Mapping tenascin-C interaction with toll-like receptor 4 reveals a new subset of endogenous inflammatory triggers.
ABSTRACT: Pattern recognition underpins innate immunity; the accurate identification of danger, including infection, injury, or tumor, is key to an appropriately targeted immune response. Pathogen detection is increasingly well defined mechanistically, but the discrimination of endogenous inflammatory triggers remains unclear. Tenascin-C, a matrix protein induced upon tissue damage and expressed by tumors, activates toll-like receptor 4 (TLR4)-mediated sterile inflammation. Here we map three sites within tenascin-C that directly and cooperatively interact with TLR4. We also identify a conserved inflammatory epitope in related proteins from diverse families, and demonstrate that its presence targets molecules for TLR detection, while its absence enables escape of innate immune surveillance. These data reveal a unique molecular code that defines endogenous proteins as inflammatory stimuli by marking them for recognition by TLRs.
Project description:The factors responsible for maintaining persistent organ fibrosis in systemic sclerosis (SSc) are not known but emerging evidence implicates toll-like receptors (TLRs) in the pathogenesis of SSc. Here we show the expression, mechanism of action and pathogenic role of endogenous TLR activators in skin from patients with SSc, skin fibroblasts, and in mouse models of organ fibrosis. Levels of tenascin-C are elevated in SSc skin biopsy samples, and serum and SSc fibroblasts, and in fibrotic skin tissues from mice. Exogenous tenascin-C stimulates collagen gene expression and myofibroblast transformation via TLR4 signalling. Mice lacking tenascin-C show attenuation of skin and lung fibrosis, and accelerated fibrosis resolution. These results identify tenascin-C as an endogenous danger signal that is upregulated in SSc and drives TLR4-dependent fibroblast activation, and by its persistence impedes fibrosis resolution. Disrupting this fibrosis amplification loop might be a viable strategy for the treatment of SSc.
Project description:Endogenous molecules generated upon pathogen invasion or tissue damage serve as danger signals that activate host defense; however, their precise immunological role remains unclear. Tenascin-C is an extracellular matrix glycoprotein that is specifically induced upon injury and infection. Here, we show that its expression is required to generate an effective immune response to bacterial lipopolysaccharide (LPS) during experimental sepsis in vivo. Tenascin-C enables macrophage translation of proinflammatory cytokines upon LPS activation of toll-like receptor 4 (TLR4) and suppresses the synthesis of anti-inflammatory cytokines. It mediates posttranscriptional control of a specific subset of inflammatory mediators via induction of the microRNA miR-155. Thus, tenascin-C plays a key role in regulating the inflammatory axis during pathogenic activation of TLR signaling.
Project description:In order to investigate what signalling pathways are turned on by tenascin-C, we generated Mouse Embryonic Fibroblasts (MEFs) deficient for tenascin-C and compared their gene expression profile to MEFs proficient for tenascin-C. Overall design: TNC-KO MEFs as well as WT MEFs in which tenascin-C was knocked-down (following stable transfection of a short hairpin RNA) were compared to WT MEFs (expressing strong endogenous levels of tenascin-C).
Project description:In order to investigate what signalling pathways are turned on by tenascin-C, we generated Mouse Embryonic Fibroblasts (MEFs) deficient for tenascin-C and compared their gene expression profile to MEFs proficient for tenascin-C. TNC-KO MEFs as well as WT MEFs in which tenascin-C was knocked-down (following stable transfection of a short hairpin RNA) were compared to WT MEFs (expressing strong endogenous levels of tenascin-C).
Project description:The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLC?2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.
Project description:The extracellular matrix molecule tenascin-C is highly expressed during embryonic development, tissue repair and in pathological situations such as chronic inflammation and cancer. Tenascin-C interacts with several other extracellular matrix molecules and cell-surface receptors, thus affecting tissue architecture, tissue resilience and cell responses. Tenascin-C modulates cell migration, proliferation and cellular signaling through induction of pro-inflammatory cytokines and oncogenic signaling molecules amongst other mechanisms. Given the causal role of inflammation in cancer progression, common mechanisms might be controlled by tenascin-C during both events. Drugs targeting the expression or function of tenascin-C or the tenascin-C protein itself are currently being developed and some drugs have already reached advanced clinical trials. This generates hope that increased knowledge about tenascin-C will further improve management of diseases with high tenascin-C expression such as chronic inflammation, heart failure, artheriosclerosis and cancer.
Project description:Chick embryo fibroblasts cultured on a collagen matrix exert tractional forces leading to the contraction of unrestrained, floating collagen gels and to the development of tension in attached, restrained gels. On a restrained, attached collagen gel the fibroblasts synthesize large quantities of tenascin-C, whereas in a floating, contracting gel tenascin-C synthesis is decreased. This regulation of tenascin-C synthesis can be observed by the secretion of metabolically labeled tenascin-C into the conditioned medium, as well as by the deposition of tenascin-C into the collagen matrix as judged by immunofluorescence. Regulation appears to occur at the transcriptional level, because when cells on attached or floating collagen gels are transfected with promoter constructs of the tenascin-C gene, luciferase expression driven by the tenascin-C promoter parallels the effects measured for endogenous tenascin-C synthesis, whereas luciferase expression under the control of the SV40 promoter does not depend on the state of the collagen gel. The promoter region responsible for tenascin-C induction on attached collagen gels is distinct from the region important for the induction of tenascin-C by serum, and may define a novel kind of response element. By joining this tenascin-C sequence to the SV40 promoter of a reporter plasmid, its activity can be transferred to the heterologous promoter. We propose that the tenascin-C promoter is directly or indirectly activated in fibroblasts generating and experiencing mechanical stress within a restrained collagen matrix. This may be an important aspect of the regulation of tenascin-C expression during embryogenesis as well as during wound healing and other regenerative and morphogenetic processes.
Project description:CA-125 has been a valuable marker for the follow-up of ovarian cancer patients but it is not sensitive enough to be used as diagnostic marker. We had already used secretomic methods to identify proteins differentially secreted by serous ovarian cancer cells compared to healthy ovarian cells. Here, we evaluated the secretion of these proteins by ovarian cancer cells during the follow-up of one patient. Proteins that correlated with CA-125 levels were screened using serum samples from ovarian cancer patients as well as benign and healthy controls. Tenascin-X secretion was shown to correlate with CA-125 value in the initial case study. The immunohistochemical detection of increased amount of tenascin-X in ovarian cancer tissues compared to healthy tissues confirms the potent interest in tenascin-X as marker. We then quantified the tenascin-X level in serum of patients and identified tenascin-X as potent marker for ovarian cancer, showing that secretomic analysis is suitable for the identification of protein biomarkers when combined with protein immunoassay. Using this method, we determined tenascin-X as a new potent marker for serous ovarian cancer.
Project description:Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors (PRR) that play a key role in innate and adaptive immunity by recognizing structural components unique to bacteria, fungi and viruses. TLR4 is the most studied of the TLRs, and its primary exogenous ligand is lipopolysaccharide, a component of Gram-negative bacterial walls. In the absence of exogenous microbes, endogenous ligands including damage-associated molecular pattern molecules from damaged matrix and injured cells can also activate TLR4 signaling. In humans, single nucleotide polymorphisms of the TLR4 gene have an effect on its signal transduction and on associated risks of specific diseases, including cirrhosis. In liver, TLR4 is expressed by all parenchymal and non-parenchymal cell types, and contributes to tissue damage caused by a variety of etiologies. Intact TLR4 signaling was identified in hepatic stellate cells (HSCs), the major fibrogenic cell type in injured liver, and mediates key responses including an inflammatory phenotype, fibrogenesis and anti-apoptotic properties. Further clarification of the function and endogenous ligands of TLR4 signaling in HSCs and other liver cells could uncover novel mechanisms of fibrogenesis and facilitate the development of therapeutic strategies.
Project description:Extracellular matrix proteins of the tenascin family resemble each other in their domain structure, and also share functions in modulating cell adhesion and cellular responses to growth factors. Despite these common features, the 4 vertebrate tenascins exhibit vastly different expression patterns. Tenascin-R is specific to the central nervous system. Tenascin-C is an "oncofetal" protein controlled by many stimuli (growth factors, cytokines, mechanical stress), but with restricted occurrence in space and time. In contrast, tenascin-X is a constituitive component of connective tissues, and its level is barely affected by external factors. Finally, the expression of tenascin-W is similar to that of tenascin-C but even more limited. In accordance with their highly regulated expression, the promoters of the tenascin-C and -W genes contain TATA boxes, whereas those of the other 2 tenascins do not. This article summarizes what is currently known about the complex transcriptional regulation of the 4 tenascin genes in development and disease.