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Using a series of fusion proteins that span almost all of the thrombospondin-1 (TSP-1) molecule, we observed in this study that Chinese hamster ovary (CHO) K1 cells strongly attached to the N-terminus but not to the other domains of TSP-1 (e.g. the C-terminus, and type 1, type 2 and type 3 repeats). In addition, attachment to the N-terminus of CHO S745 cells defective in cell-surface glycosaminoglycans (GAGs) was decreased by 47% compared with that observed with CHO K1 cells, indicating the presence of GAG-dependent cell adhesive sites. With the aim of identifying these cell adhesive sites, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), MKKTRG (residues 79-84) and TRDLASIARLRIAKGVNDNF (residues 170-189), were synthesized and tested for their ability to support CHO cell attachment. Using both centrifugation and cell-attachment assays, MKKTRG-containing peptides promoted CHO K1 cell adhesion, while ARKGSGRR-containing peptides and peptide TRDLASIARLRIAKGVNDNF did not. CHO S745 cell attachment to MKKTRG-containing peptides was partially decreased. A 36% decrease in CHO K1 cell attachment to the N-terminus was also observed when the heparin-binding consensus sequence KKTR was mutated to QNTR. In addition, peptide MKKTRG partially inhibited (25% inhibition) CHO K1 cell attachment to the N-terminus. However, peptide MKKTRG was not sufficient to fully promote cell attachment to the N-terminus of TSP-1. Peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS also supported CHO K1 cell attachment in a GAG-dependent and -independent manner respectively. Moreover, CHO K1 cell attachment to MKKTRG was found to be markedly enhanced when flanked with the sequences VDAVRTEKGFLLLASLRQ and TLLALERKDHS. Peptide VDAVRTEKGFLLLASLRQMKKTRG nearly abolished (98% inhibition) CHO K1 cell attachment to the N-terminus, while peptides MKKTRG, MKKTRGTLLALERKDHS and VDAVRTEKGFLLLASLRQ had only a moderate inhibitory effect (25, 27 and 53% inhibition respectively). These data indicate that the sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS (residues 60-94) constitutes a GAG-dependent cell adhesive site in the N-terminus of TSP-1. Moreover, a GAG-independent site, encompassing residues 189-200 (FQGVLQNVRFVF), has been identified. These two adhesive sites supported the attachment of a wide variety of cells (human breast carcinoma, melanoma and osteosarcoma cells), and a high degree of sequence homology was found between TSP-1 and TSP-2 between residues 60 and 94 (48% identity) and 189-200 (67% identity), further suggesting the functional importance of these two cell adhesive sites in the N-terminus of TSP-1.

Thrombospondin-1 (TSP1) is a Mr 450,000 extracellular matrix glycoprotein that modulates tumor growth, angiogenesis, and metastasis. Of the five structurally different TSPs described to date, only TSP2 is similar to TSP1 in terms of its molecular architecture, and TSP2 also modulates angiogenesis. Angiogenesis plays a relevant role in the biological aggressiveness of breast cancer, and TSP1 is present in the tumor stroma (termed desmoplasia) of invasive human breast ductal carcinoma not otherwise specified (NOS). The present study was designed to identify and quantify TSP1 and TSP2 mRNAs in normal, benign, and neoplastic human breast tissues using the reverse transcriptase PCR technique. We found that TSP2, like TSP1, was expressed in human breast tissues, and that TSP1 and TSP2 mRNA expression in invasive breast carcinoma NOS was significantly increased compared to that observed in normal and benign tissues. The expression of TSP1 and TSP2 in invasive breast ductal carcinoma NOS did not significantly correlate with any of the prognostic factors studied (tumor size, lymph node status, morphology, and hormone receptor status). However, when our study population was divided according to the quantity of tumor stroma, TSP1 (and possibly TSP2) mRNA expression and microvessel counts in desmoplastic-rich stroma of breast carcinoma NOS were significantly increased compared to those observed in desmoplastic-poor stromata.

The ultrastructural distribution of thrombospondin (TSP) and its cell surface receptor, integrin alpha V, was studied in two cases of human breast carcinoma: one of ductal carcinoma in situ (DCIS) with an invasive component, and one of invasive lobular carcinoma. In DCIS, moderate immunolabelling for TSP and integrin alpha V was observed in the rough endoplasmic reticulum and at the plasma membrane of intraductal carcinoma cells. TSP was also associated with extracellular matrix collagen fibrils surrounding in situ carcinoma cells. In the invasive part of this ductal carcinoma, most of the malignant cells were negative for TSP, while integrin alpha V was moderately expressed in these cells. In sharp contrast, typical strands of invasive lobular carcinoma cells in "Indian file" showed moderate TSP immunostaining in the rough endoplasmic reticulum and strong immunoreactivity for TSP at the plasma membrane and in the extracellular matrix. Moderate to strong immunoreactivity for integrin alpha V was also observed in invasive lobular carcinoma cells. Because of the role of TSP during cancer cell invasion, the different expression patterns of TSP in invasive ductal versus lobular carcinoma may well reflect biological differences between these two types of breast carcinoma and could account for the peculiar diffuse invasive behaviour of breast lobular carcinoma cells.

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 a 450-kDa extracellular matrix glycoprotein which supports the growth of human MG-63 osteoblastic cells [Abbadia et al., FEBS Lett., 329 (1993) 341-346]. In this study, we describe the effect of the glucocorticoid, dexamethasone, on cell proliferation and TSP expression by MG-63 cells. Using a serum-free mitogenesis assay, dexamethasone (25 to 500 nM) caused a dose-dependent decrease in [3H]thymidine incorporation by MG-63 cells in culture, reaching 40% inhibition of cell proliferation at a concentration of 250 nM. Similarly, the stimulatory effect of TSP (500 ng/ml) on proliferation of MG-63 cells was totally abolished in the presence of dexamethasone (250 nM). In situ hybridization indicated that TSP mRNA level in dexamethasone-treated MG-63 cells decreased compared to quiescent cells. As judged by fluorescence-activated cell sorting analysis, dexamethasone treatment of MG-63 cells resulted in a 50 to 70% decrease in TSP cell surface expression compared to quiescent cells. Secretion of TSP in the culture fluid of dexamethasone-treated MG-63 cells also decreased by 40% while, under similar experimental conditions, a 180% increase in alkaline phosphatase activity was observed in dexamethasone-treated cells. Because glucocorticoids induce osteoporosis in vivo and reduce proliferation of osteoblasts in vitro, our results argue for an important role of TSP during bone formation.

Thrombospondin (TSP) is a 450-kDa glycoprotein synthesized and secreted by human MG-63 osteoblastic cells. In this study, we have first studied the effect of alpha-thrombin on TSP expression by human MG-63 cells. In situ hybridization indicated that TSP mRNA level in thrombin-treated MG-63 cells was increased when compared to unstimulated cells. As judged by immunofluorescence, thrombin-treatment of MG-63 cells resulted in increased cell surface expression of TSP when compared to quiescent cells. Because thrombin stimulates proliferation of osteoblastic cells, the involvement of TSP in proliferation of thrombin-stimulated osteoblastic cells was then investigated using a serum-free mitogenesis assay. Both alpha-thrombin (0.01 to 0.15 U/ml) and TSP (5 to 600 ng/ml) caused a dose-dependent increase in [3H]thymidine incorporation by MG-63 cells. Proliferation of osteoblastic cells induced by alpha-thrombin or TSP was specifically and totally inhibited by anti-TSP monoclonal antibodies (3-10 micrograms/ml) or by indomethacin (1 microM), an inhibitor of prostaglandin synthesis. Anti-TSP antibodies which inhibited cell proliferation also inhibit TSP expression to the surface of these cells. Our experiments support the existence of a mechanism whereby TSP bound to the cell surface of thrombin-treated MG-63 cells stimulates secretion of prostaglandins which, in turn, allow cell proliferation to proceed.

In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets. Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts. SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells. Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen. Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen. Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.

Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (Kd) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (Kd = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81% inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calcium-dependent as shown by a 50-80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A--Sepharose 4B. Elution of the anti-thrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.