Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis in vitro. However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPAR gamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Co15a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations. (c) 2005 Elsevier B.V. All rights reserved.

Renal allografts with IF/TA display distinct expression profiles of metzincins and related genes

Chronic renal allograft injury is often reflected by interstitial fibrosis (IF) and tubular atrophy (TA) without evidence of specific etiology. In most instances, IF/TA remains an irreversible disorder, representing a major cause of long-term allograft loss. As members of the protease family metzincins and functionally related genes are involved in fibrotic and sclerotic processes of the extracellular matrix (ECM), we hypothesized their deregulation in IF/TA. Gene expression and protein level analyses using allograft biopsies with and without Banff'05 classified IF/TA illustrated their deregulation. Expression profiles of these genes differentiated IF/TA from Banff'05 classified Normal biopsies in three independent microarray studies and demonstrated histological progression of IF/TA I to III. Significant upregulation of matrix metalloprotease-7 (MMP-7) and thrombospondin-2 (THBS-2) in IF/TA biopsies and sera was revealed in two independent patient sets. Furthermore, elevated THBS-2, osteopontin (SPP1) and beta-catenin may play regulatory roles on MMP. Our findings further suggest that deregulated ECM remodeling and possibly epithelial to mesenchymal transition (EMT) are implicated in IF/TA of kidney transplants, and that metzincins and related genes play an important role in these processes. Profiling of these genes may be used to complement IF/TA diagnosis and to disclose IF/TA progression in kidney transplant recipients.

The Thrombospondin Receptor CD47 (IAP) Modulates and Associates with α2β1 Integrin in Vascular Smooth Muscle Cells

The carboxyl-terminal domain of thrombospondin-1 enhances the migration and proliferation of smooth muscle cells. Integrin-associated protein (IAP or CD47) is a receptor for the thrombospondin-1 carboxyl-terminal cell-binding domain and binds the agonist peptide 4N1K (kRFYVVMWKk) from this domain. 4N1K peptide stimulates chemotaxis of both human and rat aortic smooth muscle cells on gelatin-coated filters. The migration on gelatin is specifically blocked by monoclonal antibodies against IAP and a β1 integrin, rather than αvβ3 as found previously for 4N1K-stimulated chemotaxis of endothelial cells on gelatin. Both human and rat smooth muscle cells displayed a weak migratory response to soluble type I collagen; however, the presence of 4N1K peptide or intact thrombospondin-1 provoked a synergistic chemotactic response that was partially blocked by antibodies to α2 and β1 integrin subunits and to IAP. A combination of antiα2 and IAP monoclonal antibodies completely blocked chemotaxis. RGD peptide and antiαvβ3 mAb were without effect. 4N1K and thrombospondin-1 did not augment the chemotactic response of smooth muscle cells to fibronectin, vitronectin, or collagenase-digested type I collagen. Complex formation between α2β1 and IAP was detected by the coimmunoprecipitation of both α2 and β1 integrin subunits with IAP. These data suggest that IAP can associate with α2β1 integrin and modulate its function.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca(2+) entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca(2+) entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca(2+)-sensing role of STIM1 is served by the protein in the ER.

Deficiency of thrombospondin-1 reduces Th17 differentiation and attenuates experimental autoimmune encephalomyelitis

Transforming growth factor beta (TGF-beta) plays a role both in the induction of Treg and in the differentiation of the IL-17-secreting T cells (Th17) which drive inflammation in experimental autoimmune encephalomyelitis (EAE). We investigated the role that thrombospondin-1 (TSP-1) dependent activation of TGF-beta played in the generation of an encephalitic Th17 response in EAE. Upon immunization with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)), TSP-1 deficient (TSP-1(null)) mice and MOG(35-55) TCR transgenic mice that lack of TSP-1 (2D2.TSP-1(null)) exhibited an attenuated form of EAE, and secreted lower levels of IL-17. Adoptive transfer of in vitro-activated 2D2.TSP-1(null) T cells induced a milder form of EAE, independent of TSP-1 expression in the recipient mice. Furthermore, in vitro studies demonstrated that anti-CD3/anti-CD28 pre-activated CD4+ T cells transiently upregulated latent TGF-beta in a TSP-1 dependent way, and such activation of latent TGF-beta was required for the differentiation of Th17 cells. These results demonstrate that TSP-1 participates in the differentiation of Th17 cells through its ability to activate latent TGF-beta, and enhances the inflammatory response in EAE. (C) 2009 Elsevier Ltd. All rights reserved.

10 mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin

Objective
Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects.

Design
HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells.

Results
Ten mM glucosamine and 5–10 mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn387 and Asn440, abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells.

Conclusions
Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120Cas. These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.