Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.

Regulation of tie receptor expression on human endothelial cells by protein kinase C-mediated release of soluble tie.

The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.

Thrombospondin mediates migration and potentiates platelet-derived growth factor-dependent migration of calf pulmonary artery smooth muscle cells.

A precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet-derived growth factor (PDGF)-mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin-binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an alpha v-containing integrin receptor, but not alpha v beta 1 or alpha v beta 3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50-100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation.

Activated T-cell adhesion to thrombospondin is mediated by the alpha 4 beta 1 (VLA-4) and alpha 5 beta 1 (VLA-5) integrins.

T lymphocytes utilize adhesion receptors in a regulated manner to interact with other cells and with components of the extracellular matrix. These cell-cell and cell-matrix interactions serve a critical role in T cell recognition of foreign Ag and in the migration of T cells to various anatomic sites in vivo. Thrombospondin is an extracellular matrix protein that is transiently expressed at high concentration in damaged and inflamed tissue. Given recent evidence implicating a role for the extracellular matrix in modulating T-cell migration and function, we analyzed T-lymphocyte interactions with thrombospondin. We show here that CD4+ T cells specifically adhere to thrombospondin predominantly via the 70 kDa core region of the thrombospondin molecule. Antibody blocking and affinity chromatography analysis suggest that T-cell adhesion to thrombospondin involves three distinct receptors: an activation-independent receptor that mediates adhesion of resting T cells, and the alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate a rapid increase in adhesion to thrombospondin upon activation. These three molecules appear to be novel thrombospondin receptors, as other receptors previously implicated in the adhesion of nonlymphoid cells to thrombospondin appear not to be involved in T-cell/thrombospondin interactions. The up-regulation of alpha 4 beta 1 and alpha 5 beta 1 functional activity upon activation is associated with the preferential adhesion of memory T cells to thrombospondin. Our results thus define three novel thrombospondin receptors, and provide additional evidence that extracellular matrix proteins play an important role in lymphocyte migration into, and retention in, inflammatory sites.

Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains.

The interactions of tumorigenic cells with the extracellular matrix play a critical role in the establishment of metastases. Thrombospondin (TSP) is prominent at sites of tissue injury and promotes the attachment, spreading, and motility of several cell types. We have investigated the relationship between human carcinoma cell metastatic potential and TSP-mediated cell motility by comparing highly metastatic 11B carcinoma cells with a nonmetastatic counterpart, 22B carcinoma cells. 11B cells demonstrated motility in response to soluble TSP with a maximal effect observed at 1 microM TSP. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. Monoclonal antibody C6.7, specific for the distal COOH terminus of TSP, inhibited chemotaxis by 60%. Studies with TSP fragments demonstrated that the M(r) 140,000 COOH-terminal domain (140K) supported chemotaxis to the same extent as intact TSP. The NH2-terminal heparin-binding domain was ineffective in stimulating chemotaxis. Substrate-bound TSP also elicited 11B cell motility with a maximal response at 100 nM TSP. Directionality of this response was confirmed by checkerboard analysis. Interestingly, as in chemotaxis, haptotaxis was mediated exclusively by 140K as demonstrated by TSP fragment studies and inhibition with monoclonal antibody C6.7. Therefore, 140K appeared to mediate both chemotaxis and haptotaxis. Compared with 11B cells, 22B carcinoma cells are nonmetastatic and synthesize and secrete low levels of TSP. Immunoprecipitation and Northern blot analysis confirmed that 11B cells expressed much higher levels of TSP than 22B cells. Although 22B cells are able to attach to TSP, they did not exhibit either chemotaxis or haptotaxis in response to TSP or TSP fragments. Similarly, an antisense TSP cell line responded poorly in chemotaxis assays to TSP and 140K. These data suggest that the ability of metastatic cells in vivo to establish secondary sites of proliferation may be related to their ability to migrate in response to extracellular matrix proteins such as TSP incorporated into basement membranes or interstitial matrices.

The A20 zinc finger protein protects cells from tumor necrosis factor cytotoxicity.

Resistance against the cytotoxic actions of tumor necrosis factor alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc finger protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.

Mapping the binding domain of a myosin II binding protein.

The way in which actin and myosin II become localized to the contractile ring of dividing cells resulting in cleavage furrow formation and cytokinesis is unknown. While much is known about actin binding proteins and actin localization, little is known about myosin localization. A 53-kDa (53K) polypeptide present in the sea urchin egg binds to myosin II in a nucleotide-dependent manner and mediates its solubility in vitro [Yabkowitz, R., & Burgess, D.R. (1987) J. Cell Biol. 105, 927-936]. The binding site of 53K on the myosin molecule was examined in an effort to understand the mechanism of 53K-induced myosin solubility and its potential function in myosin regulation. Blot overlay and chemical cross-linking techniques utilizing myosin proteolytic fragments indicate that 53K binds to fragments proximal to the head-rod junction of myosin. Fragments distal to the head-rod junction do not bind 53K. In addition, the binding of 53K to myosin largely inhibits protease digestion that produces the head and rod fragments. The binding of 53K to the head-rod domain of myosin may be critical in regulation of myosin conformation, localization, assembly, and ATPase activity.

Human carcinoma cells bind thrombospondin through a Mr 80,000/105,000 receptor.

The metastatic 11B squamous carcinoma cell line synthesizes and secretes high levels of the extracellular matrix glycoprotein thrombospondin (TSP) and displays aggressive invasiveness in a nude mouse model forming highly undifferentiated tumors. The importance of adhesion events involving extracellular matrix proteins and the tumorigenic cell surface in metastasis led us to investigate the nature of the 11B cell surface receptor for TSP. Using TSP affinity chromatography, a cell surface complex of molecular weight 80,000 and 105,000 was isolated that appears to function as a receptor for TSP. Binding was specific for the COOH-terminal Mr 140,000 fragment of TSP. TSP and the Mr 140,000 fragment competed for the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP-coated microtiter wells in a dose-dependent manner with half-maximal inhibition observed at 16 and 40 micrograms/ml, respectively. In contrast, the NH2-terminal heparin-binding domain did not inhibit binding in a dose-dependent manner. Other extracellular matrix proteins, such as laminin, vitronectin, or type I collagen, were also unable to inhibit the binding of the 125I-labeled Mr 80,000/105,000 cell surface complex to TSP. The specificity of the Mr 80,000/105,000 receptor for the Mr 140,000 fragment of TSP was further confirmed through the use of monoclonal antibodies. Monoclonal antibody C6.7 specific for the distal COOH terminus of TSP, but not monoclonal antibody A2.5 specific for the heparin-binding domain, inhibited binding. Binding was observed to be strongly Ca2+ dependent, slightly Mg2+ dependent, and independent of Mn2+. Immunoprecipitation analyses demonstrated no apparent cross-reactivity between the Mr 80,000/105,000 TSP receptor and members of the beta 1 or beta 3 integrin receptor families. Additionally, V-8 protease mapping demonstrated that the Mr 80,000 and 105,000 polypeptide bands are not related to each other through proteolytic processing. This initial identification and characterization of a carcinoma cell TSP receptor should allow a more detailed examination of the role of TSP in metastatic adhesion and motility.

Human carcinoma cells express receptors for distinct domains of thrombospondin.

Thrombospondin (TSP), an adhesive glycoprotein, is incorporated into the extracellular matrix, mediates cell attachment and spreading, chemotaxis, haptotaxis, and may participate in the directed movement of cells in metastasis. Evidence from several model systems suggests that these functions may be mediated by different domains within the TSP molecule. Radioligand binding assays on 11B squamous carcinoma cells with 125I-Radioligand binding assays on 11B squamous carcinoma cells with 125I-TSP demonstrated the presence of 1.2 x 10(6) sites/cell with an apparent Kd of 74 nM. Binding studies using TSP fragments demonstrated that both the NH2 terminal heparin-binding domain (HBD) and the COOH terminal fragment with a molecular weight of 140,000 (140K) retained the ability to bind 11B cells in a time-dependent, dose-dependent, saturable, and specific manner. The HBD bound to 11B cells with an apparent Kd of 1.2 microM at 1.4 x 10(6) sites/cell. Binding of 140K to cells demonstrated half-maximal binding at 36 nM and a Bmax of 1.9 x 10(5) sites/cell. The binding of 140K also showed a high degree of positive cooperativity with a Hill slope of +3.5, suggesting that binding one 140K molecule to cells leads to increased binding of additional 140K molecules. In addition, the HBD and 140K showed no cross-competition in binding assays. Therefore, it appears likely that these distinct TSP domains bind to separate sites on the cell surface. Neither vitronectin or the peptide RGDS were able to inhibit the binding of TSP or 140K to 11B cells. Based on these data, there appears to be more than one distinct receptor on 11B cells for TSP; one receptor class which mediates the binding of the HBD and a second receptor class which mediates the binding of the Mr 140,000 fragment.

Expression and initial characterization of a recombinant human thrombospondin heparin binding domain.

Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000. It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation. TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix. Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD). In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli. A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6. In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide. Milligram quantities of this protein were purified to homogeneity from E. coli lysates. The structure of the recombinant HBD was confirmed by protein sequencing. The protein was further characterized by analysis of its conformation and function. The recombinant HBD binds [3H]heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM). Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells. These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD. They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin.

Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein.

We identify a novel myosin-binding protein, designated 53K, which appears to mediate the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein possesses a subunit molecular mass on SDS-PAGE of 53 kD, an S value of 7, may be organized into disulfide-linked oligomers, and is associated with myosin in egg extracts. Both myosin and 53K co-precipitate from extract upon the addition of nucleoside triphosphates and co-sediment with an S value of 24 by sedimentation velocity centrifugation. Myosin in extracts not associated with 53K has an S value of 10. Further, myosin can be immunoprecipitated from extract with antibody to 53K and the 53K in extracts binds to a myosin affinity column. When extract is depleted of 53K, a majority of the myosin precipitates out of extract in a nucleotide-independent manner. Whereas purified myosin precipitates in the absence of nucleotide when recombined with dialysis buffer or myosin-depleted extract, reconstituting 53K and myosin before addition to buffer or myosin-depleted extract partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts. The 53-kD protein may represent a new class of authentic myosin-binding proteins that may regulate the supramolecular organization of myosin in nonmuscle cells.