syndecan modulate proteolytic balance in airway inflammation

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Abstracts

Atrioventricular (AV) valves of the heart develop from undifferentiated mesenchymal endocardial cushions (EC) that later remodel into stratified valves with diversified extracellular matrix (ECM). Although some ECM components and remodeling enzymes have been identified, a complete gene expression profile of heart valve maturation and ECM remodeling program has not been established. In order to identify gene expression profile of developmental valve remodeling, Affymetrix gene expression profiling analysis was performed on murine embryonic day (E)12.5 AV cushion compared with E17.5 AV valves. The most highly upregulated family of genes detected in the E17.5 and adult valves compared to E12.5 EC were members of the small leucine-rich proteoglycan (SLRP) family including Asporin and Osteoglycin. Interestingly, Asporin is a known negative regulator of matrix mineralization and could be important in valve disease mechanisms. Consistent with ECM maturation and remodeling, collagens associated with specific types of connective tissue, including Col14a1 and Col1a1 are increased in late E17.5 AV valves. In contrast, cartilage-associated type IX Col9a3 and Col2a1 are increased in early E12.5 EC. Several ECM remodeling enzymes and tissue inhibitor of matrix metalloproteinases (TIMPs) are also differentially expressed during mouse AV valve development. Adamts12 is identified as one of the most increased genes in E17.5 AV valves, whereas Adamts15, Mmp11 and Adamtsl1 are increased in early E12.5 EC. The differential regulation of multiple ECM proteins and remodeling enzymes is critical for normal valve structure and function with implications in valve degeneration and disease mechanisms. doi:10.1016/j.matbio.2008.09.405

191 HS/syndecan modulate proteolytic balance in airway inflammation Chi Hang Chana,b, Valeria On Yue Leunga, Mary Sau Man Ipb, Daisy Kwok Yan Shum a Department of Biochemistry, the University of Hong Kong, Hong Kong b Department of Medicine, the University of Hong Kong, Hong Kong Persistent tissue injury in chronic airway inflammation is contributed by the unopposed neutrophil elastase (NE) activity. In sputum sols of patients with bronchiectasis, alpha-1-antitrypsin (α1-AT) was found in excess of unopposed NE at molar ratios that averaged 16:1, suggesting the existence of imbalance between protease and anti-protease. Western blot analysis of sputum sol samples however found NE in a supramolecular complex with syndecan-1 and as such, inhibition of NE activity was incomplete even with addition of α1-AT. To confirm that NE binding to heparan sulfate (HS) moieties of syndecan-1 limits the anti-elastase effect, recombinant syndecan-1 was recovered from stable syndecan-1 transfectants of ARH-77 cells. Western ligand blot confirmed that NE bound only to HS moieties of syndecan-1. Inhibition of NE activity by standard additions of α1-AT was incomplete unless syndecan-1 had been deglycanated by heparitinase treatment. SPR analysis revealed that NE binding to HS could be competed out by heparin variants. To test if heparin fragments can displace NE from the complex, heparin oligosaccharides were incubated with sputum sol samples. Western blot analysis indicated that heparin oligosaccharides (dp ≥ 4) displaced NE from the complex and the displaced NE became accessible to inhibition by endogenous α1-AT. MALDI TOF-MS analysis further revealed subunit composition of the heparin oligosaccharides. Our results therefore suggest the possible use of heparin oligosaccharides in restoring the balance between protease/anti-protease in chronic airway inflammation. doi:10.1016/j.matbio.2008.09.406

192 Role of serglycin in acute lung injury and repair Michelle Lina, Peter Chena, Magnus Åbrinkb, Qinglang Lia, Ying Wanga, Bill C. Parksa a Center for Lung Biology, University of Washington, Seattle, WA, United States b Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden

Proteoglycans influence cell–cell and cell–matrix interactions, as well as the functions of chemokines, enzymes, and growth factors through sequestration, transport, and activation of substrates or target cells. Serglycin is a heparin/ heparan- and chondroitin-sulfated proteoglycan involved in the retention and maturation of several proteases inside storage granules and secretory vesicles of hematopoietic cells and endothelial cells. In addition, secreted serglycin can further modulate activity of proteins through their interactions. Using immunohistochemistry and qRT-PCR, we determined that serglycin is expressed by alveolar type II cells, and we hence assessed if this proteoglycan functions in the response to acute lung injury. We found an increase in the number of leukocytes, particularly macrophages, in the bronchoalveolar lavage (BAL) fluid of serglycin-null mice compared to wildtype animals after bleomycin-induced injury. Furthermore, serglycin expression was increased in response to mechanical injury in organotypic airway epithelial cell cultures. In the absence of serglycin, the epithelial cells had decreased wound closure and increased total MMP activity as measured with a fluorogenic substrate. These data suggest that serglycin is involved in modulating inflammation and injury repair. doi:10.1016/j.matbio.2008.09.407

193 Confocal microscopy study of decorin binding to collagen fibrils Elena Makareevaa, Mary B. Suttera, Angela M. DeRiddera, Antonella Forlinob, Antonio Rossib, Ruggero Tennib, Sergey Leikina a Section on Physical Biochemistry, NICHD, National Institutes of Health, Bethesda, MD 20892, United States b University of Pavia, Italy Most studies of protein and proteoglycan binding to collagen are based on techniques that do not account for the supramolecular organization of collagen fibers and physiological environment. To overcome these limitations, we developed a confocal microscopy binding assay with differential fluorescent labeling of collagen and its ligands. For the present study, purified type I human fibroblast collagen was labeled with AlexaFluor488 (A488) and recombinant human decorin was labeled with AlexaFluor546 (A546). Decorin solution was added to reconstituted collagen fibrils at 37 °C. Three dimensional confocal microscopy imaging revealed a non-uniform binding pattern of decorin with more proteoglycan bound at fibril edges, kinks, junctions, and within poorly organized collagen aggregates. Such binding heterogeneity was caused by variation in the exposed collagen surface. The amount of decorin bound to isolated fibrils was directly proportional to the fibril surface area. The A546/A488 intensity ratio normalized to the surface area followed the classical binding isotherm with the dissociation constant Kd ~ 50 nM. From competitive binding experiments, we found the effect of decorin labeling to be minimal. The isotherms on fibrils reconstituted from collagen with intact and pepsin-cleaved telopeptides were indistinguishable, indicating that telopeptides do not play a significant role in decorin binding. We are currently investigating the role of GAG chains and different cofactors in decorin binding. doi:10.1016/j.matbio.2008.09.408

194 Estrogen and progesterone modulate versican in the mouse uterus Renato M. Salgadoa, Fernanda F. Fernandesa, Rodolfo R. Favaroa, Jocelyn D. Glazierb, John D. Aplinb, Telma M. Zorna a Department of Cell and Developmental Biology, University of Sao Paulo, Brazil b Maternal and Fetal Health Research Group, University of Manchester, UK Versican is a hyaluronan-binding proteoglycan found in various soft tissues. This molecule undergoes RNA alternative splicing, originating four distinct isoforms, which play important roles in physiological and pathological conditions. In this study, we analyze the synthesis and distribution of versican in mouse uterine tissues during the estrous cycle, in ovariectomized animals and after hormonal replacement. Uteri samples were fixed in methacarn and embedded in paraplast for immunoperoxidase, or immersed