BSCB Newsletter Winter 2000 |
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The biology of thrombospondins and other modulatory extracellular matrix proteins N. Anilkumar and Soren Prag Page 1 - Page 2 The thrombospondin type 1 repeat (TSR) superfamily Over the last couple of years, many new additional proteins that contain TSR have been identified. The question of whether they have properties in common with thrombospondin-1 and thrombospondin-2 is of more than academic interest because of the anti-angiogenic activities of the TSR of thrombospondins. A second theme at the meeting thus related to the biological roles of these new relatives. In her Plenary Talk, Judith Kimble (University of Wisconsin, USA) gave an overview of her lab’s work on GON-1 metalloprotease in C. elegans. The gon-1 gene encodes a secreted metalloprotease with a metalloprotease domain and multiple TSRs. The TSRs are postulated to anchor GON-1 to the extracellular matrix and thereby localise its metalloprotease activity. In gon-1 mutants, gonadogenesis is impaired such that no arm elongation occurs and somatic gonadal structures are severely malformed. Kimble described how Gon-1 is expressed in two sites, leader cells and muscle, and expression in each site has a unique role in forming the gonad. The gon-1 gene is crucial for migration of the gonadal leader cells. In wild-type animals, the leader cells migrate hundreds of microns, whereas in gon-1 null mutants, the leader cells do not move at all. The gon-1 mutants also have defects in gonadogenesis that are unrelated to leader cell migration, suggesting that gon-1 might have a second role in gonadogenesis. Following from this talk, it was interesting to hear the effects of a TSR relative being knocked-out in mice. Kauji Kuno (Kanazawa, Japan) described the work in his lab on a matrix-anchored metalloprotease, ADAMTS-1. He reported that the precursor and processed forms of ADAMTS-1 are secreted from cells and bind to the extracellular matrix (ECM) in a heparin-sensitive manner. Analyses of deletion mutants of the ADAMTS-1 protein revealed that a spacer region and the three TSR were important for a tight interaction with the ECM. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with malformation of adipose tissue, kidneys and the adrenal medullary gland. Interestingly, in relation to the gon-1 mutants, female mice had malformed ovaries and low fertility. ADAMTS-1 thus appears necessary for normal growth, female gonadogenesis and the morphology and function of certain organs. Luisa Iruela-Arispe (UCLA, USA) described studies on the secretion and processing of ADAMTS1. She showed that human ADAMTS-1 is secreted, then processed in two consecutive steps to release two active forms. Matrix metalloproteinases 2, 8 and 15 were able to release the smaller form from the larger one. This second processing step removes two TSR from the carboxyl terminal end of ADAMTS-1 and alters the affinity of the protein for heparin. ADAMTS-1 has anti-angiogenic activity in culture assays, and she hypothesises that processing could be relevant for modulating the anti-angiogenic properties. Sunil Apte (Cleveland Clinic, USA) gave an overview of the whole ADAMTS family, of which there are now around 25 members, and introduced a new subfamily of TSR proteins, the ADAMTS-like (ADAM-TSL) proteins. This family includes Punctin, a novel extracellular matrix component of muscle tissues. There were two interesting talks that related to the structure of TSR. The first NMR-structure of a TSR-containing protein - heparin-binding growth-associated molecule or HB-GAM — was presented by Ilkka Kilpeläinen (Helsinki, Finland). HB-GAM is an extracellular matrix-associated protein implicated in the development and plasticity of neuronal connections in the brain which contains two repeats distantly related to TSR at primary sequence level. The NMR structure shows two domains of antiparallel b-sheets separated by a long flexible hinge. This structure opens the way to new studies of TSR and their functions. There has been some debate about whether TSR bind heparin directly. An interesting talk from Jan Hofsteenge (FMI, Switzerland) cleared up some of the contradictions. He described a new protein modification, C-mannosylation of tryptophan residues, in which carbohydrate is attached to the indole moiety of tryptophan within a specific recognition sequence. This modification is not made in plants, yeast or eubacteria. Analysis of several TSR revealed multiple C-mannosylation sites within the TSR. The functional significance of this modification remains mysterious, yet when using synthetic peptides in comparison to native protein, one should be aware of the possibilities for different properties due to the absence of these post-translational modifications.
Cellular actions of modulatory matrix proteins The cell biology and signalling mechanisms initiated by thrombospondins emerged as another hot topic of discussion in the meeting. The diversity in cellular responses to thrombospondin-1 is explained by differential expression by different cell types of multiple thrombospondin-1 receptors that induce distinct signals. David Roberts (NIH, USA) reported that integrin a3b1 is an activation-dependent adhesion receptor for thrombospondin-1 in breast carcinoma cells and small cell lung carcinoma cells. This integrin is also expressed in endothelial cells but its ability to recognize thrombospondin-1 is suppressed in confluent cells. When activated in subconfluent cells, it contributes to cell spreading on TSP-1. Bill Frazier (Washington University, USA) discussed the role of CD47 in cross-talk to integrin a2b1. He described how activation of a2b1 by thrombospondin-1 or a CD47-binding peptide stimulate smooth muscle migration by activation of a heterotrimeric G-protein. The co-organiser, Jo Adams (UCL, UK), described cellular responses to thrombospondin-1. In contrast to fibronectin, collagen and various other extracellular matrix proteins, cell adherence to thrombospondin-1 promotes the formation of actin-containing microspikes. These microspikes contains the actin-bundling protein, fascin and are negatively regulated by protein kinase C-dependent phosphorylation of fascin. The formation of these spikes depends on activities of Rac and Cdc42, but not of Rho GTPase. Mark Ginsberg (TSRI, USA) presented a poster of recent data describing how the a4 integrin cytoplasmic domain binds to paxillin. Fusion of the a4-tail to the aIIb-integrin resulted in a reduction in the formation of focal adhesion and stress fiber formation, indicating that the association of paxillin and a4 promotes integrin signalling that leads to focal adhesion disassembly. Paul Bornstein (University of Washington, USA) presented interesting studies of thrombospondin-2 null fibroblasts. These skin fibroblasts show altered adhesion properties in culture. Compared to wild-type cells, the KO cells show increased activity of metalloproteinase-2 (gelatinase-A). When KO cells are treated with the specific gelatinase-A inhibitor, TIMP2, or with neutralising antibodies against metalloproteinase 2, they regain full spreading on several matrix proteins. Alan Rapraeger (Madison, USA) discussed the mechanisms by which heparan sulphate chains on proteoglycans mediate stable high affinity binding of fibroblast growth factors (FGFs) to their receptors and regulate FGF activity. Basement membrane and cell surface heparan sulphate proteoglycans differ in their ability to bind individual FGF family members, presumably due to domain structure differences in the heparan sulfate chains.
Tenascins, SPARC and New Modulatory Proteins
Jean Schwarzbauer (Princeton, USA) discussed cellular effects of tenascin-C in a matrix gel designed to provide a matrix similar to that of healing wounds. Inclusion of tenascin-C in the gel alters actin cytoskeletal organisation in cells by modulating Rho GTPase activity. Helen Sage (Washington, USA) described the phenotype of SPARC-null mice. A prominent phenotype is aberrant connective tissue responses to injury. In culture, cells from these mice exhibit significantly enhanced rates of proliferation. Most strikingly, all strains of mice lacking the sparc gene develop early-onset of cataracts associated with aberrant proliferation and differentiation of lens epithelial cells. Additional phenotypic alterations associated with aging, connective tissue, wound healing, and/or angiogenesis suggest unanticipated roles for SPARC in the maintenance of certain organs and tissues through life. In his stimulating talk, Matthias Chiquet (Bern, Switzerland) presented data about the regulation of tenascin-C and collagen XII production by mechanical stress. It is well known that connective tissue cells adapt their ECM to changes in mechanical load. A feedback mechanism must exist, by which cells that sense mechanical stress from their matrix respond by an altered pattern of protein expression, and thus remodel the ECM to adapt to changing mechanical requirements. The production of tenascin-C and collagen XII, ECM proteins typical of tendons and ligaments, was found to be high in fibroblasts attached to a stretched collagen matrix, but suppressed in cells on a relaxed matrix. The response to a change in stress was rapid and reversible, and was reflected at the mRNA level. Both the tenascin-C and the collagen XII gene promoters contain ‘stress-responsive’ enhancer regions with similarity to ‘shear stress response elements’ found in other genes. Richard Tucker (Davis, USA) gave us an entertaining and educational talk about his work on tenascin-Y, the avian homologue to the mammalian tenascin-X. Tenascin-Y is localised with the ependymal cells in the spinal cord and in the brain. In the adult, the protein levels are high at boundaries between the Schwann cells in the peripheral nervous system and the central nervous system. Tenascin-Y inhibits Schwann cell motility in vitro and thus might define a boundary in vivo. The meeting ended with dinner and dancing to a local jazz band. This second ad-hoc meeting on modulatory matrix proteins generated a lot of enthusiastic discussion amongst the participants and we anticipate will be continued in a series of meetings.
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Winter 2000 Contents News UKLSC Database Features Meeting Reports |
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The
other major theme of the meeting was advances in understanding the biology
and mechanisms of other modulatory extracellular matrix proteins. Ruth
Chiquet-Ehrismann (FMI, Switzerland) reported a novel family of proteins,
teneurins, which are predominantly expressed in the nervous system. Chicken
teneurin-1 and teneurin-2 were found as two homologues of the Drosophila pair-rule gene product Ten-m and Drosophila Ten-a. By in situ hybridisation, they showed that teneurin-1 and -2 are expressed by distinct
populations of neurons during the time of axonal growth, most prominently
in the developing visual system. The expression of teneurin-2 in neuronal
cells leads to a significant increase in the number of filopodia and to
the formation of enlarged growth cones. These proteins might have a function
in the formation of neuronal connections. 
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