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2.
Int J Mol Sci ; 21(2)2020 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-31963938

RESUMEN

Matrilins (MATN1, MATN2, MATN3 and MATN4) are adaptor proteins of the cartilage extracellular matrix (ECM), which bridge the collagen II and proteoglycan networks. In humans, dominant-negative mutations in MATN3 lead to various forms of mild chondrodysplasias. However, single or double matrilin knockout mice generated previously in our laboratory do not show an overt skeletal phenotype, suggesting compensation among the matrilin family members. The aim of our study was to establish a mouse line, which lacks all four matrilins and analyze the consequence of matrilin deficiency on endochondral bone formation and cartilage function. Matn1-4-/- mice were viable and fertile, and showed a lumbosacral transition phenotype characterized by the sacralization of the sixth lumbar vertebra. The development of the appendicular skeleton, the structure of the growth plate, chondrocyte differentiation, proliferation, and survival were normal in mutant mice. Biochemical analysis of knee cartilage demonstrated moderate alterations in the extractability of the binding partners of matrilins in Matn1-4-/- mice. Atomic force microscopy (AFM) revealed comparable compressive stiffness but higher collagen fiber diameters in the growth plate cartilage of quadruple mutant compared to wild-type mice. Importantly, Matn1-4-/- mice developed more severe spontaneous osteoarthritis at the age of 18 months, which was accompanied by changes in the biomechanical properties of the articular cartilage. Interestingly, Matn4-/- mice also developed age-associated osteoarthritis suggesting a crucial role of MATN4 in maintaining the stability of the articular cartilage. Collectively, our data provide evidence that matrilins are important to protect articular cartilage from deterioration and are involved in the specification of the vertebral column.

3.
Sci Rep ; 9(1): 19851, 2019 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-31882701

RESUMEN

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

4.
Nat Protoc ; 14(12): 3395-3425, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31705125

RESUMEN

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.


Asunto(s)
Matriz Extracelular/metabolismo , Imagenología Tridimensional/métodos , Coloración y Etiquetado/métodos , Animales , Anticuerpos/metabolismo , Matriz Extracelular/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especificidad de Órganos , Perfusión/métodos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química
5.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-31615030

RESUMEN

The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFß)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.


Asunto(s)
Matriz Extracelular/genética , Macrófagos/metabolismo , Piel/metabolismo , Cicatrización de Heridas/genética , Animales , Biología Computacional , Elastina/genética , Perfilación de la Expresión Génica , Humanos , Macrófagos/patología , Análisis por Micromatrices , Piel/patología
6.
J Biol Chem ; 294(37): 13769-13780, 2019 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-31346034

RESUMEN

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.

7.
JCI Insight ; 4(6)2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-30895940

RESUMEN

The clinical application of advanced next-generation sequencing technologies is increasingly uncovering novel classes of mutations that may serve as potential targets for precision medicine therapeutics. Here, we show that a deep intronic splice defect in the COL6A1 gene, originally discovered by applying muscle RNA sequencing in patients with clinical findings of collagen VI-related dystrophy (COL6-RD), inserts an in-frame pseudoexon into COL6A1 mRNA, encodes a mutant collagen α1(VI) protein that exerts a dominant-negative effect on collagen VI matrix assembly, and provides a unique opportunity for splice-correction approaches aimed at restoring normal gene expression. Using splice-modulating antisense oligomers, we efficiently skipped the pseudoexon in patient-derived fibroblast cultures and restored a wild-type matrix. Similarly, we used CRISPR/Cas9 to precisely delete an intronic sequence containing the pseudoexon and efficiently abolish its inclusion while preserving wild-type splicing. Considering that this splice defect is emerging as one of the single most frequent mutations in COL6-RD, the design of specific and effective splice-correction therapies offers a promising path for clinical translation.

8.
Biochem Biophys Res Commun ; 503(3): 1273-1277, 2018 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-30001809

RESUMEN

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 (P = 0.033). The serum of 8/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4 or COMP (P = 0.005). The serum of 9/10 OA patients but only 1/10 HD exhibited IgG isotype antibodies against TSP-4, COMP or CLEC3A (P = 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.


Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Osteoartritis/inmunología , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/terapia , Biomarcadores/sangre , Proteína de la Matriz Oligomérica del Cartílago/sangre , Proteína de la Matriz Oligomérica del Cartílago/inmunología , Colágeno Tipo II/sangre , Colágeno Tipo II/inmunología , Colágeno Tipo VI/sangre , Colágeno Tipo VI/inmunología , Fibrilina-2/sangre , Fibrilina-2/inmunología , Humanos , Lectinas Tipo C/sangre , Lectinas Tipo C/inmunología , Proteínas Matrilinas/sangre , Proteínas Matrilinas/inmunología , Persona de Mediana Edad , Osteoartritis/diagnóstico , Osteoartritis/terapia , Trombospondinas/sangre , Trombospondinas/inmunología
9.
J Immunol ; 201(3): 1007-1020, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29925677

RESUMEN

Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A-like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI-derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.


Asunto(s)
Antibacterianos/inmunología , Colágeno Tipo VI/inmunología , Péptidos/inmunología , Bacterias/inmunología , Infecciones Bacterianas/inmunología , Humanos , Inmunidad Innata/inmunología , Dominios Proteicos/inmunología , Piel/inmunología , Piel/microbiología , Enfermedades Cutáneas Bacterianas/inmunología
10.
Methods Cell Biol ; 143: 429-446, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29310791

RESUMEN

Marilins mediate interactions between macromolecular components of the extracellular matrix, e.g., collagens and proteoglycans. They are composed of von Willebrand factor type A and epidermal growth factor-like domains and the subunits oligomerize via coiled-coil domains. Matrilin-1 and -3 are abundant in hyaline cartilage, whereas matrilin-2 and -4 are widespread but less abundant. Mutations in matrilin genes have been linked to chondrodysplasias and osteoarthritis and recently characterization of matrilin-deficient mice revealed novel functions in mechanotransduction, regeneration, or inflammation. Due to their intrinsic adhesiveness and partially also low abundance, the study of matrilins is cumbersome. In this chapter, we describe methods for purification of matrilins from tissue, analysis of matrilins in tissue extracts, recombinant expression, and generation of matrilin-specific antibodies.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Cromatografía de Afinidad/métodos , Matriz Extracelular/metabolismo , Proteínas Matrilinas/aislamiento & purificación , Animales , Anticuerpos/inmunología , Anticuerpos/aislamiento & purificación , Cartílago/química , Técnicas de Cultivo de Célula/instrumentación , Cromatografía de Afinidad/instrumentación , Colágeno/metabolismo , Inmunización/métodos , Proteínas Matrilinas/análisis , Proteínas Matrilinas/química , Proteínas Matrilinas/fisiología , Mecanotransducción Celular , Dominios Proteicos/fisiología , Proteoglicanos/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Regeneración
11.
PLoS One ; 13(1): e0191224, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29351342

RESUMEN

Congenital anomalies of the kidney and urinary tract (CAKUT) are the most common cause (40-50%) of chronic kidney disease (CKD) in children. About 40 monogenic causes of CAKUT have so far been discovered. To date less than 20% of CAKUT cases can be explained by mutations in these 40 genes. To identify additional monogenic causes of CAKUT, we performed whole exome sequencing (WES) and homozygosity mapping (HM) in a patient with CAKUT from Indian origin and consanguineous descent. We identified a homozygous missense mutation (c.1336C>T, p.Arg446Cys) in the gene Von Willebrand factor A domain containing 2 (VWA2). With immunohistochemistry studies on kidneys of newborn (P1) mice, we show that Vwa2 and Fraser extracellular matrix complex subunit 1 (Fras1) co-localize in the nephrogenic zone of the renal cortex. We identified a pronounced expression of Vwa2 in the basement membrane of the ureteric bud (UB) and derivatives of the metanephric mesenchyme (MM). By applying in vitro assays, we demonstrate that the Arg446Cys mutation decreases translocation of monomeric VWA2 protein and increases translocation of aggregated VWA2 protein into the extracellular space. This is potentially due to the additional, unpaired cysteine residue in the mutated protein that is used for intermolecular disulfide bond formation. VWA2 is a known, direct interactor of FRAS1 of the Fraser-Complex (FC). FC-encoding genes and interacting proteins have previously been implicated in the pathogenesis of syndromic and/or isolated CAKUT phenotypes in humans. VWA2 therefore constitutes a very strong candidate in the search for novel CAKUT-causing genes. Our results from in vitro experiments indicate a dose-dependent neomorphic effect of the Arg446Cys homozygous mutation in VWA2.


Asunto(s)
Biomarcadores de Tumor/genética , Síndrome de Fraser/genética , Mutación Missense , Anomalías Urogenitales/genética , Reflujo Vesicoureteral/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Biomarcadores de Tumor/química , Niño , Consanguinidad , Secuencia Conservada , Exones , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Humanos , Masculino , Ratones , Modelos Animales , Modelos Moleculares , Linaje , Homología de Secuencia de Aminoácido , Sistema Urogenital/crecimiento & desarrollo , Sistema Urogenital/metabolismo
12.
J Biol Chem ; 293(1): 203-214, 2018 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-29146595

RESUMEN

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.


Asunto(s)
Lectinas Tipo C/metabolismo , Activadores Plasminogénicos/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Secuencia de Aminoácidos , Animales , Cartílago/metabolismo , Cromatografía en Gel , Células HEK293 , Humanos , Inmunohistoquímica , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/genética , Ratones , Ratones Endogámicos C57BL , Plasminógeno/metabolismo , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética
13.
Hum Mol Genet ; 26(15): 2897-2911, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28475764

RESUMEN

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.


Asunto(s)
Osteogénesis Imperfecta/genética , Fenilbutiratos/metabolismo , Animales , Calcificación Fisiológica , Células Cultivadas , Colágeno/genética , Colágeno Tipo I/genética , Fibroblastos , Modelos Animales , Chaperonas Moleculares/metabolismo , Mutación , Osteoblastos , Osteogénesis Imperfecta/metabolismo , Fenilbutiratos/uso terapéutico , Pliegue de Proteína , Ácido Tauroquenodesoxicólico/metabolismo , Pez Cebra/genética
14.
J Exp Med ; 213(10): 1961-71, 2016 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-27573814

RESUMEN

During homeostasis, hematopoietic stem cells (HSCs) are mostly kept in quiescence with only minor contribution to steady-state hematopoiesis. However, in stress situations such as infection, chemotherapy, or transplantation, HSCs are forced to proliferate and rapidly regenerate compromised hematopoietic cells. Little is known about the processes regulating this stress-induced proliferation and expansion of HSCs and progenitors. In this study, we identified the extracellular matrix (ECM) adaptor protein Matrilin-4 (Matn4) as an important negative regulator of the HSC stress response. Matn4 is highly expressed in long-term HSCs; however, it is not required for HSC maintenance under homeostasis. In contrast, Matn4 is strongly down-regulated in HSCs in response to proliferative stress, and Matn4 deficiency results in increased proliferation and expansion of HSCs and progenitors after myelosuppressive chemotherapy, inflammatory stress, and transplantation. This enhanced proliferation is mediated by a transient down-regulation of CXCR4 in Matn4(-/-) HSCs upon stress, allowing for a more efficient expansion of HSCs. Thus, we have uncovered a novel link between the ECM protein Matn4 and cytokine receptor CXCR4 involved in the regulation of HSC proliferation and expansion under acute stress.


Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Receptores CXCR4/metabolismo , Estrés Fisiológico , Animales , Trasplante de Médula Ósea , Proliferación Celular , Regulación hacia Abajo , Femenino , Hematopoyesis , Proteínas Matrilinas/metabolismo , Ratones Endogámicos C57BL
15.
Matrix Biol ; 55: 90-105, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26944560

RESUMEN

In response to injury, tendon fibroblasts are activated, migrate to the wound, and contribute to tissue repair by producing and organizing the extracellular matrix. Collagen VI is a microfibrillar collagen enriched in the pericellular matrix of tendon fibroblasts with a potential regulatory role in tendon repair mechanism. We investigated the molecular basis of the interaction between collagen VI and the cell membrane both in tissue sections and fibroblast cultures of human tendon, and analyzed the deposition of collagen VI during migration and myofibroblast trans-differentiation, two crucial events for tendon repair. Tendon fibroblast displayed a collagen VI microfibrillar network closely associated with the cell surface. Binding of collagen VI with the cell membrane was mediated by NG2 proteoglycan, as demonstrated by in vitro perturbation of collagen VI-NG2 interaction with a NG2-blocking antibody. Cultures subjected to wound healing scratch assay displayed collagen VI-NG2 complexes at the trailing edge of migrating cells, suggesting a potential role in cell migration. In fact, the addition of a NG2-blocking antibody led to an impairment of cell polarization and delay of wound closure. Similar results were obtained after in vitro perturbation of collagen VI extracellular assembly with the 3C4 anti-collagen VI antibody and in collagen VI-deficient tendon cultures of a Ullrich congenital muscular dystrophy patient carrying mutations in COL6A2 gene. Moreover, in vitro treatment with transforming growth factor ß1 (TGFß1) induced a dramatic reduction of NG2 expression, both at protein and mRNA transcript level, and the impairment of collagen VI association with the cell membrane. Instead, collagen VI was still detectable in the extracellular matrix in association with ED-A fibronectin and collagen I, which were strongly induced by TGFß1 treatment. Our findings reveal a critical role of the NG2 proteoglycan for the binding of collagen VI to the surface of tendon fibroblasts. By interacting with NG2 proteoglycan and other extracellular matrix proteins, collagen VI regulates fibroblasts behavior and the assembly of tendon matrix, thereby playing a crucial role in tendon repair.


Asunto(s)
Antígenos/metabolismo , Colágeno Tipo VI/fisiología , Fibroblastos/metabolismo , Proteoglicanos/metabolismo , Adolescente , Movimiento Celular , Transdiferenciación Celular , Células Cultivadas , Humanos , Persona de Mediana Edad , Unión Proteica , Transporte de Proteínas , Tendones/citología , Factor de Crecimiento Transformador beta1/fisiología , Adulto Joven
16.
J Invest Dermatol ; 136(6): 1150-1160, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26945878

RESUMEN

Elastin microfibril interface-located proteins (EMILINs) 1 and 2 belong to a family of structurally related extracellular glycoproteins with unique functions in the extracellular space, such as modulation of pro-transforming growth factor-ß processing, activation of the extrinsic apoptotic pathway, and regulation of Hedgehog and Wnt ligand bioavailability. However, little is known about how EMILINs may exert their extracellular functions. We therefore investigated the spatiotemporal localization and deposition of EMILIN-1 and -2 within the extracellular space. By using immunoelectron and immunofluorescence microscopy together with biochemical extraction, we showed that EMILIN-1 and -2 are targeted to fibrillin microfibrils in the skin. In addition, during skin wound healing and in vitro matrix fiber assembly by primary dermal fibroblasts, EMILIN-1 and -2 are deposited on and coregulated with fibrillin. Analysis of wounds and mouse embryonic fibroblast cultures showed that EMILIN-1 and -2 network formation also requires the presence of fibronectin. Disruption of microfibrils in fibrillin-1-deficient mice leads to fragmentation of the EMILIN-1 and -2 networks, suggesting an involvement of EMILINs in fibrillin-related skin disorders. The addition of EMILINs to the ligand repertoire of fibrillin strengthens the concept of fibrillin microfibrils as extracellular scaffolds integrating cellular force transmission and growth factor bioactivity.


Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Heridas y Traumatismos/patología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Fibrilina-1/genética , Fibrilinas/metabolismo , Fibroblastos/citología , Ratones , Ratones Mutantes , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Piel/lesiones , Cicatrización de Heridas/fisiología
17.
J Biol Chem ; 291(10): 5247-58, 2016 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-26742845

RESUMEN

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.


Asunto(s)
Colágeno Tipo IV/química , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Terciaria de Proteína
18.
Matrix Biol ; 49: 106-119, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26235539

RESUMEN

Collagen XXVIII is the last discovered member of the collagen superfamily and thus has been only sparsely investigated. We studied collagen XXVIII in zebrafish to gain insight into its structure, evolution and expression. In contrast to human and mouse, the zebrafish genome contains four collagen XXVIII genes, col28a1a and -b, and col28a2a and -b. Genomic context and phylogenetic analysis revealed that the a2 branch was lost during evolution of mammals, whereas the duplication of the a1 and a2 branches results from the whole genome duplication in the teleost lineage. Sequence analysis revealed conservation of domain structure and the unique imperfections in the triple helical domain. Two major forms of collagen XXVIII were identified, Col28a1b in adult and Col28a2a in 3-5dpf zebrafish. Composite agarose/polyacrylamide gel electrophoresis revealed that both these chains mainly form dimers of trimers, although Col28a1b appears to be more polydisperse. Homodimers are abundant, although it is possible that complexes consisting of Col28a2a and Col28a1a or -a2b occur. Peptide mass fingerprint analysis revealed that the C-terminal Kunitz domain is often proteolytically processed. In contrast to murine collagen XXVIII, the zebrafish orthologs are widely expressed and not only present in the nervous system. They are differentially expressed in the liver, thymus, muscle, intestine and skin. Altogether our results point to a unique nature of collagen XXVIII within the collagen family.


Asunto(s)
Colágeno/química , Colágeno/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/genética , Animales , Colágeno/genética , Evolución Molecular , Duplicación de Gen , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Filogenia , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , Distribución Tisular , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
19.
Immunity ; 43(4): 803-16, 2015 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-26474656

RESUMEN

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-ß induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.


Asunto(s)
Cicatriz/etiología , Colágeno/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Macrófagos/metabolismo , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Cicatrización de Heridas/fisiología , Animales , Cicatriz/metabolismo , Cicatriz/patología , Técnicas de Cocultivo , Dermatitis/metabolismo , Dermatitis/patología , Fibroblastos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Interleucinas/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Microfibrillas/metabolismo , Microfibrillas/ultraestructura , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/biosíntesis , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Receptores de Superficie Celular/deficiencia , Esclerodermia Localizada/metabolismo , Esclerodermia Localizada/patología , Piel/lesiones , Piel/metabolismo , Piel/patología
20.
Protein Expr Purif ; 107: 20-8, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25462806

RESUMEN

VWA domains are the predominant independent folding units within matrilins and mediate protein-protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems.


Asunto(s)
Proteínas Matrilinas/química , Proteínas Matrilinas/genética , Animales , Dicroismo Circular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Matrilinas/aislamiento & purificación , Proteínas Matrilinas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Pez Cebra
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