ABSTRACT
The blood-brain barrier (BBB) is a unique set of properties of the brain vasculature which severely restrict its permeability to proteins and small molecules. Classic chick-quail chimera studies have shown that these properties are not intrinsic to the brain vasculature but rather are induced by surrounding neural tissue. Here, we identify Spock1 as a candidate neuronal signal for regulating BBB permeability in zebrafish and mice. Mosaic genetic analysis shows that neuronally expressed Spock1 is cell non-autonomously required for a functional BBB. Leakage in spock1 mutants is associated with altered extracellular matrix (ECM), increased endothelial transcytosis, and altered pericyte-endothelial interactions. Furthermore, a single dose of recombinant SPOCK1 partially restores BBB function in spock1 mutants by quenching gelatinase activity and restoring vascular expression of BBB genes including mcamb. These analyses support a model in which neuronally secreted Spock1 initiates BBB properties by altering the ECM, thereby regulating pericyte-endothelial interactions and downstream vascular gene expression.
Subject(s)
Blood-Brain Barrier , Proteoglycans , Zebrafish , Animals , Mice , Biological Transport , Blood-Brain Barrier/metabolism , Brain , Endothelium/metabolism , Proteoglycans/metabolismABSTRACT
Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/ß-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor ß (TGF-ß), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/ß-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/ß-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/ß-catenin pathway.
Subject(s)
Follistatin-Related Proteins , Frizzled Receptors , Kidney , Wnt Signaling Pathway , Animals , Follistatin-Related Proteins/genetics , Follistatin-Related Proteins/metabolism , Frizzled Receptors/metabolism , Humans , Kidney/metabolism , Kidney/physiopathology , Ligands , Mice , Wnt3A ProteinABSTRACT
Tungsten is a naturally occurring transition element used in a broad range of applications. As a result of its extensive use, we are increasingly exposed to tungsten from our environment, including potable water, since tungsten can become bioaccessible in ground sources. The kidneys are particularly susceptible to tungsten exposure as this is the main site for tungsten excretion. In this study, we investigated the prolonged effects of tungsten on the kidneys and how this may impact injury and function. When mice were exposed to tungsten in their drinking water for 1 mo, kidney function had not significantly changed. Following 3-mo exposure, mice were presented with deterioration in kidney function as determined by serum and urine creatinine levels. During 3 mo of tungsten exposure, murine kidneys demonstrated significant increases in the myofibroblast marker α-smooth muscle actin (αSMA) and extracellular matrix products: fibronectin, collagen, and matricellular proteins. In addition, Masson's trichrome and hematoxylin-eosin (H&E) staining revealed an increase in fibrotic tissue and vacuolization of tubular epithelial cells, respectively, from kidneys of tungsten-treated mice, indicative of renal injury. In vitro treatment of kidney fibroblasts with tungsten led to increased proliferation and upregulation of transforming growth factor ß1 (TGFß1), which was consistent with the appearance of fibroblast-to-myofibroblast transition (FMT) markers. Our data suggest that continuous exposure to tungsten impairs kidney function that may lead to the development of chronic kidney disease (CKD).
Subject(s)
Myofibroblasts/drug effects , Myofibroblasts/pathology , Renal Insufficiency, Chronic/chemically induced , Renal Insufficiency, Chronic/pathology , Tungsten/administration & dosage , Tungsten/toxicity , Administration, Oral , Animals , Dose-Response Relationship, Drug , Fibrosis , Male , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Toxicity Tests, Subchronic/methodsABSTRACT
The extracellular matrix (ECM) surrounding cells is indispensable for regulating their behavior. The dynamics of ECM signaling are tightly controlled throughout growth and development. During tissue remodeling, matricellular proteins (MCP) are secreted into the ECM. These factors do not serve classical structural roles, but rather regulate matrix proteins and cell-matrix interactions to influence normal cellular functions. In the tumor microenvironment, it is becoming increasingly clear that aberrantly expressed MCPs can support multiple hallmarks of carcinogenesis by interacting with various cellular components that are coupled to an array of downstream signals. Moreover, MCPs also reorganize the biomechanical properties of the ECM to accommodate metastasis and tumor colonization. This realization is stimulating new research on MCPs as reliable and accessible biomarkers in cancer, as well as effective and selective therapeutic targets.
Subject(s)
Carcinogenesis/pathology , Cell Communication , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Neoplasms/pathology , Tumor Microenvironment , Animals , Carcinogenesis/metabolism , Humans , Neoplasms/metabolism , Signal TransductionABSTRACT
The assembly of collagen VI microfibrils is a multistep process in which proteolytic processing within the C-terminal globular region of the collagen VI α3 chain plays a major role. However, the mechanisms involved remain elusive. Moreover, C5, the short and most C-terminal domain of the α3 chain, recently has been proposed to be released as an adipokine that enhances tumor progression, fibrosis, inflammation, and insulin resistance and has been named "endotrophin." Serum endotrophin could be a useful biomarker to monitor the progression of such disorders as chronic obstructive pulmonary disease, systemic sclerosis, and kidney diseases. Here, using biochemical and isotopic MS-based analyses, we found that the extracellular metalloproteinase bone morphogenetic protein 1 (BMP-1) is involved in endotrophin release and determined the exact BMP-1 cleavage site. Moreover, we provide evidence that several endotrophin-containing fragments are present in various tissues and body fluids. Among these, a large C2-C5 fragment, which contained endotrophin, was released by furin-like proprotein convertase cleavage. By using immunofluorescence microscopy and EM, we also demonstrate that these proteolytic maturations occur after secretion of collagen VI tetramers and during microfibril assembly. Differential localization of N- and C-terminal regions of the collagen VI α3 chain revealed that cleavage products are deposited in tissue and cell cultures. The detailed information on the processing of the collagen VI α3 chain reported here provides a basis for unraveling the function of endotrophin (C5) and larger endotrophin-containing fragments and for refining their use as biomarkers of disease progression.
Subject(s)
Bone Morphogenetic Protein 1/metabolism , Collagen Type VI/metabolism , Proprotein Convertases/metabolism , Fibrosis , Furin/metabolism , HEK293 Cells , Humans , Insulin Resistance , Microfibrils/metabolism , Peptide Fragments/metabolism , ProteolysisABSTRACT
We investigated the presence of autoantibodies against the extracellular matrix proteins thrombospondin-4 (TSP-4), cartilage oligomeric matrix protein (COMP), C-type lectin domain family 3 member A (CLEC3A), collagen II, collagen VI, matrilin-3, and fibrillin-2 in the serum of osteoarthritis (OA) patients. We compared those results with the presence of such antibodies in rheumatoid arthritis (RA) patients and in healthy donors (HD). Our study examines whether antibodies against extracellular proteins can be used as potential biomarkers to support the clinical diagnosis of OA. 10 OA, 10 RA patients and 10 HD were enrolled in this explorative cross-sectional study. SDS-PAGE and immunoblot were used to investigate the presence of antibodies against extracellular matrix proteins. The serum of 5/10 OA patients but 0/10 HD exhibited TSP-4 IgG isotype antibodies (Pâ¯=â¯0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (Pâ¯=â¯0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (Pâ¯=â¯0.005). We found strong evidence for the presence of IgG isotype autoantibodies against the cartilage extracellular matrix proteins TSP-4, COMP and CLEC3A in OA. The detection of IgG isotype autoantibodies against TSP-4, COMP and CLEC3A may support the clinical diagnosis of OA. OA with autoantibodies against cartilage extracellular matrix proteins defines a new OA subgroup suggesting that patients with high concentrations of autoantibodies may benefit from an immune suppressive therapy.
Subject(s)
Arthritis, Rheumatoid/immunology , Autoantibodies/immunology , Osteoarthritis/immunology , Arthritis, Rheumatoid/diagnosis , Arthritis, Rheumatoid/therapy , Biomarkers/blood , Cartilage Oligomeric Matrix Protein/blood , Cartilage Oligomeric Matrix Protein/immunology , Collagen Type II/blood , Collagen Type II/immunology , Collagen Type VI/blood , Collagen Type VI/immunology , Fibrillin-2/blood , Fibrillin-2/immunology , Humans , Lectins, C-Type/blood , Lectins, C-Type/immunology , Matrilin Proteins/blood , Matrilin Proteins/immunology , Middle Aged , Osteoarthritis/diagnosis , Osteoarthritis/therapy , Thrombospondins/blood , Thrombospondins/immunologyABSTRACT
C-type lectin domain family 3 member A (CLEC3A) is a poorly characterized protein belonging to the superfamily of C-type lectins. Its closest homologue tetranectin binds to the kringle 4 domain of plasminogen and enhances its association with tissue plasminogen activator (tPA) thereby enhancing plasmin production, but whether CLEC3A contributes to plasminogen activation is unknown. Here, we recombinantly expressed murine and human full-length CLEC3As as well as truncated forms of CLEC3A in HEK-293 Epstein-Barr nuclear antigen (EBNA) cells. We analyzed the structure of recombinant CLEC3A by SDS-PAGE and immunoblot, glycan analysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy; compared the properties of the recombinant protein with those of CLEC3A extracted from cartilage; and investigated its tissue distribution and extracellular assembly by immunohistochemistry and immunofluorescence microscopy. We found that CLEC3A mainly occurs as a monomer, but also forms dimers and trimers, potentially via a coiled-coil α-helix. We also noted that CLEC3A can be modified with chondroitin/dermatan sulfate side chains and tends to oligomerize to form higher aggregates. We show that CLEC3A is present in resting, proliferating, and hypertrophic growth-plate cartilage and assembles into an extended extracellular network in cultures of rat chondrosarcoma cells. Further, we found that CLEC3A specifically binds to plasminogen and enhances tPA-mediated plasminogen activation. In summary, we have determined the structure, tissue distribution, and molecular function of the cartilage-specific lectin CLEC3A and show that CLEC3A binds to plasminogen and participates in tPA-mediated plasminogen activation.
Subject(s)
Lectins, C-Type/metabolism , Plasminogen Activators/metabolism , Tissue Plasminogen Activator/metabolism , Amino Acid Sequence , Animals , Cartilage/metabolism , Chromatography, Gel , HEK293 Cells , Humans , Immunohistochemistry , Lectins, C-Type/biosynthesis , Lectins, C-Type/genetics , Mice , Mice, Inbred C57BL , Plasminogen/metabolism , Protein Binding , Recombinant Proteins/biosynthesis , Recombinant Proteins/geneticsABSTRACT
Mitochondria play a pivotal role in energy metabolism, but whether insulin signaling per se could regulate mitochondrial function has not been identified yet. To investigate whether mitochondrial function is regulated by insulin signaling, we analyzed muscle and liver of insulin receptor (IR)+/--insulin receptor substrate-1 (IRS-1)+/- double heterozygous (IR-IRS1dh) mice, a well described model for insulin resistance. IR-IRS1dh mice were studied at the age of 6 and 12 months and glucose metabolism was determined by glucose and insulin tolerance tests. Mitochondrial enzyme activities, oxygen consumption, and membrane potential were assessed using spectrophotometric, respirometric, and proton motive force analysis, respectively. IR-IRS1dh mice showed elevated serum insulin levels. Hepatic mitochondrial oxygen consumption was reduced in IR-IRS1dh animals at 12 months of age. Furthermore, 6-month-old IR-IRS1dh mice demonstrated enhanced mitochondrial respiration in skeletal muscle, but a tendency of impaired glucose tolerance. On the other hand, 12-month-old IR-IRS1dh mice showed improved glucose tolerance, but normal muscle mitochondrial function. Our data revealed that deficiency in IR/IRS-1 resulted in normal or even elevated skeletal muscle, but impaired hepatic mitochondrial function, suggesting a direct cross-talk between insulin signaling and mitochondria in the liver.
Subject(s)
Insulin Receptor Substrate Proteins/metabolism , Insulin/metabolism , Mitochondria/metabolism , Mitochondria/pathology , Receptor, Insulin/metabolism , Animals , Blood Glucose/metabolism , Glucose/metabolism , Insulin Receptor Substrate Proteins/genetics , Liver/metabolism , Mice , Muscle, Skeletal/metabolism , Receptor, Insulin/genetics , Signal Transduction/genetics , Signal Transduction/physiologyABSTRACT
The mechanisms leading to amyloid-ß (Aß) accumulation in sporadic Alzheimer disease (AD) are unknown but both increased production or impaired clearance likely contribute to aggregation. To understand the potential roles of the extracellular matrix proteoglycan Testican-1 in the pathophysiology of AD, we used samples from AD patients and controls and an in vitro approach. Protein expression analysis showed increased levels of Testican-1 in frontal and temporal cortex of AD patients; histological analysis showed that Testican-1 accumulates and co-aggregates with Aß plaques in the frontal, temporal and entorhinal cortices of AD patients. Proteomic analysis identified 10 fragments of Testican-1 in cerebrospinal fluid (CSF) from AD patients. HEK293T cells expressing human wild type or mutant Aß precursor protein (APP) were transfected with Testican-1. The co-expression of both proteins modified the sorting of Testican-1 into the endocytic pathway leading to its transient accumulation in Golgi, which seemed to affect APP processing, as indicated by reduced Aß40 and Aß42 levels in APP mutant cells. In conclusion, patient data reflect a clearance impairment that may favor Aß accumulation in AD brains and our in vitro model supports the notion that the interaction between APP and Testican-1 may be a key step in the production and aggregation of Aß species.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/physiology , Brain/metabolism , Brain/pathology , Proteoglycans/metabolism , Adult , Aged , Alzheimer Disease/cerebrospinal fluid , Biomarkers/cerebrospinal fluid , Biomarkers/metabolism , Female , HEK293 Cells , Humans , Male , Middle AgedABSTRACT
The ability of cancer cells to establish lethal metastatic lesions requires the survival and expansion of single cancer cells at distant sites. The factors controlling the clonal growth ability of individual cancer cells remain poorly understood. Here, we show that high expression of the transcription factor ARNTL2 predicts poor lung adenocarcinoma patient outcome. Arntl2 is required for metastatic ability in vivo and clonal growth in cell culture. Arntl2 drives metastatic self-sufficiency by orchestrating the expression of a complex pro-metastatic secretome. We identify Clock as an Arntl2 partner and functionally validate the matricellular protein Smoc2 as a pro-metastatic secreted factor. These findings shed light on the molecular mechanisms that enable single cancer cells to form allochthonous tumors in foreign tissue environments.
Subject(s)
ARNTL Transcription Factors/genetics , Adenocarcinoma/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , ARNTL Transcription Factors/metabolism , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Blotting, Western , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice, 129 Strain , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Neoplasm Metastasis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Survival AnalysisABSTRACT
BACKGROUND: SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. RESULTS: In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. CONCLUSIONS: Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development.
Subject(s)
Calcium-Binding Proteins/metabolism , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Myelopoiesis/genetics , Myelopoiesis/physiology , Zebrafish Proteins/metabolism , Zebrafish/embryology , Animals , Embryo, Nonmammalian/drug effects , Gene Expression Regulation, Developmental/genetics , Hematopoiesis/physiology , Mesoderm/metabolism , Myelopoiesis/drug effectsABSTRACT
BACKGROUND & AIMS: To determine if diabetic and insulin-resistant states cause mitochondrial dysfunction in liver or if there is long term adaptation of mitochondrial function to these states, mice were (i) fed with a high-fat diet to induce obesity and T2D (HFD), (ii) had a genetic defect in insulin signaling causing whole body insulin resistance, but not full blown T2D (IR/IRS-1(+/-) mice), or (iii) were analyzed after treatment with streptozocin (STZ) to induce a T1D-like state. METHODS: Hepatic lipid levels were measured by thin layer chromatography. Mitochondrial respiratory chain (RC) levels and function were determined by Western blot, spectrophotometric, oxygen consumption and proton motive force analysis. Gene expression was analyzed by real-time PCR and microarray. RESULTS: HFD caused insulin resistance and hepatic lipid accumulation, but RC was largely unchanged. Livers from insulin resistant IR/IRS-1(+/-) mice had normal lipid contents and a normal RC, but mitochondria were less well coupled. Livers from severely hyperglycemic and hypoinsulinemic STZ mice had massively depleted lipid levels, but RC abundance was unchanged. However, liver mitochondria isolated from these animals showed increased abundance and activity of the RC, which was better coupled. CONCLUSIONS: Insulin resistance, induced either by obesity or genetic manipulation and steatosis do not cause mitochondrial dysfunction in mouse liver. Also, mitochondrial dysfunction is not a prerequisite for liver steatosis. However, severe insulin deficiency and high blood glucose levels lead to an enhanced performance and better coupling of the RC. This may represent an adaptation to fuel overload and the high energy-requirement of an unsuppressed gluconeogenesis.
Subject(s)
Adaptation, Physiological , Diabetes Mellitus, Type 2/physiopathology , Insulin Resistance/physiology , Mitochondria, Liver/physiology , Animals , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 2/etiology , Diet, High-Fat/adverse effects , Fatty Liver/etiology , Fatty Liver/physiopathology , Gene Expression , Insulin Receptor Substrate Proteins/deficiency , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Ion Channels/metabolism , Liver/metabolism , Liver/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondrial ADP, ATP Translocases/metabolism , Mitochondrial Proteins/metabolism , Obesity/etiology , Obesity/physiopathology , Oxidative Phosphorylation , Proton-Motive Force , Receptor, Insulin/deficiency , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Signal Transduction , Uncoupling Protein 2ABSTRACT
The testicans are a three-member family of secreted proteoglycans structurally related to the BM-40/secreted protein acidic and rich in cystein (SPARC) osteonectin family of extracellular calcium-binding proteins. In vitro studies have indicated that testicans are involved in the regulation of extracellular protease cascades and in neuronal function. Here, we describe the biochemical characterization and tissue distribution of mouse testican-3 as well as the inactivation of the corresponding gene. The expression of testican-3 in adult mice is restricted to the brain, where it is located diffusely within the extracellular matrix, as well as associated with cells. Brain-derived testican-3 is a heparan sulphate proteoglycan. In cell culture, the core protein is detected in the supernatant and the extracellular matrix, whereas the proteoglycan form is restricted to the supernatant. This indicates possible interactions of the testican-3 core protein with components of the extracellular matrix which are blocked by addition of the glycosaminoglycan chains. Mice deficient in testican-3 are viable and fertile and do not show an obvious phenotype. This points to a functional redundancy among the different members of the testican family or between testican-3 and other brain heparan sulphate proteoglycans.
Subject(s)
Osteonectin/metabolism , Proteoglycans/genetics , Proteoglycans/metabolism , Amino Acid Sequence , Animals , Brain/anatomy & histology , Brain/metabolism , Cell Line, Tumor , Cloning, Molecular , Extracellular Matrix/metabolism , Fibrosarcoma/pathology , Gene Expression Regulation, Developmental/genetics , Humans , Immunoprecipitation , Mice , Mice, Inbred C57BL , Mice, Knockout , Proteoglycans/deficiency , TransfectionABSTRACT
Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.
Subject(s)
Heparin/metabolism , Osteonectin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Adhesion/physiology , Heparin/chemistry , Humans , Mice , Models, Molecular , Molecular Docking Simulation , Molecular Sequence Data , Mutation , Osteonectin/chemistry , Osteonectin/genetics , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , Proteoglycans/metabolism , Sequence Alignment , Tryptophan/chemistryABSTRACT
Tenascin-C is an extracellular matrix protein over-expressed in a large variety of cancers. In the present study, we aimed at identifying new interactors of tenascin-C by purifying secreted proteins on a tenascin-C affinity column. Analysis of eluates by mass spectrometry revealed phosphoglycerate kinase 1, clusterin, fibronectin, SPARC-related modular calcium-binding protein 1 (SMOC1) and nidogen-2 as potential interactors of tenascin-C. The interaction between tenascin-C and SMOC1 was confirmed by co-immunoprecipitation and further analyzed by Surface Plasmon Resonance Spectroscopy, which revealed an apparent dissociation constant (K(D)) value of 2.59∗10(-9)M. Further analyses showed that this binding is reduced in the presence of EDTA. To investigate whether SMOC1 itself could be over-expressed in the context of tumorigenesis, we analyzed data of two independent RNA profiling studies and found that mRNA levels of SMOC1 are significantly increased in oligodendrogliomas compared to control brain samples. In support of these data, western blot analysis of protein extracts from 12 oligodendrogliomas, 4 astrocytomas and 13 glioblastomas revealed elevated levels compared to healthy brain extract. Interestingly, cell migration experiments revealed that SMOC1 can counteract the chemo-attractive effect of tenascin-C on U87 glioma cells. The present study thus identified SMOC1 as a new cancer-associated protein capable of interacting with tenascin-C in vitro.
Subject(s)
Biomarkers, Tumor/metabolism , Brain Neoplasms/metabolism , Glioma/metabolism , Osteonectin/metabolism , Tenascin/metabolism , Up-Regulation , Adult , Aged , Brain Neoplasms/pathology , Case-Control Studies , Cell Movement , Gene Expression Regulation, Neoplastic , Glioma/pathology , Humans , Immunoprecipitation , Middle Aged , Protein Binding , Surface Plasmon ResonanceABSTRACT
AIM: To determine the distribution of major basement membrane constituents, particularly nidogen 1 and 2, in young and aging mouse retinae. METHODS: The specificity of antibodies against basement membrane proteins was ascertained by immunoblotting with proteins extracted from mouse retinae. The same antibodies were used in indirect immunofluorescence microscopy to localize basement membrane proteins in paraffin sections of retinae from 1-, 12- and 18-month-old C57BL/6 mice. RESULTS: At a young age, laminin, perlecan and collagen IV were most abundant in Bruch's membrane. Later, the proteins were clearly detected in capillary basement membranes and the inner limiting membrane. In both of these basement membranes, a massive increase in protein amount was seen upon aging, whereas in Bruch's membrane the staining intensity was less drastically changed. Both nidogen 1 and 2 were present in vascular basement membranes and Bruch's membrane throughout the age periods studied. In the inner limiting membrane, the nidogens were more strongly expressed at higher ages, with an earlier and more extensive deposition of nidogen 1. CONCLUSIONS: All major basement membrane constituents are present in the mouse retina, but the onset of deposition differs among the different proteins and between the various retinal basement membranes. In general, basement membrane protein deposition increases with age.
Subject(s)
Aging/physiology , Extracellular Matrix Proteins/metabolism , Membrane Glycoproteins/metabolism , Retina/metabolism , Animals , Basement Membrane/metabolism , Blotting, Western , Bruch Membrane/metabolism , Calcium-Binding Proteins , Cell Adhesion Molecules , Fluorescent Antibody Technique, Indirect , Mice , Mice, Inbred C57BL , Microscopy, FluorescenceABSTRACT
SMOC-2 is a recently discovered member of the BM-40/SPARC/osteonectin family of extracellular multidomain proteins of so far unknown function. While we have shown earlier that the homologous protein SMOC-1 is associated with basement membranes, in this study we demonstrate that, in the mouse, SMOC-2 could be detected in a large number of non-basement membrane localizations, often showing a diffuse tissue distribution. A more distinct expression pattern was seen in skin where SMOC-2 is mainly present in the basal layers of the epidermis. Functionally, recombinant SMOC-2 stimulated attachment of primary epidermal cells as well as several epidermal-derived cell lines but had no effect on the attachment of non-epidermal cells. Inhibition experiments using blocking antibodies against individual integrin subunits allowed the identification of alphavbeta6 and alphavbeta1 integrins as important cellular receptors for SMOC-2. Cell attachment as well as the formation of focal adhesions could be attributed to the extracellular calcium-binding domain. The calcium-binding domain also stimulated migration, but not proliferation of keratinocyte-like HaCaT cells. We conclude that SMOC-2, like other members of the BM40/SPARC family, acts as a regulator of cell-matrix interactions.
Subject(s)
Calcium-Binding Proteins/physiology , Cell Movement , Keratinocytes/physiology , Animals , Animals, Newborn , Antigens/chemistry , Antigens/metabolism , Antigens/physiology , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Cell Adhesion , Cell Proliferation , Cells, Cultured , Embryo, Mammalian , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/physiology , Guinea Pigs , Humans , Keratinocytes/metabolism , Mice , Mice, Inbred C57BL , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Skin/embryology , Skin/metabolism , Tissue DistributionABSTRACT
Testicans are proteoglycans belonging to the BM-40/SPARC/osteonectin family of extracellular calcium-binding proteins. Testican-1 is strongly expressed in the brain and has been reported to modulate neuronal attachment and matrix metalloproteinase activation. Characterization of the mouse testican-1 gene (Ticn1), consisting of 12 exons out of which exon 3 is alternatively spliced, allowed the construction of a gene targeting construct. Mice deficient in testican-1 showed no obvious morphological or behavioral abnormalities, were fertile, and had normal life spans. Despite the fact that neither of the testican-1 homologues expressed in the brain, testican-2, testican-3 and SC1/hevin, showed an increased expression in Ticn1 null mice, these results, together with those from other gene targetings, indicate extensive functional redundancy among brain proteoglycans.
Subject(s)
Gene Expression Regulation, Developmental , Proteoglycans/physiology , Animals , Blotting, Northern , Brain/metabolism , Exons , Extracellular Matrix/metabolism , Immunohistochemistry , Mice , Models, Genetic , Neurons/metabolism , Osteonectin/metabolism , Phenotype , Proteoglycans/genetics , Proteoglycans/metabolismABSTRACT
Mouse testican-2 was cloned, sequenced, and shown to be a proteoglycan with a multidomain structure closely similar to that of the human ortholog, previously described as a calcium binding extracellular matrix molecule of the BM-40/SPARC/osteonectin family (Vannahme, C., Schübel, S., Herud, M., Gösling, S., Hülsmann, H., Paulsson, M., Hartmann, U., and Maurer, P. (1999). J. Neurochem. 73, 12-20). Recombinant mouse testican-2 was used to prepare specific antibodies that allowed the detection of testican-2 in various brain structures but also in lung, testis, and in several endocrine glands. Although the testican-2 expressed in EBNA-293 cells carried both heparan sulfate and chondroitin/dermatan sulfate glycosaminoglycan chains, the tissue form always contained only heparan sulfate. Both tissue-derived and recombinant testican-2 carried N-linked glycans. Tissue-derived forms of testican-2 were detected as proteoglycans of varying size, whereas a portion of the molecules produced by EBNA-293 cells were core proteins, lacking glycosaminoglycans. Both the proteoglycan and core protein forms of testican-2 inhibited neurite extension from cultured primary cerebellar neurons and may play regulatory roles in the development of the central nervous system.
Subject(s)
Neurites/physiology , Proteoglycans/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Fluorescent Antibody Technique, Indirect , Glycosylation , Mice , Molecular Sequence Data , Neurites/drug effects , Proteoglycans/genetics , Proteoglycans/pharmacology , Proteoglycans/physiology , Recombinant Proteins/biosynthesis , Recombinant Proteins/pharmacologyABSTRACT
We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.