Publications

Thrombospondin is an adhesive glycoprotein that promotes breast cancer cell adhesion to human vascular endothelial cells (Incardona et al., 1995). In this study, we have identified the molecular domains of thrombospondin that mediate its binding to specific receptors on the human breast adenocarcinoma cell line, MDA-MB-231. Two recombinant fragments from the amino-terminus (TSPN18 and TSPN28), and the fusion proteins of the type 1 and type 2 repeats of human thrombospondin, inhibited binding of radiolabeled thrombospondin to MDA-MB-231 cells in suspension by 40-60% at 50 micrograms/ml whereas the type 3 repeat, carboxy-terminus and unfused glutathione-S-transferase as well as the synthetic peptide Gly-Arg-Gly-Asp-Ser (500 micrograms/ml) had little or no effect. Heparin and various glycosaminoglycans as heparan sulfate, chondroitin sulfates A, B or C, and fucoidan inhibited thrombospondin binding to MDA-MB-231 cells by more than 60% whereas dextran sulfate had only little effect. Treatment of cells with heparitinase, chondroitinase ABC, and hyaluronidase, but not with neuraminidase, induced 30-50% inhibition of thrombospondin binding suggesting the participation of both heparan sulfate and chondroitin sulfate cell surface-associated molecules. Inhibition of proteoglycan sulfation by chlorate or inhibition of glycosaminoglycan chain formation by two beta-D-xylosides also led to a substantial inhibition of thrombospondin binding. Our results indicate that several domains within the thrombospondin molecule, namely the amino-terminus, type 1 and type 2 repeats, participate in its binding to specific receptors bearing sulfated glycosaminoglycans on MDA-MB-231 cells. Biological assays have indicated that, in addition to these domains, the peptide Gly-Arg-Gly-Asp-Ser inhibited MDA-MB-231 cell attachment to thrombospondin suggesting that the last type 3 repeat of the molecule may also contribute to its cell adhesive activity.

The thrombospondins are a family of extracellular calcium binding proteins that are involved in cell proliferation, adhesion, and migration. We have sequenced full-length human thrombospondin-4 and characterized the recombinant protein. In contrast to Xenopus laevis thrombospondin-4, the human protein contains an RGD cell binding sequence in the third type 3 repeat. Transfection of mouse NIH3T3 fibroblasts or C2C12 myoblasts with a full-length human thrombospondin-4 cDNA results in the expression of a polypeptide with a reduced molecular weight of 140,000. In the absence of reducing agent, the expressed protein has an apparent molecular weight of 550,000. Recombinant thrombospondin-4 has been purified from the culture supernatant by heparin-Sepharose and anti-thrombospondin-4 antibody-Affi-gel affinity chromatography. Electron microscopy indicates that thrombospondin-4 is composed of five subunits with globular domains at each end. The observation of a calcium-dependent change in the electron microscopic appearance of thrombospondin-4 is consistent with limited tryptic digestion data that indicate that thrombospondin-4 is resistant to digestion in the presence of calcium. These data indicate that thrombospondin-4 is a pentameric protein that binds to heparin and calcium.

The thrombospondins are a family of related glycoproteins found in the embryonic extracellular matrix. To date, five members of this family have been identified. Thrombospondin-1 and thrombospondin-2 have similar primary structure, but are expressed in different tissues at different times during development. Thrombospondins-3, -4, and cartilage oligomeric protein belong to a second thrombospondin subgroup in which the carboxyl-half of each molecule is most similar to thrombospondin-1 and -2. Here we report the cloning and sequencing of a novel probe to avian thrombospondin-4. We have used this probe to determine the origins of thrombospondin-4 in the chick embryo by in situ hybridization. Thrombospondin -4 transcripts first appear in the mesenchyme surrounding bone anlage coinciding with the initial stages of osteogenesis. The expression in osteogenic tissues is transient: thrombospondin-4 mRNAs are not seen in the osteoblasts of bone collars in developing long bones. This pattern is distinct from avian thrombospondin-2 which is expressed in perichondrium and embryonic fibrous connective tissues. Our observations indicate that connective tissues are the principal site of thrombospondin-4 expression in the chick. The diverse origins of different thrombospondin gene family members imply distinctive roles for these proteins related to the growth and differentiation of cartilage, tendons, and bone.

The thrombospondins (TSPs) are a family of 5 distinct gene products designated TSP1, -2, -3, -4, and COMP, for cartilage oligomeric matrix protein. TSP1, the prototypical member, is a trimeric extracellular matrix molecule implicated in cell migration and development. TSP1 trimer formation is mediated by interchain disulfide linkage involving two NH2-terminal cysteines. TSP3, a recent addition to the family, is a developmentally regulated heparin binding protein that is similar in sequence to the COOH terminus of TSP1 but has a distinct NH2 terminus. This has raised the question of the oligomeric nature of TSP3 and identification of the cysteine residues involved in oligomer formation. We demonstrate, using a combination of deletional and site-directed mutagenesis and rotary shadowing electron microscopy, that TSP3, like TSP4 and COMP, is a pentameric molecule. TSP3 is held together by interchain disulfide linkage involving just two cysteine residues, Cys-245 and Cys-248.

Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.

The thrombospondins (TSPs) are a growing family of cell surface and extracellular matrix molecules composed of multiple repeating elements. Thrombospondin 3 is a recently described member that possesses the calcium binding Type 3 repeats, has 4 epidermal growth factor receptor-like Type 2 repeats, a complete absence of the complement-like Type 1 repeats, and a distinct N terminus that has no significant homology to the other TSPs. Metabolic labeling and immunoprecipitation analysis of cells transfected with a TSP3 expression vector revealed it to be an oligomeric heparin binding protein present in both the cell layer and medium. Finally, a combination of in situ hybridization and immunocytochemistry demonstrated TSP3 to be expressed in a temporal and spatial manner during murine embryogenesis, especially in the gut, cartilage, lung, and central nervous system.

Thrombospondin (TSP) is a 450 kDa extracellular matrix glycoprotein expressed in normal, hyperplastic, and neoplastic human breast. In this study, the patterns of expression of TSP were determined during development of the human fetal mammary gland between the 15th and the 39th week of gestation. Using immunohistochemistry, TSP is found in the dense mesenchyme immediately adjacent to the mammary bud, and at the membrane of budding epithelial cells invading the surrounding mesenchyme. As formation of the ductal tree system occurs, TSP is deposited at the myoepithelial-stromal junction of mammary ducts. Such an immunolocalization of TSP in buds and ducts of the fetal mammary gland has been confirmed at the mRNA level using in situ hybridization. Presence of TSP transcripts in nascent breast tissue has been also demonstrated by polymerase chain reaction assay. Comparison of TSP immunolocalization with that of two known TSP cell surface receptors, CD36 and CD51, reveals no codistribution of TSP with these receptors during mammary gland development. As opposed to TSP, CD36 is strongly expressed at the membrane of preadipocytes present in the fat pad tissue, but absent from budding epithelial cells. CD51 is only weakly expressed by malpighian epithelial cells and does not colocalize with TSP. In lactating ducts of a newborn, TSP disappears from the myoepithelial-stromal junction of ducts and is synthesized at the apices of secretory epithelial cells of lactating ducts together with CD36. In conclusion, our findings support the existence of an important role for TSP during development of the human fetal mammary gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombospondin (TSP) is complexed with transforming growth factor-beta (TGF-beta) in the alpha-granules of stimulated platelets. TSP stripped of associated TGF-beta activity (sTSP) activates latent TGF-beta secreted by bovine aortic endothelial cells (BAE) in culture. To better understand the interactions of TSP with TGF-beta, we investigated which region of sTSP interacts with TGF-beta. The chymotrypsin-resistant core of TSP, which contains the procollagen-like region and the properdin-like type 1 repeats, activated both latent TGF-beta secreted by BAE and a recombinant form of the small latent TGF-beta complex at levels similar to or better than sTSP. The core fragment bound 125I-TGF-beta in solution and shifted the elution profile of 125I-TGF-beta in gel permeation chromatography. Fusion constructs of the type 1, 2, and 3 repeats and the COOH terminus of TSP1 were tested for their ability to activate latent TGF-beta. Only the type 1 construct, containing the three properdin-like repeats of TSP found in the 50-kDa fragment, activated latent TGF-beta. In addition, a polyclonal antibody against the type 1 construct inhibits activation of latent TGF-beta by intact TSP, suggesting that this region is exposed in the intact molecule. These results show that the type 1 properdin-like repeats of TSP are responsible for activating recombinant and endothelial cell-derived latent TGF-beta and that this site is exposed in intact TSP.

The vascular endothelium lines the entire cardiovascular system and serves as a nonthrombogenic and selectively permeable boundary between the blood-stream and extravascular space. Endothelial cells are polar cells that are continuously subjected to fluid-generated forces on their luminal surface whereas their abluminal surface resides on basement membranes/extracellular matrix. The integrin family of cell-surface heterodimeric glycoproteins is located along both of these surfaces and participates in maintaining the normal endothelium and in the dynamic changes associated with the pathophysiology of the endothelium. Endothelial cell beta 1 and beta 3 integrins function together with other families of adhesion molecules during vasculogenesis, angiogenesis, inflammation, and wound healing. Leukocyte beta 1 and beta 2 integrins, in conjunction with members of the Ig and selectin gene families expressed on endothelium, mediate leukocyte recruitment to sites of inflammation. The structural and functional properties of integrins make them uniquely suited to mediate these essential and complex processes in the vasculature.