Tenascin-X: beyond the architectural function

Abstract

Tenascin-X is the largest member of the tenascin (TN) family of evolutionary conserved extracellular matrix glycoproteins, which also comprises TN-C, TN-R and TN-W. Among this family, TN-X is the only member described so far to exert a crucial architectural function as evidenced by a connective tissue disorder (a recessive form of Ehlers-Danlos syndrome) resulting from a loss-of-function of this glycoprotein in humans and mice. However, TN-X is more than an architectural protein, as it displays features of a matricellular protein by modulating cell adhesion. However, the cellular functions associated with the anti-adhesive properties of TN-X have not yet been revealed. Recent findings indicate that TN-X is also an extracellular regulator of signaling pathways. Indeed, TN-X has been shown to regulate the bioavailability of the Transforming Growth Factor (TGF)-β and to modulate epithelial cell plasticity. The next challenges will be to unravel whether the signaling functions of TN-X are functionally linked to its matricellular properties.

Keywords: ECM, extracellular matrix; EDS, Ehlers-Danlos syndrome; EGF, epidermal growth factor; EMT, epithelial-to-mesenchymal transition; Ehlers-Danlos syndrome (EDS); FAK, focal adhesion kinase; FBG, fibrinogen-like domain; FNIII, fibronectin type III module; LAP, latency associated peptide; MMP, matrix metalloproteinase; SLC, small latent complex; TGF-β; TGF-β activation; TN, tenascin; TSP-1, thrombospondin-1; VEGF, vascular endothelial growth factor; cell signaling; epithelial-to-mesenchymal transition (EMT); integrin α11β1; matricellular protein; tenascin-X; transforming growth factor-β.

Figure 1.

TN-X is a tribrachion. (A) Schematic representation of human, mouse and bovine TN-X monomers. Each molecule is composed of the N-terminal oligomerization domain (tenascin assembly domain), followed by 18.5 epidermal growth factor (EGF)-like repeats, between 30 to 32 fibronectin type III (FNIII) repeats and with a fibrinogen (FBG)-like domain at the C-terminus. The FNIII repeats are potentially interrupted by a serine/proline-rich (SPX) region that was identified in the TN-Y molecule, the avian TN-X ortholog. For each TN-X ortholog, FNIII repeats (Hu1-32, M1-31 and b0-29) are numbered according to the nomenclature used in the original publications. The alternatively spliced FNIII repeats found in the mouse TN-X are in yellow (M3, and M15-M22). Cell-surface receptors of the bovine TN-X are depicted under the corresponding monomer. The heparin-binding site is underlined. Note that the human and bovine TN-X glycoproteins, but not the mouse ortholog, have a RGD sequence in a conserved FNIII module (Hu11 and b10, respectively). (B) Electron Micrographs of rotary shadowed purified recombinant bovine TN-X molecules (upper panels) and their respective putative schematic representation (lower panels). Bars, 50 nm.

Figure 2.

Tissue distribution of TN-X (A) in fetal bovine tissues and (B) during wound healing in mice. (A) Indirect immunofluorescence of TN-X performed on cryostat sections of fetal bovine tissues (at the indicated weeks of gestation) using a monoclonal antibody (8F2) recognizing the FNIII b10 domain of the bovine glycoprotein. (B) Indirect immunofluorescence of TN-X performed on cryostat sections of mouse skin after incisional wound (*). TN-X was detected using polyclonal antibodies directed against the bovine TN-X FNIII b9-b10 repeats. Note that TN-X is hardly detected in the wound (*) even after the completion of the re-epithelialization process (day 7). TN-X staining is re-observed in the deep layers of the wound 13 days after incision. Bars, 50 μm.

Figure 3.

Model of TN-X integration within the collagenous network. TN-X regulates the spacing and cohesiveness between collagen fibrils through direct interaction with collagen molecules and/or through molecular associations with several ECM components interacting with collagen fibrils. Type XII collagen interacts with TN-X through its non-collagenous N-terminal NC3 domain. The binding site of this NC3 domain has not been identified in the full-length TN-X. The dermatan sulfate chains of decorin ensure the binding to the heparin-binding site included within the FNIII b10 and b11 domains of bovine TN-X. TN-X might also anchor collagen fibrils at the vicinity of cell surface through its interaction with (not yet identified) heparin-sulfate proteoglycan receptor(s) and/or the α11β1 integrin.

Figure 4.

Model of TGF-β activation by the FBG-like domain of TN-X. The small latent TGF-β complex (SLC) interacts with the FBG-like domain of TN-X. In this full-length glycoprotein, the LAP·TGF-β thus constitutes a reservoir of signaling molecule within the ECM. During physiological or pathological events of ECM remodeling, we hypothesized that some proteases would cleave the FBG-like domain of TN-X from the full-length molecule. At the vicinity of cell membrane, the FBG-like domain interacts with the α11β1 integrin receptor, allowing a conformational change of the latent complex, facilitating the presentation of mature TGF-β to TβRII and TβRI receptors. Latent TGF-β activation by the FBG-like domain results in the induction of Smad signaling and subsequent TGF-β response, such as EMT.

Figure 5.

The FNIII region of TN-X antagonizes the induction of EMT by the FBG-like domain. (A) Actin direct fluorescence in normal murine mammary epithelial (NMuMG) cells seeded onto non-coated dishes or dishes containing equimolar quantities of immobilized recombinant full-length TN-X, FBG-like domain, FNIII repeats (TN-XΔEΔF) or both FNIII repeats and FBG-like domain. Note that compared to the uncoated condition, where almost all cells exhibited a cortical actin staining, the full-length TN-X induced a mild EMT, visualized by a partial delocalization of actin cytoskeleton from cell junctions (*). In contrast, the FBG-like domain caused a full EMT, as illustrated by the acquisition of elongated cell morphology and the organization of actin cytoskeleton into stress fibers. Recombinant human TGF-β1, used here as a positive control, gave similar results to those obtained with the FBG-like domain. However, the FNIII region of TN-X fully inhibited the EMT triggered by the FBG-like domain. Bars, 15 μm. (B) Model representing the regulation of epithelial cell plasticity by TN-X. When separated from the intact TN-X molecule, the FBG-like domain is able to induce a robust EMT response, through its ability to activate latent TGF-β. This response relies on the presence of TβRII/I receptors and α11β1 integrin at the cell surface. In the intact TN-X molecule, the FNIII region antagonizes the EMT induced by the FBG-like domain, most likely via distinct intracellular cues that are initiated by the interaction of certain FNIII repeats to a yet unidentified receptor.