b,c,d,e a

Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Krakow, Poland Department of Dermatology, CHU-Brugmann, Belgium c Department of Dermatology, Erasme Hospital, Belgium d Department of Medical Oncology, Jules Bordet Institute, Brussels, Belgium e Laboratiore d'Oncologie Chirurgicale et Expérimentale (LOCE), Bordet Institute, Belgium b

a r t i c l e

i n f o

Article history: Received 22 January 2010 Received in revised form 11 March 2010 Accepted 17 March 2010 Available online 20 March 2010 Keywords: Migration α3β1 integrin α5β1 integrin αvβ3 integrin N-oligosaccharides

a b s t r a c t Background: Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell–ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding. Scope of review: The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation. General significance: A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized. © 2010 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: APC protein, adenomatous polyposis coli protein; ATRA, all-trans retinoic acid; BM, basal membrane; CMP-NeuAc, cytidine monophosphate-sialic acid; Csk, C-terminal Src kinase; ECM, extracellular matrix; EGF, epidermal growth factor; ER, endoplasmic reticulum; ERK, extracellular signal regulated kinase; FA, focal adhesion; FAK, focal adhesion kinase; FN, fibronectin; Fut8, α1,6-fucosyltransferase; Gal, galactose; GlcNAc, N-acetylglucosamine; GnT, N-acetylglucosaminyltransferase; GSK3β, glycogen synthase kinase-3β; GTP, guanosine triphosphate; HGF, hepatocyte growth factor; ILK, integrin linked kinase; JEB, junctional epidermolysis bullosa; JNK, c-Jun protein kinase; LEF-1, lymphoid enhancer-binding factor 1; LN-332, laminin 332; MAA, Maackia amurensis agglutinin; Man, mannose; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; MT1-MMP, membrane type 1 matrix metalloproteinase; p130Cas, Crk-associated substrate; PAK, p21 activated kinase; PHA-L, Phaseolus vulgaris leucoagglutinin; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PYK2, proline-rich tyrosine kinase-2; RGD, arginine-glycin-aspartic acid; RTK, receptor tyrosine kinase; SNA, Sambucus nigra lectin; ST6GalI, α2-6 sialyltransferase I; TCF, T-cell factor; TGF-β1, transforming growth factor-β1; VN, vitronectin ⁎ Corresponding author. Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, ul. Ingardena 6, 30-060 Krakow, Poland. Fax: + 48 12 634 37 16. E-mail address: [email protected] (M.E. Janik). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.013

Cell migration plays an essential role in a wide variety of biological phenomena. Cell migration is involved in normal as well as pathological events. In the adult, cell migration is essential in homeostatic processes such as the immune response and repair of injured tissues [1]. Skin is renewed continuously from precursors which migrate from the basal layer; leukocytes migrate from the circulation into the surrounding tissues to ingest bacteria. The pathological processes to which migration can contribute include vascular disease, chronic inflammatory diseases and tumor metastasis. Tumor development is accompanied by the formation of blood vessels which arise from proliferation and migration of their endothelium. In metastatic cancer some tumor cells acquire the ability to migrate out of the primary tumor to a distant organ where they form secondary tumors [2]. Understanding the molecular mechanisms that govern metastatic progression may lead to new therapeutic approaches targeting adhesion molecules and the cancer microenvironment [3]. Among them are growth factor signaling molecules, chemokines [4], cell–cell adhesion molecules (cadherins, integrins) [5,6] and extracellular proteases (matrix metalloproteinases) [7–9].

546

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

2. Phases of cell migration process Cell migration is a very complicated process requiring precise regulation and integration of multiple signaling pathways [10,11]. Cell migration is a dynamic and cyclical process. It starts from polarization (a clear distinction between the cell front and rear) and the formation of a protrusion in the direction of movement [12]. Next, the adhesion receptors bind to the ECM ligands, forming links to the cytoskeleton. These sites of cell–ECM attachment serve as traction points for migration but also stabilize the protrusion via structural connections to actin filaments, and they mediate signaling by the Rho family of GTPases, ERK/MAP kinases and other regulatory molecules. Finally, the cells contract from the edges toward the nucleus and the adhesion receptors are released from the cytoskeleton, detaching the cell from the previous attachment sites [2,7,13,14]; or else detachment is associated with the release of cell vesicles and fragments including their plasma membrane, cell surface constituents/receptors and cytoplasm [15]. 3. Cell adhesion receptors in migration—integrins Cell adhesion receptors play important roles in promoting migration. The establishment of strong adhesion inhibits migration, which is fastest at optimum adhesion strength: strong enough to support traction but weak enough to allow rapid detachment of the rear of the cell. This modulation of adhesion strength is realized by a wide spectrum of adhesion molecules present in a cellular membrane. Many of the known adhesion receptors belong to the integrins, a major family of cell–cell and cell–ECM adhesion proteins [16]. In addition to adhesive function, integrins are also involved in intracellular signaling and in regulation of cytoskeletal formation [17,18]. The integrins have been the subject of intense study because their expression is up-regulated in migratory cells and their activities have been linked to physiological differentiation of cells [19] and pathological processes such as tumor metastasis [20–22]. Each integrin consists of non-covalently linked α and β subunits. Integrin subunits are composed of an extracellular domain, a single membrane-spanning domain, and a short cytoplasmic tail (Fig. 1). The extracellular part of the subunit consist of three β-sandwich domains – one known as an Ig-like “thigh”, and another two, very similar β-sandwiches, known as “calf” [23,24]. In total there are 18 α and 8 β subunits [25], giving rise to 24 distinct integrin molecules. They all differ in substrate specificity [17,26], as revealed by knockout experiments [27,28]. The ligand-

Fig. 1. Domain structure of integrin α and β subunits: based on literature: [23,26,76].

binding site of integrins is present in the globular head domain of the extracellular part of the molecule [24,29] and is formed by both integrin subunits. In α subunits the seven repeat motifs fold into a propeller structure which forms a globular domain with the ligand binding site on the upper surface. Several integrin α subunits contain an insertion of 200 aas known as the I-domain, which is capable of binding ligands and plays an important role in ligand binding by intact integrins [30]. A similar domain called the I-like domain is present in the extracellular part of the β subunit. Both of these I-domains take part in ligand binding by integrins [31,32]. The transmembrane helices of both α and β subunits are thought to help stabilize heterodimer formation, but the interactions do not specifically associate particular pairs of α and β subunits; they rather constrain the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association [33], while the cytoplasmic tail interacts with intracellular protein complexes – focal adhesions (FAs) [34,35]. FAs control the linkage between the cytoskeleton and integrins, and mediate intracellular signaling. Integrins direct the forces generated by the cytoskeleton onto the ECM to produce the traction necessary for cell adhesion and migration [36]. In general, integrins recognize short linear peptide sequences on adhesion proteins, the most prevalent being arginine-glycin-aspartic acid (RGD), which is found in many ECM proteins such as fibronectin, collagens and vitronectin. For this reason the integrins were classified as RGD-dependent and RGD-independent. Ligand binding to the extracellular integrin domain induces conformational changes and integrin clustering for activation of signaling cascades and recruitment of multiprotein complexes to FAs. Focal adhesions are dynamic structures which assemble, disperse and recycle during cell migration. They transmit force or tension to maintain strong attachment to the ECM and act as signaling centers regulating many intracellular pathways of different cell functions. These multiprotein complexes, which consist of integrins, integrin-associated adaptors and signaling proteins, growth factor receptors and their related downstream targets [37,38], control various signaling pathways leading to diverse cell behaviors. Most of the proteins in FAs have several potential interaction partners. One of the signaling events occurring at FAs is tyrosine phosphorylation, which leads to the formation of docking sites for binding of SH2-containing proteins and regulates the subsequent activation of additional kinases and phosphatases. Focal adhesion kinase (FAK) and Src are two of the major kinases found in FAs, binding to different partners and regulating FA dynamics and behavior. Also found in FAs are other tyrosine kinases such as Ab1, Csk and PYK2, and Ser/Thr kinases such as ILK, PAK and PKC. In addition FAs contain several adaptor proteins such as p130Cas, paxillin and Crk. These molecules function as signaling scaffolds for components of FAs. The non-receptor bound tyrosine kinase FAK is involved in mediating both integrin and RTK signaling for regulation of adhesion, cell shape and cell motility [38,39]. The C-terminal region of FAK is responsible for binding of ILK and integrin-associated proteins like paxillin and talin, and for localization of FAK to integrins and FAs [40,41]. FAK activation occurs after external integrin, growth factor receptor or G-protein-linked stimulation, and starts with autophosphorylation at tyrosine 397 followed by the recruitment of Src-family kinases and the binding and phosphorylation of p130 Cas and paxillin [37]. The intracellular tail of the β subunit can interact with a number of protein kinases such as FAK, integrin-linked kinase (ILK), Src and protein kinase C (PKC) [42]. FA and FAK represent one of the possible mechanisms of integrin-mediated signaling events [43]. It leads to activation of ERK and cJun kinase (JNK) in the MAP kinase pathway, which itself is not sufficient to push the cell into the cell cycle but can prolong and intensify signaling from growth factor receptors. There is also evidence that integrin-mediated signaling modulates the activity of Rho/Rac/Cdc42 [44–46], small GTPase signaling molecules which are particularly important in regulation of the actin cytoskeleton [34] and which play a role in controlling cell motility [47,48]. Interestingly,

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

although the cell requires some adhesion to the ECM to generate traction, cell movement rates are maximal only at intermediate levels of adhesion. At low adhesion the traction generated is not enough to facilitate movement, whereas strong adhesion to the ECM effectively blocks any motility [49,50]. The situation is therefore optimal somewhere between the two states. To allow forward motion there should be some adhesion at the leading edge of the cell and the release of contact at the back of the cell. In this circumstance, integrins are critical in controlling adhesion to and release from the ECM. We see that integrins contribute in multiple ways to the process of cell migration. First, integrins’ affinity for the ECM can be modulated by intracellular signaling in a process called inside-out signaling. Second, activated integrins mobilize a multiprotein complex at the cell-substratum interface to generate a variety of intracellular signals in a process known as outside-in signaling [51,52]. Third, the bridge that integrins form between the ECM and the intracellular actin cytoskeleton allows cells to exert force on their environment [53,54]. Finally, the recycling of integrins in the plasma membrane takes part in cell motility, and above all is required in cells with relatively low levels of integrin expression [55]. Adhesion to a solid surface via integrin heterodimers is the first step in cell migration. It is well known that antibodies that interfere with integrin-ligand binding not only inhibit cell attachment to the ECM but also inhibit cell migration in vitro and reduce in vivo tumor growth [56]. Palecek et al. [50] established a mathematical relationship between the extent of cell adhesion and cell migration. They determined that the rate of cell migration could be plotted as an approximately bell-shaped curve versus the extent of cell adhesion. Cell–matrix interaction via integrins is essential for adhesion and migration [10,43]. Increased cellsubstratum adhesion is achieved by redistribution of β1 integrin receptors on the plasma membrane. When integrins are randomly diffused on the cell surface and are not well connected with the actin cytoskeleton, they are weakly bound to the ECM. The overall strength of cell adhesion is known to be regulated by either the affinity (interaction of a single integrin with its ligand) or the avidity (due to the association of the receptors in cell membrane) for a ligand. Changes in integrin affinity are the result of conformational changes in the heterodimer, while avidity depends on their arrangement and ability to move (either actively or passively by diffusion) from another part of the cell into the zone of cell adhesion [28,57]. After binding with the ligand and subsequent integrin clustering, the intracellular protein kinase cascade is activated, starting with autophosphorylation of FAK. Cell migration is a process requiring precise regulation of integrin-mediated adhesion and its release [2]. In a migrating cell, integrins are well suited to allow firm attachment at the front and fast detachment at the rear. The integrin-dependent mechanisms by which these opposite processes are regulated at the same time within cells are just about to be disclosed [58]. Glycosylation of integrins, an underappreciated regulatory mechanism, may well be found to participate in both these processes. Integrins are glycoproteins, and both their subunits are subjected to these post-translational modifications. 4. Glycosylation of proteins Glycosylation is one of the most frequent post-translational modifications of proteins [59]. Most cell surface receptors are glycosylated, so that heterogeneous glycan moieties decorate the extracellular regions of membrane-bound proteins. Biosynthesis of glycoprotein oligosaccharides is catalyzed by glycosyltransferases, tissue-specific enzymes which transfer a sugar residue from a nucleotide-sugar donor to an acceptor [60–62]. Oligosaccharides of glycoproteins, called glycans, are classified according to their linkage to the protein, as N-linked (through C-1 binding of N-acetylglucosamine to the amide side chain of an asparagine) or O-linked (through C-1 binding of N-acetylgalactosamine to the hydroxyl of threonine/

547

serine) [63,64]. The structural diversity of glycans is very wide and depends not only on the number and sequence of monomeric units but also on the position, anomeric configuration and branching of glycosidic units. All N-linked glycans contain the pentasaccharide Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc as the common core structure [65]. On the basis of the structure and location of oligosaccharides added to this core, N-glycans are classified into three groups: complex type, high-mannose type, and hybrid type. The complex type has the largest structural variation among the three N-glycan subgroups. This variation is due to the different numbers of outer chains linked to the core structure, resulting in the formation of mono-, di-, tri-, tetra- and penta-antennary glycans. The combination of different antennary structures and various outer chains is responsible for the large number of different complex type glycans. N-glycan processing in the Golgi involves the action of several glycosidases and glycosyltransferases, resulting in further glycan trimming and elaboration. The biosynthetic basis for such diversity is alteration of the activity of various transferases and competition between enzymes for acceptor intermediates during glycan elongation (Fig. 2). Branched glycans such as bisected GlcNAc or β1-6GlcNAc are enzymatic products of GlcNAc transferases: N-acetylglucosaminyltransferase III and V (GnT-III, GnT-V). These branched structures are highly associated with various biological functions including cell adhesion, migration and cancer metastasis [66–71]. Sialylation terminates the maturation of glycans and is performed by the sialyltransferases, a family of anabolic enzymes which transfer sialic acid from CMP-NeuAc to glycoconjugates. Sialic acids are nine-carbon monosaccharides linked to the terminal galactose, N-acetylglucosamine (GlcNAc), or another sialic acid in the carbohydrate chain. In N-linked glycoproteins, sialic acids are attached to Galβ1-(3)-4GlcNAc with α2-6 and α2-3 linkages. Because sialic acid bears a net negative charge at physiological pH [72] and is located terminally, sialic acid has the potential to inhibit interactions between molecules and cells [73]. 5. Ways in which glycosylation could affect the function of proteins Glycoproteins can contain both N-linked (Asn-linked) and O-linked (Ser/Thr-linked) glycans of variable length and composition. Complex, hybrid and high-mannose N-glycans are usually present in glycoproteins. Variations in the carbohydrate structure linked to individual sites are responsible for the heterogeneity of glycoprotein composition. It is generally accepted that glycans shield the protein surface and prevent nonspecific protein-protein interactions, providing protease protection and increasing glycoprotein stability. Some glycans have a great influence on the conformation of the receptor protein to which they are linked, modulating their ligand binding activity [26,70,74–79]. Glycosylation of proteins can affect their folding, intracellular trafficking, localization and rate of degradation. It also influences cell–cell interactions [80] as well as the interactions of cells with ECM proteins and soluble signaling molecules [81]. Due to their location, cell surface glycoconjugates contribute to a complex array of intercellular interactions, among them adhesion and migration [82]. In the case of integrins the oligosaccharides’ influence on protein conformation may also affect their activity. Integrins have been shown to be present in the cell membrane in an inactive state, requiring activation accompanied by conformational changes of the integrin dimer. This has been described as a switchblade-like motion of the headpiece, producing a highly extended, active integrin conformation [83,84]. Much experimental data on human and animal tumors have shown that malignant transformation of cells is associated with various and complex alterations of the protein glycosylation process [64,85,86]. Generally the most frequently occurring cancer-related changes in the glycosylation pattern include synthesis of highly

548

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

Fig. 2. Schema of glycosylation reactions. The first phase takes place in ER where oligosaccharide is trimmed by α-glucosidase I and II (αG I/II), and α1-2 mannosidase I (α1-2 Man I) ER. Then, in Golgi Apparatus, another α1-2 mannosidase I (α1-2 Man I-Golgi) continues trimming, forming high mannose type. In the next steps, oligosaccharide is processed by β-Nacetylglucosaminyltransferase I (GnT I) and III (GnT III) as well as α mannosidase II (α Man II) leading to hybrid and/or bisected type oligosaccharide. After that, the biosynthesis of N-glycan is govern by β-N-acetylglucosaminyltransferase II (GnT II), IV (GnT IV), V (GnT V), β1,4 galactosyltransferase (GalT) and α sialyltransferase (ST) or/and α fucosyltransferase (Fuc T) producing complex type glycans.

branched glycans elongated by poly-N-acetyllactosamine chains and highly sialylated, premature termination of biosynthesis, and reexpression of fetal-type antigens [64,85,87,88]. Expression of sialic acids, especially those α2-6-linked to terminal galactose, is an accepted hallmark of tumor malignancy [89–92]. The oligosaccharide chains of glycoproteins are essential for maintenance of the “social behavior” of cells, among them adhesion and migration; alterations of the glycans are the molecular basis for abnormal behavior of tumor cells, such as invasion and metastasis [93]. These changes provide a selective advantage for tumor cells during progression to a more invasive and metastatic phenotype [66,80,86,94,95]. Seales et al. [91] have shown that integrin sialylation is controlled by activation of ras oncogen and ras-dependent signal transduction cascades, implying that the expression of variant glycoforms is inducible and moreover that cells may synthesize variant glycoforms in response to extracellular agents that stimulate these signal transduction cascades. Ras-dependent up-regulated expression of sialyltransferase ST6Gal I – adding sialic acids in the α2-6 position as was observed in colon carcinoma [91] and in ovarian adenocarcinoma [92] – is correlated with increased tumor malignancy. The effect of hypersialylation was observed in the β1 but not in the β3 or β5 integrin subunit, leading to enhanced cell adhesion to collagens and laminins as well as elevated migration of cancer cells.

The presence of β1-6-branched complex type N-oligosaccharides is positively correlated with increased cancer cell motility and in turn their increasing malignancy [70,94,96–100]. It has been suggested that growth factor receptors with N-glycans modified by poly N-acetyllactosamine residues could be recognized by galectins, resulting in the formation of a lattice that opposes endocytosis. This would enhance intracellular signaling and consequently cell migration and tumor metastasis [101]. There are many examples illustrating that integrin function depends on its glycosylation and that variant glycosylation may be a regulatory mechanism for β1 integrins [70,74,102,103]. All integrins are prominent carriers of N-glycans on the cell surface as they contains over 20 potential glycosylation sites [26,102,104,105]. Among them, glycans of α3β1, α5β1 and αvβ3 have been studied. The majority of studies on integrin-mediated functions have used cells that adhere to fibronectin (FN) through the integrin α5β1, one of the bestcharacterized integrins. The association between α5β1 integrin and FN is involved in regulating not only cell adhesion and migration but also ECM remodeling [106]. Another integrin, α3β1, mediates adhesion to basement membrane laminins [107] and preferentially promotes cell migration [108]. The integrin αvβ3, a cellular receptor of adhesion to vitronectin, is engaged in angiogenesis [109] and cancer metastasis, and takes part in migration events [110,111].

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

549

6. Integrin α5β1—a glycoprotein participant in cell motility Because expression of variant β1 glycoforms is associated with significant changes in the cell phenotype, the potential role of glycosylation in regulating integrin function has drawn great interest. In particular, glycosylation of integrin α5β1 appears to be important to both its function and structure [26,70,76–78,112]. Integrin α5β1 contains 26 potential N-glycosylation sites, 14 in the α subunit (Fig. 3) and 12 in the β subunit (Fig. 4). The first findings suggested that the presence of N-glycans on integrin α5β1 is required for αβ heterodimer formation and also for proper interactions with the ECM. Treatment of purified α5β1 with N-glycanase F, which cleaves N-linked glycans from glycoproteins, resulted in the blocking of α5β1mediated binding to FN and inhibition of subunit association [113]. Also, it has been shown that the presence of N-oligosaccharides can modulate α5β1 integrin activation. In human fibroblasts cultured in the presence of N-linked oligosaccharide processing inhibitor, immature integrin α5β1 appeared at the cell surface, and FNdependent adhesion was greatly reduced [114]. Zhang et al. [115] demonstrated that when glycans are processed from high-mannose type to complex type they increase their ability to modulate cell adhesion and migration processes. In human hepatocarcinoma H7721 cell, differential processing of oligosaccharides attached to integrin α5β1 strongly influenced cell–FN and cell–HUVEC adhesion and migration [115]. Sialylation of α5β1 N-glycans has been shown to play an important role in cell adhesion. Hyposialylation of integrin β1 enhanced binding to FN by myeloid cells [73]. Similar results have been obtained in hematopoietic and epithelial cells. Increased sialylation of the β1 subunit was correlated with decreased adhesion [74,93,116]. In human melanoma cell line G361, however, oligosialic

Fig. 3. Domain structure and 14 potential N-glycosylation sites of human integrin α5 subunit. This is the best analyzed integrin subunit and there is a large body of evidence concerning the role of N-glycans for its structure and function.

Fig. 4. Domain structure and 12 potential N-glycosylation sites of human integrin β1 subunit.

acid on the α5 subunit was necessary for binding to FN [117]. Experiments have been designed to determine whether GnT-V overexpression can affect cell migration, assayed by wound healing on FN and by haptotaxis toward FN. Several research groups have reported that alterations in integrin α5β1 oligosaccharides, modulated by the expression of GnT-V, GnT-III and ST6GalI, regulate α5β1mediated cell migration and cell spreading [70,94,118,119]. GnT-V expression in lung epithelial cell MvLu caused decreased adhesion to FN and collagen type IV. At the same time these cells migrated rapidly into the scratch wound and had well developed microfilaments and FA [118]. Similarly, in human fibrosarcoma HT1080 cells, with increased GnT-V expression, Guo et al. [94] observed reduced α5β1 integrin clustering and increased cell migration. On the other hand, modification of N-glycan structure by overexpression of GnT-III blocked α5β1-mediated cell spreading, cell migration and FAK phosphorylation. The binding affinity of α5β1 for FN was significantly reduced after introduction of bisecting GlcNAc into the α5 subunit. These results strongly suggested that N-oligosaccharides attached to integrins play a crucial role in the conformational changes of integrins [70,119]. There is growing evidence suggesting that aberrant N-glycosylation of α5β1 integrin observed during cancer metastatic transformation leads to modulation of integrin-mediated functions [26]. As mentioned before, this could be a result of the influence of glycans on the activation state of integrins. In a work on the possible mechanism of glycans’ influence on integrin conformation, sequential side-directed mutagenesis was carried out to remove single or combined putative N-glycosylation sites on the α5 integrin subunit [76]. The absence of N-glycosylation sites 1–5 on the β-propeller resulted in persistent association of the integrin subunit with calnexin in the endoplasmic reticulum (ER), and subsequently blocked heterodimerization and expression on the cell surface. For cell spreading and migration, only three (3–5) glycosylation sites on the β-propeller were necessary. These results clearly demonstrated that N-glycans on the β-propeller domain of α5 are essential for maturation, heterodimer formation and the stability of integrin α5β1. Then Sato et al. [78] found that site 4 on the α5 subunit

550

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

is the most important site for its biological functions and can be specifically modified by GnT-III. So an individual N-glycosylation site can have unique functions. This observation is in accordance with results Seales et al. [120] reported for the I-like domain on the β subunit, which could be the partner of the β-propeller of the α5 subunit. The exact mechanism by which glycosylation influences integrins’ biological functions is unknown, but conformational changes in key functional regions of the subunits may be implicated, and the work of Luo et al. [83], Seales et al. [120] and Liu et al. [112] and Isaji et al. [77] seems to support this suggestion. The influence of glycosylation on the β1 integrin subunit's conformation was shown by Luo et al. [83]. They engineered an artificial N-glycosylation site at position 333 on the C-terminal end of the β1 I-like domain. Upon transfection of this construct in CHO-K1 cells, this variant glycoform had increased ligand binding activity, establishing that changes in glycan structure within a key region of the integrin molecule can alter its conformation and activity. Then Seales et al. [120] showed that the β1 integrin subunit is typically N-glycosylated at 10 of 12 possible sites, including sites within the β1 I-like domain, a region crucial for ligand binding. They also showed that differentiating myeloid cells down-regulate ST6GalI sialyltransferase via a protein kinase C/Ras/ERK signaling cascade. In consequence the β1 integrin subunit becomes hyposialylated, which stimulates the ligand binding activity of α5β1 fibronectin receptors. Consistent with the enhanced activity of hyposialylated cell surface integrins, purified integrin α5β1 binds FN more strongly upon enzymatic desialylation, an effect completely reversed by resialylation of this receptor. These results, along with the previous observation that synthesis of hyposialylated integrins are temporally correlated with enhanced adhesion to FN, strongly support variant β1 sialylation as a mechanism for activation of the myeloid fibronectin receptor during monocyte differentiation. Seales et al. [120] hypothesize that sialic acid, a negatively charged sugar, either alters the overall conformation of the integrin or more directly regulates ligand binding. Binding of divalent cations is an important event in integrin activation, so the presence of sialic acid within this domain could influence the coordination of divalent cations or else alter the positioning of the ligand. Analyzing HD3 colonocyte sialylation and its influence on colonocyte adhesion to collagen, Shaikh et al. [121] reported a totally different trend: forced ST6Gal-I down-regulation, leading to decreased α2-6 sialylation of integrins, inhibited cell adhesion to collagen I, a β1 ligand; these cells with forced ST6Gal-I down-regulation also exhibited decreased migration on collagen I and diminished invasion through Matrigel. Very recently, Isaji et al. [77] performed combined substitution at putative N-glycosylation sites by replacement of asparagine residues with glutamine residues, and showed that N-glycosylation sites 4–6 (S4–6) on the β1 subunit I-like domain are essential to both heterodimerization and biological functions such as cell spreading. At the same time, Liu et al. [112] used molecular modeling to analyze the effect of the presence of α2-6 sialic acid on the structure of the β1 integrin I-like domain. Altered glycosylation caused significant conformational changes in most of its key functional regions, including the metal ion-dependent adhesion and ligand binding sites. N-linked oligosaccharides exhibited extensive interactions with the I-like domain of β1 integrin, including the formation of hydrogen bonds. Sialylation affected the interactions through significant changes in the number of hydrogen bonds and in the orientation of oligosaccharides relative to the I-like domain. The unsialylated variant was oriented away from the specificity-determining loop, which allowed its exposure for binding with the ligand. These results explain earlier experimental observations that hyposialylated β1 integrin influence cell binding to FN [73,91]. Taken together, these results suggest that glycosylation affects integrins’ biological functions. One proposed mechanism lies in conformational changes in the key regions for dimerization. Another possible mechanism in α/β interactions involves an unknown lectin domain that may exist in the β subunit, as was shown for αMβ2 integrin [122,123] and L1

protein [124]. Finally, integrin glycosylation may affect complex formation with other membrane proteins such as growth factor receptors [125] or tetraspanins [126,127]. Those supramolecular complexes on the cell surface control intracellular signal transduction [125]. Having reviewed a variety of experimental approaches here, it is also important to note that there is extensive literature showing that naturally occurring changes in integrins’ glycan structures are associated with dramatic alterations of cell behavior such as adhesion and migration. 7. Integrin α3β1—glycoprotein participant in cell motility Among the integrins, α3β1 is particularly interesting due to its role in development, tumorigenesis [128–130] and wound healing [131,132]. Integrin α3β1, the major laminin-332 receptor, is also highly associated with tumor metastasis [130,133]. Recently it was reported that its activity and function can be modified by the glycosyltransferases GnT-III, GnT-V and Fut8 [70,71,79,134,135], enzymes responsible for synthesis of GlcNAc bisected, β1,6-branched and core fucosylated structures. Integrin α3β1 is abundant in keratinocytes in the basal layer of skin epidermis. These cells form close contacts with the basal membrane (BM), consisting mainly of laminin-332 (LN-332), a member of the laminin family composed of α3β3γ2 chains. LN-332 is reported to be bound by integrins α3β1, α6β1 and α6β4, and to be involved in the formation of hemidesmosomes [136,137]. The absence of LN-332 is associated with a decreased number of hemidesmosomes and in consequence detachment of the epidermis from the BM, a cause of junctional epidermolysis bullosa (JEB). The changed expression of laminin-332 and its integrin receptors was observed in certain cancers and is thought to promote invasion of breast, colon and skin cancer cells. On the other hand, cleaved by matrix metalloproteinases, the proteolitic fragments of LN-332 has been shown to induce and stimulate cell migration. Therefore it promote the dissemination of cancer cell [138]. In immortalized keratinocytes – MK cells cultured on laminin-332-rich ECM – the expression of integrin α3β1 was required for MMP-9 secretion; this was not observed in primary keratinocytes. It suggested a significant role for α3β1 in the invasion ability of transformed adherent cells [139]. This dual role of LN-332 was also seen during the process of wound healing: LN-332 expression increased in the skin wound, and keratinocytes migrated into the wound bed using integrin α3β1 [140]. Based on their results, Goldfinger et al. [131] proposed a model to explain the role of LN-332 and its integrin receptors in epithelial wound healing. The α3 subunit of LN-332 can be present as a 190 kDa form or a processed 160 kDa form. In “resting” skin, α6β1 integrin in hemidesmosomes is bound to the processed (160 kDa) form of LN-332. Upon wounding, production of the unprocessed (190 kDa) form is up-regulated, increasing the presence of 190 kDa LN-332 at the wound edge. Integrin α3β1 binds to the 190 kDa LN-332, initiating migration of keratinocytes over the wound bed [131]. Decline et al. [141] showed that transforming growth factor-β1 (TGF-β1), inducing keratinocyte motility, was accompanied by markedly decreased adhesion to processed LN-332. TGF-β1 induced keratinocyte migration by switching the repertoire of integrins, with decreased expression of the ones that bind LN-332. Choma et al. [142] confirmed the role of integrin α3β1 in epithelial cell migration: they found integrin α3β1 at the leading edge of migrating keratinocytes, and inhibition of α3β1 binding to LN-332 prevented the formation of stable leading lamellipodia. Formation of lamellipodia also required α3β1-dependent activation of Rac1. These observations identified a crucial role for integrin α3β1-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. Lamellipodium formation is a crucial event in epithelial wound closure, indicating that the cell has become polarized to migrate. So the interaction between integrin α3β1 and its principal epidermal ligand,

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

551

LN-332, seems of particular importance in keratinocyte function. In migrating keratinocytes, integrin α3β1 dramatically relocates from cell– cell contacts to the basal surface, suggesting that its interaction with the ECM is enhanced during cutaneous wound healing. Indeed, α3β1blocking antibodies inhibit closure of scrape-wound monolayers of primary human keratinocytes. Similarly to other integrins, α3β1 is a glycoprotein with 14 and 12 potential N-glycosylation sites on the α and β subunits (Fig. 5), respectively. N-linked glycosylation of human α3β1 has been studied by a number of groups [102,104,134,143,144], mostly in cancer cell lines. In colon cancer, α3β1 integrin is a sialoglycoprotein containing β1-6-branched N-linked oligosaccharides and short poly-N-acetyllactosamine structures [144]. Using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), we compared the glycosylation profiles of integrin α3β1 from different human cell lines [102,104,145]. Integrin α3β1 isolated from human ureter epithelium cells possessed complex type oligosaccharides with wide heterogeneity of glycans in both subunits. The adhesion test revealed that non-malignant and malignant ureter cancer cells interact with collagen IV, laminin and fibronectin with cell-linedependent intensity and in a glycosylation-dependent manner. The involvement of particular integrins in this process was cell-linedependent, except that in T-24 cells the α3β1 integrin apparently was high-affinity receptor for all analyzed ECM proteins. Decreased adhesion to LN and FN after neuraminidase treatment was observed in all analyzed cell lines, suggesting that despite the metastatic potential of the cell, sialylated N-oligosaccharides strongly influence its adhesion abilities [146]. In primary and metastatic melanoma models, β1-6-branched N-oligosaccharides contributed to α3β1

integrin-dependent cell adhesion to FN [147]. Treatment of cultures with the processing inhibitor swainsonine, which blocks mannosidase II, reduced the rate of cell migration into the scratch wound by 11% in A375 and 38% in WM239 cells. In a Boyden chamber, migration of A375 cells was reduced by 53% in the presence of swainsonine. Kremser et al. [104] described the heterogeneity of integrin α3β1 glycosylation status in two metastatic melanoma cell lines (WM9, WM239) of different origin. N-glycans strongly influenced the interaction of this receptor with its primary ligand, LN-332, in both cell lines. The effect was more pronounced in WM9 cells, where this integrin was more variously glycosylated. Those results are in good agreement with previous work pointing to the involvement of integrin α3β1 glycosylation in ureter cell adhesion [145]. It was also reported that α3β1, which is highly associated with tumor metastasis, can be modified by either GnT-III or GnT-V [134]; GnT-III inhibits GnT-Vstimulated α3β1-mediated cell migration, and GnT-III and GnT-V competitively modify the same target glycoprotein. This competition may regulate the function of the target protein. It can be speculated that, as with integrin α5β1, the effect is due to conformational changes in regions important for dimerization.

Fig. 5. Domain structure and 14 potential N-glycosylation sites of human integrin α3 subunit.

Fig. 6. Domain structure and 13 potential N-glycosylation sites of human integrin αv subunit.

8. Integrin αvβ3—glycoprotein participant in cell motility Because it plays an essential role in angiogenesis and vascular remodeling, integrin αvβ3 is one of the best-characterized integrins (Fig. 6). Normally its expression is minimal in endothelial cells,

552

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

Fig. 7. Localization of oligosaccharides on integrin subunits – comparison of α5β1, α3β1 and αv integrins. On integrin α5β1 the 1–5 glycosylation sites were suggested to play a role in heterodimer formation, and only three of them (3–5) were shown to influence α5β1-mediated cell migration and spreading [76].

intestinal cells, leukocytes and macrophages, but it has been shown to be up-regulated in many types of cancers, including melanoma, breast, lung, colon and pancreas carcinomas [148,149]. Based on their clinical study, Neto et al. [150] suggested that high levels of its expression could be an initial metabolic step for in situ preparation of melanoma for invasion. This integrin forms complexes with other proteins present in cell membranes, and takes part in such cell processes as migration and invasion. It has been recognized as a partner for MMP-2 and suggested as a factor locating MMP activity at the leading edge of migrating cells. In this way it enables them to degrade and remodel the ECM and thus orchestrate effective invasion events. Especially in the presence of VN, melanoma cells expressing αvβ3 have been shown to increase MMP-2 secretion and invasion activity [151]. Moreover, MT1-MMP, which exhibits integrin convertase activity, is able to specifically cleave pro-αv in tumor cells, leading to the formation of an active form of integrin αvβ3. In breast carcinoma MCF7 the co-expression of αvβ3 and MT-MMP1 was correlated with efficient stimulation of the FAK signaling pathway, which in turn promoted cell adhesion and migration on VN [152,153]. Integrin αvβ3 is a primary vitronectin receptor, but it is known to bind a variety of ECM proteins such as fibronectin, laminin, fibrinogen and collagens. Vitronectin is a 75 kDa plasma glycoprotein which plays a role in blood coagulation and wound healing. It is also found in the ECM, particularly at sites of injury or remodeling. In circulation, vitronectin is in closed conformation and does not interact with integrins, but upon binding to the surface of collagens at sites of vascular injury it undergoes a conformational change which exposes RGD integrin binding sites [154–156]. The interaction between vitronectin and its receptors (including αvβ3 integrin) is of special interest because it can accelerate the migration of vascular cells. During metastatic progression, altered expression of integrins is a hallmark of changed cell behavior. Factors affecting αvβ3 expression have been shown to cause changes in cell adhesion and migration of melanoma cells. One of them is ATRA, which lowered α v β 3 expression in melanoma and caused decreased cell adhesion and reduction of cell motility on VN [157]. Little is yet known about the role of glycosylation in integrin αvβ3 function because almost nothing is known about its glycosylation profile. Work by Kremser et al. [104] showed that in two metastatic melanoma cell lines, WM9 and WM239, this integrin is glycosylated in a cell-line-dependent manner and that it is recognized among other

possessed N-oligosaccharides as cancer-related: the migratory ability of WM239 cells, presenting integrin αvβ3 with more diverse glycans, was affected by the presence of PHA-L, SNA and MAA lectins in wound healing assays on VN; in WM9 cells only PHA-L affected this process. 9. Summary Glycosylation is a frequent post-translational protein modification which strongly influences the formation of protein 3D structure and modulates interaction between proteins, thereby regulating their functions. An increasing body of evidence suggests that integrin glycosylation plays an essential role in the formation and biological function of heterodimers (Fig. 7). It is well documented that during carcinogenesis the repertoire of N-glycans attached to integrins is altered. Such integrin-mediated cellular behavior as adhesion and migration are affected by the presence of some characteristic oligosaccharide structures (highly branched complex type N-glycans, long poly-N-acetyllactosamine chains, sialic acids), described as cancer-related glycans. A better understanding of the glycosylation process and its influence on protein function may help to clarify the molecular mechanism of cancer transformation and metastasis. References [1] B. Steffensen Proteolytic, events of wound-healing coordinated interaction among Matrix Metalloproteinases (MMPs), integrins, and extracellular matrix molecules, Crit. Rev. Oral Biol. Med. 12 (2001) 373–398. [2] D.A. Lauffenburger, A.F. Horwitz, Cell migration: a physically integrated molecular process, Cell 84 (1996) 359–369. [3] T. Bogenrieder, M. Herlyn, Axis of evil: molecular mechanisms of cancer metastasis, Oncogene 22 (2003) 6524–6536. [4] D. Kedrin, J. van Rheenen, L. Hernandez, J. Condeelis, J.E. Segall, Motility and cytoskeletal regulation in invasion and metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 143–152. [5] G. Agiostratidou, J. Hulit, G.R. Phillips, R.B. Hazan, Differential cadherin expression: potential markers for epithelial to mesenchymal transformation during tumor progression, J. Mammary Gland Biol. Neoplasia 12 (2007) 127–133. [6] M.A. Arnaout, S.L. Goodman, J.P. Xiong, Structure and mechanics of integrin-based cell adhesion, Curr. Opin. Cell Biol. 19 (2007) 495–507. [7] A.C. Staff, An introduction to cell migration and invasion, Scan. J. Clin. Lab. Investig. 61 (2001) 257–268. [8] H. Yamaguchi, J. Wyckoff, J. Condeelis, Cell migration in tumors, Curr. Opin. Cell Biol. 17 (2005) 559–564. [9] K. Hotary, X.-Y. Li, E. Allen, S.L. Stevens, S.J. Weiss1, A cancer cell metalloprotease triad regulates the basement membrane transmigration program, Genes Dev. 20 (2006) 2673–2686.

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555 [10] C. Brakebusch, D. Bouvard, F. Stanchi, T. Sakai, R. Fässler, Integrins in invasive growth, J. Clin. Invest. 109 (2002) 999–1006. [11] E. Sahai, Illuminating the metastatic process, Nat. Rev. Cancer 7 (2007) 737–749. [12] A. Grande-Garcia, M.A. del Pozo, Caveolin-1 in cell polarization and directional migration, Eur. J. Cell Biol. 87 (2008) 641–647. [13] A.R. Horwitz, J.T. Parsons, Cell migration – movin’ on, Science 286 (1999) 1102–1103. [14] J.G. Lock, B. Wehrle-Haller, S. Strömblad, Cell–matrix adhesion complexes: master control machinery of cell migration, Semin. Cancer Biol. 18 (2008) 65–76. [15] C. Mayer, K. Maaser, N. Daryab, K.S. Zänker, E.-B. Bröcker, P. Friedl, Release of cell fragments by invading melanoma cells, Eur. J. Cell Biol. 83 (2004) 709–715. [16] R.C. Liddington, L.A. Bankston, The structural basis of dynamic cell adhesion: heads, tails, and allostery, Exp. Cell Res. 261 (2000) 37–43. [17] R.O. Hynes, Integrin: bidirectional allosteric signalling machines, Cell 110 (2002) 673–687. [18] M.A. Schwartz, D.W. DeSimon, Cell adhesion receptors in mechanotransduction, Curr. Opin. Cell Biol. 20 (2008) 551–556. [19] C.M. Meighan, J.E. Schwarzbauer, Temporal and spatial regulation of integrins during development, Curr. Opin. Cell Biol. 20 (2008) 520–524. [20] G.J. Mizejewski, Role of integrins in cancer: survey of expression patterns, P. S. E. B. M. 222 (1999) 124–138. [21] H. Jin, J. Varner, Integrins: roles in cancer development and as treatment targets, Br. J. Cancer 90 (2004) 561–565. [22] D.E. White, W.J. Muller, Multifaceted roles of integrins in breast cancer metastasis, J. Mammary Gland Biol. Neoplasia 12 (2007) 135–142. [23] J.P. Xiong, T. Stehle, B. Diefenbach, R. Zhang, R. Dunker, D.L. Scott, A. Joachimiak, S.L. Goodman, M.A. Arnaout, Crystal structure of the extracellular segment of integrin αVβ3, Science 294 (2001) 339–345. [24] C.G. Gahmberg, S.C. Fagerholm, S.M. Nurmi, T. Chavakis, S. Marchesan, M. Grönholm, Regulation of integrin activity and signalling, Biochim. Biophys. Acta 1790 (2009) 431–444. [25] J.S. Kerr, A.M. Slee, S.A. Mousa, The αv integrin antagonists as novel anticancer agents: an update, Expert Opin. Investig. Drugs 11 (2002) 1765–1774. [26] J. Gu, T. Isaji, Y. Sato, Y. Kariya, T. Fukuda, Importance of N-Glycosylation on α5β1 integrin for iportance of N-Glycosylation on α5β1 integrin for its biological functions, Biol. Pharm. Bull. 32 (2009) 780–785. [27] M.J. Humphries, Integrin structure, Biochem. Soc. Trans. 28 (2000) 311–339. [28] A. van der Flier, A. Sonnenberg, Function and interactions of integrins, Cell Tissue Res. 305 (2001) 285–298. [29] E.F. Plow, T.A. Haas, L. Zhang, J. Loftusi, J.W. Smith, Ligand binding to integrins, J. Biol. Chem. 275 (2000) 21785–21788. [30] S.K. Dickeson, S.A. Santoro, Ligand recognition by the I domain-containing integrins, Cell. Mol. Life Sci. 54 (1998) 556–566. [31] J. Takagi, B.M. Petre, T. Walz, T.A. Springer, Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling, Cell 110 (2002) 599–611. [32] J. Takagi, Structural basis for ligand recognition by integrins, Curr. Opin. Cell Biol. 19 (2007) 557–564. [33] D. Schneider, D.M. Engelman, Involvement of transmembrane domain interactions in signal transduction by α /β integrins, J. Biol. Chem. 279 (2004) 9840–9846. [34] S.K. Sastry, K. Burridge, Focal adhesions: a nexus for intracellular signaling and cytoskeletal dynamics, Exp. Cell Res. 261 (2000) 25–36. [35] A.E. Berman, N.I. Kozlova, G.E. Morozevich, Integrins: structure and signaling, Biochemistry (Moscow) 68 (2003) 1284–1299. [36] C.T. Mierke, D. Rösel, B. Fabry, J. Brábek, Contractile forces in tumor cell migration, Eur. J. Cell Biol. 87 (2008) 669–676. [37] M.A. Wozniak, K. Modzelewska, L. Kwong, P.J. Keely, Focal adhesion regulation of cell behavior, Biochim. Biophys. Acta 1692 (2004) 103–119. [38] S. Hehlgans, M. Haase, N. Cordes, Signalling via integrins: implications for cell survival and anticancer strategies, Biochim. Biophys. Acta 1775 (2007) 163–180. [39] M.J. van Nimwegen, B. van de Water, Focal adhesion kinase: a potential target in cancer therapy, Biochem. Pharmacol. 73 (2007) 597–609. [40] I. Zachary, Focal adhesion kinase, Int. J. Biochem. Cell Biol. 29 (1997) 929–934. [41] D.D. Schlaepfer, S.K. Mitra, D. Ilic, Control of motile and invasive cell phenotypes by focal adhesion kinase, Biochim. Biophys. Acta 1692 (2004) 77–102. [42] S. Kuphal, R. Bauer, A.-K. Bosserhoff, Integrin signaling in malignant melanoma, Cancer Metastasis Rev. 24 (2005) 195–222. [43] C. Brakebusch, R. Fässler, The integrin-actin connection, an eternal love affair, EMBO J. 22 (2003) 2324–2333. [44] Y.D. Benoit, C. Lussier, P.-A. Ducharme, S. Sivret, L.M. Schnapp, N. Basora, J.-F. Beaulieu, Integrin α8β1 regulates adhesion, migration and proliferation of Human Intestinal Crypt Cells via a predominant RhoA/ROCK dependent mechanism, Biol. Cell. 101 (2009) 695–708. [45] S. Fernandez-Sauze, D. Grall, B. Cseh, E. Van Obberghen-Schilling, Regulation of fibronectin matrix assembly and capillary morphogenesis in endothelial cells by Rho family GTPases, Exp. Cell Res. 315 (2009) 2092–2104. [46] M.Z. Gilcrease, X. Zhou, X. Lu, W.A. Woodward, B.E. Hall, P.J. Morrissey, α6β4 integrin crosslinking induces EGFR clustering and promotes EGF-mediated Rho activation in breast cancer, J. Exp. Clin. Cancer Res. 26 (28) (2009) 67, doi:10.1186/1756-9966-28-67. [47] P.J. Keely, E.V. Rusyn, A.D. Cox, L.V. Parise, R-Ras signals through specific integrin a cytoplasmic domains to promote migration and invasion of breast epithelial cells, J. Cell Biol. 145 (1999) 1077–1088. [48] R.A. Cairns, R. Khokha, R.P. Hill, Molecular mechanisms of tumour invasion and metastasis: an integrated view, Curr. Mol. Med. 3 (2003) 659–671. [49] S.P. Palecek, A. Huttenlocher, A.F. Horwitz, D.A. Lauffenburger, Physical and biochemical regulation of integrin release during rear detachment of migrating cells, J. Cell Sci. 111 (1998) 929–940.

553

[50] S.P. Palecek, A.F. Horwitz, D.A. Lauffenburger, Kinetic model for integrin-mediated adhesion release during cell migration, Ann. Biomed. Eng. 27 (1999) 219–235. [51] M. Kim, C.V. Carman, T.A. Springer, Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins, Science 301 (2003) 1720–1725. [52] M.H. Ginsberg, A. Partridge, S.J. Shattil, Integrin regulation, Curr. Opin. Cell Biol. 17 (2005) 509–516. [53] F.G. Giancotti, E. Ruoslahti, Integrin signaling, Science 285 (1999) 1028–1032. [54] S.P. Holly, M.K. Larson, L.V. Parise, Multiple roles of integrins in cell motility, Exp. Cell Res. 261 (2000) 69–74. [55] P.T. Caswell, J.C. Norman, Integrin trafficking and the control of cell migration, Traffic 7 (2006) 14–21. [56] O. Engebraaten, M. Trikha, S. Juell, S. Garman-Vik, Ø. Fodstad, Inhibition of in vivo tumour growth by the blocking of host αvβ3 and αIIbβ3 integrins, Anticancer Res. 29 (2009) 131–138. [57] D.A. Calderwood, Integrin activation, J. Cell Sci. 117 (2004) 657–666. [58] J.A. Eble, J. Haier, Integrins in cancer treatment, Curr. Cancer Drug Targets 6 (2006) 89–105. [59] R. Apweiler, H. Hermjakob, N. Sharon, On the frequency of protein glycosylation deduced from analysis of the SWISS-PROT database, Biochim. Biophys. Acta 1473 (1999) 4–8. [60] H. Schachter, Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharide, Biochem. Cell Biol. 64 (1986) 163–181. [61] M.A. Kukuruzinska, K. Lennon, Protein N-glycosylation: molecular genetics and functional significance, Crit. Rev. Oral Biol. Med. 9 (1998) 415–448. [62] S. Kawano, K. Hashimoto, T. Miyama, S. Goto, M. Kanehisa, Prediction of glycan structures from gene expression data based on glycosyltransferase reactions, Bioinformatics 21 (2005) 3976–3982. [63] P. van den Steen, P.M. Rudd, R.A. Dwek, G. Opdenakker, Concepts and principles of O-linked glycosylation, Crit. Rev. Biochem. Mol. Biol. 33 (1998) 151–208. [64] J.W. Dennis, M. Granovsky, C.E. Warren, Review: glycoprotein glycosylation and cancer progression, Biochim. Biophys. Acta 1473 (1999) 21–34. [65] M. Fukuda, O. Hindsqaul, Molecular glycobiology, Oxford University Press, New York, 1994. [66] D. Bironaite, J.M. Nesland, B. Risberg, M. Bryne, N-glycans influence the in vitro adhesive and invasive behavior of the three metastatic cell lines, Tumor Biol. 21 (2000) 165–175. [67] H. Yamamoto, J. Swoger, S. Greene, T. Saito, J. Hurh, C. Sweeley, J. Leestma, E. Mkrdichian, L. Cerullo, A. Nishikawa, Y. Ihara, N. Taniguchi, J.R. Moskal, β1, 6-NAcetylglucosamine-bearing N-Glycans in human gliomas: implications for a role in regulating invasivity, Cancer Res. 60 (2000) 134–142. [68] A. Kobata, J. Amano, Altered glycosylation of proteins produced by malignant cells, and application for the diagnosis and immunotherapy of tumours, Immunol. Cell Biol. 83 (2005) 429–439. [69] N. Taniguchi, E. Miyoshi, G. Jianguo, K. Honke, A. Matsumoto, Decoding sugar functions by identifying target glycoproteins, Curr. Opin. Struct. Biol. 16 (2006) 561–566. [70] Y. Zhao, Y. Sato, T. Isaji, T. Fukuda, A. Matsumoto, E. Miyoshi, J. Gu, N. Taniguchi, Branched N-glycans regulate the biological functions of integrins and cadherins, FEBS J. 275 (2008) 1939–1948. [71] Y.Y. Zhao, M. Takahashi, J.G. Gu, E. Miyoshi, A. Matsumoto, S. Kitazume, N. Taniguchi, Functional roles of N-glycans in cell signaling and cell adhesion in cancer, Cancer Sci. 99 (2008) 1304–1310. [72] R. Schauer, K. Soerge, G. Reuter, P. Roggentin, L. Shaw, Biochemistry and role of sialic acids, in: A. Rosenberg (Ed.), Biology of the sialic acids, Plenum, New York, 1995, pp. 7–49. [73] A.C. Semel, E.C. Seales, A. Singhal, E.A. Eklund, K.J. Colley, S.L. Bellis, Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors, J. Biol. Chem. 277 (2002) 32830–32836. [74] S. Bellis, Variant glycosylation: an underappreciated regulatory mechanism for β1 integrins, Biochim. Biophys. Acta 1663 (2004) 52–60. [75] C.J. Bosques, S.M. Tschampel, R.J. Woods, B. Imperiali, Effects of glycosylation on peptide conformation: a synergistic experimental and computational study, J. Am. Chem. Soc. 126 (2004) 8421–8425. [76] T. Isaji, Y. Sato, Y. Zhao, E. Miyoshi, Y. Wada, N. Taniguchi, J. Gu, N-glycosylation of the β-propeller domain of the integrin α5 subunit is essential for α5β1 heterodimerization, expression on the cell surface and its biological function, J. Biol. Chem. 281 (2006) 33258–33267. [77] T. Isaji, Y. Sato, T. Fukuda, J. Gu, N-glycosylation of the I-like domain of β1 integrin is essential for β1 integrin expression and biological function: identification of the minimal N-glycosylation requirement for α5β1, J. Biol. Chem. 284 (2009) 12207–12216. [78] Y. Sato, T. Isaji, M. Tajiri, S. Yoshida-Yamamoto, T. Yoshinaka, T. Somehara, T. Fukuda, Y. Wada, J. Gu, An N-glycosylation site on the β-propeller domain of the integrin α5 subunit plays key roles in both its function and site-specific modification by β1, 4-N-acetylglucosaminyltransferase III, J. Biol. Chem. 284 (2009) 11873–11881. [79] M. Takahashi, Y. Kuroki, K. Ohtsubo, N. Taniguchi, Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins, Carbohydr. Res. 344 (2009) 1387–1390. [80] M. Ono, S. Hakomori, Glycosylation defining cancer cell motility and invasiveness, Glycoconj. J. 20 (2004) 71–78. [81] A. Varki, Biological roles of oligosaccharides: all of the theories are correct, Glycobiology 3 (1993) 97–130. [82] R.C. Casey, T.R. Oegema Jr., K.M. Skubitz, S.E. Pambuccian, S.M. Grindle, A.P.N. Skubitz, Cell membrane glycosylation mediates the adhesion, migration, and invasion of ovarian carcinoma cells, Clin. Exp. Metastasis 20 (2003) 143–152.

554

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555

[83] B.-H. Luo, T.A. Springer, J. Takagi, Stabilizing the open conformation of the integrin headpiece with a glycan wedge increases affinity for ligand, PNAS 100 (2003) 2403–2408. [84] N. Nishida, C. Xie, M. Shimaoka, Y. Cheng, T. Walz, T.A. Springer, Activation of leukocyte β2 integrins by conversion from bent to extended conformations, Immunity 25 (2006) 583–594. [85] S. Hakomori, Glycosylation defining cancer malignancy. New wine in an old bottle, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 10231–10233. [86] C. Couldrey, J.E. Green, Metastases: the glycan connection, Breast Cancer Res. 2 (2000) 321–323. [87] E. Gorelik, U. Galili, A. Raz, On the role of cell surface carbohydrates and their binding proteins (lectins) in tumor metastasis, Cancer Metastasis Rev. 20 (2001) 245–277. [88] K.S. Lau, J.W. Dennis, N-glycans in cancer progression, Glycobiology 18 (2008) 750–760. [89] F. Dall'Olio, The sialyl-α2, 6-lactosaminyl-structure: biosynthesis and functional role, Glycoconj. J. 17 (2000) 669–676. [90] P.H. Wang, Altered glycosylation in cancer: sialic acids and sialyltransferases, J. Cancer Mol. 1 (2005) 73–81. [91] E.C. Seales, G.A. Jurado, B.A. Brunson, J.K. Wakefield, A.R. Frost, S.L. Bellis, Hypersialylation of β1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility, Cancer Res. 65 (2005) 4546–4552. [92] D.R. Christie, F.M. Shaikh, J.A. Lucas IV, J.A. Lucas III, S.L. Bellis, ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function, J. Ovarian Res. 1 (2008) 3, doi:10.1186/1757-2215-1-3. [93] B.V.V.G. Reddy, R.D. Kalraiya, Sialilated β1, 6 branched N-oligosaccharides modulate adhesion, chemotaxis and motility of melanoma cells: Effect on invasion and spontaneous metastasis properties, Biochim. Biophys. Acta 1760 (2006) 1393–1402. [94] H.-B. Guo, I. Lee, M. Kamar, S.K. Akiyama, M. Pierce, Aberrant N-glycosyaltion of β1 integrin cause reduced α5β1 integrin clustering and stimulates migration, Cancer Res. 62 (2002) 6837–6845. [95] V. Krishnan, S.M. Bane, P.D. Kawle, K.N. Naresh, R.D. Kalraiya, Altered melanoma cell surface glycosylation mediates organ specific adhesion and metastasis via lectin receptors on the lung vascular endothelium, Clin. Exp. Metastasis 22 (2005) 11–24. [96] H.B. Guo, F. Liu, H.L. Chen, Increased susceptibility to apoptosis of human hepatocarcinoma cells transfected with antisense N-acetylglucosaminyltransferase V cDNA, Biochem. Biophys. Res. Commun. 264 (1999) 509–517. [97] S.F. Siddiqui, J. Pawelek, T. Handerson, C.Y. Lin, R.B. Dickson, D.L. Rimm, R.L. Camp, Coexpression of β1,6-N-acetylglucosaminyltransferase V glycoprotein substrates defines aggressive breast cancers with poor outcome, Cancer Epidemiol. Biomark. Prev. 14 (2005) 2517–2523. [98] H.B. Guo, M. Randolph, M. Pierce, Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase, J. Biol. Chem. 282 (2007) 22150–22162. [99] L. Wang, Y. Liang, Z. Li, X. Cai, W. Zhang, G. Wu, J. Jin, Z. Fang, Y. Yang, X. Zha, Increase in β1-6 GlcNAc branching caused by N-acetylglucosaminyltransferase V directs integrin β1 stability in human hepatocellular carcinoma cell line SMMC7721, J. Cell. Biochem. 100 (2007) 230–241. [100] Y. Zhao, J. Li, Y. Xing, J. Wang, C. Lu, X. Xin, M. Geng, N-acetylglucosaminyltransferase V mediates cell migration and invasion of mouse mammary tumor cells 4TO7 via RhoA and Rac1 signaling pathway, Mol. Cell Biochem. 309 (2008) 199–208. [101] E.A. Partridge, C. Le Roy, G.M. Di Guglielmo, J. Pawling, P. Cheung, M. Granovsky, I.R. Nabi, J.L. Wrana, J.W. Dennis, Regulation of cytokine receptors by Golgi N-glycans processing and endocytosis, Science 306 (2004) 120–124. [102] E. Pocheć, A. Lityńska, A. Amoresano, A. Casbarra, Glycosylation profile of integrin α3β1 changes with melanoma progression, Biochim. Biophys. Acta 1643 (2003) 113–123. [103] J. Gu, N. Taniguchi, Regulation of integrin functions by N-glycans, Glycoconj. J. 21 (2004) 9–15. [104] M.E. Kremser, M. Przybyło, D. Hoja-Łukowicz, E. Pocheć, A. Amoresano, A. Carpentieri, M. Bubka, A. Lityńska, Characterisation of α3β1 and αvβ3 integrin Noligosaccharides in metastatic melanoma WM9 and WM239 cell lines, Biochim. Biophys. Acta 1780 (2008) 1421–1431. [105] E. Yamamoto, K. Ino, E. Miyoshi, K. Inamori, A. Abe, S. Sumigama, A. Iwase, H. Kajiyama, K. Shibata, A. Nawa, F. Kikkawa, N-acetylglucosaminyltransferase V regulates extravillous trophoblast invasion through glycosylation of α5β1 integrin, Endocrinology 150 (2009) 990–999. [106] A. Lagana, J.G. Goetz, P. Cheung, A. Raz, J.W. Dennis, I.R. Nabi, Galectin binding to Mgat5- modified N-glycans regulates fibronectin matrix remodeling in tumor cells, Mol. Cell. Biol. 26 (2006) 3181–3193. [107] G.W. DeHart, K.E. Healy, J.C.R. Jones, The role of α3β1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes, Exp. Cell Res. 283 (2003) 67–79. [108] M. Morini, M. Mottolese, N. Ferrari, F. Ghiorzo, S. Buglioni, R. Mortarini, D.M. Noonan, P.G. Natali, A. Albini, The α3β1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity, Int. J. Cancer 87 (2000) 336–342. [109] G. Serini, D. Valdembri, F. Bussolino, Integrins and angiogenesis: a sticky business, Exp. Cell Res. 312 (2006) 651–658. [110] B. Felding-Habermann, E. Fransvea, T.E. O'Toole, L. Manzuk, B. Faha, M. Hensler, Involvement of tumor cell integrin αvβ3 in hematogenous metastasis of human melanoma cells, Clin. Exp. Metastasis 19 (2002) 427–436. [111] B. Felding-Habermann, Integrin adhesion receptors in tumor metastasis, Clin. Exp. Metastasis 20 (2003) 203–213.

[112] Y. Liu, D. Pan, S.L. Bellis, Y. Song, Effect of altered glycosylation on the structure of the I-like domain of β1 integrin: a molecular dynamics study, Proteins 73 (2008) 989–1000. [113] M. Zheng, H. Fang, S. Hakomori, Functional role of N-glycosylation in α5β1 integrin receptor. De-N-glycosylation induces dissociation or altered association of α5β1 subunits and concomitant loss of fibronectin binding activity, J. Biol. Chem. 269 (1994) 12325–12331. [114] S.K. Akiyama, S.S. Yamada, K.M. Yamada, Analysis of the role of glycosylation of the human fibronectin receptor, J. Biol. Chem. 264 (1989) 18011–18018. [115] Y. Zhang, J. Zhao, X. Zhang, H. Guo, F. Liu, H. Chen, Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions, Mol. Cell. Biochem. 260 (2004) 137–146. [116] A. Passaniti, G.W. Hart, Cell sialylation and tumor metastasis: metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures, J. Biol. Chem. 263 (1988) 7591–7603. [117] S. Nadanaka, Ch. Sato, K. Kitajima, S. Irie, T. Yamagata, Occurrence of oligosialic acids on integrin α5 subunit and their involvement in cell adhesion to fibronectin, J. Biol. Chem. 276 (2001) 33657–33664. [118] M. Demetriou, I.R. Nabi, M. Coppolino, S. Dedher, J.W. Dennis, Reduced contactinhibition and substratum adhesion in epithelial cells expressing GlcNActransferase V, J. Cell Biol. 130 (1995) 383–392. [119] T. Isaji, J. Gu, R. Nishiuchi, Y. Zhao, M. Takahashi, E. Miyoshi, K. Honke, K. Sekiguchi, N. Taniguchi, Introduction of bisecting GlcNAc into integrin α5β1 reduces ligand binding and down-regulates cell adhesion and cell migration, J. Biol. Chem. 279 (2004) 19747–19754. [120] E.C. Seales, F.M. Shaikh, A.V. Woodard-Grice, P. Aggarwal, A.C. McBrayer, K.M. Hennessy, S.L. Bellis, A protein kinase C/Ras/ERK signaling pathway activates myeloid fibronectin receptors by altering β1 integrin sialylation, J. Biol. Chem. 280 (2005) 37610–37615. [121] F.M. Shaikh, E.C. Seales, W.C. Clem, K.M. Hennessy, Y. Zhuo, S.L. Bellis, Tumor cell migration and invasion are regulated by expression of variant integrin glycoforms, Exp. Cell Res. 314 (2008) 2941–2950. [122] K.M. Hoffmeister, E.C. Josefsson, N.A. Issac, H. Clause, J.H. Hartwig, T.P. Stossel, Glycosylation restores survival of chilled blood platelets, Science 301 (2003) 1531–1534. [123] E.C. Josefsson, H.H. Gebhard, T.P. Stossel, J.H. Hartwig, K.M. Hoffmeister, The macrophage αΜβ2 integrin αΜ lectin domain mediates the phagocytosis of chilled platelets, J. Biol. Chem. 280 (2005) 18025–18032. [124] M. Heller, M. von der Ohe, R. Kleene, H. Mohajeri, M. Schachner, The immunoglobulin-superfamily molecule basigin is a binding protein for oligomannosidic carbohydrates: an anti-idiotypic approach, J. Neurochem. 84 (2003) 557–565. [125] Ch.H. Streuli, N. Akhtar, Signal co-operation between integrins and other receptor systems, Biochem. J. 418 (2009) 491–506. [126] R. Nishiuchi, N. Sanzen, S. Nada, Y. Sumida, Y. Wada, M. Okada, J. Takagi, H. Hasegawa, K. Sekiguchi, Potentiation of the ligand binding activity of integrin α3β1 via association with tetraspanin CD151, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 1939–1944. [127] L. Liu, B. He, W.M. Liu, D. Zhou, J.V. Cox, X.A. Zhang, Tetraspanin CD151 promotes cell migration by regulating integrin trafficking, J. Biol. Chem. 282 (2007) 31631–31642. [128] J.A. Kreidberg, Functions of α3β1 integrin, Curr. Opin. Cell Biol. 12 (2000) 548–553. [129] G. Giannelli, S. Astigiano, S. Antonaci, M. Morini, O. Barbieri, D.M. Noonan, A. Albini, Role of the α3β1 and α6β4 integrins in tumor invasion, Clin. Exp. Metastasis 19 (2002) 217–223. [130] T. Tsuji, Physiological and pathological roles of α3β1 integrin, J. Membr. Biol. 200 (2004) 115–132. [131] L.E. Goldfinger, S.B. Hopkinson, G.W. deHart, S. Collawn, J.R. Couchman, J.C. Jones, The α3-laminin subunit, α6β4 and α3β1 integrin coordinately regulate wound healing in cultured epithelial cells and in the skin, J. Cell Sci. 112 (1999) 2615–2629. [132] R. Grose, C. Hutter, W. Bloch, I. Thorey, F.M. Watt, R. Fässler, C. Brakebusch, S. Werner, A crucial role of β1 integrins for keratinocyte migration in vitro and during cutaneous wound repair, Development 129 (2002) 2303–2315. [133] A. Melchiori, R. Mortarini, S. Carlos, P.C. Marchisio, A. Anichini, D.M. Noonan, A. Albuni, The α3β1 integrin is involved in melanoma cell migration and invasion, Exp. Cell Res. 219 (1995) 233–242. [134] Y. Zhao, T. Nakagawa, S. Itoh, K. Inamori, T. Isaji, Y. Kariya, A. Kondo, E. Miyoshi, K. Miyazaki, N. Kawasaki, N. Taniguchi, J. Gu, N-Acetylglucosaminyltransferase III antagonizes the effect of N-Acetylglucosaminyltransferase V on α3β1 integrinmediated cell migration, J. Biol. Chem. 281 (2006) 32122–32130. [135] Y. Zhao, S. Itoh, X. Wang, T. Isaji, E. Miyoshi, Y. Kariya, K. Miyazaki, N. Kawasaki, N. Taniguchi, J. Gu, Deletion of core fucosylation on α3β1 integrin down-regulates its functions, J. Biol. Chem. 281 (2006) 38343–38350. [136] M. Aumailley, P. Roussel, Laminins of the dermo-epidermal junction, Matrix Biol. 18 (1999) 19–28. [137] G. Giannelli, S. Antonaci, Biological and clinical relevance of Laminin-5 in cancer, Clin. Exp. Metastasis 18 (2000) 439–443. [138] D. Tsuruta, H. Kobayashi, H. Imanishi, K. Sugawara, M. Ishii, J.C.R. Jones, Laminin332-integrin interaction: a target for cancer therapy? Curr. Med. Chem. 15 (2008) 1968–1975. [139] C.M. DiPersio, M. Shao, L. Di Costanzo, J.A. Kreidberg, R.O. Hynes, Mouse keratinocytes immortalized with large T antigen acquire α3β1 integrin-dependent secretion of MMP-9/gelatinase B, J. Cell Sci. 113 (2000) 2909–2921. [140] H. Larjava, T. Salo, K. Haapasalmi, R.H. Kramer, J. Heino, Expression of integrins and basement membrane components by wound keratinocyes, J. Clin. Invest. 92 (1993) 1425–1435.

M.E. Janik et al. / Biochimica et Biophysica Acta 1800 (2010) 545–555 [141] F. Decline, O. Okamoto, F. Mallein-Gerin, B. Helbert, J. Bernaud, D. Rigal, P. Rousselle, Keratinocyte motility induced by TGF-β1 is accompanied by dramatic changes in cellular interactions with lamin 5, Cell Motil. Cytoskeleton 54 (2003) 64–80. [142] D.P. Choma, K. Pumiglia, C.M. DiPersio, Integrin α3β1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1, J. Cell Sci. 117 (2004) 3947–3959. [143] J.A. Eble, K.W. Wucherpfennig, L. Gauthier, P. Dersch, E. Krukonis, R.E. Isberg, M.E. Hemler, Recombinant Soluble Human α3β1 integrin: purification, regulation, and specific binding to laminin-5 and invasion in a mutually exclusive manner, Biochemistry 37 (1998) 10945–10955. [144] N.L. Prokopishyn, W. Puzon-McLaughlin, Y. Takada, S. Laferte, Integrin α3β1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes, J. Cell Biochem. 72 (1999) 189–209. [145] A. Lityńska, E. Pocheć, D. Hoja-Łukowicz, E. Kremser, P. Laidler, A. Amoresano, Ch. Monti, The structure of the oligosaccharides of α3β1 integrin from human uterer epithelium (HCV29) cell line, Acta Biochim. Pol. 49 (2002) 491–500. [146] A. Lityńska, M. Przybyło, E. Pocheć, P. Laidler, Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation, Acta Biochim. Pol. 49 (2002) 643–650. [147] A. Lityńska, M. Przybyło, E. Pocheć, E. Kremser, D. Hoja-Łukowicz, U. Sulowska, Does glycosylation of melanoma cells influence their interactions with fibronectin? Biochimie 88 (2006) 527–534. [148] S.A. Ahmad, Y.D. Jung, W. Liu, N. Reinmuth, A. Parikh, O. Stoeltzing, F. Fan, L.M. Ellis, The role of the microenvironment and intercellular cross-talk in tumor angiogenesis, Cancer Biol. 12 (2002) 105–112. [149] N.K. Haass, K.S.M. Smalley, L. Li, M. Herlyn, Adhesion, migration and communication in melanocytes and melanoma, Pigment Cell Res. 18 (2005) 150–159.

555

[150] D.S. Neto, L. Pantaleão, B.C. Soares de Sá, G. Landman, Alpha-v-beta3 integrin expression in melanocytic nevi and cutaneous melanoma, J. Cutan. Pathol. 34 (2007) 851–856. [151] L.M. Bafetti, T.N. Young, Y. Itoh, M. Sharon, Stack intact vitronectin induces Matrix Metalloproteinase-2 and Tissue Inhibitor of Metalloproteinases-2 expression and enhanced cellular invasion by melanoma cells, J. Biol. Chem. 273 (1998) 143–149. [152] E.I. Deryugina, B.I. Ratnikov, T.I. Postnova, D.V. Rozanov, A.Y. Strongin, Processing of integrin αv subunit by Membrane Type 1 Matrix Metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of Focal Adhesion Kinase, J. Biol. Chem. 277 (2002) 9749–9756. [153] B.I. Ratnikov, D.V. Rozanov, T.I. Postnova, P.G. Baciu, H. Zhang, R.G. DiScipio, G.G. Chestukhina, J.W. Smith, E.I. Deryugina, A.Y. Strongin, An alternative processing of integrin αv subunit in tumor cells by Membrane Type-1 Matrix Metalloproteinase, J. Biol. Chem. 277 (2002) 7377–7385. [154] D. Seiffert, J.W. Smith, The cell adhesion domain in plasma vitronecitn is cryptic, J. Biol. Chem. 272 (1997) 13705–13710. [155] T.J. Podor, S. Campbell, P. Chindemi, D.M. Foulon, D.H. Farrell, P.D. Walton, J.I. Weitz, C.B. Peterson, Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gammaA/gamma' fibrinogen, J. Biol. Chem. 277 (2002) 7520–7528. [156] S. Stefansson, E.J. Su, S. Ishigami, J.M. Cale, Y. Gao, N. Gorlatova, D.A. Lawrence, The contributions of integrin affinity and integrin-cytoskeletal engagement in endothelial and smooth muscle cell adhesion to vitronectin, J. Biol. Chem. 282 (2007) 15679–15689. [157] A. Baroni, I. Paoletti, I. Silvestri, E. Buommin, M.V. Carriero, Cutaneous biology: early vitronectin receptor downregulation in a melanoma cell line during all-trans retinoic acid-induced apoptosis, Br. J. Dermatol. 148 (2003) 424–433.