Tenascin-C (TNC, Tnc)
Tenascins are extracellular matrix glycoproteins with subunits composed of epidermal growth factor (EGF)-like repeats, fibronectin type III (FNIII) domains, and a C-terminal fibrinogen-related domain (Chiquet-Ehrismann and Tucker 2011). Tenascins trimerize via heptad repeats found near their N-terminus, and, in at least some cases, two tenascin trimers can form a hexabrachion. In most vertebrates there are four paralogous tenascins: tenascin-C, tenascin-R, tenascin-W, and tenascin-X. The best studied and first to be discovered is tenascin-C, which was independently identified as an antigen or extract in tumors, tendons, muscles, bones, and developing brain. The name tenascin was adopted for this protein by Chiquet-Ehrismann and colleagues from the Latin verbs tenere (to hold) and nasci (to be born) reflecting its prominent expression in tendons and embryos, respectively (Chiquet-Ehrismann et al. 1986). The “C” of tenascin-C corresponds to cytotactin, which was the name given this protein by Grumet and colleagues (Grumet et al. 1985).
In 1975, Yamada and colleagues described the agglutination of fixed sheep erythrocytes by fibronectin extracted with urea from chicken embryo fibroblasts (Yamada et al. 1975). However, when fibronectin purified in this way was examined in the electron microscope, it was clear that the preparations were contaminated with a large, previously undescribed six-armed protein (Erickson and Inglesias 1984). The contaminating hexabrachion (Fig. 1b) is now known to be tenascin-C and much, if not all, of the hemagglutinating activity initially attributed to fibronectin was due to the presence of tenascin-C, which is able to agglutinate fixed erythrocytes at concentrations as low as 1.5 μg/ml (Chiquet-Ehrismann et al. 1986). With hindsight it is not surprising that tenascin-C was an undetected contaminant of the fibronectin preparations: tenascin-C and fibronectin are both expressed by chicken embryo fibroblasts, they bind to each other (Chiquet-Ehrismann et al. 1988), and they have almost identical subunit molecular weights, so they migrate together under reducing conditions on polyacrylamide gels.
Tenascin-C was the first extracellular matrix protein to have anti-adhesive or adhesion modulatory properties. When cells are cultured on adhesive glycoproteins like fibronectin or laminin, they typically attach and spread, but when cultured on tenascin-C-coated dishes, cells typically fail to spread and instead remain rounded or form only small lamellipodia (Fig. 1b, c) (Chiquet-Ehrismann et al. 1988). Remarkably, tenascin-C added to the medium over cells cultured on fibronectin-coated substrata inhibits both attachment and spreading in a dose-dependent fashion (Fig. 1d) (Chiquet-Ehrismann et al. 1988). Determining the molecular basis underlying this adhesion modulation became an important research objective during the following decade (see below). Cells plated on tenascin-C-coated substrata were not only less spread; they were often more motile (Mackie et al. 1988). Early studies also showed that exogenous tenascin-C can promote chondrogenesis in vitro and increase the proliferation of Swiss 3T3 and NIH 3T3 cells (Chiquet-Ehrismann and Tucker 2011).
Patterns of Expression
Adhesion Modulation, Integrin Signaling, and Growth Factor Interactions
In addition to influencing cell behavior indirectly by blocking fibronectin/syndecan-4 interactions, tenascin-C can act directly on cell behavior as an integrin ligand. Tenascin-C from most, but not all, species has an RGD motif in FNIII3 that is found in the same exposed loop as the integrin-binding RGD motif of fibronectin’s FNIII10 (Tucker and Chiquet-Ehrismann 2015). There is experimental evidence that α8β1, αVβ6, and αVβ3 integrins may recognize this RGD motif. A second, more highly conserved integrin-binding tripeptide motif, IDG, is found in a loop between the B and C beta sheets of the same FNIII3 of tenascin-C. This IDG motif is recognized by α9β1 integrin. In addition, the binding of tenascin-C to α7β1 integrin was mapped to the one the variable FNIII domains. Tenascin-C can promote cell proliferation by activating α9β1 and αVβ3 integrins, and activation of α9β1 and α7β1 integrins can influence cell differentiation. These latter two integrins also appear to play roles in promoting cell motility. The effects of integrin-mediated tenascin-C signaling on cell behavior are summarized in Fig. 3b.
Tenascin-C can bind to a number of growth factors via its FNIII5, including members of the FGF, PDGF, and TGFβ family (De Laporte et al. 2013). Tenascin-C also co-immunoprecipitates with the signaling molecule Wnt3a (Hendaoui et al. 2014). When tenascin-C and Wnt3a are added to the medium of cells containing a reporter construct, there is reduced β-catenin-mediated Wnt3a signaling. However, when Wnt3a is added to tenascin-C coating the surface of tissue culture plastic, Wnt3a signaling is enhanced (Hendaoui et al. 2014). These observations indicate that binding of growth and signaling factors by tenascin-C can both inhibit and promote the properties of these factors, depending on whether or not the tenascin-C is soluble or bound to a substratum.
Regulation of Expression
During embryogenesis, tenascin-C is frequently distributed in segmental patterns. Prrx1 (paired related homeobox 1; formerly called Prx1 or Mhox) is a transcription factor important for limb and craniofacial morphogenesis, with a similar embryonic expression pattern as tenascin-C. Prrx1 transactivates the mouse tenascin-C gene by recognizing a conserved homeodomain binding sequence in its promoter. Two more homeobox transcription factors, Pou3F2 and Otx2, regulate tenascin-C gene expression by direct binding to its promoter. Pou3F2 stimulates whereas Otx2 suppresses transcriptional activity. In contrast, Evx1 (even-skipped homeobox 1) acts in synergy with Jun/Fos transcription factors and indirectly activates the tenascin-C gene; the latter factors target an AP1 site in the promoter. Finally, Sox4 is overexpressed in many human tumors together with tenascin-C and stimulates its expression. The regulation of tenascin-C expression was recently reviewed (Chiovaro et al. 2015).
Tenascin-C is strongly expressed de novo in inflammation, wound healing, and cancer (Chiquet-Ehrismann and Chiquet 2003). Inflammatory cytokines have been shown to induce the protein, but it has not been studied yet how they control tenascin-C transcription on the promoter level. Transforming growth factor-β (TGF-β), which is important for tissue regeneration, signals via Smad transcription factors, and two Smad2/3 binding sites were shown to be responsible for activation of the tenascin-C promoter by TGF-β in human fibroblasts. During wound healing, platelet-derived growth factor regulates tenascin-C expression via the PI3K/Akt signaling pathway, which induces binding of SP1 and Ets transcription factors to the respective consensus sequences in its gene promoter. In addition to soluble growth factors, cell-bound ligands Delta and Jagged induce tenascin-C by binding and activating their transmembrane receptor Notch. The cleaved intracellular domain of Notch translocates to the nucleus and stimulates transcription of the tenascin-C gene. Constitutive Notch2 signaling is presumably responsible for the large accumulation of tenascin-C in glioblastomas. Finally, tenascin-C expression is known to be strongly suppressed by glucocorticoids, but the mechanism has not been elucidated on the gene promoter level. These studies have recently been reviewed (Chiquet-Ehrismann and Tucker 2011).
As expected from its occurrence in weight-bearing tissues such as ligaments and tendons, tenascin-C expression is clearly regulated by tensile stress. For example, tenascin-C was shown to accumulate in arterial walls of hypertensive rats and to be induced de novo in skeletal muscle endomysium in response to eccentric overload (for review, see Chiquet et al. 2009). In vitro, the mechanotransduction pathway that leads from cyclic stretching of embryonic fibroblasts to the induction of the tenascin-C gene has been fully elucidated. In these cells, cyclic strain applied to the elastic culture substrate is transduced into activation of small GTPase RhoA and the downstream Rho-dependent kinase, ROCK. Gene knockout experiments showed that mechanical activation of this pathway depends on pericellular fibronectin, cellular a5b1 integrin, and integrin-linked kinase, which is required for the formation of fibrillar adhesions (Chiquet et al. 2009). Strain-activated RhoA/ROCK induces the formation of actin stress fibers in fibroblasts. The resulting depletion of cytoplasmic G-actin is monitored by the actin sensor and transcriptional regulator megakaryocytic leukemia-1 (MKL-1), which translocates to the nucleus. In response to cyclic strain, nuclear MKL-1 initiates tenascin-C gene transcription both indirectly as a co-factor of SRF (serum response factor) and directly by binding to the proximal gene promoter region (Fig. 3c) (Asparuhova et al. 2011). In other cell types, tenascin-C was shown to be induced by alternative mechanotransduction pathways. In cardiomyocytes, for example, cyclic strain leads to release of reactive oxygen species and activation of NFkB (nuclear factor kappa B), which stimulates transcription of the tenascin-C gene by binding to a consensus sequence in its promoter. In arterial smooth muscle cells, stretch primarily activates the Jun kinase pathway. This induces the synthesis of transcription factor NFAT5, which consequently binds to and activates the tenascin-C gene promoter (reviewed in Chiovaro et al. 2015).
Rheumatoid Arthritis and Inflammation
Tenascin-C is elevated in joints during rheumatoid arthritis (RA) and is necessary for inflammation in a mouse model of RA, and tenascin-C can activate Toll-like receptor 4 (TLR4) in macrophages (Midwood et al. 2009; Goh et al. 2010). TLR4 recognizes lipopolysaccharides (LPS) on the surface of Gram-negative bacteria, which leads to the upregulation of proinflammatory cytokines to fight infection. When sepsis is induced in mice by the injection of LPS, the mice experience weight loss, reduced motility, and diarrhea. However, tenascin-C knockout mice fail to develop these symptoms (Piccinini and Midwood, 2012). The levels of TNF-α, IL-6, CXCL1, and IL-10 are elevated in bone marrow-derived macrophages from wild-type mice following exposure to LPS, but they remain unchanged in cells from tenascin-C knockout animals (Piccinini and Midwood, 2012). These and other observations support the hypothesis that tenascin-C is an early response gene that mediates signaling by TLR4 during inflammation both during infection and RA.
Tenascin-C is found in the stroma of most solid tumors (Orend and Chiquet-Ehrismann 2006, Chiquet-Ehrismann and Tucker 2011). In glioblastomas and melanomas, the source of tenascin-C are the tumors cells themselves, but in carcinomas tumor-associated fibroblasts are producing tenascin-C. In high-grade tumors tenascin-C with variable FNIII domains predominates. Experimental evidence supports roles for tenascin-C promoting both angiogenesis and metastasis (Chiquet-Ehrismann and Tucker 2011, Lowy and Oskarsson 2015). miR335 can inhibit metastasis in a mouse model via the downregulation of Sox4 and tenascin-C, and tenascin-C is a signature gene mediating lung metastasis of breast cancer. Clinical studies using iodine-131 or yttrium-90 labeled anti-tenascin-C for intracavitary radioimmunotherapy showed significant increases in postsurgery longevity in patients with astrocytoma or glioblastoma (Reulen et al. 2015).
Tenascin-C is found in the extracellular matrix of vertebrate embryos, and in vitro it typically promotes cell proliferation and migration. Tenascin-C acts both directly as an integrin ligand and by interfering with fibronectin/syndecan-4 interactions, thus altering fibronectin-mediated cell signaling. Tenascin-C reappears during inflammation, wound healing, and cancer, and its expression is upregulated by tensile stress as well as by TGF-β, Notch, and PI3K/Akt signaling pathways. Future challenges include refining the use of anti-tenascin-C as a tumor marker and a target for radioimmunotherapy.
- Asparuhova MB, Ferralli J, Chiquet M, Chiquet-Ehrismann R. The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress. FASEB J. 2011;25(10):3477–88. doi:10.1096/fj.11-187310.PubMedCrossRefGoogle Scholar
- Chiquet-Ehrismann R, Tucker RP. Tenascins and the importance of adhesion modulation. Cold Spring Harb Perspect Biol. 2011;3(5). pii: a004960. doi: 10.1101/cshperspect.a004960.
- Midwood K, Sacre S, Piccinini AM, Inglis J, Trebaul A, Chan E, Drexler S, Sofat N, Kashiwagi M, Orend G, Brennan F, Foxwell B. Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease. Nat Med. 2009;15(7):774–80. doi: 10.1038/nm.1987.PubMedCrossRefGoogle Scholar
- Reulen HJ, Poepperl G, Goetz C, Gildehaus FJ, Schmidt M, Tatsch K, Pietsch T, Kraus T, Rachinger W. Long-term outcome of patients with WHO Grade III and IV gliomas treated by fractionated intracavitary radioimmunotherapy. J Neurosurg. 2015;123(3):760–70. doi: 10.3171/2014.12.JNS142168.PubMedCrossRefPubMedCentralGoogle Scholar