HEPARAN SULFATE BIOSYNTHETIC FEEDBACKS AND EXTRACELLULAR SULFATASE EXPRESSION
Ai Xingbin, Ph.d.
Boston University Medical Campuscity: Boston country: United States (us)
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Sulfs are a family of extracellular endosulfatases that modify heparan sulfate (HS) chains at cell surfaces and in extracellular matrices. They release sulfate groups at the 6-O-position of a subset of glucosamine residues in HS chains. The activity of Sulf enzymes serves to potentiate activities of growth factors including GDNF, BMP, Shh, and Wnt and to reduce activities of FGF2, HP-EGF, HGF, and TGF-¿. As a result of these activities and depending on the context, Sulfs have been observed to serve as oncogenic effectors and as tumor suppressors. Sulf activities appear to induce changes in expression of biosynthetic enzymes, resulting in changes in expressed HS structure that do not necessary reflect the direct Sulf enzymatic activity. As a result, the influences of Sulf activity on cellular phenotype are complex. The goals of this work are to (1) define the HS structural phenotype in mouse embryonic fibroblast cells as a function of Sulf enzyme knockout at both the HS disaccharide and oligosaccharide levels. These results will be compared against those obtained using recombinant Sulf enzymes on purified HS
Keywords: Cell surface; Cells; Complex; Computer Retrieval of Information on Scientific Projects Database; Disaccharides; Embryo; Enzymes; extracellular; Extracellular Matrix; Family; FGF2 gene; Fibroblasts; Funding; GDNF gene; Glucosamine; Goals; Grant; Growth Factor; Heparitin Sulfate; Inorganic Sulfates; Institution; Knock-out; Mus; Oligosaccharides; Oncogenic; Phenotype; Positioning Attribute; Recombinants; Research; Research Personnel; Resources; Source; Structure; Sulfatases; Tumor Suppressor Proteins; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Work
Project start date: 2010-06-01
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
5P41RR010888-14_6860 (2010): $14845
Sponsored Links Excellgen http://Excellgen.com
POST-BIOSYNTHETIC MODIFICATION OF HEPARAN SULFATE BY REACTIVE NITROGEN SPECIES
Ai Xingbin, Ph.d.
Boston University Medical Campuscity: Boston country: United States (us)
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Heparan sulfate (HS) chains on cell surfaces and in extracellular matrices are acted upon by enzymes and chemical species to alter their biological activities. Reactive nitrogen species (RNS) give rise to conditions for deaminative cleavage of HS chains in vascular tissue. It is likely that aldehyde-containing HS chains so liberated become reactive toward cellular proteins. Structural investigations to elucidate these mechanisms are the subject of this project. Our results using a mass spectrometry glycomics platform show the presence of a disaccharide of structure (¿HexA-GlcNH26S). These results are consistent with the presence of HS repeats corresponding to HexA-GlcNH26S that are not detectable using other methods. It is likely that disaccharides with free amino groups are difficult to detect using standard disaccharide analysis methods because its zwitterionic nature causes poor retention on ion exchange resins or weak pairing with ion-pairing agents. The presence of disaccharide units containing free glucosamine amino groups in mature HS chains seems to be related to the mechanisms of action of N-deacetylase/N-sulfotransferase enzymes and the availability of 3´-phosphoadenosine-5´-phosphosulfate. Our work shows that disaccharide units containing HexA-GlcNH26S are widely expressed in organ tissue. HS chains containing such disaccharide units would be susceptible to NO-mediated cleavage as has been described for glypican-1 cycling. Conditions for NO-mediated cleavage may exist in other biological processes. Glucosamine amino groups of HS are also known to react rapidly with oxidizing agents such as HOCl under conditions that are likely to be present at inflammation. It is clear that the HexA-GlcNH26S repeat is a widely expressed and functionally relevant HS sub-structure
Keywords: Aldehydes; amino group; Biological; Biological Process; Blood Vessels; Cell surface; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Deacetylase; Disaccharides; Enzymes; Extracellular Matrix; Funding; Glucosamine; Glypican; Grant; Heparitin Sulfate; Inflammation; Institution; Investigation; Ion Exchange Resins; Ions; Mass Spectrum Analysis; Mediating; Methods; Modification; Nature; Organ; Oxidants; Proteins; Reactive Nitrogen Species; Research; Research Personnel; Resources; Source; Structure; sulfotransferase; Tissues; United States National Institutes of Health; Work
Project start date: 2010-06-01
Project end date: 2011-05-31
Budget start date: 1-JUN-2010
Budget end date: 31-MAY-2011
5P41RR010888-14_6819 (2010): $24277
Grants awarded to Ai Xingbin
IDENTIFYING THE MOLECULAR PHENOTYPE OF NORMAL AND ASTHMATIC BRONCHIAL SMOOTH MUSC
Ai Xingbin
Boston University Medical Campuscity: Boston country: United States (us)
Grant 1R21HL112619-01 from National Heart, Lung, And Blood Institute
Abstract: Asthma is a common disease with several effective treatments, including inhaled steroids and ¿-agonists. Despite this, a small subpopulation of patients has a severe unrelenting course associated with airway remodeling. A central feature of airway remodeling is alteration in bronchial smooth muscle (BSM) phenotype, which is classically characterized by expansion of cell number and size, and increased hyper- reactivity to specific and non-specific agonists. While fundamental to asthma pathogenesis and its clinical manifestations, a lack of knowledge regarding the basis for a deranged BSM phenotype is an ongoing, unresolved, issue in the field. This is manifested by the paucity of information regarding the molecular signals underlying bronchial hyper-reactivity and by the lack of treatments directed specifically at reversing the asthmatic BSM phenotype. One major contributing factor to this state-of-affairs is the lack of tools/methodologies that support the high fidelity isolation of pure BSM cells from asthmatic lungs for analysis. To overcome this, we developed a unique transgenic mouse in which BSM singly express a green fluorescent protein (hrGFP) whereas vascular smooth muscle express green (hrGFP) and red fluorescent proteins (dsRed); thereby providing for the first time a reliable methodology for separating each of these two smooth muscle cell populations from one another, and from other lung cells using flow cytometry. Using this unique mouse, our plan is to perform comprehensive mRNA and miRNA profiling of BSM RNA to test the following broad based hypothesis 1) BSM express a distinct genetic signature and 2) alterations in this signature mediate asthmatic BSM phenotypes. Our plan is to use the profiling data to generate lists of complete and differentially expressed mRNAs and miRNAs in normal and asthmatic BSM. Relationships between deregulated miRNAs, mRNA expression, and the identity of active signaling pathways in asthmatic BSM will be examined by bioinformatic and functional studies. At the end of this work, we will have initiated a process to fill a marked knowledge void in the asthma field and will have established a foundation for a variety of future studies. Asthma involves changes in the function and properties of the muscle that surrounds the bronchial tube. While central to the many of the symptoms associated with asthma including wheezing and shortness of breath, the molecular signals that cause changes in bronchial muscle are poorly understood. The objective of this proposal is to use several unique tools and methodologies that we developed to identify key molecular signals that underlay the change in bronchial smooth muscle in asthma
Keywords: Address; Agonist; airway remodeling; Asthma; base; Bioinformatics; Biological Assay; Breathing; Cell Count; Cell Size; Cells; Chronic Airflow Obstruction; Clinical; Computer Simulation; Computer software; Data; Disease; DsRed; effective therapy; Flow Cytometry; Foundations; Functional RNA; Future; Gene Expression; Genes; Genetic; Goals; Green Fluorescent Proteins; In Situ Hybridization; Individual; Knowledge; Logic; Luciferases; Lung; Mediating; meetings; Messenger RNA; Methodology; MicroRNAs; Molecular; molecular phenotype; mRNA Expression; Mus; Muscle; Muscle, Smooth, Vascular; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Pattern; Phenotype; Physiological; Population; Process; Property; Protein Family; red fluorescent protein; Relative (related person); Rest; Reverse Transcriptase Polymerase Chain Reaction; RNA; Role; Shortness of Breath; Signal Pathway; Signal Transduction; Smooth muscle (tissue); Smooth Muscle Myocytes; Specificity; Steroids; Students; Symptoms; Testing; therapeutic target; Time; tool; Transfection; Transgenic Mice; Translational Repression; Tube; Wheezing; Work
Relevance: Asthma involves changes in the function and properties of the muscle that surrounds the bronchial tube. While central to the many of the symptoms associated with asthma including wheezing and shortness of breath, the molecular signals that cause changes in bronchial muscle are poorly understood. The objective of this proposal is to use several unique tools and methodologies that we developed to identify key molecular signals that underlay the change in bronchial smooth muscle in asthma
Project start date: 2012-01-01
Project end date: 2013-12-31
Budget start date: 1-JAN-2012
Budget end date: 31-DEC-2012
1R21HL112619-01 (2012): $245400
IDENTIFICATION OF SULFS AS THERAPEUTIC TARGETS FOR THE TREATMENT OF AGE-IMPAIRED
Ai Xingbin, Ph.d.
Boston University Medical Campuscity: Boston country: United States (us)
Grant 5R01AG034939-02 from National Institute On Aging
Abstract: Aging is inevitably associated with diminished regeneration capacity of stem cells. We choose the skeletal muscle as a model system to study mechanisms that regulate the influence from aged environment on stem cell function. Aged skeletal muscle has reduced levels of FGF2 and elevated Wnts and TGF2, leading to decreased proliferation of resident stem cells (so called satellite cells) and increased fibrosis during regeneration. The bioavailability of FGF2, Wnts and TGF2 is regulated by sulfated heparan sulfate. This proposal investigates heparan sulfate-dependent mechanisms that regulate the transmission of age-related environmental signals to satellite cells during skeletal muscle regeneration. The first set of experiments focuses on roles of two extracellular heparan sulfate 6-O-endosulfatases (Sulfs) in differential regulation of the bioavailability of age-related signals. Sulfs enzymatically remodel heparan sulfate 6-O-sulfation, thereby Sulfs reduce Wnts and TGF2 binding to heparan sulfate, while disrupting FGF2 interaction with the receptor. Therefore, Sulfs are hypothesized to promote age-augmented Wnt and TGF2 signaling, while repressing age-reduced FGF2 signaling, leading to impaired function of satellite cells. This hypothesis will be tested by comparing the efficiency of myogenesis, fibrosis and age-related regeneration signaling in aged control, systemic and satellite cell-specific Sulf double mutant mice using a combination of in vivo regeneration and in vitro culture assays. The second set of experiments will test whether heparin, which is similar to heparan sulfate of Sulf-deficient mice in the structure and signaling function, will improve the efficiency of skeletal muscle regeneration in an aged environment. The results of these investigations are expected to lead to the discovery of Sulf- and heparan sulfate-dependent mechanisms that regulate signal communication between satellite cells and the aged muscle environment. Such knowledge may open new venues for prevention and therapy of impaired muscle regeneration by age. Aging is inevitably associated with diminished capacity of stem cells to regenerate. In the skeletal muscle, age-related changes of environmental signals have a major impact on impaired function of resident stem cells, so-called satellite cells. Disruption of the communication between satellite cells and the aged muscle environment may lead to effective therapies for age-impaired skeletal muscle regeneration. This proposal investigates regulatory mechanisms during the transmission of age-related environmental signals into satellite cells. We have identified two Sulf enzymes that regulate the bioavailability of age-related signals in the regeneration environment and satellite cells. Proposed studies will test whether Sulfs are candidate regulators of the communication between satellite cells and the aged muscle environment. Our findings may open new venues for prevention and therapy of aging-related impairment of skeletal muscle regeneration
Keywords: Abbreviations; Age; age related; aged; Aging; Agonist; Animal Model; base; Basement membrane; Binding (Molecular Function); Biological Assay; Biological Availability; Biological Models; cell age; Cell Aging; Cell Communication; Cell Culture Techniques; Cell physiology; Cells; Coagulants; Communication; effective therapy; Environment; environmental change; Enzymes; extracellular; FGF2 gene; Fibroblast Growth Factor; Fibrosis; Heparin; Heparitin Sulfate; Hepatocyte Growth Factor; Impairment; improved; In Vitro; in vivo regeneration; Individual; Injury; Inorganic Sulfates; Investigation; Knowledge; Lead; Mediating; Mus; Muscle; muscle aging; muscle regeneration; Mutant Strains Mice; myogenesis; Natural regeneration; Pathway interactions; Phenotype; Population; Prevention therapy; Process; public health relevance; receptor; receptor binding; Regulation; research study; response; Rest; Role; Saline; satellite cell; Signal Pathway; Signal Transduction; Skeletal Muscle Satellite Cells; Skeletal muscle structure; small molecule; Somatomedins; Stem cells; Structure; sulfation; Testing; Therapeutic; therapeutic target; Transforming Growth Factor beta; transmission process; Unspecified or Sulfate Ion Sulfates
Relevance: Aging is inevitably associated with diminished capacity of stem cells to regenerate. In the skeletal muscle, age-related changes of environmental signals have a major impact on impaired function of resident stem cells, so-called satellite cells. Disruption of the communication between satellite cells and the aged muscle environment may lead to effective therapies for age-impaired skeletal muscle regeneration. This proposal investigates regulatory mechanisms during the transmission of age-related environmental signals into satellite cells. We have identified two Sulf enzymes that regulate the bioavailability of age-related signals in the regeneration environment and satellite cells. Proposed studies will test whether Sulfs are candidate regulators of the communication between satellite cells and the aged muscle environment. Our findings may open new venues for prevention and therapy of aging-related impairment of skeletal muscle regeneration
Project start date: 2010-03-15
Project end date: 2015-01-31
Budget start date: 15-FEB-2011
Budget end date: 31-JAN-2012
PFA/PA: PA-07-070
5R01AG034939-02 (2011): $308776