Thrombospondin Repeats Research Articles

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase

AbstractCD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A–induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A–induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Regulated expression of ADAMTS-12 in human trophoblastic cells: a role for ADAMTS-12 in epithelial cell invasion?

Metastatic carcinoma cells exploit the same molecular machinery that allows human placental cytotrophoblasts to develop an invasive phenotype. As altered expression levels of ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) subtypes have been associated with cancer progression, we have examined the function and regulation of members of this gene family in epithelial cell invasion using cultures of highly invasive extravillous cytotrophoblasts and the poorly invasive JEG-3 cytotrophoblast cell line as model systems. Of the multiple ADAMTS subtypes identified in first trimester human placenta and these two trophoblastic cell types, only ADAMTS-12 was preferentially expressed by extravillous cytotrophoblasts. Transforming growth factor-β1 and interleukin-1β, two cytokines that promote and restrain cytotrophoblast invasion in vitro, were also found to differentially regulate trophoblastic ADAMTS-12 mRNA levels. Loss- or gain-of-function studies confirmed that ADAMTS-12, independent of its proteolytic activity, plays a specific, non-redundant role in trophoblast invasion. Furthermore, we demonstrated that ADAMTS-12 regulated cell-extracellular matrix adhesion and invasion through a mechanism involving the αvβ3 integrin heterodimer. This study identifies a novel biological role for ADAMTS-12, and highlights the importance and complexity of its non-proteolytic domain(s) pertaining to its function.

Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.

Molecular characterization and structure determination of human ADAMTSL4

The thrombospondin type 1 repeat (TSR) is an ancient extracellular protein domain that is commonly found in invertebrate and vertebrate proteins. The ADAMTSL4 protein, also known as TSRC1, belongs to the TSR superfamily and has multiple thrombospondin repeats, most of which are clustered at the C-terminus. It has been reported that some TSP1 domain-containing proteins, e.g. thrombospondin 1 and thrombospondin 2, could induce apoptosis of endothelial cells but it is not clear how these proteins operate in death pathways. Our recent work shows that mutations in ADAMTSL4 are responsible for autosomal-recessive isolated ectopia lentis, i.e. abnormal positioning of the lens of the eye and affect the development of the zonular fiber. However, little is known about the function of ADAMTSL4. To shed more light on the function of the ADMTSL4 and its roles in different tissues we extended our work to carry out molecular characterization of this gene and its variants. We have obtained cDNA clones encoding the full ADAMTSL4 protein and its truncated isoform. Both are subcloned into the pET28a vector for expression in E. coli. In anticipation of possible expression challenges in this host, e.g. formation of inclusion bodies or lack of expression, we subcloned each fragment into the yeast vector, pYES2. The gene in both cases has been fused in frame with a region encoding an N-terminal His-Tag to facilitate the purification of the recombinant protein. DNA analysis indicates that each fragment has been correctly cloned into the pYES2 vector. Each construct was transformed into Saccharomyces cerevisiae for protein expression. Our preliminary analysis indicates that one of the genes is expressed in S. cerevisiae but at a very low level. Work to optimize the expression of ADAMTSL4 in yeast as well as in E. coli is in progress. We also extracted the seven domains of the ADAMTSL4 using PCR. Each domain was subcloned into the E. coli overexpression vector, pET28a. Expression studies of all the constructs have shown that the seven domains have been successfully overexpressed in E. coli. The overexpression was confirmed using Western blot techniques. The recombinant protein of each domain is purified for NMR and x-ray studies.

Thrombospondin-1 Inhibits VEGF Receptor-2 Signaling by Disrupting Its Association with CD47

Thrombospondin-1 (TSP1) can inhibit angiogenic responses directly by interacting with VEGF and indirectly by engaging several endothelial cell TSP1 receptors. We now describe a more potent mechanism by which TSP1 inhibits VEGF receptor-2 (VEGFR2) activation through engaging its receptor CD47. CD47 ligation is known to inhibit downstream signaling targets of VEGFR2, including endothelial nitric-oxide synthase and soluble guanylate cyclase, but direct effects on VEGFR2 have not been examined. Based on FRET and co-immunoprecipitation, CD47 constitutively associated with VEGFR2. Ligation of CD47 by TSP1 abolished resonance energy transfer with VEGFR2 and inhibited phosphorylation of VEGFR2 and its downstream target Akt without inhibiting VEGF binding to VEGFR2. The inhibitory activity of TSP1 in large vessel and microvascular endothelial cells was replicated by a recombinant domain of the protein containing its CD47-binding site and by a CD47-binding peptide derived from this domain but not by the CD36-binding domain of TSP1. Inhibition of VEGFR2 phosphorylation was lost when CD47 expression was suppressed in human endothelial cells and in murine CD47-null cells. These results reveal that anti-angiogenic signaling through CD47 is highly redundant and extends beyond inhibition of nitric oxide signaling to global inhibition of VEGFR2 signaling.

PGF2α-F-prostanoid receptor signalling via ADAMTS1 modulates epithelial cell invasion and endothelial cell function in endometrial cancer

BackgroundAn increase in cancer cell invasion and microvascular density is associated with a poorer prognosis for patients with endometrial cancer. In endometrial adenocarcinoma F-prostanoid (FP) receptor expression is elevated, along with its ligand prostaglandin (PG)F2α, where it regulates expression and secretion of a host of growth factors and chemokines involved in tumorigenesis. This study investigates the expression, regulation and role of a disintegrin and metalloproteinase with thrombospondin repeat 1 (ADAMTS1) in endometrial adenocarcinoma cells by PGF2α via the FP receptor.MethodsHuman endometrium and adenocarcinoma tissues were obtained in accordance with Lothian Research Ethics Committee guidance with informed patient consent. Expression of ADAMTS1 mRNA and protein in tissues was determined by quantitative RT-PCR analysis and immunohistochemistry. Signal transduction pathways regulating ADAMTS1 expression in Ishikawa cells stably expressing the FP receptor to levels seen in endometrial cancer (FPS cells) were determined by quantitative RT-PCR analysis. In vitro invasion and proliferation assays were performed with FPS cells and human umbilical vein endothelial cells (HUVECs) using conditioned medium (CM) from PGF2α-treated FPS cells from which ADAMTS1 was immunoneutralised and/or recombinant ADAMTS1. The role of endothelial ADAMTS1 in endothelial cell proliferation was confirmed with RNA interference. The data in this study were analysed by T-test or ANOVA.ResultsADAMTS1 mRNA and protein expression is elevated in endometrial adenocarcinoma tissues compared with normal proliferative phase endometrium and is localised to the glandular and vascular cells. Using FPS cells, we show that PGF2α-FP signalling upregulates ADAMTS1 expression via a calmodulin-NFAT-dependent pathway and this promotes epithelial cell invasion through ECM and inhibits endothelial cell proliferation. Furthermore, we show that CM from FPS cells regulates endothelial cell ADAMTS1 expression in a rapid biphasic manner. Using RNA interference we show that endothelial cell ADAMTS1 also negatively regulates cellular proliferation.ConclusionsThese data demonstrate elevated ADAMTS1 expression in endometrial adenocarcinoma. Furthermore we have highlighted a mechanism whereby FP receptor signalling regulates epithelial cell invasion and endothelial cell function via the PGF2α-FP receptor mediated induction of ADAMTS1.

Preclinical Testing of Human Recombinant von Willebrand Factor: ADAMTS13 Cleavage Capacity in Animals as Criterion for Species Suitability

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 ( A Disintegrin and Metalloproteinase with Thrombo Spondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested. Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects. These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.

Abstract 2325: Thrombospondin 1 inhibits angiogenesis in liver metastases but not lung metastases due to differential cleavage activity of ADAMTS1

Abstract Background: Metastases to any organ site require angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors including thrombospondin 1 (TSP1). The principal anti-angiogenic activity of TSP1 resides in a domain containing 3 TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. Methods and Results: Wild-type B16F10 melanoma cells formed increased liver metastases in Tsp1-null mice compared to controls but equivalent lung metastases in Tsp1-null mice and controls. Stable cell lines of B16F10, CT26 colon carcinoma, and RenCa renal carcinoma overexpressing full-length TSP1 or 3TSR were generated. Metastases from CT26 and RenCa cell lines over-expressing TSP1 were reduced in liver but not in lung. To determine why TSP1 blocked metastatic tumor growth in the liver but not the lung, we examined cleavage of recombinant TSP1 protein by liver and lung lysates and found that liver lysate cleaved TSP1 into its 3TSR domain much more efficiently that lung lysate. TSP1-mediated inhibition of CT26 lung metastases was restored when only the 3TSR domain was over-expressed rather than full-length TSP1. Liver lysate cleavage activity was significantly abrogated when ADAMTS1 activity was (1) neutralized using ADAMTS-1 antibody, (2) knocked down by lentiviral shRNA, or (3) absent in Adamts1-null liver lysate. Livers metastases generated from B16F10 cells over-expressing TSP1 in Adamts1-haploinsufficient mice demonstrated accelerated growth compared to growth in wild-type mice. Conclusions: TSP1 inhibits the growth of liver metastases but not lung metastases secondary to differential cleavage of TSP1 into anti-angiogenic fragments by ADAMTS1. This study emphasizes the importance of host organ environment in the regulation of tumor angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2325.

Thrombospondins and Novel TSR-containing Proteins, R-spondins, Regulate Bone Formation and Remodeling

Thrombospondins (TSPs) are a family of five secreted multimeric matricellular proteins that share homology in the type II and III repeats and carboxy-terminal region. Type I repeats, also known as properdin or thrombospondin repeats (TSRs), are found in TSP1/2, but not TSP3-5. A variety of other secreted proteins contain TSRs, including the novel extracellular molecules, R-spondins. TSP family and many TSR-containing proteins, including R-spondins, are highly expressed in skeletal tissues during development and postnatal. TSP2 regulates the osteoblast lineage, influencing bone mass and geometry, as well as response to fracture healing, ovariectomy, and mechanical loading. Compound knockout mice of TSPs have revealed important mechanistic insights. TSP1/2 knockout mice have craniofacial dysmorphism, and TSP1/3/5 compound knockout mice display growth plate abnormalities. R-spondins promote osteoblast differentiation and R-spondin-2 deficiency results in skeletal developmental defects. Overall, TSP and other TSR molecules influence multiple aspects of bone development and remodeling.

Characterization of a novel thrombospondin-related protein in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades a wide range of host cells. The parasite releases a large variety of proteins from a secretory organelle, microneme, and the secretion is essential for the parasite invasion. We cloned a secreted protein with an altered thrombospondin repeat of Toxoplasma gondii (TgSPATR), which was the homologue of Plasmodium SPATRs. Immunofluorescence double staining experiment revealed that TgSPATR was co-localized with a microneme protein, MIC2, and immuno-electron microscopic (IEM) analysis detected TgSPATR in the microneme-like structure. TgSPATR secretion was induced by ethanol, while an intracellular Ca 2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), suppressed the ethanol-induced secretion, suggesting the secretion was Ca 2+-dependent, similarly to known microneme proteins. Furthermore, TgSPATR, existed on outer surface of the parasites, was detected by incomplete membrane permeabilization by saponin and immunofluorescent antibody test (IFAT). Both TgSPATR and MIC2 were detected on outer surface of extracellular parasites, but not of intracellular single parasites, suggesting they were similarly secreted during early stages of parasite invasion. Therefore, TgSPATR is probably new member of microneme protein and maybe involved in parasite invasion.

RETRACTED: Variable inhibition of thrombospondin 1 against liver and lung metastases through differential activation of metalloproteinase ADAMTS1.

Metastasis relies on angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors, including thrombospondin 1 (TSP1). The principal antiangiogenic activity of TSP1 resides in a domain containing three TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. In this study, we examined the role of TSP1 and ADAMTS1 in controlling metastatic disease in the liver and lung. TSP1 overexpression inhibited metastatic growth of colon or renal carcinoma cells in liver but not lung. Metastatic melanoma in liver grew more rapidly in Tsp1-null mice compared with controls, whereas in lung grew similarly in Tsp1-null mice or controls. Recombinant TSP1 was cleaved more efficiently in lysates from liver than lung. ADAMTS1 inhibition by neutralizing antibody, small interfering RNA, or genetic deletion abrogated cleavage activity. To confirm that lack of cleavage of TSP1 ablated its antiangiogenic function in the lung, we generated colon cancer cells stably secreting only the 3TSR domain and found that they inhibited formation of both liver and lung metastases. Collectively, our results indicate that the antiangiogenic activity of TSP1 is differentially regulated by ADAMTS1 in the liver and lung, emphasizing the concept that regulation of angiogenesis is varied in different tissue environments.

Endothelial cell ADAMTS-13 and VWF: production, release, and VWF string cleavage

AbstractHuman umbilical vein endothelial cell (HUVEC)–released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca2+/Zn2+-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y1605-M1606 decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.

Homozygous Mutations in ADAMTS10 and ADAMTS17 Cause Lenticular Myopia, Ectopia Lentis, Glaucoma, Spherophakia, and Short Stature

Weill-Marchesani syndrome (WMS) is a well-characterized disorder in which patients develop eye and skeletal abnormalities. Autosomal-recessive and autosomal-dominant forms of WMS are caused by mutations in ADAMTS10 and FBN1 genes, respectively. Here we report on 13 patients from seven unrelated families from the Arabian Peninsula. These patients have a constellation of features that fall within the WMS spectrum and follow an autosomal-recessive mode of inheritance. Individuals who came from two families and met the diagnostic criteria for WMS were each found to have a different homozygous missense mutation in ADAMTS10. Linkage analysis and direct sequencing of candidate genes in another two families and a sporadic case with phenotypes best described as WMS-like led to the identification of three homozygous mutations in the closely related ADAMTS17 gene. Our clinical and genetic findings suggest that ADAMTS17 plays a role in crystalline lens zonules and connective tissue formation and that mutations in ADAMTS17 are sufficient to produce some of the main features typically described in WMS.

Multi-step binding of ADAMTS-13 to von Willebrand factor.

ADAMTS-13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS-13 distant from the active site. We hypothesized that not all binding sites for ADAMTS-13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS-13. Immunoprecipiation of VWF-ADAMTS-13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Co-immunoprecipitation demonstrated that ADAMTS-13 binds to native multimeric VWF (K(d) of 79 +/- 11 nmol L(-1)) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50-200). Moreover, results with ADAMTS-13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS-13 spacer, probably thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS-13 to VWF stoichiometry of 0.0040 +/- 0.0004 (mean +/- SEM, n = 10). ADAMTS-13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.

A peptide derived from type 1 thrombospondin repeat-containing protein WISP-1 inhibits corneal and choroidal neovascularization.

Ocular neovascularization is the primary cause of blindness in a wide range of prevalent ocular diseases including proliferative diabetic retinopathy, exudative age-related macular degeneration, and retinopathy of prematurity, among others. Antiangiogenic therapies are starting to give promising results in these diseases. In the present study the antiangiogenic potential of an 18-mer peptide derived from type 1 thrombospondin repeat-containing protein WISP-1 (wispostatin-1) was analyzed in vitro with human retinal endothelial cell proliferation and migration assays. The peptide was also tested in vivo in the corneal micropocket and the laser-induced choroidal neovascularization (CNV) mouse models. Human retinal endothelial cells were treated with the WISP-1 peptide and in vitro migration and proliferation assays were performed. Also evaluated was the antiangiogenic effect of this peptide in vivo using the corneal micropocket assay and the laser-induced CNV model. Wispostatin-1 derived peptide demonstrated antimigratory and antiproliferative activity in vitro. Wispostatin-1 completely abolished bFGF-induced neovascularization in the corneal micropocket assay. The peptide also demonstrated significant inhibition of laser-induced CNV. An inhibitory effect of Wispostatin-1 on ocular neovascularization was found in vitro and in vivo. The identification of novel and potent endogenous peptide inhibitors provides insight into the pathogenesis of corneal and choroidal neovascularization. The results demonstrate potential for therapeutic application in prevalent ocular disease.

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.

The Role of N-Linked Glycosylation in Expression and Function of ADAMTS13

N-linked glycosylation is an important co-translational protein modification affecting protein folding, secretion, stability & function. We have previously demonstrated that N-linked glycosylation of Von Willebrand Factor (VWF) modulates its interaction with ADAMTS13, the plasma metalloprotease responsible for controlling VWF multimeric size. In the present study we have investigated the role of N-linked glycosylation in modulating ADAMTS13 expression, secretion & function.Lectin analysis of plasma derived & recombinant ADAMTS13 (rAD13) demonstrated that ADAMTS13 presents sialic acid & galactose residues, indicating the presence of complex glycans structures. Analysis with the lectin GNL, specific for high mannose structures showed that plasma derived ADAMTS13 also contained a small portion of basic high mannose glycans & this was increased in rAD13. Enzymatic modification of rAD13 with PNGase F to remove whole N-linked glycans & neuraminidase to remove terminal sialic residues were completely effective. However, neither modification affected the enzymatic activity of ADAMTS13 towards VWF, indicating that following secretion ADAMTS13 N-linked glycans are expendable.Using an ADAMTS13 ELISA and western blotting, we showed that expression of rAD13 in the presence of Tunicamycin prevented N-linked glycosylation & prevented secretion, indicating that N-linked glycosylation is essential for secretion of ADAMTS13. Expression in the presence of the glucosidase inhibitor castanospermine, that inhibits binding to the endoplasmic lectin chaperones calnexin & calreticulin, also prevented secretion of ADAMTS13 suggesting that glycan dependent interaction with these chaperones is essential for the correct cellular processing of ADAMTS13To further explore these observations, 10 individual point mutations encoding asparagine to glutamine changes were generated to prevent glycosylation at each of the 10 N-linked glycans sites in ADAMTS13: N462 & N46Q in the metalloprotease (MP) domain, N552Q in the cysteine rich domain, N579Q, N614Q & N667Q in the spacer domain, N707Q in the second Thrombospondin repeat (TSR), N828Q in the forth TSR and N1235Q and N1354Q in the CUB domains. Mutations N42Q, N579Q, N614Q, N707Q, N828Q and N1354Q caused only a slight to moderate reduction in ADAMTS13 secretion; 57%, 62%, 50%, 84%, 70% and 68% of wild type respectively. Mutations N552Q and N667Q significantly reduced ADAMTS13 secretion to 23% and 25% of wild type respectively indicating that glycosylation of the cysteine rich & spacer domains is required for efficient secretion. Significantly, mutation N46Q in the MP domain abolished secretion resulting in intracellular retention of ADAMTS13.All the secreted mutants with the exception of N828Q and N1235Q were able to effectively proteolyse the short VWF-115 substrate and full length VWF (FL-VWF). Interestingly, N828Q was demonstrated to have reduced activity towards VWF-115 and FL-VWF under both static and flow conditions. Although binding to immobilised VWF A2 domain and FL-VWF was not altered, a difference was observed in the binding of N828Q to globular FL-VWF. Conversely, mutation of N1235 increased activity of ADAMTS13 towards VWF.Together these data demonstrate that N-linked glycosylation of ADAMTS13, in particular at N46, is essential for efficient cellular processing & secretion of the enzyme. Following secretion the N-linked glycans are not required for proper function; however glycosylation of TSR 4 and the CUB domains are required for ADAMTS13 to assume a correct conformation for proper enzymatic activity towards VWF.

Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation

Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.

Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels

To ascertain, using specific inhibitors, the potential role of calcium-related signal transduction pathways in the mechanism of cartilage matrix protein gene induction and metalloproteinase gene suppression by capacitively coupled electric fields. Articular chondrocytes were isolated from adult bovine patellae and cultured in high density for 7 days. To study matrix protein expression, cells cultured in the absence or presence of specific calcium pathway inhibitors were exposed to a capacitively coupled electrical field (60 kHz, 20 mV/cm): for aggrecan 1h at 50% duty cycle and for type II collagen 6h at 8.3% duty cycle. To study metalloproteinase expression in the presence of interleukin 1 beta (IL-1beta), cells were cultured as above but exposed for only 30 min to a 100% duty cycle signal. At harvest, total mRNA was isolated and aggrecan, type II collagen, matrix metalloproteinase (MMP-1, -3 and -13) and aggrecanase [a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTS-4 and -5)] mRNA expression were measured by quantitative real-time polymerase chain reaction (qPCR). (1) In the absence of inhibitors, appropriate electrical stimulation induces a 3-4-fold up-regulation of both aggrecan and type II collagen mRNA and a 3.7-9.6-fold down-regulation of IL-1beta-induced metalloproteinases; (2) the presence of inhibitors alone does not affect any target mRNA levels; (3) inhibitors of intracellular calcium regulation and inositol 1,4,5-triphosphate (IP(3)) formation [8-(diethylamino)octyl-3,4,5,-trimethoxybenzoate hydrochloride (TMB-8) and neomycin, respectively] have no effect on regulation of target mRNA levels by electrical stimulation; and (4) inhibitors of voltage-gated calcium channels (verapamil), calmodulin activation (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride, W-7), calcineurin activity (cyclosporin A), phospholipase C activity (bromophenacyl bromide, BPB) and prostaglandin E(2) (PGE(2)) synthesis (indomethacin) completely inhibit the effects of electrical stimulation. The results are consistent with the effects of electrical stimulation involving a pathway of extracellular Ca(2+) influx via voltage-gated calcium channels rather than from intracellular Ca(2+) repositories; and with downstream roles for calmodulin, calcineurin and nuclear factor of activated T-cells (NF-AT) rather than for phospholipase C and IP(3).

Thrombospondin-1 in Von Willebrand Factor Function

Thrombospondin-1 (TSP1), expressed in many cells and tissues is abundantly present in platelet alpha-granules, from where it is released upon platelet activation. Murine Tsp1(-/-) platelet studies have revealed that TSP1 is redundant for platelet aggregation, but that it reinforces platelet aggregate stabilization, especially in a shear field. von Willebrand factor (VWF), synthesized by megakaryocytes and endothelial cells is stored both in platelet alpha-granules and in endothelial Weibel-Palade bodies as ultralarge multimers. When released from endothelial cells, these multimers are temporarily retained on the endothelium, to be cleaved by the plasma protease ADAMTS13 into smaller and hemostatically less reactive multimers, released in plasma. This protease shows partial sequence identity with the type 1 (TSR1) and type 2 (TSR2) repeats of TSP1 and contains 1 TSR1 and 6 TSR2 repeats. TSP1, locally released by platelets, competes with ADAMTS13 during VWF proteolysis and controls the degree of VWF multimer processing. In addition, TSP1 and VWF both interact with the platelet GPIb/V/IX membrane complex, primarily in flow. These interactions control the recruitment of platelets to (sub) endothelial VWF and TSP1, exposed to the circulation, as a consequence of vascular inflammation and endothelial injury. TSP1-VWF interactions do not strictly enhance platelet recruitment and secreted TSP1 even weakly competes with the dynamic platelet rolling and adhesion onto VWF. Hence, TSP1 and VWF show partially related hemostatic functions, the most important one being the TSP1 role in the ADAMTS13 operated VWF multimer processing, in pro-inflammatory and thrombogenic conditions.