Identification and characterization of a tumor cell receptor for CSVTCG, a thrombospondin adhesive domain

We have previously shown that peptides derived from the thrombospondin sequence, CSVTCG, promoted tumor cell adhesion. To further investigate this observation, the CSVTCG-tumor cell adhesion receptor from A549 human lung adenocarcinoma cells was isolated and characterized. A single protein peak was isolated by CSVTCG affinity chromatography which also analyzed as a single peak by anion exchange chromatography. The purified protein had a pI of 4.7 and analyzed on SDS-gels as a single band of M(r) = 50,000 under nonreducing conditions and as two protein bands of M(r) = 50,000, and 60,000 under reducing conditions. Purified CSVTCG binding protein (CBP) bound either CSVTCG- or TSP- Sepharose but showed little interaction with either VCTGSC- or BSA- Sepharose. CBP was cell surface exposed. CSVTCG derivatized with [125I] Bolton-Hunter reagent was taken up by cells in a dose-dependent manner and the cell association was inhibited with a monospecific polyclonal anti-CBP antibody. Examination of the cell proteins crosslinked to labeled CSVTCG by SDS-gel electrophoresis revealed one band that comigrated with purified CPB. Using an in vitro binding assay, purified CBP bound mannose, galactose, and glucosamine-specific lectins. CBP bound TSP saturably and reversibly. The binding was Ca+2/Mg+2 ion dependent and inhibited with fluid phase TSP and anti-CBP. Little or no binding was observed on BSA, fibronectin, GRGES, and GRGDS. Heparin, but not lactose, inhibited binding. Anti-CBP IgG and anti-CSVTCG peptide IgG inhibited A549 cell spreading and adhesion on TSP but not on fibronectin and laminin. These results indicate that CBP and the CSVTCG peptide domain of TSP can mediate TSP-promoted tumor cell adhesion.     

Thrombin-Stimulated Calcium Mobilization Is Inhibited by Thrombospondin via CD36

Activation of the G-protein-linked thrombin receptor in endothelial cells normally leads to an increase in free intracellular calcium, [Ca²⁺]_i, which is the proximate stimulus for many important cell functions. We present evidence showing that signals from CD36, the thrombospondin (TSP) receptor, can inhibit this thrombin-mediated calcium response. Human endothelial cells preloaded with Indo-1 exhibited rapid calcium mobilization in response to thrombin. The presence of TSP inhibited the thrombin-stimulated calcium response in CD36-positive microvascular endothelial cells but not in CD36-negative umbilical vein endothelial cells. This TSP effect was mimicked by anti-CD36 antibodies and a TSP peptide (CSVTCG), but not by an alternative CD36 ligand (collagen IV) or an antibody to an alternative TSP receptor (α_vβ₃). TSP also inhibited the calcium response to the thrombin receptor-tethered ligand peptide, SFLLRN. In addition, TSP and anti-CD36 antibodies inhibited the calcium response of a closely related receptor, the trypsin/SLIGKVD-activated receptor PAR-2. TSP did not indiscriminately inhibit all calcium release pathways, since histamine- or VEGF-stimulated calcium responses were not inhibited by TSP. We conclude that cross-talk from the CD36 receptor influences the responsive state of the endothelial thrombin receptor family and/or its signaling pathway.     

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

CD36 (glycoprotein IV or IIIB) is an integral plasma membrane protein of wide cellular distribution and functions as a receptor site for thrombospondin (TSP), an adhesive protein important in cell-cell and cell-matrix interactions. OKM5, a monoclonal anti-CD36 antibody, has been reported to block CD36 cell adhesive functions suggesting that the OKM5 epitope on CD36 is functionally important. A panel of 10 synthetic CD36 peptides was made. One peptide, P139-155, specifically inhibited the immunoadsorption of CD36 by OKM5, and P139-155 was directly immunoadsorbed by OKM5, indicating that CD36 sequence 139-155 represents part of the OKM5 epitope. TSP bound to immobilized P139-155 in a dose-dependent and saturable manner. Surprisingly, P139-155 significantly augmented, instead of inhibited, binding of CD36 to TSP. This peptide did not induce platelet aggregation but augmented ADP- and collagen-induced aggregation in platelet-rich plasma. Another CD36 peptide, P93-110, which had no effect on OKM5 immunoadsorption, blocked binding of CD36 to immobilized TSP and partially inhibited collagen-induced platelet aggregation. P93-110 by itself did not bind to TSP; however, in the presence of P139-155, there was a marked enhancement of P93-110 binding to TSP, with a stoichiometry consistent with the trimeric nature of TSP. The data suggest that CD36-TSP interaction is a two-step process; the sequence 139-155 region of CD36 binds first to TSP, triggering a change in TSP to reveal a second site, which binds the 93-110 region of CD36 with high affinity. CD36 peptides can be used as stimulators or inhibitors in cellular adhesive events involving TSP-CD36 interaction. Conformational changes leading to the exposure or activation of high affinity binding sites may occur in both the receptor and the ligand upon cell-cell and cell-matrix adhesion.     

CD36 mediates binding of soluble thrombospondin-1 but not cell adhesion and haptotaxis on immobilized thrombospondin-1

In this study, we examined the binding of soluble TSP1 (and ox-LDL) to CD36-transfected cells and the mechanisms by which immobilized TSP1 mediated attachment and haptotaxis (cell migration towards a substratum-bound ligand) of these transfected cells. CD36 cDNA transfection of NIH 3T3 cells clearly induced a dramatic increase in binding of both soluble [¹²⁵I]-TSP1 and [¹²⁵I]-ox-LDL to the surface of CD36-transfected cells, indicating that there was a gain of function with CD36 transfection in NIH 3T3 cells. Despite this gain of function, mock- and CD36-transfected NIH 3T3 cells attached and migrated to a similar extent on immobilized TSP1. An anti-TSP1 oligoclonal antibody inhibited CD36-transfected cell attachment to TSP1 while function blocking anti-CD36 antibodies, alone or in combination with heparin, did not. A series of fusion proteins encompassing cell-recognition domains of TSP1 was then used to delineate mechanisms by which NIH 3T3 cells adhere to TSP1. Although CD36 binds soluble TSP1 through a CSVTCG sequence located within type 1 repeats,^18,19 CD36-transfected NIH 3T3 cells did not attach to immobilized type 1 repeats while they did adhere to the N-terminal, type 3 repeats (in an RGD-dependent manner) and the C-terminal domain of TSP1. Conversely, Bowes melanoma cells attached to type 1 repeats and the N- and C-terminal domains of TSP1. However, CD36 cDNA transfection of Bowes cells did not increase cell attachment to type 1 repeats compared to that observed with mock-transfected Bowes cells. Moreover, a function blocking anti-CSVTCG peptide antibody did not inhibit the attachment of mock- and CD36-transfected Bowes cells to type 1 repeats. It is suggested that CD36/TSP1 interaction does not occur upon cell–matrix adhesion and haptotaxis because TSP1 undergoes conformational changes that do not allow the exposure of the CD36 binding site. © 1998 John Wiley & Sons, Ltd.     

Plasmodium falciparum-infected erythrocyte receptor(s) for CD36 and thrombospondin are restricted to knobs on the erythrocyte surface.

Adherence of Plasmodium falciparum-infected RBCs (PRBC) to endothelial cells causes PRBC sequestration in cerebral microvessels and is considered to be a major contributor to the pathogenesis of cerebral malaria. Both CD36 and thrombospondin (TSP) are glycoproteins that mediate PRBC adherence to endothelial cells in vitro. Because they are both expressed on the surface of endothelial cells, they probably contribute to PRBC sequestration and vascular occlusion in vivo. By applying affinity labeling of receptor binding sites with purified ligands, we showed for the first time that both CD36 and TSP can bind independently to the PRBC surface and that the PRBC receptor(s) for CD36 and TSP are localized specifically to the electron-dense knob protrusions of the PRBC surface. These findings may help in efforts to develop a malaria vaccine to prevent cerebral malaria.     

The anti-adhesive activity of thrombospondin is mediated by the N-terminal domain of cell surface calreticulin

Thrombospondin (TSP) induces reorganization of the actin cytoskeleton and restructuring of focal adhesions through binding of amino acids (aa) 17-35 (hep I peptide) of thrombospondin to a cell surface form of calreticulin (CRT). In this report we provide further evidence for the involvement of calreticulin in thrombospondin signaling and characterize thrombospondin-calreticulin interactions. Wild type but not crt(-/-) cells respond to hep I/TSP. Responsiveness can be restored by incubation of cells with exogenous calreticulin or by transfection with calreticulin. Thrombospondin forms complexes with the CRT-N-domain that are enhanced by physiologic levels of calcium and zinc. Consistent with thrombospondin/CRT-N-domain binding, only the CRT-N-domain blocks hep I- and thrombospondin-stimulated focal adhesion disassembly. A series of glutathione S-transferase-N-domain mutants were used to map the sequence within the N-domain that interacts with TSP/hep I. A construct containing aa 1-43 but not a construct of aa 1-31 supported thrombospondin binding and focal adhesion disassembly. A series of overlapping peptides were used to further map the thrombospondin-binding site. Peptides spanning aa 19-36 (RWIESKHKSDFGKFVLSS) blocked hep I-stimulated focal adhesion disassembly, indicating that the TSP/hep I-binding site is located to this sequence in calreticulin. A mutant fusion protein lacking aa 19-36 (glutathione S-transferase-CRTDeltahep I) failed to restore responsiveness to hep I in crt(-/-) cells, bind thrombospondin, or competitively block focal adhesion disassembly, providing evidence for the role of this calreticulin sequence in mediating thrombospondin signaling.     

Molecular mechanistic insights into the endothelial receptor mediated cytoadherence of Plasmodium falciparum-infected erythrocytes

Cytoadherence or sequestration is essential for the pathogenesis of the most virulent human malaria species, Plasmodium falciparum (P. falciparum). Similar to leukocyte-endothelium interaction in response to inflammation, cytoadherence of P. falciparum infected red blood cells (IRBCs) to endothelium occurs under physiological shear stresses in blood vessels and involves an array of molecule complexes which cooperate to form stable binding. Here, we applied single-molecule force spectroscopy technique to quantify the dynamic force spectra and characterize the intrinsic kinetic parameters for specific ligand-receptor interactions involving two endothelial receptor proteins: thrombospondin (TSP) and CD36. It was shown that CD36 mediated interaction was much more stable than that mediated by TSP at single molecule level, although TSP-IRBC interaction appeared stronger than CD36-IRBC interaction in the high pulling rate regime. This suggests that TSP-mediated interaction may initiate cell adhesion by capturing the fast flowing IRBCs whereas CD36 functions as the 'holder' for providing stable binding.     

A CD36-binding peptide from thrombospondin-1 can stimulate resorption by osteoclasts in vitro

Thrombospondin-1 (TSP-1), purified from platelets, stimulates resorption by avian osteoclasts in an in vitro resorption assay. TSP-1 binds to a number of different cellular receptors via different domains of the molecule and several short receptor-binding sequences have been identified within the TSP-1 molecule. In this study, we have used synthetic peptides representing these various sequences in order to identify the cellular receptor and TSP domain responsible for stimulation of resorption. We show that one peptide CSVTCG, which represents the CD36-binding region of TSP-1, stimulates resorption in a fashion similar to the intact molecule, while the peptides RGDS, RFYVVMWK, and RFYVVM, representing other cell-binding domains of TSP, have no effect on resorption. Using RT-PCR and immunoblotting, we further demonstrate expression of CD36 in human osteoclastoma (giant cell tumour), primary human bone derived cells, and clonal osteoblastic cells. These studies suggest that CD36 is involved in regulation of resorption by osteoclasts and is the receptor responsible for the resorption-promoting effects of TSP-1.     

Sense and antisense cDNA transfection of CD36 (glycoprotein IV) in melanoma cells. Role of CD36 as a thrombospondin receptor.

Thrombospondin (TSP) is a multifunctional matrix and platelet glycoprotein that interacts with cell surfaces and may play a role in mediating cell adhesion, platelet aggregation, platelet-monocyte interactions, cell proliferation, angiogenesis, tumor metastasis, and protease generation. To clarify and confirm the function of CD36 (glycoprotein IV) as a TSP receptor, we now describe a transfected cell model using human melanoma cells genetically manipulated by sense or antisense cDNA transfection to express either high or near zero levels of CD36. Surface expression was confirmed by flow cytometry with monoclonal anti-CD36 IgG and quantified by measuring radiolabeled antibody binding. Bowes melanoma cells, which in their wild type did not express CD36 and did not bind radiolabeled TSP, when transfected with the sense construct bound TSP in a 1:1 stoichiometric ratio with CD36 expression. Conversely, C32 melanoma cells, which in their wild type expressed high levels of CD36 and bound radiolabeled TSP at a 1:1 stoichiometric ratio, did not express CD36 and did not bind TSP when transfected with an antisense construct. In addition, transfected Bowes cells and wild type C32 cells, unlike wild type Bowes cells, adhered to activated platelets in a TSP-dependent manner. These data, i.e. the gain of function with sense cDNA transfection and loss of function with antisense transfection, strongly support the TSP receptor function of CD36. The distribution of this protein in vascular cells and tissues and observations that it may participate in signal transduction events suggest that TSP-CD36 interactions may play a role in mediating some of the pathophysiological processes associated with TSP.     

Novel CD47-dependent intercellular adhesion modulates cell migration

CD47 is a ubiquitously expressed plasma membrane protein, also known as Integrin Associated Protein, that modulates cell adhesion both through alteration of the avidity of integrin binding and through interaction with its own ligands, the extracellular matrix protein thrombospondin (TSP) and the plasma membrane response regulator SIRPalpha1. We now show that CD47 expression on fibroblasts can induce intercellular adhesion resulting in cell aggregation in the absence of active integrins, SIRPalpha1 binding, and detectable TSP. CD47-expressing cells preferentially bind to other CD47-expressing cells, and intercellular adhesion requires stimulation by serum or a CD47-binding peptide from TSP. Cell-cell adhesion is inhibited by pertussis toxin and C. difficile toxin B, and both adherent and aggregating CD47-expressing fibroblasts have more rac in the GTP bound state than CD47-deficient cells. Spontaneous migration of Jurkat lymphocytes through a fibroblast monolayer is decreased by fibroblast expression of CD47, consistent with an increased barrier function of the CD47 expressing cells. The lymphocyte chemoattractant SDF-1alpha stimulates migration of Jurkat cells through this monolayer only if both the lymphocytes and fibroblasts express CD47, and the inhibition of migration by a CD47-interacting peptide from TSP similarly requires CD47 expression on both cell types. Thus, signaling dependent on both heterotrimeric and rho family GTPases can induce CD47 to participate in cell-cell interactions independent of known ligands that enhance intercellular adhesion and modulate cell migration.     

CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes

Erythrocytes infected with P. falciparum express knob-like adhesion structures that allow the infected cells to cling to the postcapilliary endothelium of characteristic host organs. At present, the mechanism of cytoadherence is not fully understood. While parasitized erythrocytes have been shown to specifically bind to the platelet/matrix molecule thrombospondin, adherence to suitable target cells can also be blocked by monoclonal antibody OKM5, which recognizes a surface molecule expressed by hematopoietic cells and endothelium. In apparent reconciliation of these findings, it has been reported that the OKM5 antigen (CD36) is a receptor for thrombospondin. Here we report that expression of a CD36 cDNA clone in COS cells supports cytoadherence of parasitized erythrocytes but does not support increased binding of purified human thrombospondin.     

Cell contact-dependent activation of alpha3beta1 integrin modulates endothelial cell responses to thrombospondin-1

Thrombospondin-1 (TSP1) can inhibit angiogenesis by interacting with endothelial cell CD36 or proteoglycan receptors. We have now identified alpha3beta1 integrin as an additional receptor for TSP1 that modulates angiogenesis and the in vitro behavior of endothelial cells. Recognition of TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 by normal endothelial cells is induced after loss of cell-cell contact or ligation of CD98. Although confluent endothelial cells do not spread on a TSP1 substrate, alpha3beta1 integrin mediates efficient spreading on TSP1 substrates of endothelial cells deprived of cell-cell contact or vascular endothelial cadherin signaling. Activation of this integrin is independent of proliferation, but ligation of the alpha3beta1 integrin modulates endothelial cell proliferation. In solution, both intact TSP1 and the alpha3beta1 integrin-binding peptide from TSP1 inhibit proliferation of sparse endothelial cell cultures independent of their CD36 expression. However, TSP1 or the same peptide immobilized on the substratum promotes their proliferation. The TSP1 peptide, when added in solution, specifically inhibits endothelial cell migration and inhibits angiogenesis in the chick chorioallantoic membrane, whereas a fragment of TSP1 containing this sequence stimulates angiogenesis. Therefore, recognition of immobilized TSP1 by alpha3beta1 integrin may stimulate endothelial cell proliferation and angiogenesis. Peptides that inhibit this interaction are a novel class of angiogenesis inhibitors.     

Cellular attachment to thrombospondin. Cooperative interactions between receptor systems.

Tumor cell attachment to thrombospondin (TSP) in the extracellular matrix may be of critical importance in the processes of invasion and hematogenous dissemination. To determine the specific receptor systems that mediate the interaction of tumor cells with insoluble TSP, the attachment of HT1080 fibrosarcoma and C32 and G361 melanoma cells to TSP-coated discs was studied in the presence of heparin, Arg-Gly-Asp-Ser, or antibodies to glycoprotein (GP) IV (CD36, GPIIIb), a TSP receptor. HT1080 and C32 cell attachment to TSP was inhibited by the combination of heparin and a monoclonal (or polyclonal) antibody to GPIV but not by either alone. Heparin alone inhibited cell spreading. Neither control monoclonal antibodies nor the cell attachment peptide Arg-Gly-Asp-Ser inhibited tumor cell attachment to TSP, alone or in the presence of heparin. HT1080 cells attached equally as well to a 140-kDa proteolytic TSP fragment lacking the heparin-binding domain as to intact TSP. A monoclonal antibody to GPIV alone inhibited tumor cell attachment to the heparin-domainless 140-kDa TSP fragment. No attachment to the heparin-binding fragment was observed, but the addition of the heparin fragment to 140-kDa heparin-domainless TSP restored the heparin sensitivity of binding. G361 cells that lack GPIV attached well to TSP but were not inhibited by heparin or anti-GPIV alone or in combination. The combination of heparin and Arg-Gly-Asp-Ser inhibited G361 attachment to TSP. These studies suggest that tumor cells may utilize separate receptor systems in a cooperative manner to adhere to TSP. HT1080 fibrosarcoma and C32 melanoma cells utilize GPIV in concert with a heparin-modulated binding systems to attach and spread on TSP. G361 cells, which lack GPIV expression, attach and spread on TSP using an integrin system as well as a heparin-modulated system.     

Central role of thrombospondin-1 in the activation and clonal expansion of inflammatory T cells

Thrombospondin-1 (TSP) is a transiently expressed matricellular protein known to promote chemotaxis of leukocytes to inflammatory sites. However, TSP and its receptor CD36 are abundantly expressed in chronically inflamed tissues such as the rheumatoid synovium. Here, we show that TSP provides the costimulatory signal that is necessary for the activation of autoreactive T cells. Data presented reveal that TSP-mediated costimulation is achieved through its independent interaction with CD36 on APCs and with CD47 on T cells. We propose that a CD47-TSP-CD36 trimolecular complex is a novel costimulatory pathway that significantly decreases the threshold of T cell activation. Consistent with the paradigm that lesions in rheumatoid synovitis are sites of antigenic recognition, the characteristic focal expression of TSP on APCs such as macrophages and fibroblast-like synoviocytes suggest a central role of TSP in the expansion of tissue-infiltrating T cells.     

Heparan sulfate-mediated binding of epithelial cell surface proteoglycan to thrombospondin

Purified NMuMG mouse mammary epithelial cell surface proteoglycan (PG), a membrane-intercalated core protein bearing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains, binds to a thrombospondin (TSP) affinity column and is eluted by a salt gradient. Double immunofluorescence microscopy demonstrates extensive co-localization of bound exogenous TSP and cells bearing exposed cell surface PG at their apical surface. The binding, as assayed by both methods, is heparitinase-sensitive, but not chondroitinase-sensitive. Alkali-released heparan sulfate chains bind to a TSP affinity column, similarly to native PG, whereas the chrondroitin sulfate chains do not. Core protein does not bind to TSP. These results indicate that NMuMG cells bind TSP via their surface PG and that the binding is mediated by the heparan sulfate chains.     

Biological activities of peptides and peptide analogues derived from common sequences present in thrombospondin, properdin, and malarial proteins

Thrombospondin (TSP), a major platelet-secreted protein, has recently been shown to have activity in tumor cell metastasis, cell adhesion, and platelet aggregation. The type 1 repeats of TSP contain two copies of CSVTCG and one copy of CSTSCG, per each of the three polypeptide chains of TSP and show homology with peptide sequences found in a number of other proteins including properdin, malarial circumsporozoite, and a blood-stage antigen of Plasmodium falciparum. To investigate whether these common sequences functioned as a cell adhesive domain in TSP, we assessed the effect of peptides corresponding to these sequences and an antibody raised against one of these sequences, CSTSCG, in three biological assays which depend, in part, on the cell adhesive activity of TSP. These assays were TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis. We found that a number of peptides homologous to CSVTCG promoted the adhesion of a variety of cells including mouse B16-F10 melanoma cells, inhibited platelet aggregation and tumor cell metastasis, whereas control peptides had no effect. Anti-CSTSCG, which specifically recognized TSP, inhibited TSP-dependent cell adhesion, platelet aggregation, and tumor cell metastasis, whereas control IgG had no effect. These results suggest that CSVTCG and CSTSCG present in the type I repeats function in the adhesive interactions of TSP that mediate cell adhesion, platelet aggregation, and tumor cell metastasis. Peptides, based on the structure of these repeats, may find wide application in the treatment of thrombosis and in the prevention of cancer spread.     

Thrombospondin binds normally to glycoprotein IIIb deficient platelets.

Glycoprotein IIIb (GPIV, CD36) has been proposed as the platelet receptor for thrombospondin (TSP). We found two healthy blood donors, whose platelets were shown to be GPIIIb deficient. These platelets expressed endogeneous TSP as control platelets and their binding capacity for exogeneous TSP was the same. These results indicate that GPIIIb is not the major TSP receptor on platelets. However, it is not yet possible to exclude that in GPIIIb-deficient platelets other proteins may substitute for GPIIIb in TSP binding.     

Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor

Components of the extracellular matrix have been shown to modulate the interaction of endothelial cells with their microenvironment. Here we report that thrombospondin (TSP), an extracellular matrix component, induces adhesion and spreading of murine lung capillary (LE-II) and bovine aortic (BAEC) endothelial cells. This TSP-induced spreading was inhibited by heparin and fucoidan, known to bind the amino-terminal globular domain of the molecule. In addition, endothelial cells were induced to migrate by a gradient of soluble TSP (chemotaxis). The chemotactic response was inhibited by heparin and fucoidan, as well as by the mAb A2.5, which also binds to the amino-terminal domain. These data are in agreement with our previous observation that the TSP aminoterminal heparin binding region is responsible for the induction of tumor cell spreading and chemotactic motility. The inhibition of chemotaxis and spreading by antibodies against the beta 3 but not the beta 1 chain of the integrin receptor points to a role for the integrins in the interaction of endothelial cells with TSP. We also found that TSP modulates endothelial cell growth. When added to quiescent LE-II cells, it inhibited the mitogenic effects of serum and the angiogenic factor bFGF, in a dose-dependent manner. The inhibition of DNA synthesis detected in the mitogenic assay resulted in a true inhibition of BAEC and LE-II cell growth, as assessed by proliferation assay. This work indicates that TSP affects endothelial cell adhesion, spreading, motility and growth. TSP, therefore, has the potential to modulate the angiogenic process.     

Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat.

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.     

Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics.

Endothelial and other cell types synthesize thrombospondin (TSP), secrete it into their culture medium, and incorporate it into their extracellular matrix. TSP is a large multifunctional protein capable of specific interactions with other matrix components, as well as with cell surfaces, and can modulate cell adhesion to the extracellular matrix. With the aim of understanding the mechanism by which TSP exerts its effect on cell adhesion, we studied the interaction of endothelial cell TSP (EC-TSP) with three different cell types: endothelial cells, granulosa cells, and myoblasts. We find that endothelial cells specifically bind radiolabeled EC-TSP with a Kd of 25 nM, and the number of binding sites is 2.6 X 10(6)/cell. Binding is not inhibitable by the cell-adhesion peptide GRGDS, indicating that the cell-binding site of EC-TSP is not in the RGD-containing domain. Localization of the cell-binding site was achieved by testing two chymotryptic fragments representing different regions of the TSP molecule, the 70-kDa core fragment and the 27-kDa N-terminal fragment, for their ability to bind to the cells. Cell-binding capacity was demonstrated by the 70-kDa fragment but not by the 27-kDa fragment. Binding of both intact [125I]EC-TSP and of the 125I-labeled 70-kDa fragment was inhibited by unlabeled TSP, heparin, fibronectin (FN), monoclonal anti-TSP antibody directed against the 70-kDa fragment (B7-3), and by full serum, but not by heparin-absorbed serum or the cell-adhesion peptide GRGDS. The 70-kDa fragment binds to endothelial cells with a Kd of 47 nM, and the number of binding sites is 5.0 x 10(6)/cell.(ABSTRACT TRUNCATED AT 250 WORDS)