Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 μmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 μmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1β, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

<p>Abstract</p> <p>Background</p> <p>Cysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT₁or CysLT₂, mediate this effect.</p> <p>Methods</p> <p>Cultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D₄(LTD₄) and E₄(LTE₄) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.</p> <p>Results</p> <p>LTD₄and LTE₄significantly augmented the expression of Tn, whereas LTD₄, distinctly from LTE₄, was able to increase also the Ln β2 chain. Although the expression of CysLT₂prevailed over that of CysLT₁, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT₁-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.</p> <p>Conclusion</p> <p>These findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT₁receptor. The results provide a novel aspect to support the use of CysLT₁receptor antagonists in the anti-remodeling treatment of asthma.</p

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

<p>Abstract</p> <p>Background</p> <p>Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.</p> <p>Methods</p> <p>Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ <it>in vitro</it>.</p> <p>Results</p> <p>OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression <it>in vivo</it>. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our <it>in vivo </it>findings.</p> <p>Conclusions</p> <p>Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.</p

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage

<p>Abstract</p> <p>Background</p> <p>Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes <it>in vitro</it>. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.</p> <p>Methods</p> <p>TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.</p> <p>Results</p> <p>TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE₂, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.</p> <p>Conclusions</p> <p>TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.</p

Tenascin-C Enhances Pancreatic Cancer Cell Growth and Motility and Affects Cell Adhesion through Activation of the Integrin Pathway

Background: Pancreatic cancer (PDAC) is characterized by an abundant fibrous tissue rich in Tenascin-C (TNC), a large ECM glycoprotein mainly synthesized by pancreatic stellate cells (PSCs). In human pancreatic tissues, TNC expression increases in the progression from low-grade precursor lesions to invasive cancer. Aim of this study was the functional characterization of the effects of TNC on biologic relevant properties of pancreatic cancer cells. Methods: Proliferation, migration and adhesion assays were performed on pancreatic cancer cell lines treated with TNC or grown on a TNC-rich matrix. Stable transfectants expressing the large TNC splice variant were generated to test the effects of endogenous TNC. TNC-dependent integrin signaling was investigated by immunoblotting, immunofluorescence and pharmacological inhibition. Results: Endogenous TNC promoted pancreatic cancer cell growth and migration. A TNC-rich matrix also enhanced migration as well as the adhesion to the uncoated growth surface of poorly differentiated cell lines. In contrast, adhesion to fibronectin was significantly decreased in the presence of TNC. The effects of TNC on cell adhesion were paralleled by changes in the activation state of paxillin and Akt. Conclusion: TNC affects proliferation, migration and adhesion of poorly differentiated pancreatic cancer cell lines and migh

Contour models of cellular adhesion

The development of traction-force microscopy, in the past two decades, has created the unprecedented opportunity of performing direct mechanical measurements on living cells as they adhere or crawl on uniform or micro-patterned substrates. Simultaneously, this has created the demand for a theoretical framework able to decipher the experimental observations, shed light on the complex biomechanical processes that govern the interaction between the cell and the extracellular matrix and offer testable predictions. Contour models of cellular adhesion, represent one of the simplest and yet most insightful approach in this problem. Rooted in the paradigm of active matter, these models allow to explicitly determine the shape of the cell edge and calculate the traction forces experienced by the substrate, starting from the internal and peripheral contractile stresses as well as the passive restoring forces and bending moments arising within the actin cortex and the plasma membrane. In this chapter I provide a general overview of contour models of cellular adhesion and review the specific cases of cells equipped with isotropic and anisotropic actin cytoskeleton as well as the role of bending elasticity.Comment: 24 pages, 9 figures. arXiv admin note: text overlap with arXiv:1304.107

Inhibition of Fibroblast Growth by Notch1 Signaling Is Mediated by Induction of Wnt11-Dependent WISP-1

Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM). They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1Flox/Flox) embryonic fibroblasts (MEFs). Notch1-deficient (Notch1−/−) MEFs displayed faster growth and motility rate compared to Notch1Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1) in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441), which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1). Functionally, “Notch-activated” stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4) in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression

Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts

It was our objective to study the role of mechanical stimulation on fibronectin (FN) reorganization and recruitment by exposing fibroblasts to shear fluid flow and equibiaxial stretch. Mechanical stimulation was also combined with a Rho inhibitor to probe their coupled effects on FN. Mechanically stimulated cells revealed a localization of FN around the cell periphery as well as an increase in FN fibril formation. Mechanical stimulation coupled with chemical stimulation also revealed an increase in FN fibrils around the cell periphery. Complimentary to this, fibroblasts exposed to fluid shear stress structurally rearranged pre-coated surface FN, but unstimulated and stretched cells did not. These results show that mechanical stimulation directly affected FN reorganization and recruitment, despite perturbation by chemical stimulation. Our findings will help elucidate the mechanisms of FN biosynthesis and organization by furthering the link of the role of mechanics with FN

Toll-like receptor gene polymorphisms are associated with susceptibility to graves' ophthalmopathy in Taiwan males

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) are a family of pattern-recognition receptors, which plays a role in eliciting innate/adaptive immune responses and developing chronic inflammation. The polymorphisms of TLRs have been associated with the risk of various autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis and rheumatorid arthritis. The aim of this study was to evaluate whether TLR genes could be used as genetic markers for the development of Graves' ophthalmopathy (GO).</p> <p>Methods</p> <p>6 TLR-4 and 2 TLR-9 gene polymorphisms in 471 GD patients (200 patients with GO and 271 patients without GO) from a Taiwan Chinese population were evaluated.</p> <p>Results</p> <p>No statistically significant difference was observed in the genotypic and allelic frequencies of TLR-4 and TLR-9 gene polymorphisms between the GD patients with and without GO. However, sex-stratified analyses showed that the association between TLR-9 gene polymorphism and GO phenotype was more pronounced in the male patients. The odds ratios (ORs) was 2.11 (95% confidence interval [CI] = 1.14-3.91) for rs187084 AàG polymorphism and 1.97 (95% CI = 1.07-3.62) for rs352140 AàG polymorphism among the male patients. Increasing one G allele of rs287084 and one A allele of rs352140 increased the risk of GO (<it>p </it>values for trend tests were 0.0195 and 0.0345, respectively). Further, in haplotype analyses, the male patients carrying the GA haplotype had a higher risk of GO (odds ratio [OR] = 2.02, 95% confidence interval [CI] = 1.09-3.73) than those not carrying the GA haplotype.</p> <p>Conclusion</p> <p>The present data suggest that TLR-9 gene polymorphisms were significantly associated with increased susceptibility of ophthalmopathy in male GD patients.</p

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments