The role of integrin binding sites in fibronectin matrix assembly in vivo

https://doi.org/10.1016/j.ceb.2008.06.001Get rights and content

The extracellular matrix (ECM) glycoprotein fibronectin (FN) requires the help of cells to assemble into a functional fibrillar matrix, which then orchestrates the assembly of other ECM proteins and promotes cell adhesion, migration and signalling. Fibrillogenesis is initiated and governed by cell surface integrins that bind to specific sites in the FN molecule. Recent studies identified novel integrin binding sites in FN that can also participate in FN fibril formation and in morphogenetic events during development.

Introduction

The extracellular matrix (ECM), which plays crucial roles in development, tissue homeostasis and disease, is a heterogeneous meshwork of fibrillar and non-fibrillar components. ECM can be assembled into elaborate structures such as basement membranes, provide a scaffold for cell adhesion and migration, and regulate numerous cell functions by activating multiple signalling pathways at adhesion sites. A major fraction of the ECM is composed of collagens, laminins and other glycoproteins such as fibronectin (FN), which serve as substrates for different adhesion molecules including the heterodimeric cell surface receptors, integrins [1]. Importantly, these ECM components are secreted by cells as non-functional building units, which are assembled into functional supramolecular structures in a highly regulated manner [2, 3, 4].

The functional properties of the FN fibrillar matrix are diverse and represent a prime example of how ECM protein assembly functions. First, FN fibrils possess binding sites for multiple ECM components (Figure 1), which are used to orchestrate the assembly of several other ECM proteins, including collagen I and III, fibulin-1, fibrinogen, thrombospondin-1 (for a review see [5]), latent Transforming Growth Factor-β (TGF-β) binding protein-1 (LTBP-1) [6], decorin and biglycan (Chen et al., in press). Second, FN fibrils provide structural support for cell adhesion at the same time as the adhesion receptors, most notably integrins, transduce signals that promote actin dynamics, cell migration, cell proliferation and apoptosis [2, 3]. Finally, FN controls the availability of growth factors, for example by regulating their activation from latent complexes as shown for TGF-β [7]. Therefore, it is not surprising that a constitutive FN gene ablation in mice has dramatic consequences in vivo that are characterized by severe mesodermal, vascular and neural tube defects leading to lethality at around embryonic day (E) 8.5 (E8.5) [8].

FN is a modular protein, which consists of an array of type I, II and III domains and is characterized by several FN-specific features. FN is secreted as a disulfide-bonded dimer, and the dimerization seems to be required to assemble FN into a fibrillar matrix (see Figure 1; [9]). Furthermore, the FN gene can be alternatively spliced allowing the expression of up to 20 possible monomeric isoforms in man and up to 12 in mouse, which may result in an even larger variety of FN dimers [10]. Finally, FN exists in two forms; cellular FN (cFN), which is present in tissues where it is assembled into a fibrillar matrix, and plasma FN (pFN), which is produced by hepatocytes and is secreted into the blood where it remains in a non-fibrillar, soluble form. Interestingly, in a recent report pFN was shown to diffuse into tissues where it is incorporated into the fibrillar matrix [11]. The existence of fibrillar cFN and soluble pFN largely results from the fact that the assembly of the FN fibrils is a cell-driven process, in which integrins play a central role [12]. Integrins shift between a low affinity (also called inactive) and a high affinity (active) conformation [13]. In tissues, cells express activated integrins that immediately bind cFN that is secreted as a compact globular structure harbouring hidden, cryptic sites for binding other FN molecules. Binding is followed by generation of mechanical tension via the actin cytoskeleton, which leads to stretching of the bound FN molecule, unravelling of the cryptic ‘self-association’ sites and finally the binding to other FN molecules, resulting in a chain-reaction of self-assembly and fibril formation [14, 15]. In blood, however, haematopoietic cells express their integrins in an inactive conformation, which prevents cells from binding pFN and assembling FN fibrils. As soon as the blood cell integrins shift into the high affinity conformation, for example when platelets are activated in response to a vascular injury, pFN is bound and assembled into fibrils, which in the case of platelets are required for thrombus growth and stability [16, 17].

To date, 11 different integrin heterodimers are reported to be capable of binding to FN (see Figure 1), and four of them, α5β1, αvβ3, α4β1 and αIIbβ3 trigger FN fibril formation in vitro [18, 19]. In addition, recent studies have implicated non-integrin cell surface receptors in FN binding and fibrillogenesis [20, 21]. In this review we will summarize the current knowledge about the function of FN–integrin interactions in the process of FN fibril assembly and place it in the context of recent in vivo observations.

Section snippets

RGD motif and synergy site: the central cell binding motifs of FN

More than two decades ago, Ruoslahti and colleagues demonstrated that cell adhesion to FN depends on the RGD motif located in the 10th type III domain (see Figure 1) [22] and that the RGD motif is bound by α5β1 integrin [23]. Since then, several additional integrins were shown to bind to the RGD motif of FN, including all members of the αv subfamily, α8β1, α9β1 and the platelet-specific αIIbβ3 integrin (see Figure 1; [24]; for a review, see [25]). Among the FN binding integrins, α5β1 is

The discovery of new αv integrin binding site(s) in the N-terminus of FN

Since the addition of RGD-containing peptides to cultured cells abolishes assembly of FN fibrils [42], it was proposed that the RGD motif of FN is required for FN binding to integrins and subsequent FN matrix assembly to proceed. This notion was supported by in vivo studies that demonstrated that FN fibrillogenesis can proceed in mice null for either α5 or αv, while deletion of both genes dramatically reduced the amount of the FN matrix in vivo [32]. On the basis of this in vitro and in vivo

The variable region of FN

Some integrin-interaction sites within the FN meshwork can be regulated by alternative splicing within three different regions of FN (see Figure 1). One of them is the variable-region (v-region), which can give rise to five different FN splice variants in man. The majority of cFN harbours a v-region in both subunits, while pFN contains only one subunit with a v-region. The v-region can associate with the two non-RGD-binding integrins, α4β1 and α4β7, both of which bind the two minimal sequences

The extra domains (ED) A and B

The alternatively spliced extra domain A (EDA) and extra domain B (EDB) are exclusively present in cFN (see Figure 1). Their expression levels are high during development and they progressively decrease after birth [54, 55, 56]. In pathological situations including cancer, atherosclerosis, thrombosis and pulmonary fibrosis the expression of ED-containing FN is reactivated [57, 58, 59, 60, 61]. The EDGIHEL sequence of the EDA can facilitate binding to α4β1 and α9β1 integrins [24, 62] (Figure 1).

Heparan sulfate binding sites

FN has two heparin binding domains that mapped to the first five type I domains (FN-I1–5) and the three type III domains preceding the v-region (FN-III13–15), respectively [10] (see Figure 1). Both domains facilitate the binding of proteoglycan cell surface receptors, most notably members of the syndecan family. Syndecan-2 and syndecan-4 promote FN assembly via at least two mechanisms. Firstly, they activate kinases such as PKCα and FAK that trigger integrin clustering and focal adhesion

The search for novel integrin-binding sites in FN

The identification of novel αv integrin binding motifs in the N-terminus of FN raises the questions why the site(s) remained undetected for such a long time, and how many additional integrin binding sites are still to be discovered in FN. The discovery of the novel integrin-binding site(s) in the N-terminus of FN was hampered by the fact that they share the same ligand binding pocket in integrins with RGD, and thus can be efficiently blocked with soluble RGD-containing peptides. Furthermore,

References and recommended reading

Papers of particular interest published within the period of review have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We thank Harold Erickson, Deane Mosher, Roy Zent and Ambra Pozzi for carefully reading the manuscript, Iain Campbell and Chris Millard for providing structural data, Brian Hoffmann and Deane Mosher for communicating unpublished work, and Max Iglesias for artwork. We apologize to all whose papers should but have not been cited owing to space constraints. The work was supported by an EMBO long-term fellowship to KB, the Deutsche Forschungsgemeinschaft (SFB576) and the Max Planck Society.

References (72)

  • J.T. Yang

    Overlapping and independent functions of fibronectin receptor integrins in early mesodermal development

    Dev Biol

    (1999)
  • S. Aota et al.

    The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function

    J Biol Chem

    (1994)
  • D. Chada et al.

    The synergy site of fibronectin is required for strong interaction with the platelet integrin αIIbβ3

    Ann Biomed Eng

    (2006)
  • T. Nagai

    Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

    J Cell Biol

    (1991)
  • S. Takahashi

    The RGD motif in fibronectin is essential for development but dispensable for fibril assembly

    J Cell Biol

    (2007)
  • V. Wilson et al.

    Cell fate and morphogenetic movement in the late mouse primitive streak

    Mech Dev

    (1996)
  • E.A. Wayner

    Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin

    J Cell Biol

    (1989)
  • G.C. Gurtner

    Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation

    Genes Dev

    (1995)
  • J.T. Yang et al.

    Cell adhesion events mediated by α4 integrins are essential in placental and cardiac development

    Development

    (1995)
  • A. Grazioli

    Defective blood vessel development and pericyte/pvSMC distribution in α4 integrin-deficient mouse embryos

    Dev Biol

    (2006)
  • M.H. Tan

    Deletion of the alternatively spliced fibronectin EIIIA domain in mice reduces atherosclerosis

    Blood

    (2004)
  • V.R. Babaev

    Absence of regulated splicing of fibronectin EDA exon reduces atherosclerosis in mice

    Atherosclerosis

    (2007)
  • A.K. Chauhan

    Prothrombotic effects of fibronectin isoforms containing the EDA domain

    Arterioscler Thromb Vasc Biol

    (2008)
  • A. Villa

    A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo

    Int J Cancer

    (2008)
  • M.R. Morgan et al.

    Synergistic control of cell adhesion by integrins and syndecans

    Nat Rev Mol Cell Biol

    (2007)
  • A. Woods

    Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

    Arch Biochem Biophys

    (2000)
  • D.J. Leahy et al.

    2.0 A crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region

    Cell

    (1996)
  • M.J. Humphries

    Mechanisms of integration of cells and extracellular matrices by integrins

    Biochem Soc Trans

    (2004)
  • X. Zhou

    Fibronectin fibrillogenesis regulates three-dimensional neovessel formation

    Genes Dev

    (2008)
  • L. Fontana

    Fibronectin is required for integrin αvβ6-mediated activation of latent TGF-β complexes containing LTBP-1

    FASEB J

    (2005)
  • E.L. George

    Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin

    Development

    (1993)
  • J.E. Schwarzbauer

    Identification of the fibronectin sequences required for assembly of a fibrillar matrix

    J Cell Biol

    (1991)
  • R. Pankov et al.

    Fibronectin at a glance

    J Cell Sci

    (2002)
  • F.A. Moretti

    A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived

    J Biol Chem

    (2007)
  • Y. Mao et al.

    Fibronectin fibrillogenesis, a cell-mediated matrix assembly process

    Matrix Biol

    (2005)
  • R. Pankov

    Integrin dynamics and matrix assembly: tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis

    J Cell Biol

    (2000)
  • Cited by (220)

    View all citing articles on Scopus
    3

    Both authors contributed equally.

    View full text