The multifaceted role of Matricellular Proteins in health and cancer, as biomarkers and therapeutic targets.

The extracellular matrix (ECM) is composed of a mesh of proteins, proteoglycans, growth factors, and other secretory components. It constitutes the tumor microenvironment along with the endothelial cells, cancer-associated fibroblasts, adipocytes, and immune cells. The proteins of ECM can be functionally classified as adhesive proteins and matricellular proteins (MCP). In the tumor milieu, the ECM plays a major role in tumorigenesis and therapeutic resistance. The current review encompasses thrombospondins, osteonectin, osteopontin, tenascin C, periostin, the CCN family, laminin, biglycan, decorin, mimecan, and galectins. The matrix metalloproteinases (MMPs) are also discussed as they are an integral part of the ECM with versatile functions in the tumor stroma. In this review, the role of these proteins in tumor initiation, growth, invasion and metastasis have been highlighted, with emphasis on their contribution to tumor therapeutic resistance. Further, their potential as biomarkers and therapeutic targets based on existing evidence are discussed. Owing to the recent advancements in protein targeting, the possibility of agents to modulate MCPs in cancer as therapeutic options are discussed.

Functions of Matricellular Proteins in Dental Tissues and Their Emerging Roles in Orofacial Tissue Development, Maintenance, and Disease.

Matricellular proteins (MCPs) are defined as extracellular matrix (ECM) associated proteins that are important regulators and integrators of microenvironmental signals, contributing to the dynamic nature of ECM signalling. There is a growing understanding of the role of matricellular proteins in cellular processes governing tissue development as well as in disease pathogenesis. In this review, the expression and functions of different MP family members (periostin, CCNs, TSPs, SIBLINGs and others) are presented, specifically in relation to craniofacial development and the maintenance of orofacial tissues, including bone, gingiva, oral mucosa, palate and the dental pulp. As will be discussed, each MP family member has been shown to have non-redundant roles in development, tissue homeostasis, wound healing, pathology and tumorigenesis of orofacial and dental tissues.

Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaired closure of an epithelial defect and accelerated infiltration of neutrophils into the wound periphery as compared to the response in WT tissue. Expression of wound healing-related proinflammatory and reparative components, i.e., interleukin-6, transforming growth factor ß, matrix metalloproteinases, were unaffected by the loss of tenascin X expression. Marked accumulation of malondialdehyde (a lipid peroxidation-derived product) was observed in KO healing epithelia as compared with its WT counterpart. Neutropenia induced by systemic administration of a specific antibody rescued the impairment of epithelial healing in KO corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels.

ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting.

The composition and physical properties of the extracellular matrix (ECM) critically influence tumor progression, but the molecular mechanisms underlying ECM layering are poorly understood. Tumor-stroma interaction critically depends on cell communication mediated by exosomes, small vesicles generated within multivesicular bodies (MVBs). We show that caveolin-1 (Cav1) centrally regulates exosome biogenesis and exosomal protein cargo sorting through the control of cholesterol content at the endosomal compartment/MVBs. Quantitative proteomics profiling revealed that Cav1 is required for exosomal sorting of ECM protein cargo subsets, including Tenascin-C (TnC), and for fibroblast-derived exosomes to efficiently deposit ECM and promote tumor invasion. Cav1-driven exosomal ECM deposition not only promotes local stromal remodeling but also the generation of distant ECM-enriched stromal niches in vivo. Cav1 acts as a cholesterol rheostat in MVBs, determining sorting of ECM components into specific exosome pools and thus ECM deposition. This supports a model by which Cav1 is a central regulatory hub for tumor-stroma interactions through a novel exosome-dependent ECM deposition mechanism.

The "other" 15-40%: The Role of Non-Collagenous Extracellular Matrix Proteins and Minor Collagens in Tendon.

Extracellular matrix (ECM) determines the physiological function of all tissues, including musculoskeletal tissues. In tendon, ECM provides overall tissue architecture, which is tailored to match the biomechanical requirements of their physiological function, that is, force transmission from muscle to bone. Tendon ECM also constitutes the microenvironment that allows tendon-resident cells to maintain their phenotype and that transmits biomechanical forces from the macro-level to the micro-level. The structure and function of adult tendons is largely determined by the hierarchical organization of collagen type I fibrils. However, non-collagenous ECM proteins such as small leucine-rich proteoglycans (SLRPs), ADAMTS proteases, and cross-linking enzymes play critical roles in collagen fibrillogenesis and guide the hierarchical bundling of collagen fibrils into tendon fascicles. Other non-collagenous ECM proteins such as the less abundant collagens, fibrillins, or elastin, contribute to tendon formation or determine some of their biomechanical properties. The interfascicular matrix or endotenon and the outer layer of tendons, the epi- and paratenon, includes collagens and non-collagenous ECM proteins, but their function is less well understood. The ECM proteins in the epi- and paratenon may provide the appropriate microenvironment to maintain the identity of distinct tendon cell populations that are thought to play a role during repair processes after injury. The aim of this review is to provide an overview of the role of non-collagenous ECM proteins and less abundant collagens in tendon development and homeostasis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:23-35, 2020.

Roles of the matricellular protein Tenascin-C in T-lymphocyte trafficking and etiopathogenesis of Oral Lichen Planus.

OBJECTIVE: This study was aimed at examining the role of Tenascin-C in T cell trafficking in Oral Lichen Planus (OLP). DESIGN: For the in vivo immunohistochemical analyses, 115 OLP samples were collected from patients and immunostaining was performed. The intensity and distribution of TN-C expression were quantified and correlated with histological analyses of basement membrane integrity and presence of inflammatory infiltrate. For the in vitro study, TN-C and collagen were coated on culture plates and migration of T lymphocytes was assessed. RESULTS: TN-C immunoexpression was increased in terms of both distribution and intensity along the basement membrane zone. These changes were significantly associated with basement membrane duplication (distribution p < 0.002 and intensity p < 0.001) and bands of inflammation (distribution p < 0.002 and intensity p < 0.001) assessed by Chi-square test. T lymphocytes demonstrated significant migration towards TN-C as compared to collagen (n = 3, p < 0.05). CONCLUSIONS: These findings indicate TN-C may have a key role in promoting T cell migration at the epithelial-mesenchymal junction in OLP. These observations suggest TN-C could be a good target for therapeutic intervention, either in itself or synergistically with anti-inflammatory directed strategies in this chronic disease management.

Selective Filopodia Adhesion Ensures Robust Cell Matching in the Drosophila Heart.

The ability to form specific cell-cell connections within complex cellular environments is critical for multicellular organisms. However, the underlying mechanisms of cell matching that instruct these connections remain elusive. Here, we quantitatively explored the dynamics and regulation of cell matching processes utilizing Drosophila cardiogenesis. We found that cell matching is highly robust at boundaries between cardioblast (CB) subtypes, and filopodia of different CB subtypes have distinct binding affinities. Cdc42 is involved in regulating this selective filopodia binding adhesion and influences CB matching. Further, we identified adhesion molecules Fasciclin III (Fas3) and Ten-m, both of which also regulate synaptic targeting, as having complementary differential expression in CBs. Altering Fas3 expression changes differential filopodia adhesion and leads to CB mismatch. Furthermore, only when both Fas3 and Ten-m are lost is CB alignment severely impaired. Our results show that differential adhesion mediated by selective filopodia binding efficiently regulates precise and robust cell matching.

Investigating cell-type specific functions of tenascin-C.

The extracellular matrix molecule tenascin-C (TNC) has received a lot of attention since its discovery 30 years ago because of its multiple roles in tissue repair, and in pathologies such as chronic inflammation, fibrosis, and cancer. Mouse models with high or no TNC expression have enabled the validation of key roles for TNC in immunity and angiogenesis. In parallel, many approaches including primary cell or organ cultures have shed light on the cellular and molecular mechanisms by which TNC exerts its multiple actions in vivo. Here, we will describe assays that investigate its antiadhesive properties and that measure the effect of TNC on the actin cytoskeleton, cell survival, proliferation, and migration. We will also describe assays to assess the impact of TNC on endothelial and immune cells in cell and organ culture, and to compare the responses of fibroblasts from normal and diseased tissues.

Extracellular matrix and traumatic brain injury.

The brain extracellular matrix (ECM) plays a crucial role in both the developing and adult brain by providing structural support and mediating cell-cell interactions. In this review, we focus on the major constituents of the ECM and how they function in both normal and injured brain, and summarize the changes in the composition of the ECM as well as how these changes either promote or inhibit recovery of function following traumatic brain injury (TBI). Modulation of ECM composition to facilitates neuronal survival, regeneration and axonal outgrowth is a potential therapeutic target for TBI treatment.

Bone Marrow-Derived Tenascin-C Attenuates Cardiac Hypertrophy by Controlling Inflammation.

BACKGROUND: Tenascin-C (TNC) is a highly conserved matricellular protein with a distinct expression pattern during development and disease. Remodeling of the left ventricle (LV) in response to pressure overload leads to the re-expression of the fetal gene program. OBJECTIVES: The aim of this study was to investigate the function of TNC in cardiac hypertrophy in response to pressure overload. METHODS: Pressure overload was induced in TNC knockout and wild-type mice by constricting their abdominal aorta or by infusion of angiotensin II. Echocardiography, immunostaining, flow cytometry, quantitative real-time polymerase chain reaction, and reciprocal bone marrow transplantation were used to evaluate the effect of TNC deficiency. RESULTS: Echocardiographic analysis of pressure overloaded hearts revealed that all LV parameters (LV end-diastolic and -systolic dimensions, ejection fraction, and fractional shortening) deteriorated in TNC-deficient mice compared with their wild-type counterparts. Cardiomyocyte size and collagen accumulation were significantly greater in the absence of TNC. Mechanistically, TNC deficiency promoted rapid accumulation of the CCR2+/Ly6Chi monocyte/macrophage subset into the myocardium in response to pressure overload. Further, echocardiographic and immunohistochemical analyses of recipient hearts showed that expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling of the pressure-overloaded heart. CONCLUSIONS: TNC deficiency further impaired cardiac function in response to pressure overload and exacerbated fibrosis by enhancing inflammation. In addition, expression of TNC in the bone marrow, but not the myocardium, protected the myocardium against excessive remodeling in response to mild pressure overload.

Quantitative proteomics identify Tenascin-C as a promoter of lung cancer progression and contributor to a signature prognostic of patient survival.

The extracellular microenvironment is an integral component of normal and diseased tissues that is poorly understood owing to its complexity. To investigate the contribution of the microenvironment to lung fibrosis and adenocarcinoma progression, two pathologies characterized by excessive stromal expansion, we used mouse models to characterize the extracellular matrix (ECM) composition of normal lung, fibrotic lung, lung tumors, and metastases. Using quantitative proteomics, we identified and assayed the abundance of 113 ECM proteins, which revealed robust ECM protein signatures unique to fibrosis, primary tumors, or metastases. These analyses indicated significantly increased abundance of several S100 proteins, including Fibronectin and Tenascin-C (Tnc), in primary lung tumors and associated lymph node metastases compared with normal tissue. We further showed that Tnc expression is repressed by the transcription factor Nkx2-1, a well-established suppressor of metastatic progression. We found that increasing the levels of Tnc, via CRISPR-mediated transcriptional activation of the endogenous gene, enhanced the metastatic dissemination of lung adenocarcinoma cells. Interrogation of human cancer gene expression data revealed that high TNC expression correlates with worse prognosis for lung adenocarcinoma, and that a three-gene expression signature comprising TNC, S100A10, and S100A11 is a robust predictor of patient survival independent of age, sex, smoking history, and mutational load. Our findings suggest that the poorly understood ECM composition of the fibrotic and tumor microenvironment is an underexplored source of diagnostic markers and potential therapeutic targets for cancer patients.

The extracellular matrix glycoprotein tenascin-C and matrix metalloproteinases modify cerebellar structural plasticity by exposure to an enriched environment.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

Composition and adaptation of human myotendinous junction and neighboring muscle fibers to heavy resistance training.

The myotendinous junction (MTJ) is a common site of strain injury and yet understanding of its composition and ability to adapt to loading is poor. The main aims of this study were to determine the profile of selected collagens and macrophage density in human MTJ and adjoining muscle fibers, and to investigate whether heavy exercise loading would alter this profile. Fifteen individuals scheduled for anterior cruciate ligament repair surgery were randomized into three groups: control, acute or 4 weeks heavy resistance training. MTJ samples were collected from the semitendinosus and gracilis muscles and were sectioned and stained immunohistochemically for collagen types I, III, VI, XII, XIV, XXII, Tenascin-C and CD68. Macrophage density and distribution was evaluated and the amount of each collagen type in muscle and MTJ was graded. Collagen XXII was observed solely at the MTJ, while all other collagens were abundant at the MTJ and in muscle perimysium or endomysium. The endomysial content of collagen XIV, macrophages and Tenascin-C increased following 4 weeks of training. These findings illustrate the heterogeneity of collagen type composition of human MTJ. The increase in collagen XIV following 4 weeks of training may reflect a training-induced protection against strain injuries in this region.

Tenascin-C Is a Major Component of the Fibrogenic Niche in Kidney Fibrosis.

Kidney fibrosis initiates at certain focal sites in which the fibrogenic niche provides a specialized microenvironment that facilitates fibroblast activation and proliferation. However, the molecular identity of these fibrogenic niches is poorly characterized. Here, we determined whether tenascin-C (TNC), an extracellular matrix glycoprotein, is a component of the fibrogenic niche in kidney fibrosis. In vivo, TNC expression increased rapidly in kidneys subjected to unilateral ureteral obstruction or ischemia/reperfusion injury and predominantly localized at the foci rich in fibroblasts in renal interstitium. In vitro, TNC selectively promoted renal interstitial fibroblast proliferation, bromodeoxyuridine incorporation, and the expression of proliferation-related genes. The mitogenic activity of TNC required the integrin/focal adhesion kinase/mitogen-activated protein kinase signaling cascade. Using decellularized extracellular matrix scaffolds, we found that TNC-enriched scaffolds facilitated fibroblast proliferation, whereas TNC-deprived scaffolds inhibited proliferation. Matrix scaffold prepared from fibrotic kidney also promoted greater ex vivo fibroblast proliferation than did scaffolds prepared from healthy kidney. Conversely, small interfering RNA-mediated knockdown of TNC in vivo repressed injury-induced fibroblast expansion and renal fibrosis. These studies identify TNC as a major constituent of the fibrogenic niche that promotes fibroblast proliferation, and illustrate a pivotal role for the TNC-enriched microenvironment in kidney fibrogenesis.

High expression of intratumoral stromal proteins is associated with chemotherapy resistance in breast cancer.

We studied the changes of intratumoral stromal proteins including THBS1, TNC, FN, SPARC and α-SMA, following neoadjuvant chemotherapy. The underlying mechanisms by which THBS1 and TNC regulated resistance to docetaxel were further studied using functional studies. 100 patients with newly diagnosed breast cancer were treated with alternating sequential doxorubicin and docetaxel. Immunohistochemistry (IHC) staining for stromal proteins was performed on pre- and post-treatment core biopsies respectively. THBS1 and TNC were further validated with IHC in an independent cohort of 31 patients. A high baseline combined expression score of the 5 stromal proteins predicted independently for poor progression-free (HRadjusted 2.22, 95% CI 1.06-4.64) and overall survival (HRadjusted 5.94, 95% CI 2.25-15.71). After 1-2 cycles of chemotherapy, increased expression of THBS1, TNC, FN, SPARC and α-SMA was seen in patients with subsequent pathological lymph node involvement at surgery. Increased expression of THBS1 and TNC compared to baseline was also seen in intrinsically resistant tumors, but not in sensitive ones. Both THBS1 and TNC-associated chemoresistance were confirmed in an independent validation cohort. Exogenous THBS1 and TNC protected MCF-7 cells against proliferation inhibition induced by docetaxel through activating integrin ß1/mTOR pathway. Thus, up-regulation of THBS1, TNC, FN, SPARC and α-SMA following neoadjuvant chemotherapy was associated with chemotherapy resistance in breast cancer patients. Functional studies showed THBS1 and TNC to mediate chemoresistance through the integrin ß1/mTOR pathway, suggesting that therapies targeting integrin ß1/mTOR pathway may be a promising strategy to overcome chemotherapy resistance.

Tenascin-C promotes angiogenesis in fibrovascular membranes in eyes with proliferative diabetic retinopathy.

PURPOSE: We previously demonstrated that tenascin-C was highly expressed in the fibrovascular membranes (FVMs) of patients with proliferative diabetic retinopathy (PDR). However, its role in the pathogenesis of FVMs has not been determined. The purpose of this study was to investigate what role tenascin-C plays in the formation and angiogenesis of FVMs. METHODS: The level of tenascin-C was determined by sandwich enzyme-linked immunosorbent assay in the vitreous samples collected from patients with PDR and with a macular hole as control. The locations of tenascin-C, α- smooth muscle actin (SMA), CD34, glial fibrillary acidic protein (GFAP), and integrin αV in the FVMs from PDR patients were determined by immunohistochemistry. We also measured the in vitro expression of the mRNA and protein of tenascin-C in vascular smooth muscle cells (VSMCs) stimulated by interleukin (IL)-13. The effects of tenascin-C on cell proliferation, migration, and tube formation were determined in human retinal endothelial cells (HRECs) in culture. RESULTS: The mean vitreous levels of tenascin-C were significantly higher in patients with PDR than in patients with a macular hole (p<0.001). Double immunofluorescence analyses of FVMs from PDR patients showed that tenascin-C co-stained FVMs with α-SMA, CD34, and integrin αV but not with GFAP. In addition, IL-13 treatment increased both the expression and secretion of tenascin-C by VSMCs in a dose-dependent manner. Tenascin-C exposure promoted proliferation, migration, and tube formation in HRECs. Tenascin-C neutralizing antibody significantly blocked the tube formation by HRECs exposed to VSMC-IL-13-conditioned medium. CONCLUSIONS: Our findings suggest that tenascin-C is secreted from VSMCs and promotes angiogenesis in the FVMs associated with PDR.

Immunohistochemical localization of tenascin-C in rat periodontal ligament with reference to alveolar bone remodeling.

We investigated the immunohistochemical localization of tenascin-C in 8-week-old rat periodontal ligaments. Tenascin-C immunoreactivity was detected in zones along with cementum and alveolar bone, and more intensely on the resorption surface of alveolar bone than on the formation surface. On the resorbing surface, tenascin-C immunoreactivity was detected in Howship's lacunae without osteoclasts, and in the interfibrous space of the periodontal ligaments, indicating that this molecule works as an adhesion molecule between bone and fibers of periodontal ligaments. Upon experimental tooth movement by inserting elastic bands (Waldo method), the physiological resorption surface of alveolar bone under compressive force showed enhanced bone resorption and enhanced tenascin-C immunoreactivity. However, on the physiological bone formation surface under compressive force, bone resorption was seen only occasionally, and no enhanced tenascin-C immunoreactivity was noted. In an experiment involving excessive occlusal loading to rat molars, transient bone resorption occurred within interradicular septa, but no enhanced tenascin-C immunoreactivity was seen in the periodontal ligaments. These results indicate that tenascin-C works effectively on the bone resorbing surface of physiological alveolar bone remodeling sites, rather than on the non-physiological transient bone resorbing surface. Fibronectin immunoreactivity was distributed evenly in the periodontal ligaments under experimental conditions. Co-localization of tenascin-C and fibronectin immunoreactivity was observed in many regions, but mutually exclusive expression patterns were also seen in some regions, indicating that fibronectin might not be directly involved in alveolar bone remodeling, but may play a role via interaction with tenascin-C.

Carbon Monoxide Inhibits Tenascin-C Mediated Inflammation via IL-10 Expression in a Septic Mouse Model.

Tenascin-C (TN-C), an extracellular matrix (ECM) glycoprotein, is specifically induced upon tissue injury and infection and during septic conditions. Carbon monoxide (CO) gas is known to exert various anti-inflammatory effects in various inflammatory diseases. However, the mechanisms underlying the effect of CO on TN-C-mediated inflammation are unknown. In the present study, we found that treatment with LPS significantly enhanced TN-C expression in macrophages. CO gas, or treatment with the CO-donor compound, CORM-2, dramatically reduced LPS-induced expression of TN-C and proinflammatory cytokines while significantly increased the expression of IL-10. Treatment with TN-C siRNA significantly suppressed the effects of LPS on proinflammatory cytokines production. TN-C siRNA did not affect the CORM-2-dependent increase of IL-10 expression. In cells transfected with IL-10 siRNA, CORM-2 had no effect on the LPS-induced expression of TN-C and its downstream cytokines. These data suggest that IL-10 mediates the inhibitory effect of CO on TN-C and the downstream production of proinflammatory cytokines. Additionally, administration of CORM-2 dramatically reduced LPS-induced TN-C and proinflammatory cytokines production while expression of IL-10 was significantly increased. In conclusion, CO regulated IL-10 expression and thus inhibited TN-C-mediated inflammation in vitro and in vivo.

[Advances in molecular mechanisms of tenascin-C in promoting tumor metastasis].

Tenascin-C (TNC) is an extracellular matrix glycoprotein, which is usually highly expressed in embryonic tissues and tumor tissues, but is not expressed or just lowly expressed in mature tissues. TNC is involved in various complex signaling pathways during tumor metastasis, especially through modulating FAK, RhoA, Wnt and Notch pathways by interacting with syndecan-4, integrin α5ß1, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). As a result, TNC affects epithelial mesenchymal transition, tumor cell adhesion, proliferation and angiogenesis, which eventually enhances the invasion and metastasis ability of many tumors. Further studies have demonstrated that TNC could be used as prognosis or metastasis marker of patients with malignant tumor.