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Cell–Matrix interactions, the role of fibronectin and integrins. A survey
Pathologie Biologie 60 (2012) 15–19
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50th anniversary – French Society for Connective Tissue Research
Cell–Matrix interactions, the role of fibronectin and integrins. A survey Interactions cellules–matrice extracellulaire, roˆle de la fibronectine et des inte´grines. Une revue J. Labat-Robert Laboratoire de recherche ophtalmologique, universite´ Paris-V, hoˆpital Hoˆtel-Dieu, 1, place du Parvis-Notre-Dame, 75181 Paris cedex 04, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2011 Accepted 16 September 2011 Available online 21 January 2012
In this review, we present several aspects of cell-matrix interactions, especially the role of fibronectin and integrins in the mediation of these interactions. As this field of investigations literally exploded over the last decades, we had to limit this review to some aspects of this field. We cited experiments giving details on the modifications of fibronectin molecules during their interactions with cells as well as on recent progress of the molecular mechanisms of fibronectin–integrin interactions. We insisted on the molecular details which were shown to play a role in the bi-directional signals ‘‘sent’’ by cells to the surrounding matrix (inside-out and outside-in). A number of recent publications confirmed the physiopathological importance of these messages both for the normal function of tissues as well as for the understanding of their pathological modifications. We insist also on the importance of fibronectin-fragments during some pathologies. ß 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Extracellular matrix Connective Tissues Fibronectin Integrins Remodeling Proteases Fibronectin-fragments Matricryptins Mots cle´s: Matrice extracellulaire Tissus conjonctifs Fibronectine Inte´grines Remodelage Prote´ases Fragments de fibronectine Matricryptines
R E´ S U M E´
Nous pre´sentons dans cette revue plusieurs aspects de l’interaction entre les cellules et la matrice extracellulaire et, plus particulie`rement, le roˆle de la fibronectine et des inte´grines dans ces processus. Comme ce domaine de recherche a litte´ralement explose´ au cours des dernie`res anne´es, nous avons e´te´ amene´s a` restreindre cette revue a` quelques aspects des modifications de la conformation de la fibronectine au cours de son interaction avec les cellules, ainsi qu’aux derniers de´tails des changements de conformation au cours de l’interaction entre fibronectine et inte´grines et leur roˆle dans la transduction bilate´rale des signaux entre cellule et matrice. Les de´tails mole´culaires de ces interactions me´diatrices de signaux jouent, en effet, un roˆle important dans la biologie des tissus conjonctifs ainsi que dans leurs modifications pathologiques. Nous insistons aussi sur l’importance des fragments de fibronectine au cours de certaines pathologies. ß 2011 Elsevier Masson SAS. Tous droits re´serve´s.
1. Introduction Not such a long time ago, extracellular matrix (ECM) was considered as an inert support for the cells it surrounds. We now know that on the contrary, ECM represents a dynamic and complex environment. There is between cells and ECM a permanent crosstalk through receptors, mainly the integrin family. Doing so, ECM delivers to cells signals controlling their shape, migration, proliferation, differentiation and survival. The molecular components of ECM belong to four families of macromolecules: collagen(s), elastin(s), glycosaminoglycans and
Adresse e-mail : [email protected]. 0369-8114/$ – see front matter ß 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.patbio.2011.10.003
structural or matrix glycoproteins. Collagens and elastin(s) are fibrous proteins, glycosaminoglycans and proteoglycans are proteinpolysaccharides or pure polysaccharides (hyaluronan) which fill the meshes of the fibrillar lattice formed by the collagens and elastin fibers whereas structural glycoproteins play an organizing role in the ECM construction. This organization is not static. ECM composition varies as a function of the differentiated state of tissues and as a function of age. It changes during development, maturity, pathologies and aging. Thus the organization of ECM is a dynamic process. Connective tissues are ECM-rich but all tissues contain variable amounts of ECM. ECM can be calcified to form bones or teeth, but in cornea, it is transparent. Its structure takes a variety of shapes and confers to tissues strength and elasticity in varying proportions. Between epithelia
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and connective tissues, it forms a basal lamina which is a specialized form of ECM.
2. Fibronectin Among structural glycoproteins, fibronectin (FN) was the first to be intensively studied [1]. This glycoprotein family is composed of high molecular weight molecules. The peptides which compose the subunits comprise three types of repeats, types I, II and III, assembled into domains with autonomous and specific functions. Glycoproteins like FN are often designated as mosaic proteins [2]. In FN, there are domains with binding sites for bacteria, collagen, fibrin, FN itself and heparin. Thus, FN can play a role in the organization of ECM. But, there is also in the FNIII10 repeat an important cell-binding site (RGD) interacting with the main FN receptor a5b1 integrin. This site needs, for complete activity, the presence in the FNIII9 motif of a synergy sequence (PHSRN) of amino acids. Thus, FN plays an important role in matrix assembly, in cell adhesion and spreading, migration, morphology, cytoskeletal organization and also in oncogenic transformation. FN is an ubiquitous protein present from the first pluricellular eukaryotes, the sponges [3]. FN is present in a soluble form in plasma and biological fluids. Plasma fibronectin (FNp) is synthesized by hepatocytes [4]. FN is also present in tissues in an insoluble form synthesized by fibroblasts, epithelial cells and other differentiated cell types. Nevertheless, FNp can diffuse into tissues and be incorporated in the fibrillar matrix [5]. There are a number of isoforms issued from a single gene by alternative splicing of the primary transcript generating up to 20 different variants in humans [6]. Splicing was shown to occur in three regions: EIIIA and EIIIB where the exon can be included or skipped during transcription. FNp never contains these two exons [1,7–9]. However, small quantities of FN containing EIIIA (A+FN) are present in the circulation of healthy individuals [10]. In tissues, EIIIA and EIIIB are present in various combinations; during development, they are present at an elevated level but they are low in adult tissues, increasing during some pathologies such as, rheumatoid arthritis [11], wound healing [12]. Peptide sequences have been identified which mediate integrin a9b1 dependent cellular activities [13]. During wound healing, EIIIA is expressed transiently by wound cells (day 4 to 7), whereas EIIIB persists through day 14 (rat). Similar processing of FN was shown also in rheumatoid arthritis and tumors. A third region shows alternative splicing: V/IIICS (for the type III connecting segment). Here, the exon can be subdivided giving rise to several variants of FN, generating new cell-binding site, LDV [14], especially for a4b1 integrin expressed by leukocytes and lymphocytes and REDV for a4b7 integrin. FN is synthesized as a monomer followed very rapidly by dimerisation in the rough endoplasmic reticulum. The IIICS region is required for formation and secretion of native FN dimers. FNp are 50% V0, according to the species of origin. It lacks V0 – V0 dimers and consists of V0 – V+ and V+ – V+ combinations. Fibroblasts produce very few V0 subunits. FN in solution forms a compact dimer which does not undergo fibril assembly, but in culture and in tissues, FN assembles through a cell-mediated process. Cell-associated FN is first distributed diffusely over the cell surface. This dimeric ligand induces integrin clustering by binding to two integrins [15], this produces FN selfassociation [16] and connection of FN to the cytoskeleton. The cysteins forming the dimerisation site are essential for assembly, they play a role in integrin clustering and FN–FN interactions. On each subunit, there are four sites for FN–FN interactions. An RGDindependent mechanisms implies a4b1 to the CS1 site. The amino-terminal type I repeats (II–5) are an essential assembly domain. FN is secreted as a disulfide-bonded dimer in a compact
inactive form. Integrin (a5b1) binding will convert FN in an active, extended dimer, through the RGD binding sequence in the type III10 module [15] and the synergy sequence located in type III9 module [16]. Then fibril formation will take place. Functions of FN critically depend on the assembly of secreted FN dimers into a fibrillar network in order to FN assembly to occur. As assembly progresses, dimeric FN forms short deoxycholate-soluble fibrils, later converted in a dense detergent-insoluble network in a stepwise manner [17–21]. More recently, Takahashi et al. [22] could show that the RGD motif is dispensable for fibril assembly. Using mutant embryos or mutant cells where RGD motif was replaced with RGE, they could show that in the absence of RGD motif FN assembly was not compromised to form FN matrix. anb3 integrin assembles FN-RGE by binding an iso-DGR motif in FN1-5 which is generated by the non-enzymatic rearrangement of asparagines into an iso-aspartate. The RGD motif is however essential during development. During matrix assembly, cryptic binding sites are exposed by unfolding of individual type III modules. FN fibrils are quite elastic as shown by Ohashi et al. [23]. A good review on FN matrix assembly was published by Wierzbicka-Patynowski and Schwarzbauer [24].
3. Integrins, receptors for fibronectin Integrins are adhesion receptors which transmit signals bidirectionally across the plasma membrane [25]. They are also evolutionary old [26]. They comprise non-covalently bound two subunits, a and b which form a head and two long legs in the ectodomain and span the membrane to end in the cytoplamic compartment. In mammals, 18-a and 8-b subunits combine to give 24 specific dimers with different ligand binding properties. The extracellular domain comprises a ligand binding head domain and two long legs. a-subunit contains a seven-bladed head domain that forms the head, then a thigh domain, followed by calf-1 and -2 domains. Half of the a-subunits contain an I domain which contains the ligand binding site. It also contains a metal-iondependent-site (MIDAS) for divalent cations. The b-subunit is composed of a hybrid domain that connects to the b1 domain homologous to the I domain of a-subunit, a PSI domain (plexin/ semaphorin/integrin), four EGF domains, a membrane proximal btail domain. When integrin does not contain an I domain, ligands bind to a crevice between the a- b-subunit interface. The TM domains are poorly defined. The short a- and b-cytoplasmic domains are very similar. In the membrane proximal region, there are GFFKR and HDR (R/K) E sequences conserved in the a- and bsubunits. Almost all b-tails have recognition sequences for phosphotyrosine-binding (PTB) domains with a membrane proximal NPxY motif where x can represent any amino acid and a membrane distal NxxY motif. These last motifs are binding sites for integrin-binding proteins as talin and kindlins. Integrins bind ECM proteins through their large ectodomain and connect to the cytoskeleton through their short cytoplasmic tail [27,28,29,30]. Only a subset on integrins (eight out of 24) bind to the RGD sequence. The collagen-binding integrins recognize a triple helical sequence GFOGER. LDV is another sequence present in the IIICS alternatively spliced region of FN. Integrins exist in low-, intermediate and high-affinity stage [27]. They are quite unusual receptors because they transmit signals bi-directionally. In a first step, they encounter ECM macromolecules which induce activation. This activation increases the affinity of individual integrins for ECM ligands and induces clustering of integrins leading to immediate effects, up-regulation of lipid kinase activity and phosphorylation of protein substrates (for a review, see [28,29]). Signals are transduced from ECM to the cytoplasm (outside-in signaling). Affinity regulation plays a role in integrin priming
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(inside-out signaling), induces the lateral redistribution (clustering) which strengthens adhesion. Both integrin conformation change and clustering are necessary for outside-in signaling. There are changes in conformation of the cytoplasmic tails of integrins, linking to talin and kindlin. Talin binding to integrin b-tail produces an activation signal followed by kindlin cooperation with talin, behaving as adaptor proteins regulating integrin activation [31–33] and couple the integrin family to the actin cytoskeleton [34]. Activated and clustered integrins are then able to transmit outside-in signaling which regulates cell shape, migration, growth and survival.
4. Plasma fibronectin FNp concentration (330 mg/L, range 360–380) is higher in man than in women (280 mg/L average) [1]. This difference disappears after menopausis suggesting a hormonal regulation. In adults, FNp concentration increases exponentially as a function of age after 60 years [35,36]. However, during a study on the FNp level of centenarians in relatively good health, 268 persons (average 101.40 1.38 years) comprising 238 women (average age 101.37 years) and 30 men (101.66 1.34 years), were compared first to a population consisting of 31 patients, six men and 25 women between 60 and 96 years, residing in a geriatric ward at the Paul-Brousse Hospital in Villejuif, France. This population described previously suffered of a variety of pathologies, 11 patients had clinically diagnosed Alzheimer disease, five suffered from other dementias and 15 were cognitively normal with a variety of clinical problems and with a variety of pathologies. The results were also compared to those of a third population, from the EVA study (E´tude du vieillissement arterial: Study of brain aging) consisting of a group of men (520) and women (742) between 59 and 70 years average in good health as controls (average age 65 3) home-living, included in an extensive epidemiological study on vascular aging and brain aging [37]. We found that in centenarians FNp was in the lower range as compared to controls, 58.1 18.8 mg % vs 65.2 19.5 mg % (P = 0.002). No fibronectin-fragments (FN-fs) were found in centenarian plasma [38].
5. Role of nutrition pFN level diminished dramatically as a function of malnutrition. Malnutrition is a major health problem in less developed countries and also in industrialized countries [39]. In poor areas, it is more frequent among preschool children, in more developed areas it is most prevalent among elderly people and hospitalized patients. In a study comprising more than 10,000 elderly persons, the mean prevalence of malnutrition was found to be 1% in communitydwelling healthy elderly persons, 4% in out-patients receiving home care, 5% in Alzheimer patients living at home, 20% in hospitalized old patients and 37% in institutionalized elderly persons [40]. Studying FNp concentration in malnourished Liberian children, with a clinical diagnosis of marasmus or kwashiorkor, Sandberg et al. [41,42] found FNp levels lower than normal. The mean FNp level for controls was 253 mg/L. The mean level for the malnourished group was 96 mg/L (P < 0.0001). They also found that malnourished children with initial FNp levels above 100 mg/L had a higher survival rate than those with levels less than 100 mg/L (92 vs 69%). With successful therapy, FNp levels rose quickly in most children often before detectable changes were noted in clinical and other laboratory parameters. An overshoot of the mean normal levels was observed with successful treatment wherein the mean levels rose to 315 mg/L (P < 0.05). FNp determinations on malnourished children can serve as an important prognostic marker as well as a reliable indicator of successful therapy and recovery.
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In a study of FN in institutionalized elderly people, we also found in some cases a significant decrease of FNp < 100 mg/L, closely followed by death of these patients. It was also shown that FNp can be the hallmark of a fat overload of the liver. In this case, FNp was significantly increased and decreased after a successful diet. In pregnant women, FNp was significantly higher in women who developed pre-eclampsia than those who remained normotensive (383.68 mg/L 19.07 vs 227.65 mg/L 97.39, P < 0.0001). Plasma total FN 360 mg/L is predictive for the development of preeclampsia. FN level may serve also to predict clinical symptoms of pre-eclampsia [43]. Studying the expression of FNp molecular forms in an Alzheimer patient group compared with vascular dementia and age-matched controls by immunoblotting, the authors found, in the plasma of elderly individuals with and without dementia, a mixture of heterogeneous molecules with increasing molecular masses. Besides the bands usually found in plasma of 240 kDa and 220 kDa, there were more frequently high molecular FN forms of 280 kDa and 320 kDa in the plasma of the Alzheimer group and at a higher proportion than in vascular dementia and in age-matched non-demented controls suggesting that the molecular status of FNp could be an additional biomarker for the assessment of dementia risk [44]. 6. Tissue fibronectin Like FNp, tissue FN increases exponentially as a function of age. We studied the synthesis of tissue FN in mice [45] as correlated to the level of mRNA coding for FN. Similar results were obtained on fibroblast cultures as a function of passage number for cellular fibronectin (FNc) and the corresponding mRNA. During aging also, we noticed an exponential increase of proteolytic enzymes in tissues capable to degrade FN. Exposure to UVA and B radiation, at sub-erythemal doses, provoked in mice, an increase of FN biosynthesis and of the coding mRNA, comparable to the modifications observed during aging in mice and humans. During these experiments, we have observed also an increased expression of MMPs. FN is very sensitive to proteases and FN-degradation products were shown to exhibit a variety of biological properties not found for intact FN. Some of these properties are harmful and shown to form a vicious circle with age-dependent up-regulations [46]. These findings will be developed later. 7. Biological activities of fibronectin-fragments An original observation was made by Barlati et al. [47] which opened a new field in matrix biology and beyond in biochemistry. The Italian team showed that transformation-enhancing factors (TEF) were present in the culture media of tumor-virus-transformed cells. This activity was also found in human plasma cryoprecipitates from patients affected with different neoplastic diseases. TEF activity was obtained from plasminolytic fragments of FN and from Cathepsin G-treated FN [48]. Defined fragments have transformation-promoting activity and may serve as markers for tumor progression [49] among them, a FN13 amino acid peptide could be used as anti-tumoregenic agent [50]. The FN13 peptide inhibits tumor cell invasion through the modulation of anb3 integrin organization and the inactivation of ILK pathway [51]. At about the same time, the team of V. Keil at the Pasteur Institute in Paris published experiments [52–54] showing that FNfs obtained by treating FN with cathepsin-D have properties that the intact molecule did not possess: it contains a latent proteinase that after activation cleaves gelatin and FN. The purified gelatinase
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was a 35 kDa fragment from the N-terminal part and had a fibronectinase activity. Another fragment of 27 kDa from the beginning of the central part of the molecule showed a collagenase activity and a terminal 25 kDa fragment was able to cleave laminin. In cartilage, a 29 kDa gelatin-binding fragment was shown to increase gelatinolytic and collagenolytic activities and proteoglycan release in bovine articular cartilage explant cultures. FN is very sensitive to proteolytic degradation. Activation of extracellular proteolysis in osteoarthritis and rheumatoid arthritis may lead to FN fragmentation. Increased levels of FN-fs of 30 to 200 kDa are found in cartilage and synovial fluids [55,56,57,58]. An aminoterminal fragment of 29 kDa, very potent in term of cartilage damage, show proteoglycan degradation activity. Other fragments: 50 kDa, 140 kDa showed also proteoglycanase activity but to a lesser extent, enhanced phosporylation of ERK1/2, p38 and JNK1/2. Other fragments: a gelatin-binding 50 kDa and a central 140 kDa FN-fs activates ERK1/2. These results suggest that these FN-fs regulate MMP-3 and MMP-13 through MAP kinases by comparison of three fragments and native FN [59,60]. The same fragments mediated MMPs up-regulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-kappaB and protein kinase C-delta [61] providing a better knowledge of FN-fs involved in cartilage damage. FN-fs can also degrade aggrecan, type II collagen [62]. Up-regulation of MMPs is implicated in numerous pathologic processes including osteoarthritis and rheumatoid arthritis. MMPs comprises gelatinases, collagenases types I, II, III. Chondrocytes express MMP-1 collagenase 1. MMP-8 (collagenase 2), MMP-13. FN-fs alters MMP expression [63]. Some FN-fs, especially the COOH-terminal heparin-binding fragment induces nitric oxide production [64]. Cytokine production (IL-1b) and TNF-a promote cartilage degradation by stimulating the production of MMPs [65]. Similar observations were published on the generation of biologically active fragments from other matrix components besides FN: laminin, collagens, proteoglycans. . . In the context of wound healing, it was shown that FN was degraded more extensively in fluids from poorly healing diabetic wounds compared to normal wounds [66]. Elastase could be the key enzyme [67]. Other enzymes up-regulated in diabetic wounds are MMPs which are also able to cleave FN. Fragments of FN have been detected in severe periodontitis. We note that 40, 68 and 120 kDa FN-fs were more frequently present in severe periodontal disease. This can alter cellular behavior [68]. During pathological circumstances, there is a remodeling of extracellular matrices and exposure of cryptic sites normally hidden in the molecules with revelation of new properties of the generated peptides. ECM is a support for cells, but it is also a reserve of cytokines, growth factors, enzymes able to generate changes of conformation. These observations initiated the proposition to designate matrix derived biologically active peptides: matricryptins and even for some of them matrikins. As a matter of fact, their action resembles those of cytokines. In contradistinction, however, from cytokines, matrikins do not derive from the activation of distinct genes coding for them. They all are derived from matrix components, macromolecules of the ECM by proteolytic attack. These discoveries opened therefore a new field of investigations in the biology and pathology of ECM [69].
8. Conclusions We presented in this review some of the recent results on cellmatrix interactions in general and more specifically on the role of FN and integrins. Since its start with the last decades of the 20th
century, this field of matrix biology was the subject of a rapid expansion. Besides FN, a number of other matrix glycoproteins were identified and shown to fulfill similar functions however, in a more restricted field of cell-matrix interactions. The number of significant publications on FN and also on integrins exploded also during the last decades. For these reasons, we restricted our review to some observations considered to be of particular relevance to the recent extension of the mechanisms and roles of cell-matrix interactions. Before closing, we should mention another recent and important field of interest concerning the mediation of mechanical forces on the modulation of cell phenotype and cell-matrix interactions. This field started with the observation of the effect of blood flow through blood vessels on the phenotype of endothelial cells. Recent reviews suggest that mechanotransduction is involved in several important functions of matrix-rich (connective) tissues. The recent findings, together with those concerning the role of cellular and molecular components of the Biomatrix in the malignant process strongly contributed to the rapid extension of matrix biology and more especially to the importance of cellmatrix interactions. Disclosure of interest The author declares that he has no conflicts of interest concerning this article. References [1] Hynes RO. Fibronectins. Springer; 1990. [2] Engel J. Domains in proteins and proteoglycans of the extracellular matrix with functions in assembly and cellular activities. Int J Biol Macromol 1991;13:147–51. [3] Labat-Robert J, Robert L, Auger C, Lethias C, Garrone R. A fibronectin-like protein in porifera; its role in cell aggregation. Proc Natl Acad Sci U S A 1981;78:6261–5. [4] Tamkun JW, Hynes RO. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem 1983;258:4641–7. [5] Moretti FA, Chauhan AL, Iaconcig A, Porro F, Baralle FE, Muro AF. A major fraction of fibronectin present in the extracellular matrix of tissues is plasmaderived. J Biol Chem 2007;282:28057–62. [6] Muro AF, Chauhan AK, Galovic S, Iaconcig A, Porro F, Stanta G, et al. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan. J Cell Biol 2003;162:149–51. [7] Kornblihtt AR, Vibe-Petersen K, Baralle FE. Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain. EMBO J 1984;3:221–6. [8] Gutman A, Kornblihtt AR. Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA. Proc Natl Acad Sci U S A 1987;84:7179–82. [9] Schwarzbauer JE, Tamkun JW, Lemischka JR, Hynes RO. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell 1983;35:421–31. [10] Peters JH, Greasby T, Lane N, Woolf A. Correlations between plasma levels of a fibronectin isoform subpopulation and C-reactive protein in patients with systemic inflammatory disease. Biomarkers 2009;14:250–7. [11] Hino K, Shiozawa S, Kuroki Y, Ishikawa H, Shiozawa K, Sekiguchi K, et al. EDAcontaining fibronectin is synthesized from rheumatoid synovial fibroblast-like cells. Arthritis Rheum 1995;38:678–83. [12] Singh P, Reimer C, Peters JH, Stepp MA, Hynes RO, Van de Water L. J The spatial and temporal expression patterns of integrin a9b1 and one of its ligands, the EIIIA segment of fibronectin, in cutaneous wound healing. J Invest Dermatol 2004;123:793–801. [13] Shinde AV, Bystroff C, Wang C, Vogelezang M, Vincent PA, Hynes RO, et al. Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin a9b1-dependent cellular activities. J Biol Chem 2008;283:2858–70. [14] Guan JL, Hynes RO. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor a4b1. Cell 1990;60:53–61. [15] Hynes RO. Integrins: versatility, modulation and signaling in cell adhesion. Cell 1992;44:517–8. [16] Ruoslahti E, Pierschbacher M. RGD, a versatile cell recognition signal. Cell 1986;44:517–8. [17] Schwarbauer JE, Sechler JL. Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly. Curr Opin Cell Biol 1999;11:622–7. [18] Sechler JL, Cumiskey AM, Gazzola DM, Scharzbauer JE. A novel RGD-independent fibronectin assembly pathway initiated by a4b1 integrin-binding to the alternatively spliced V region. J Cell Sci 2000;113:1491–8.
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[46] Labat-Robert J. Cell-matrix interactions in aging: role of receptors and matricryptins. Aging Res Rev 2004;3:233–47. [47] de Petro G, Barlati S, Vartio T, Vaheri A. Transformation-enhancing activity of gelatin-binding fragments of fibronectin. Proc Natl Acad Sci U S A 1981;78:4965–9. [48] De Petro, Tavian D, Copeta A, Fortolani N, Giulini SM, Barlati S. Expression of urokinase-type plasminogren activator (u-PA), u-PA receptor and tissue-type PA messenger RNAs in human hepatocellular carcinoma. Cancer Res 1998;58:2234–9. [49] Vartio T, Vaheri A, de Petro G, Barlati S. Fibronectin and its proteolytic fragments. Potential as cancer markers. Invasion Metastasis 1983;3:125–38. [50] Colombi M, Zoppi N, de Petro G, Marchina E, Gardella R, Tavian D, et al. Matrix assembly induction and cell migration and invasion inhibition by a 13-amino acid fibronectin peptide. J Biol Chem 2003;16:14346–55. [51] Zoppi N, Ritelli M, Salvi A, Colombi M, Barlati S. The FN13 peptide inhibits human tumor cells invasion through the modulation of anb3 integrins organization and the inactivation of ILK pathway. Biochem Biophys Acta 2007;1773:747–63. [52] Keil-Dlouha V, Planchenault T, Imhoff JM, Emod I, Blondeau X, LambertVidmar S. Proteolytic potential of fibronectin and degradation of extracellular matrix. Pathol Biol 1990;38:993–8. [53] Planchenault T, Lambert-Vidmar, Imhoff JM, Blondeau C, Emod I, Lottspeich F, et al. Potential proteolytic activity of human plasma fibronectin: fibronectin gelatinase. Biol Chem Hoppe-Seyler 1990;371:117–28. [54] Emod I, Lafaye P, Planchenault T, Lambert-Vidmar S, Imhoff JM, Keil-Dlouha V. Potential proteolytic activity of fibronectin: fibronectin laminase and its substrate specificity. Biol Chem Hoppe-Seyler 1990;371:129–35. [55] Homandberg GA, Davis G, Maniglia C, Shrikhande A. Cartilage chondrolysis by fibronectin-fragments causes cleavage of aggrecan at the same site as found in osteoarthritic cartilage. Osteoarthritis Cartilage 1999;6:450–3. [56] Xie D, Homandberg GA. Fibronectin-fragments bind to and penetrate cartilage tissue resulting in proteinase expression and cartilage damage. Biochim Biophys Acta 1993;1882:189–96. [57] Homandberg GA. Potential regulation of cartilage metabolism in osteoarthritis by fibronectin-fragments. Front Biosci 1999;4(D7):13–30. [58] Homandberg GA. Cartilage damage by matrix degradation products: fibronectin-fragments. Clin Orthop Relat Res 2001;391 S:100–7. [59] Purple CR, Untermann TG, Pichika R, Homandberg GAG. Fibronectin-fragments up-regulate insulin-like growth factor binding proteins in chondrocytes. Osteoarthritis Cartilage 2002;9:734–46. [60] Ding L, Guo D, Homandberg GA. The cartilage chondrolytic mechanism of fibronectin-fragments involves MAP kinases: comparison of three fragments and native fibronectin. Osteoarthritis Cartilage 2008;10:1253–62. [61] Ding L, Guo D, Homandberg GA. Fibronectin-fragments mediate matrix metalloproteinases up-regulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-kappaB and protein kinase C-delta. Osteoarthritis Cartilage 2009;10:1385–13892. [62] Homandberg GA, Meyers R, Williams M. Intra-articular injection of fibronectin-fragments causes severe deletion of cartilage proteoglycan in vivo. J Rheumatol 1993;20:1378–82. [63] Werb Z, Tremble PM, Berhendtsen O, Crowley E, Damski DH. Signal transduction through the fibronectin receptor induces collagenase and stromelysine gene expression. J Cell Biol 1989;109:877–89. [64] Yasuda T, Kakinuma T, Julovi SM, Yoshida M, Hiramitsu T, Akiyoshi M. COOHterminal heparin-binding fibronectin-fragment induces nitric oxide production in rheumatoid cartilage through CD44. Rheumatology 2004;43:1116– 11120. [65] Yasuda T. Cartilage destruction by matrix degradation products. Mod Rheumatol 2006;16:197–205. [66] Wysocki AB, Grinnell F. Fibronectin profiles in normal and chronic wound fluid. Lab Invest 1990;63:825–31. [67] Grinnell F, Zhu M. Fibronectin degradation in chronic wounds depends on the relative levels of elastase, a1-proteinase inhibitor, and a2-macroglobulin. J Invest Dermatol 1996;106:335–41. [68] Stanley CM, Wang Y, Pal S, Klebe RJ, Harkless LB, Xu X, et al. Fibronectin fragmentation is a feature of both periodontal disease sites and diabetic foot and leg wounds and modifies cell behavior. J Periodontol 2008;79:861–75. [69] Davis GE, Bayless KJ, Davis MJ, Meininger GA. Regulation of tissue injury response by the exposure of matricryptic sites within extracellular matrix molecules. Proc Natl Acad Sci U S A 2000;78:4965–9.
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50th anniversary – French Society for Connective Tissue Research
Cell–Matrix interactions, the role of fibronectin and integrins. A survey Interactions cellules–matrice extracellulaire, roˆle de la fibronectine et des inte´grines. Une revue J. Labat-Robert Laboratoire de recherche ophtalmologique, universite´ Paris-V, hoˆpital Hoˆtel-Dieu, 1, place du Parvis-Notre-Dame, 75181 Paris cedex 04, France
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2011 Accepted 16 September 2011 Available online 21 January 2012
In this review, we present several aspects of cell-matrix interactions, especially the role of fibronectin and integrins in the mediation of these interactions. As this field of investigations literally exploded over the last decades, we had to limit this review to some aspects of this field. We cited experiments giving details on the modifications of fibronectin molecules during their interactions with cells as well as on recent progress of the molecular mechanisms of fibronectin–integrin interactions. We insisted on the molecular details which were shown to play a role in the bi-directional signals ‘‘sent’’ by cells to the surrounding matrix (inside-out and outside-in). A number of recent publications confirmed the physiopathological importance of these messages both for the normal function of tissues as well as for the understanding of their pathological modifications. We insist also on the importance of fibronectin-fragments during some pathologies. ß 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Extracellular matrix Connective Tissues Fibronectin Integrins Remodeling Proteases Fibronectin-fragments Matricryptins Mots cle´s: Matrice extracellulaire Tissus conjonctifs Fibronectine Inte´grines Remodelage Prote´ases Fragments de fibronectine Matricryptines
R E´ S U M E´
Nous pre´sentons dans cette revue plusieurs aspects de l’interaction entre les cellules et la matrice extracellulaire et, plus particulie`rement, le roˆle de la fibronectine et des inte´grines dans ces processus. Comme ce domaine de recherche a litte´ralement explose´ au cours des dernie`res anne´es, nous avons e´te´ amene´s a` restreindre cette revue a` quelques aspects des modifications de la conformation de la fibronectine au cours de son interaction avec les cellules, ainsi qu’aux derniers de´tails des changements de conformation au cours de l’interaction entre fibronectine et inte´grines et leur roˆle dans la transduction bilate´rale des signaux entre cellule et matrice. Les de´tails mole´culaires de ces interactions me´diatrices de signaux jouent, en effet, un roˆle important dans la biologie des tissus conjonctifs ainsi que dans leurs modifications pathologiques. Nous insistons aussi sur l’importance des fragments de fibronectine au cours de certaines pathologies. ß 2011 Elsevier Masson SAS. Tous droits re´serve´s.
1. Introduction Not such a long time ago, extracellular matrix (ECM) was considered as an inert support for the cells it surrounds. We now know that on the contrary, ECM represents a dynamic and complex environment. There is between cells and ECM a permanent crosstalk through receptors, mainly the integrin family. Doing so, ECM delivers to cells signals controlling their shape, migration, proliferation, differentiation and survival. The molecular components of ECM belong to four families of macromolecules: collagen(s), elastin(s), glycosaminoglycans and
Adresse e-mail : [email protected]. 0369-8114/$ – see front matter ß 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.patbio.2011.10.003
structural or matrix glycoproteins. Collagens and elastin(s) are fibrous proteins, glycosaminoglycans and proteoglycans are proteinpolysaccharides or pure polysaccharides (hyaluronan) which fill the meshes of the fibrillar lattice formed by the collagens and elastin fibers whereas structural glycoproteins play an organizing role in the ECM construction. This organization is not static. ECM composition varies as a function of the differentiated state of tissues and as a function of age. It changes during development, maturity, pathologies and aging. Thus the organization of ECM is a dynamic process. Connective tissues are ECM-rich but all tissues contain variable amounts of ECM. ECM can be calcified to form bones or teeth, but in cornea, it is transparent. Its structure takes a variety of shapes and confers to tissues strength and elasticity in varying proportions. Between epithelia
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and connective tissues, it forms a basal lamina which is a specialized form of ECM.
2. Fibronectin Among structural glycoproteins, fibronectin (FN) was the first to be intensively studied [1]. This glycoprotein family is composed of high molecular weight molecules. The peptides which compose the subunits comprise three types of repeats, types I, II and III, assembled into domains with autonomous and specific functions. Glycoproteins like FN are often designated as mosaic proteins [2]. In FN, there are domains with binding sites for bacteria, collagen, fibrin, FN itself and heparin. Thus, FN can play a role in the organization of ECM. But, there is also in the FNIII10 repeat an important cell-binding site (RGD) interacting with the main FN receptor a5b1 integrin. This site needs, for complete activity, the presence in the FNIII9 motif of a synergy sequence (PHSRN) of amino acids. Thus, FN plays an important role in matrix assembly, in cell adhesion and spreading, migration, morphology, cytoskeletal organization and also in oncogenic transformation. FN is an ubiquitous protein present from the first pluricellular eukaryotes, the sponges [3]. FN is present in a soluble form in plasma and biological fluids. Plasma fibronectin (FNp) is synthesized by hepatocytes [4]. FN is also present in tissues in an insoluble form synthesized by fibroblasts, epithelial cells and other differentiated cell types. Nevertheless, FNp can diffuse into tissues and be incorporated in the fibrillar matrix [5]. There are a number of isoforms issued from a single gene by alternative splicing of the primary transcript generating up to 20 different variants in humans [6]. Splicing was shown to occur in three regions: EIIIA and EIIIB where the exon can be included or skipped during transcription. FNp never contains these two exons [1,7–9]. However, small quantities of FN containing EIIIA (A+FN) are present in the circulation of healthy individuals [10]. In tissues, EIIIA and EIIIB are present in various combinations; during development, they are present at an elevated level but they are low in adult tissues, increasing during some pathologies such as, rheumatoid arthritis [11], wound healing [12]. Peptide sequences have been identified which mediate integrin a9b1 dependent cellular activities [13]. During wound healing, EIIIA is expressed transiently by wound cells (day 4 to 7), whereas EIIIB persists through day 14 (rat). Similar processing of FN was shown also in rheumatoid arthritis and tumors. A third region shows alternative splicing: V/IIICS (for the type III connecting segment). Here, the exon can be subdivided giving rise to several variants of FN, generating new cell-binding site, LDV [14], especially for a4b1 integrin expressed by leukocytes and lymphocytes and REDV for a4b7 integrin. FN is synthesized as a monomer followed very rapidly by dimerisation in the rough endoplasmic reticulum. The IIICS region is required for formation and secretion of native FN dimers. FNp are 50% V0, according to the species of origin. It lacks V0 – V0 dimers and consists of V0 – V+ and V+ – V+ combinations. Fibroblasts produce very few V0 subunits. FN in solution forms a compact dimer which does not undergo fibril assembly, but in culture and in tissues, FN assembles through a cell-mediated process. Cell-associated FN is first distributed diffusely over the cell surface. This dimeric ligand induces integrin clustering by binding to two integrins [15], this produces FN selfassociation [16] and connection of FN to the cytoskeleton. The cysteins forming the dimerisation site are essential for assembly, they play a role in integrin clustering and FN–FN interactions. On each subunit, there are four sites for FN–FN interactions. An RGDindependent mechanisms implies a4b1 to the CS1 site. The amino-terminal type I repeats (II–5) are an essential assembly domain. FN is secreted as a disulfide-bonded dimer in a compact
inactive form. Integrin (a5b1) binding will convert FN in an active, extended dimer, through the RGD binding sequence in the type III10 module [15] and the synergy sequence located in type III9 module [16]. Then fibril formation will take place. Functions of FN critically depend on the assembly of secreted FN dimers into a fibrillar network in order to FN assembly to occur. As assembly progresses, dimeric FN forms short deoxycholate-soluble fibrils, later converted in a dense detergent-insoluble network in a stepwise manner [17–21]. More recently, Takahashi et al. [22] could show that the RGD motif is dispensable for fibril assembly. Using mutant embryos or mutant cells where RGD motif was replaced with RGE, they could show that in the absence of RGD motif FN assembly was not compromised to form FN matrix. anb3 integrin assembles FN-RGE by binding an iso-DGR motif in FN1-5 which is generated by the non-enzymatic rearrangement of asparagines into an iso-aspartate. The RGD motif is however essential during development. During matrix assembly, cryptic binding sites are exposed by unfolding of individual type III modules. FN fibrils are quite elastic as shown by Ohashi et al. [23]. A good review on FN matrix assembly was published by Wierzbicka-Patynowski and Schwarzbauer [24].
3. Integrins, receptors for fibronectin Integrins are adhesion receptors which transmit signals bidirectionally across the plasma membrane [25]. They are also evolutionary old [26]. They comprise non-covalently bound two subunits, a and b which form a head and two long legs in the ectodomain and span the membrane to end in the cytoplamic compartment. In mammals, 18-a and 8-b subunits combine to give 24 specific dimers with different ligand binding properties. The extracellular domain comprises a ligand binding head domain and two long legs. a-subunit contains a seven-bladed head domain that forms the head, then a thigh domain, followed by calf-1 and -2 domains. Half of the a-subunits contain an I domain which contains the ligand binding site. It also contains a metal-iondependent-site (MIDAS) for divalent cations. The b-subunit is composed of a hybrid domain that connects to the b1 domain homologous to the I domain of a-subunit, a PSI domain (plexin/ semaphorin/integrin), four EGF domains, a membrane proximal btail domain. When integrin does not contain an I domain, ligands bind to a crevice between the a- b-subunit interface. The TM domains are poorly defined. The short a- and b-cytoplasmic domains are very similar. In the membrane proximal region, there are GFFKR and HDR (R/K) E sequences conserved in the a- and bsubunits. Almost all b-tails have recognition sequences for phosphotyrosine-binding (PTB) domains with a membrane proximal NPxY motif where x can represent any amino acid and a membrane distal NxxY motif. These last motifs are binding sites for integrin-binding proteins as talin and kindlins. Integrins bind ECM proteins through their large ectodomain and connect to the cytoskeleton through their short cytoplasmic tail [27,28,29,30]. Only a subset on integrins (eight out of 24) bind to the RGD sequence. The collagen-binding integrins recognize a triple helical sequence GFOGER. LDV is another sequence present in the IIICS alternatively spliced region of FN. Integrins exist in low-, intermediate and high-affinity stage [27]. They are quite unusual receptors because they transmit signals bi-directionally. In a first step, they encounter ECM macromolecules which induce activation. This activation increases the affinity of individual integrins for ECM ligands and induces clustering of integrins leading to immediate effects, up-regulation of lipid kinase activity and phosphorylation of protein substrates (for a review, see [28,29]). Signals are transduced from ECM to the cytoplasm (outside-in signaling). Affinity regulation plays a role in integrin priming
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(inside-out signaling), induces the lateral redistribution (clustering) which strengthens adhesion. Both integrin conformation change and clustering are necessary for outside-in signaling. There are changes in conformation of the cytoplasmic tails of integrins, linking to talin and kindlin. Talin binding to integrin b-tail produces an activation signal followed by kindlin cooperation with talin, behaving as adaptor proteins regulating integrin activation [31–33] and couple the integrin family to the actin cytoskeleton [34]. Activated and clustered integrins are then able to transmit outside-in signaling which regulates cell shape, migration, growth and survival.
4. Plasma fibronectin FNp concentration (330 mg/L, range 360–380) is higher in man than in women (280 mg/L average) [1]. This difference disappears after menopausis suggesting a hormonal regulation. In adults, FNp concentration increases exponentially as a function of age after 60 years [35,36]. However, during a study on the FNp level of centenarians in relatively good health, 268 persons (average 101.40 1.38 years) comprising 238 women (average age 101.37 years) and 30 men (101.66 1.34 years), were compared first to a population consisting of 31 patients, six men and 25 women between 60 and 96 years, residing in a geriatric ward at the Paul-Brousse Hospital in Villejuif, France. This population described previously suffered of a variety of pathologies, 11 patients had clinically diagnosed Alzheimer disease, five suffered from other dementias and 15 were cognitively normal with a variety of clinical problems and with a variety of pathologies. The results were also compared to those of a third population, from the EVA study (E´tude du vieillissement arterial: Study of brain aging) consisting of a group of men (520) and women (742) between 59 and 70 years average in good health as controls (average age 65 3) home-living, included in an extensive epidemiological study on vascular aging and brain aging [37]. We found that in centenarians FNp was in the lower range as compared to controls, 58.1 18.8 mg % vs 65.2 19.5 mg % (P = 0.002). No fibronectin-fragments (FN-fs) were found in centenarian plasma [38].
5. Role of nutrition pFN level diminished dramatically as a function of malnutrition. Malnutrition is a major health problem in less developed countries and also in industrialized countries [39]. In poor areas, it is more frequent among preschool children, in more developed areas it is most prevalent among elderly people and hospitalized patients. In a study comprising more than 10,000 elderly persons, the mean prevalence of malnutrition was found to be 1% in communitydwelling healthy elderly persons, 4% in out-patients receiving home care, 5% in Alzheimer patients living at home, 20% in hospitalized old patients and 37% in institutionalized elderly persons [40]. Studying FNp concentration in malnourished Liberian children, with a clinical diagnosis of marasmus or kwashiorkor, Sandberg et al. [41,42] found FNp levels lower than normal. The mean FNp level for controls was 253 mg/L. The mean level for the malnourished group was 96 mg/L (P < 0.0001). They also found that malnourished children with initial FNp levels above 100 mg/L had a higher survival rate than those with levels less than 100 mg/L (92 vs 69%). With successful therapy, FNp levels rose quickly in most children often before detectable changes were noted in clinical and other laboratory parameters. An overshoot of the mean normal levels was observed with successful treatment wherein the mean levels rose to 315 mg/L (P < 0.05). FNp determinations on malnourished children can serve as an important prognostic marker as well as a reliable indicator of successful therapy and recovery.
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In a study of FN in institutionalized elderly people, we also found in some cases a significant decrease of FNp < 100 mg/L, closely followed by death of these patients. It was also shown that FNp can be the hallmark of a fat overload of the liver. In this case, FNp was significantly increased and decreased after a successful diet. In pregnant women, FNp was significantly higher in women who developed pre-eclampsia than those who remained normotensive (383.68 mg/L 19.07 vs 227.65 mg/L 97.39, P < 0.0001). Plasma total FN 360 mg/L is predictive for the development of preeclampsia. FN level may serve also to predict clinical symptoms of pre-eclampsia [43]. Studying the expression of FNp molecular forms in an Alzheimer patient group compared with vascular dementia and age-matched controls by immunoblotting, the authors found, in the plasma of elderly individuals with and without dementia, a mixture of heterogeneous molecules with increasing molecular masses. Besides the bands usually found in plasma of 240 kDa and 220 kDa, there were more frequently high molecular FN forms of 280 kDa and 320 kDa in the plasma of the Alzheimer group and at a higher proportion than in vascular dementia and in age-matched non-demented controls suggesting that the molecular status of FNp could be an additional biomarker for the assessment of dementia risk [44]. 6. Tissue fibronectin Like FNp, tissue FN increases exponentially as a function of age. We studied the synthesis of tissue FN in mice [45] as correlated to the level of mRNA coding for FN. Similar results were obtained on fibroblast cultures as a function of passage number for cellular fibronectin (FNc) and the corresponding mRNA. During aging also, we noticed an exponential increase of proteolytic enzymes in tissues capable to degrade FN. Exposure to UVA and B radiation, at sub-erythemal doses, provoked in mice, an increase of FN biosynthesis and of the coding mRNA, comparable to the modifications observed during aging in mice and humans. During these experiments, we have observed also an increased expression of MMPs. FN is very sensitive to proteases and FN-degradation products were shown to exhibit a variety of biological properties not found for intact FN. Some of these properties are harmful and shown to form a vicious circle with age-dependent up-regulations [46]. These findings will be developed later. 7. Biological activities of fibronectin-fragments An original observation was made by Barlati et al. [47] which opened a new field in matrix biology and beyond in biochemistry. The Italian team showed that transformation-enhancing factors (TEF) were present in the culture media of tumor-virus-transformed cells. This activity was also found in human plasma cryoprecipitates from patients affected with different neoplastic diseases. TEF activity was obtained from plasminolytic fragments of FN and from Cathepsin G-treated FN [48]. Defined fragments have transformation-promoting activity and may serve as markers for tumor progression [49] among them, a FN13 amino acid peptide could be used as anti-tumoregenic agent [50]. The FN13 peptide inhibits tumor cell invasion through the modulation of anb3 integrin organization and the inactivation of ILK pathway [51]. At about the same time, the team of V. Keil at the Pasteur Institute in Paris published experiments [52–54] showing that FNfs obtained by treating FN with cathepsin-D have properties that the intact molecule did not possess: it contains a latent proteinase that after activation cleaves gelatin and FN. The purified gelatinase
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was a 35 kDa fragment from the N-terminal part and had a fibronectinase activity. Another fragment of 27 kDa from the beginning of the central part of the molecule showed a collagenase activity and a terminal 25 kDa fragment was able to cleave laminin. In cartilage, a 29 kDa gelatin-binding fragment was shown to increase gelatinolytic and collagenolytic activities and proteoglycan release in bovine articular cartilage explant cultures. FN is very sensitive to proteolytic degradation. Activation of extracellular proteolysis in osteoarthritis and rheumatoid arthritis may lead to FN fragmentation. Increased levels of FN-fs of 30 to 200 kDa are found in cartilage and synovial fluids [55,56,57,58]. An aminoterminal fragment of 29 kDa, very potent in term of cartilage damage, show proteoglycan degradation activity. Other fragments: 50 kDa, 140 kDa showed also proteoglycanase activity but to a lesser extent, enhanced phosporylation of ERK1/2, p38 and JNK1/2. Other fragments: a gelatin-binding 50 kDa and a central 140 kDa FN-fs activates ERK1/2. These results suggest that these FN-fs regulate MMP-3 and MMP-13 through MAP kinases by comparison of three fragments and native FN [59,60]. The same fragments mediated MMPs up-regulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-kappaB and protein kinase C-delta [61] providing a better knowledge of FN-fs involved in cartilage damage. FN-fs can also degrade aggrecan, type II collagen [62]. Up-regulation of MMPs is implicated in numerous pathologic processes including osteoarthritis and rheumatoid arthritis. MMPs comprises gelatinases, collagenases types I, II, III. Chondrocytes express MMP-1 collagenase 1. MMP-8 (collagenase 2), MMP-13. FN-fs alters MMP expression [63]. Some FN-fs, especially the COOH-terminal heparin-binding fragment induces nitric oxide production [64]. Cytokine production (IL-1b) and TNF-a promote cartilage degradation by stimulating the production of MMPs [65]. Similar observations were published on the generation of biologically active fragments from other matrix components besides FN: laminin, collagens, proteoglycans. . . In the context of wound healing, it was shown that FN was degraded more extensively in fluids from poorly healing diabetic wounds compared to normal wounds [66]. Elastase could be the key enzyme [67]. Other enzymes up-regulated in diabetic wounds are MMPs which are also able to cleave FN. Fragments of FN have been detected in severe periodontitis. We note that 40, 68 and 120 kDa FN-fs were more frequently present in severe periodontal disease. This can alter cellular behavior [68]. During pathological circumstances, there is a remodeling of extracellular matrices and exposure of cryptic sites normally hidden in the molecules with revelation of new properties of the generated peptides. ECM is a support for cells, but it is also a reserve of cytokines, growth factors, enzymes able to generate changes of conformation. These observations initiated the proposition to designate matrix derived biologically active peptides: matricryptins and even for some of them matrikins. As a matter of fact, their action resembles those of cytokines. In contradistinction, however, from cytokines, matrikins do not derive from the activation of distinct genes coding for them. They all are derived from matrix components, macromolecules of the ECM by proteolytic attack. These discoveries opened therefore a new field of investigations in the biology and pathology of ECM [69].
8. Conclusions We presented in this review some of the recent results on cellmatrix interactions in general and more specifically on the role of FN and integrins. Since its start with the last decades of the 20th
century, this field of matrix biology was the subject of a rapid expansion. Besides FN, a number of other matrix glycoproteins were identified and shown to fulfill similar functions however, in a more restricted field of cell-matrix interactions. The number of significant publications on FN and also on integrins exploded also during the last decades. For these reasons, we restricted our review to some observations considered to be of particular relevance to the recent extension of the mechanisms and roles of cell-matrix interactions. Before closing, we should mention another recent and important field of interest concerning the mediation of mechanical forces on the modulation of cell phenotype and cell-matrix interactions. This field started with the observation of the effect of blood flow through blood vessels on the phenotype of endothelial cells. Recent reviews suggest that mechanotransduction is involved in several important functions of matrix-rich (connective) tissues. The recent findings, together with those concerning the role of cellular and molecular components of the Biomatrix in the malignant process strongly contributed to the rapid extension of matrix biology and more especially to the importance of cellmatrix interactions. Disclosure of interest The author declares that he has no conflicts of interest concerning this article. References [1] Hynes RO. Fibronectins. Springer; 1990. [2] Engel J. Domains in proteins and proteoglycans of the extracellular matrix with functions in assembly and cellular activities. Int J Biol Macromol 1991;13:147–51. [3] Labat-Robert J, Robert L, Auger C, Lethias C, Garrone R. A fibronectin-like protein in porifera; its role in cell aggregation. Proc Natl Acad Sci U S A 1981;78:6261–5. [4] Tamkun JW, Hynes RO. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem 1983;258:4641–7. [5] Moretti FA, Chauhan AL, Iaconcig A, Porro F, Baralle FE, Muro AF. A major fraction of fibronectin present in the extracellular matrix of tissues is plasmaderived. J Biol Chem 2007;282:28057–62. [6] Muro AF, Chauhan AK, Galovic S, Iaconcig A, Porro F, Stanta G, et al. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan. J Cell Biol 2003;162:149–51. [7] Kornblihtt AR, Vibe-Petersen K, Baralle FE. Human fibronectin: molecular cloning evidence for two mRNA species differing by an internal segment coding for a structural domain. EMBO J 1984;3:221–6. [8] Gutman A, Kornblihtt AR. Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA. Proc Natl Acad Sci U S A 1987;84:7179–82. [9] Schwarzbauer JE, Tamkun JW, Lemischka JR, Hynes RO. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell 1983;35:421–31. [10] Peters JH, Greasby T, Lane N, Woolf A. Correlations between plasma levels of a fibronectin isoform subpopulation and C-reactive protein in patients with systemic inflammatory disease. Biomarkers 2009;14:250–7. [11] Hino K, Shiozawa S, Kuroki Y, Ishikawa H, Shiozawa K, Sekiguchi K, et al. EDAcontaining fibronectin is synthesized from rheumatoid synovial fibroblast-like cells. Arthritis Rheum 1995;38:678–83. [12] Singh P, Reimer C, Peters JH, Stepp MA, Hynes RO, Van de Water L. J The spatial and temporal expression patterns of integrin a9b1 and one of its ligands, the EIIIA segment of fibronectin, in cutaneous wound healing. J Invest Dermatol 2004;123:793–801. [13] Shinde AV, Bystroff C, Wang C, Vogelezang M, Vincent PA, Hynes RO, et al. Identification of the peptide sequences within the EIIIA (EDA) segment of fibronectin that mediate integrin a9b1-dependent cellular activities. J Biol Chem 2008;283:2858–70. [14] Guan JL, Hynes RO. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor a4b1. Cell 1990;60:53–61. [15] Hynes RO. Integrins: versatility, modulation and signaling in cell adhesion. Cell 1992;44:517–8. [16] Ruoslahti E, Pierschbacher M. RGD, a versatile cell recognition signal. Cell 1986;44:517–8. [17] Schwarbauer JE, Sechler JL. Fibronectin fibrillogenesis: a paradigm for extracellular matrix assembly. Curr Opin Cell Biol 1999;11:622–7. [18] Sechler JL, Cumiskey AM, Gazzola DM, Scharzbauer JE. A novel RGD-independent fibronectin assembly pathway initiated by a4b1 integrin-binding to the alternatively spliced V region. J Cell Sci 2000;113:1491–8.
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