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Dieter P Reinhardt - One of the best experts on this subject based on the ideXlab platform.
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adamts10 inactivation in mice leads to persistence of ocular microfibrils subsequent to reduced Fibrillin 2 cleavage
Matrix Biology, 2019Co-Authors: Lauren W Wang, Wendy E Kutz, Timothy J Mead, Lauren C Beene, Dieter P Reinhardt, Michael W. Jenkins, Shweta Singh, Sulabha S. ApteAbstract:Abstract Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects Fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance Fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on Fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10−/− mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10−/− mice, similar to Fbn1−/− mice, most likely because the mouse zonule contains Fibrillin-2 in addition to Fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10−/− zonule fibers were thicker and immunostained strongly with Fibrillin-2 antibodies into adulthood, whereas Fibrillin-1 staining was reduced. Furthermore, Fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10−/− eyes. ADAMTS10 was found to cleave Fibrillin-2, providing an explanation for persistence of Fibrillin-2 at these sites. Thus, analysis of Adamts10−/− mice led to identification of Fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular Fibrillin-2 at the end of the juvenile period.
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the Fibrillin 1 rgd integrin binding site regulates gene expression and cell function through micrornas
Journal of Molecular Biology, 2019Co-Authors: Karina A Zeyer, Rongmo Zhang, Amani Hassan, Heena Kumra, Dieter P ReinhardtAbstract:Abstract Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Fibrillin-1 contains one evolutionarily conserved RGD sequence that mediates cell–matrix interactions through cell-surface integrins. Here, we present a novel paradigm how extracellular Fibrillin-1 controls cellular function through integrin-mediated microRNA regulation. Comparative mRNA studies by global microarray analysis identified growth factor activity, actin binding and integrin binding as the most important functional groups that are regulated upon Fibrillin-1 binding to dermal fibroblasts. Many of these mRNAs are targets of miRNAs that were identified when RNA from the Fibrillin-1-ligated fibroblasts was analyzed by a miRNA microarray. The expression profile was specific to Fibrillin-1 since interaction with fibronectin displayed a partially distinct profile. The importance of selected miRNAs for the regulation of the identified mRNAs was suggested by bioinformatics prediction and the interactions between miRNAs and mRNAs were experimentally validated. Functionally, we show that miR-503 controls p-Smad2-dependent TGF-β signaling, and that miR-612 and miR-3185 are involved in the focal adhesion formation regulated by Fibrillin-1. In conclusion, we demonstrate that Fibrillin-1 interaction with fibroblasts regulates miRNA expression profiles which in turn control critical cell functions.
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Fibrillin containing microfibrils are key signal relay stations for cell function
Journal of Cell Communication and Signaling, 2015Co-Authors: Karina A Zeyer, Dieter P ReinhardtAbstract:Fibrillins constitute the backbone of microfibrils in the extracellular matrix of elastic and non-elastic tissues. Mutations in Fibrillins are associated with a wide range of connective tissue disorders, the most common is Marfan syndrome. Microfibrils are on one hand important for structural stability in some tissues. On the other hand, microfibrils are increasingly recognized as critical mediators and drivers of cellular signaling. This review focuses on the signaling mechanisms initiated by Fibrillins and microfibrils, which are often dysregulated in Fibrillin-associated disorders. Fibrillins regulate the storage and bioavailability of growth factors of the TGF-β superfamily. Cells sense microfibrils through integrins and other receptors. Fibrillins potently regulate pathways of the immune response, inflammation and tissue homeostasis. Emerging evidence show the involvement of microRNAs in disorders caused by Fibrillin deficiency. A thorough understanding of Fibrillin-mediated cell signaling pathways will provide important new leads for therapeutic approaches of the underlying disorders.
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engineered mutations in Fibrillin 1 leading to marfan syndrome act at the protein cellular and organismal levels
Mutation Research-reviews in Mutation Research, 2015Co-Authors: Karina A Zeyer, Dieter P ReinhardtAbstract:Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the Fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in Fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant Fibrillin-1 fragments. Those mutations affect Fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant Fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render Fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse Fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated Fibrillin-1.
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Human eye development is characterized by coordinated expression of Fibrillin isoforms.
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Dirk Hubmacher, Katja Schenke-layland, Thomas Plesec, Dieter P Reinhardt, Sulabha S. ApteAbstract:PURPOSE: Mutations in human Fibrillin-1 and -2, which are major constituents of tissue microfibrils, can affect multiple ocular components, including the ciliary zonule, lens, drainage apparatus, cornea, and retina. However, the expression pattern of the three human Fibrillins and an integral microfibrillar component, MAGP1, during human eye development is not known. METHODS: We analyzed sections from human eyes at gestational weeks (GWs) 6, 8, and 11 and at 1 and 3 years of age with antibodies specific for each human Fibrillin isoform or MAGP1, using immunofluorescence microscopy. RESULTS: During embryonic development, each Fibrillin isoform was detected in vascular structures bridging the ciliary body and the developing lens, hyaloid vasculature, and retina. In addition, they were present in the developing corneal basement membranes and lens capsule. MAGP1 codistributed with the Fibrillin isoforms. In contrast, the juvenile zonule was composed of Fibrillin-1 microfibrils containing MAGP1, but Fibrillin-2 was absent and Fibrillin-3 was only sparsely detected. CONCLUSIONS: Fibrillin-1, -2, and, unique to humans, Fibrillin-3 are found in various ocular structures during human embryonic eye development, whereas Fibrillin-1 dominates the postnatal zonule. We speculate that vasculature spanning the ciliary body and lens, which elaborates Fibrillin-2 and -3, may provide an initial scaffold for Fibrillin assembly and zonule formation.
Lynn Y Sakai - One of the best experts on this subject based on the ideXlab platform.
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Fibrillin‐1 in the Vasculature: In Vivo Accumulation of eGFP‐Tagged Fibrillin‐1 in a Knockin Mouse Model
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, 2019Co-Authors: N.l. Charbonneau, Eric J Carlson, Sara F Tufa, Elise C Manalo, Valerie M Carlberg, D.r. Keene, Lynn Y SakaiAbstract:: Immunolocalization studies have shown that Fibrillin-1 is distributed ubiquitously in the connective tissue space from early embryonic times through old age. When mutated, the gene for Fibrillin-1 (FBN1) causes the Marfan syndrome, a common inherited disorder of connective tissue. The multiple manifestations of the Marfan syndrome reflect the known distribution of Fibrillin-1 in cardiovascular, musculoskeletal, ocular, and dermal tissues. In this study, a mouse model of Marfan syndrome in which Fibrillin-1 is truncated and tagged with green fluorescence was used to estimate the relative abundance of Fibrillin-1 in developing tissues. In embryonic tissues, the aorta was the only tissue in which Fibrillin-1 green fluorescence was detectable. Other arteries gained detectable Fibrillin-1 green fluorescence just after birth. Fibrillin-1 fluorescence was observed at later postnatal times in the lung, skin, perichondrium, tendon, and ocular tissues, while other tissues remained negative. These results indicated that tissues most affected in the Marfan syndrome are the tissues in which Fibrillin-1 is most abundant. Focus was placed on the aorta, since aortic disease is life threatening in the Marfan syndrome and Fibrillin-1 green fluorescence was most abundant in this tissue. Fibrillin-1 green fluorescence and immunostaining showed that Fibrillin-1 is within aortic medial elastic lamellae. Endothelial-specific compared to smooth muscle-specific Fibrillin-1 green fluorescence, together with light microscopic analyses of fragmentation of aortic elastic lamellae, demonstrated that smooth muscle cell mutated Fibrillin-1 contributed most to progressive aortic fragmentation. However, these studies also indicated that other cells, possibly endothelial cells, also contribute to this aortic pathology. Anat Rec, 2019. © 2019 Wiley Periodicals, Inc.
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Fibrillin protein pleiotropy: Acromelic dysplasias
Matrix biology : journal of the International Society for Matrix Biology, 2018Co-Authors: Lynn Y Sakai, Douglas R KeeneAbstract:The Fibrillins are large extracellular matrix molecules that polymerize to form microfibrils. Fibrillin microfibrils are distinctive architectural elements that are both ubiquitous in the connective tissue space and also unique, displaying tissue-specific patterns. Mutations in the genes for Fibrillin-1 (FBN1) result in multiple distinct pleiotropic disorders. Most of the more than 3000 mutations known today in FBN1 cause the Marfan syndrome. Marfan mutations can occur in any of the 56 domains that compose Fibrillin-1. In contrast, rare mutations in FBN1 that are confined to only certain domains cause several different types of acromelic dysplasia. These genetic disorders demonstrate that specific domains of Fibrillin-1 perform roles important to musculoskeletal growth. Many of the phenotypes of acromelic dysplasias are the opposite of those found in Marfan syndrome. Knowledge of the functions and structural organization of Fibrillin molecules within microfibrils is required to understand how one protein and one gene can be the basis for multiple genetic disorders.
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Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice
PLOS Genetics, 2015Co-Authors: Gerhard Sengle, Eric J Carlson, Sara F Tufa, Valerie M Carlberg, D.r. Keene, N.l. Charbonneau, Silvia Smaldone, Francesco Ramirez, Lynn Y SakaiAbstract:Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for Fibrillin-1 cause the Marfan syndrome, while mutations in the gene for Fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of Fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the Fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that Fibrillin-2 can sequester BMP complexes in a latent state.
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The Fibrillin microfibril scaffold: A niche for growth factors and mechanosensation?
Matrix biology : journal of the International Society for Matrix Biology, 2015Co-Authors: Gerhard Sengle, Lynn Y SakaiAbstract:The Fibrillins, large extracellular matrix molecules, are polymerized to form "microfibrils." The Fibrillin microfibril scaffold is populated by microfibril-associated proteins and by growth factors, which are likely to be latent. The scaffold, associated proteins, and bound growth factors, together with cellular receptors that can sense the microfibril matrix, constitute the Fibrillin microenvironment. Activation of TGFβ signaling is associated with the Marfan syndrome, which is caused by mutations in Fibrillin-1. Today we know that mutations in Fibrillin-1 cause the Marfan syndrome as well as Weill-Marchesani syndrome (and other acromelic dysplasias) and result in opposite clinical phenotypes: tall or short stature; arachnodactyly or brachydactyly; joint hypermobility or stiff joints; hypomuscularity or hypermuscularity. We also know that these different syndromes are associated with different structural abnormalities in the Fibrillin microfibril scaffold and perhaps with specific cellular receptors (mechanosensors). How does the microenvironment, framed by the microfibril scaffold and populated by latent growth factors, work? We must await future investigations for the molecular and cellular mechanisms that will answer this question. However, today we can appreciate the importance of the Fibrillin microfibril niche as a contextual environment for growth factor signaling and potentially for mechanosensation.
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Early Fibrillin-1 assembly monitored through a modifiable recombinant cell approach.
Biomacromolecules, 2014Co-Authors: Dirk Hubmacher, Christine Fagotto-kaufmann, Lynn Y Sakai, Eric Bergeron, Dieter P ReinhardtAbstract:: Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in Fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant Fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant Fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant Fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant Fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect Fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length Fibrillin-1 assembly system that allows for rapid analysis of critical roles in Fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of Fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.
Francesco Ramirez - One of the best experts on this subject based on the ideXlab platform.
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Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice
PLOS Genetics, 2015Co-Authors: Gerhard Sengle, Eric J Carlson, Sara F Tufa, Valerie M Carlberg, D.r. Keene, N.l. Charbonneau, Silvia Smaldone, Francesco Ramirez, Lynn Y SakaiAbstract:Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for Fibrillin-1 cause the Marfan syndrome, while mutations in the gene for Fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of Fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the Fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that Fibrillin-2 can sequester BMP complexes in a latent state.
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Establishment of Fibrillin-deficient osteoprogenitor cell lines identifies molecular abnormalities associated with extracellular matrix perturbation of osteogenic differentiation
Cell and Tissue Research, 2011Co-Authors: Silvia Smaldone, Luca Carta, Francesco RamirezAbstract:Fibrillin-1 and Fibrillin-2 are structural components of the extracellular matrix which are also involved in modulating local TGFβ and BMP bioavailability. Loss of Fibrillin-1 or Fibrillin-2 is associated with perturbed osteoblast maturation principally as the result of unbalanced TGFβ and BMP signaling. Here, we demonstrated that stable expression of small hairpin RNAs against Fibrillin-1(Fbn1) or Fibrillin-2 (Fbn2) transcripts in the clonal osteoprogenitor cell line Kusa-A1 led to the same phenotypic and molecular manifestations as germline Fbn1- or Fbn2-null mutations in primary calvarial osteoblast cultures. Proof-of-concept experiments are also presented showing that Fbn1- or Fbn2-silenced Kusa-A1 cell lines are suitable models to identify candidate determinants of osteogenesis which are under the control of extracellular microfibrils. Specific findings included: the inference of a potential role for Fibrillin-1-mediated cell–matrix interactions in regulating Kusa-A1 proliferation; the possibility of Fibrillin-2 involvement in modulating the activity of transcription factor Runx2 by restricting microRNA expression and/or processing; and the suggestion that Fibrillin-1 and Fibrillin-2 influence Notch signaling indirectly by differentially regulating BMP signaling. Collectively, the data reiterated the notion that Fibrillin-1 and Fibrillin-2 exert opposite effects on osteoblast differentiation through the discrete modulation of a broad network of interacting signaling molecules.
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Material and mechanical properties of bones deficient for Fibrillin-1 or Fibrillin-2 microfibrils.
Matrix Biology, 2011Co-Authors: E. Arteaga-solis, Lee Sui-arteaga, Karl J. Jepsen, Nancy Pleshko, Mitchell B Schaffler, Francesco RamirezAbstract:The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of Fibrillin-1 or Fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in Fibrillin-1 or Fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that Fibrillin-1 plays a predominantly greater role than Fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that Fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than Fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by Fibrillin proteins. Our study therefore advance our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in Fibrillin-1 or -2.
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Fibrillin assemblies: extracellular determinants of tissue formation and fibrosis
Fibrogenesis & tissue repair, 2010Co-Authors: Jacopo Olivieri, Silvia Smaldone, Francesco RamirezAbstract:The extracellular matrix (ECM) plays a key role in tissue formation, homeostasis and repair, mutations in ECM components have catastrophic consequences for organ function and therefore, for the fitness and survival of the organism. Collagen, Fibrillin and elastin polymers represent the architectural scaffolds that impart specific mechanic properties to tissues and organs. Fibrillin assemblies (microfibrils) have the additional function of distributing, concentrating and modulating local transforming growth factor (TGF)-β and bone morphogenetic protein (BMP) signals that regulate a plethora of cellular activities, including ECM formation and remodeling. Fibrillins also contain binding sites for integrin receptors, which induce adaptive responses to changes in the extracellular microenvironment by reorganizing the cytoskeleton, controlling gene expression, and releasing and activating matrix-bound latent TGF-β complexes. Genetic evidence has indicated that Fibrillin-1 and Fibrillin-2 contribute differently to the organization and structural properties of non-collagenous architectural scaffolds, which in turn translate into discrete regulatory outcomes of locally released TGF-β and BMP signals. Additionally, the study of congenital dysfunctions of Fibrillin-1 has yielded insights into the pathogenesis of acquired connective tissue disorders of the connective tissue, such as scleroderma. On the one hand, mutations that affect the structure or expression of Fibrillin-1 perturb microfibril biogenesis, stimulate improper latent TGF-β activation, and give rise to the pleiotropic manifestations in Marfan syndrome (MFS). On the other hand, mutations located around the integrin-binding site of Fibrillin-1 perturb cell matrix interactions, architectural matrix assembly and extracellular distribution of latent TGF-β complexes, and lead to the highly restricted fibrotic phenotype of Stiff Skin syndrome. Understanding the molecular similarities and differences between congenital and acquired forms of skin fibrosis may therefore provide new therapeutic tools to mitigate or even prevent disease progression in scleroderma and perhaps other fibrotic conditions.
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extracellular microfibrils control osteoblast supported osteoclastogenesis by restricting tgfβ stimulation of rankl production
Journal of Biological Chemistry, 2010Co-Authors: Harikiran Nistala, Sui Leearteaga, Silvia Smaldone, Gabriella Siciliano, Francesco RamirezAbstract:Mutations in Fibrillin-1 or Fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that Fibrillin-1 and Fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGFβ and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the Fibrillins as negative regulators of bone resorption. Adult Fbn2−/− mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Although isolated cultures of Fbn2−/− preosteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type preosteoclasts were co-cultured with Fbn2−/− but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2−/− osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGFβ activity. This conclusion was based on the findings that blockade of TGFβ signaling blunts Rankl up-regulation in Fbn2−/− osteoblasts and bones and that systemic TGFβ antagonism improves locally induced osteolysis in Fbn2−/− mice. Abnormally high Rankl expression secondary to elevated TGFβ activity was also noted in cultured osteoblasts from Fbn1−/− mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.
Dirk Hubmacher - One of the best experts on this subject based on the ideXlab platform.
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Human eye development is characterized by coordinated expression of Fibrillin isoforms.
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Dirk Hubmacher, Katja Schenke-layland, Thomas Plesec, Dieter P Reinhardt, Sulabha S. ApteAbstract:PURPOSE: Mutations in human Fibrillin-1 and -2, which are major constituents of tissue microfibrils, can affect multiple ocular components, including the ciliary zonule, lens, drainage apparatus, cornea, and retina. However, the expression pattern of the three human Fibrillins and an integral microfibrillar component, MAGP1, during human eye development is not known. METHODS: We analyzed sections from human eyes at gestational weeks (GWs) 6, 8, and 11 and at 1 and 3 years of age with antibodies specific for each human Fibrillin isoform or MAGP1, using immunofluorescence microscopy. RESULTS: During embryonic development, each Fibrillin isoform was detected in vascular structures bridging the ciliary body and the developing lens, hyaloid vasculature, and retina. In addition, they were present in the developing corneal basement membranes and lens capsule. MAGP1 codistributed with the Fibrillin isoforms. In contrast, the juvenile zonule was composed of Fibrillin-1 microfibrils containing MAGP1, but Fibrillin-2 was absent and Fibrillin-3 was only sparsely detected. CONCLUSIONS: Fibrillin-1, -2, and, unique to humans, Fibrillin-3 are found in various ocular structures during human embryonic eye development, whereas Fibrillin-1 dominates the postnatal zonule. We speculate that vasculature spanning the ciliary body and lens, which elaborates Fibrillin-2 and -3, may provide an initial scaffold for Fibrillin assembly and zonule formation.
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heparin heparan sulfate controls Fibrillin 1 2 and 3 self interactions in microfibril assembly
FEBS Letters, 2014Co-Authors: Laetitia Sabatier, Dirk Hubmacher, Jelena Djokic, Dzaner Dzafik, Valentin Nelea, Dieter P ReinhardtAbstract:Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in Fibrillin assembly can result in Fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in Fibrillin-2 and -3. Multimerization of all three Fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal Fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.
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Heparin/heparan sulfate controls Fibrillin-1, -2 and -3 self-interactions in microfibril assembly
FEBS Letters, 2014Co-Authors: Laetitia Sabatier, Dirk Hubmacher, Jelena Djokic, Dzaner Dzafik, Valentin Nelea, Dieter P ReinhardtAbstract:Fibrillins form multifunctional microfibrils in most connective tissues. Deficiencies in Fibrillin assembly can result in Fibrillinopathies, such as Marfan syndrome. We demonstrate the presence of heparin/heparan sulfate binding sites in Fibrillin-2 and -3. Multimerization of all three Fibrillins drastically increased the apparent affinity of their interaction with heparin/heparan sulfate. Surprisingly, contrary to other reports heparin/heparan sulfate strongly inhibited homo- and heterotypic N-to-C-terminal Fibrillin interactions. These data suggest that heparin/heparan sulfate controls the formation of microfibrils at the bead interaction stage.
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Early Fibrillin-1 assembly monitored through a modifiable recombinant cell approach.
Biomacromolecules, 2014Co-Authors: Dirk Hubmacher, Christine Fagotto-kaufmann, Lynn Y Sakai, Eric Bergeron, Dieter P ReinhardtAbstract:: Fibrillin proteins constitute the backbone of extra-cellular macromolecular microfibrils. Mutations in Fibrillins cause heritable connective tissue disorders, including Marfan syndrome, dominant Weill-Marchesani syndrome, and stiff skin syndrome. Fibronectin provides a critical scaffold for microfibril assembly in cell culture models. Full length recombinant Fibrillin-1 was expressed by HEK 293 cells, which deposited the secreted protein in a punctate pattern on the cell surface. Cocultured fibroblasts consistently triggered assembly of recombinant Fibrillin-1, which was dependent on a fibronectin network formed by the fibroblasts. Deposition of recombinant Fibrillin-1 on fibronectin fibers occurred first in discrete packages that subsequently extended along fibronectin fibers. Mutant Fibrillin-1 harboring either a cysteine 204 to serine mutation or a RGD to RGA mutation which prevents integrin binding, did not affect Fibrillin-1 assembly. In conclusion, we developed a modifiable recombinant full-length Fibrillin-1 assembly system that allows for rapid analysis of critical roles in Fibrillin assembly and functionality. This system can be used to study the contributions of specific residues, domains, or regions of Fibrillin-1 to the biogenesis and functionality of microfibrils. It provides also a method to evaluate disease-causing mutations, and to produce microfibril-containing matrices for tissue engineering applications, for example, in designing novel vascular grafts or stents.
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nonselective assembly of Fibrillin 1 and Fibrillin 2 in the rodent ocular zonule and in cultured cells implications for marfan syndrome
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Lauren C Beene, Lauren W Wang, Dirk Hubmacher, Deane F. Mosher, Robert P Mecham, Dieter P Reinhardt, Douglas S Annis, Douglas R Keene, Elias I Traboulsi, Suneel S ApteAbstract:Purpose. Fibrillins are the major constituent of tissue microfibrils, which form the ocular zonule. In Marfan syndrome (MFS), FBN1 mutations lead to ectopia lentis. The goal of this work was to investigate zonule composition and formation in Fibrillin-deficient and wild-type mice.
Robert P Mecham - One of the best experts on this subject based on the ideXlab platform.
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identification of the growth factor binding sequence in the extracellular matrix protein magp 1
Journal of Biological Chemistry, 2020Co-Authors: Thomas J Broekelmann, Nicholas K Bodmer, Robert P MechamAbstract:Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the Fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor-binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1-TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid-long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to Fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind Fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.
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nonselective assembly of Fibrillin 1 and Fibrillin 2 in the rodent ocular zonule and in cultured cells implications for marfan syndrome
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Lauren C Beene, Lauren W Wang, Dirk Hubmacher, Deane F. Mosher, Robert P Mecham, Dieter P Reinhardt, Douglas S Annis, Douglas R Keene, Elias I Traboulsi, Suneel S ApteAbstract:Purpose. Fibrillins are the major constituent of tissue microfibrils, which form the ocular zonule. In Marfan syndrome (MFS), FBN1 mutations lead to ectopia lentis. The goal of this work was to investigate zonule composition and formation in Fibrillin-deficient and wild-type mice.
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interaction of tropoelastin with the amino terminal domains of Fibrillin 1 and Fibrillin 2 suggests a role for the Fibrillins in elastic fiber assembly
Journal of Biological Chemistry, 2000Co-Authors: Timothy M Trask, Timothy M Ritty, Barbara Crippes Trask, Joel Rosenbloom, William R Abrams, Robert P MechamAbstract:Alignment of tropoelastin molecules during the process of elastogenesis is thought to require Fibrillin-containing microfibrils. In this study, we have demonstrated that amino-terminal domains of two microfibrillar proteins, Fibrillin-1 and Fibrillin-2, interact with tropoelastin in solid phase binding assays. The tropoelastin-binding site was localized to a region beginning at the glycine-rich and proline-rich regions of Fibrillin-2 and Fibrillin-1, respectively, and continuing through the second 8-cysteine domain. Characterization of the binding requirements using the Fibrillin-2 construct found that a folded, secondary structure was necessary for binding. Furthermore, binding between tropoelastin and Fibrillin was mediated by ionic interactions involving the lysine side chains of tropoelastin. The importance of the lysine side chains was corroborated by the finding that the Fibrillin-2 construct did not bind to mature elastin, whose lysine side chains have been modified to form cross-links. Interestingly, there was no interaction between the Fibrillin constructs and tropoelastin in solution phase, suggesting that binding of tropoelastin to a solid substrate exposes a cryptic binding site. These results suggest that Fibrillin plays an important role in elastic fiber assembly by binding tropoelastin and perhaps facilitating side chain alignment for efficient cross-linking.
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n terminal domains of Fibrillin 1 and Fibrillin 2 direct the formation of homodimers a possible first step in microfibril assembly
Biochemical Journal, 1999Co-Authors: Timothy M Trask, Timothy M Ritty, Clarina Tisdale, Thomas J. Broekelmann, Robert P MechamAbstract:Aggregation of Fibrillin molecules via disulphide bonds is postulated to be an early step in microfibril assembly. By expressing fragments of Fibrillin 1 and Fibrillin 2 in a mammalian expression system, we found that the N-terminal region of each protein directs the formation of homodimers and that disulphide bonds stabilize this interaction. A large fragment of Fibrillin 1 containing much of the region downstream from the N-terminus remained as a monomer when expressed in the same cell system, indicating that this region of the protein lacks dimerization domains. This finding also confirms that the overexpression of Fibrillin fragments does not in itself lead to spurious dimer formation. Pulse-chase analysis demonstrated that dimer formation occurred intracellularly, suggesting that the process of Fibrillin aggregation is initiated early after biosynthesis of the molecules. These findings also implicate the N-terminal region of Fibrillin 1 and Fibrillin 2 in directing the formation of a dimer intermediate that aggregates to form the functional microfibril.
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cell type specific recognition of rgd and non rgd containing cell binding domains in Fibrillin 1
Journal of Biological Chemistry, 1996Co-Authors: Hiroshi Sakamoto, Joel Rosenbloom, David A Cheresh, Thomas J. Broekelmann, Francesco Ramirez, Robert P MechamAbstract:Abstract The Fibrillins are large glycoprotein components of 10-nm microfibrils found in the extracellular matrix of most tissues. Microfibrils play a role in elastic fiber assembly and serve to link cells to elastic fibers in the extracellular matrix. To determine whether Fibrillin-1 specifically interacts with receptors on cells from Fibrillin-rich tissues, we evaluated whether two cell types that produce different types of Fibrillin can adhere to purified Fibrillin-1 in cell adhesion assays. Our results indicate that both cell types attach and spread on Fibrillin-1 and that the RGD sequence in the fourth 8-cysteine motif mediates this interaction. Fibroblast attachment to Fibrillin-1 was sensitive to inhibition by antibodies to the αvβ3 receptor and by peptides encoding the RGD sequence in Fibrillin-1 and the second RGD sequence in Fibrillin-2. In contrast, adhesion of auricular chondroblasts to Fibrillin-1 was only partially inhibited by these reagents, suggesting that some cell types recognize a second, non-RGD binding site within the Fibrillin molecule. These findings confirm and extend ultrastructural studies that suggest a direct interaction between microfibrils and the cell surface and provide a functional explanation for how this association occurs.