Function-blocking antithrombospondin-1 monoclonal antibodies.

BACKGROUND: Thrombospondin-1 (TSP-1) has been implicated in many different processes based in part on inhibitory activities of anti-TSP-1 monoclonal antibodies (mAbs). OBJECTIVE: To map epitopes of 13 anti-TSP-1 mAbs to individual modules or groups of modules spanning TSP-1 and the closely related TSP-2 homolog. RESULTS: The mapping has led to assignment or reassignment of the epitopes of four mAbs, refinement of the epitopes of six mAbs, and confirmation of the epitopes of the remaining three mAbs. ESTs10, P12, and MA-II map to the N-terminal domain; 5G11, TSP127.6, and ESTs12 to the third properdin module; C6.7, HB8432, and P10 to epidermal growth factor (EGF)-like modules 1 and/or 2; and A6.1, mAb133, MA-I, and D4.6 to the calcium-binding wire module. A6.1, which recognizes a region of the wire that is identical in mouse and human TSP-1, reacts with TSP-1 from both species, and also reacts weakly with human TSP-2. Two other mouse antihuman TSP-1 mAbs, A4.1 and D4.6, also react with mouse TSP-1. CONCLUSIONS: Consideration of previous literature and mapping of epitopes of inhibitory mAbs suggest that biological activities are present throughout TSP-1, including the EGF-like modules that have not been implicated in the past. Because the epitopes for 10 of the antibodies likely are within 18 nm of one another in calcium-replete TSP-1, some of the inhibitory effects may result from steric hindrance. Such seems to be the case for mAb133, which binds the calcium-binding wire but is still able to interfere with the activation of latent TGF-beta by the properdin modules.

Actions of the soy phytoestrogen genistein in models of human chronic disease: potential involvement of transforming growth factor beta.

The structural similarity, but non-identity, between 17beta-oestradiol and the soy phytoestrogen genistein suggests that the two compounds will have actions that may be identical in some target biological systems, but different in others. Epidermal growth factor (EGF)-stimulated proliferation of human mammary epithelial cells (that do not express the oestrogen receptor) was significantly suppressed at genistein concentrations (5-10 microM) that are attainable physiologically. Others have shown previously that transforming growth factor beta (TGFbeta) has similar growth-inhibitory effects on human cells. Analysis of the conditioned medium of human mammary epithelial cells exposed to genistein plus EGF showed increased levels of TGFbeta relative to those in the medium of cells exposed to EGF or genistein alone. Related experiments in a primate model of menopause demonstrated that ingestion of soy containing isoflavones was correlated with the suppression of neurodegeneration-relevant phosphorylation of the microtubule-associated protein tau, while intake of Premarin (a hormone replacement therapy that is commonly prescribed for women) was not correlated. The results discussed here indicate that genistein, and probably other related phytoestrogens, have pleiotropic actions, some of which may involve TGFbeta activity.

Tamoxifen and estrogen effects on TGF-beta formation: role of thrombospondin-1, alphavbeta3, and integrin-associated protein.

We have found that the enhanced activation of latent TGF-beta by human breast carcinoma cell lines either treated with tamoxifen or deprived of estrogen is dependent upon thrombospondin-1 (TSP-1) since activation was blocked by anti-TSP-1 antibodies or by a TSP antagonist peptide. However, TGF-beta formation upon tamoxifen exposure to estrogen withdrawal is associated with decreased levels of soluble TSP-1. A concomitant increase in the expression of the TSP-1 receptors alphavbeta3 and integrin-associated protein (IAP) occurs under these conditions, and antibodies to TSP-1 or to these receptors inhibit increased TGF-beta formation. Therefore, increased cell surface associated TSP-1 enhances latent TGF-beta activation.

The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state?

The process of cellular de-adhesion is potentially important for the ability of a cell to participate in morphogenesis and to respond to injurious stimuli. Cellular de-adhesion is induced by the highly regulated matricellular proteins TSP1 and 2, tenascin-C, and SPARC. These proteins induce a rapid transition to an intermediate state of adhesiveness characterized by loss of actin-containing stress fibers and restructuring of the focal adhesion plaque that includes loss of vinculin and alpha-actinin, but not of talin or integrin. This process involves intracellular signaling mediators, which are engaged in response to matrix protein-receptor interactions. Each of these proteins employs different receptors and signaling pathways to achieve this common morphologic endpoint. What is the function of this intermediate adhesive state and what is the physiologic significance of this action of the matricellular proteins? Given that matricellular proteins are expressed in response to injury and during development, one can speculate that the intermediate adhesive state is an adaptive condition that facilitates expression of specific genes that are involved in repair and adaptation. Since cell shape is maintained in weakly adherent cells, this state might induce survival signals to prevent apoptosis due to loss of strong cell adhesion, but yet allow for cell locomotion. The three matricellular proteins considered here might each preferentially facilitate one or more aspects of this adaptive response rather than all of these equally. Currently, we have only preliminary data to support the specific ideas proposed in this article. It will be interesting in the next several years to continue to elucidate the biological roles of the intermediate adhesive state induced by these matricellular proteins. and focal adhesions in a cell that nevertheless maintains a spread, extended morphology and integrin clustering. TSP1, tenascin-C, and SPARC induce the intermediate adhesive state, as shown by the red arrows. The significance of each adhesive state for cell behavior is indicated beneath the cells. The weak adhesive state would be consistent with cells undergoing apoptosis during remodeling or those undergoing cytokinesis. The strong adhesive state is characteristic of a differentiated, quiescent cell, whereas cells in the intermediate adhesive state would include those involved in responding to injury during wound healing or in tissue remodeling during morphogenesis.

Glucose stimulation of transforming growth factor-beta bioactivity in mesangial cells is mediated by thrombospondin-1.

Glucose is a key factor in the development of diabetic complications, including diabetic nephropathy. The development of diabetic glomerulosclerosis is dependent on the fibrogenic growth factor, transforming growth factor-beta (TGF-beta). Previously we showed that thrombospondin-1 (TSP-1) activates latent TGF-beta both in vitro and in vivo. Activation occurs as the result of specific interactions of latent TGF-beta with TSP-1, which potentially alter the conformation of latent TGF-beta. As glucose also up-regulates TSP-1 expression, we hypothesized that the increased TGF-beta bioactivity observed in rat and human mesangial cells cultured with high glucose concentrations is the result of latent TGF-beta activation by autocrine TSP-1. Glucose-induced bioactivity of TGF-beta in mesangial cell cultures was reduced to basal levels by peptides from two different sequences that antagonize activation of latent TGF-beta by TSP, but not by the plasmin inhibitor, aprotinin. Furthermore, glucose-dependent stimulation of matrix protein synthesis was inhibited by these antagonist peptides. These studies demonstrate that glucose stimulation of TGF-beta activity and the resultant matrix protein synthesis are dependent on the action of autocrine TSP-1 to convert latent TGF-beta to its biologically active form. These data suggest that antagonists of TSP-dependent TGF-beta activation may be the basis of novel therapeutic approaches for ameliorating diabetic renal fibrosis.

Thrombospondin mediates focal adhesion disassembly through interactions with cell surface calreticulin.

Thrombospondin induces reorganization of the actin cytoskeleton and restructuring of focal adhesions. This activity is localized to amino acids 17-35 in the N-terminal heparin-binding domain of thrombospondin and can be replicated by a peptide (hep I) with this sequence. Thrombospondin/hep I stimulate focal adhesion disassembly through a mechanism involving phosphoinositide 3-kinase activation. However, the receptor for this thrombospondin sequence is unknown. We now report that calreticulin on the cell surface mediates focal adhesion disassembly by thrombospondin/hep I. A 60-kDa protein from endothelial cell detergent extracts has homology and immunoreactivity to calreticulin, binds a hep I affinity column, and neutralizes thrombospondin/hep I-mediated focal adhesion disassembly. Calreticulin on the cell surface was confirmed by biotinylation, confocal microscopy, and by fluorescence-activated cell sorting analyses. Thrombospondin and calreticulin potentially bind through the hep I sequence, since thrombospondin-calreticulin complex formation can be blocked specifically by hep I peptide. Antibodies to calreticulin and preincubation of thrombospondin/hep I with glutathione S-transferase-calreticulin block thrombospondin/hep I-mediated focal adhesion disassembly and phosphoinositide 3-kinase activation, suggesting that calreticulin is a component of the thrombospondin-induced signaling cascade that regulates cytoskeletal organization. These data identify both a novel receptor for the N terminus of thrombospondin and a distinct role for cell surface calreticulin in cell adhesion.

Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin.

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

A CD36 synthetic peptide inhibits bleomycin-induced pulmonary inflammation and connective tissue synthesis in the rat.

Transforming growth factor (TGF)-beta1 is an important regulator of inflammation and fibrosis. TGF-beta1 is usually secreted as a biologically latent protein called latent TGF-beta1 (L-TGF-beta1). L-TGF-beta1 has no biologic effect unless L-TGF-beta1 is converted to its active form. Using a well-recognized model of lung injury induced by the antineoplastic antibiotic bleomycin (Blm), we demonstrated that 7 d after intratracheal Blm administration, total lung TGF-beta was maximally increased. This induction was due to TGF-beta1 production by alveolar macrophages that, when explanted, generated increased quantities of L-TGF-beta1 complexed with the glycoprotein thrombospondin (TSP)-1. The TSP-1/L-TGF-beta1 complex was associated with CD36, a receptor for TSP-1. The association of TSP-1/L-TGF-beta1 to CD36 was critical for plasmin-mediated release of mature TGF-beta1. In this paper we show that, compared with administration of Blm by itself, when a synthetic peptide of CD36 between amino acids 93 and 110 is given concomitantly with Blm to rats, alveolar macrophages generate markedly less active TGF-beta1, the rats gain weight more rapidly, and there is less inflammation, collagen I and III, and fibronectin synthesis. These findings demonstrate a novel in vivo mechanism of activation of L-TGF-beta1 in lung injury and the importance of alveolar macrophage- derived active TGF-beta1 in the pathogenesis of pulmonary inflammation and fibrosis.

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis.

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.

Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology.

Regulation of the activation of latent TGF-beta is essential for health as too much or too little TGF-beta activity can have serious, deleterious consequences. The processes that control conversion of the precursor to the biologically active form of TGF-beta in vivo are not well characterized. We have identified a mechanism for the activation of latent TGF-beta that involves binding of the secreted and extracellular matrix protein, thrombospondin-1 (TSP-1), to the latent precursor. Specific sequences in TSP-1 and in the precursor portion (the latency associate peptide-LAP) have been determined to be essential for activation of latent TGF-beta by TSP-1. It is thought that binding of TSP-1 to the latent complex induces a conformational rearrangement of the LAP in such a manner as to prevent the LAP from conferring latency on the mature domain of TGF-beta. A TSP-dependent mechanism of activation may be locally important during wound healing and in post-natal development of epithelial structures. The possible involvement of TSP-1 in TGF-beta activation during several disease processes is also discussed.

Activation of rat alveolar macrophage-derived latent transforming growth factor beta-1 by plasmin requires interaction with thrombospondin-1 and its cell surface receptor, CD36.

Transforming growth factor-beta-1 (TGF-beta1) is secreted by cells in a latent form (L-TGF-beta1) noncovalently bound to a latency-associated peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-beta1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- beta1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- beta1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-beta1, indicating that TSP-1/L-TGF-beta1 complexes are present on alveolar macrophages. However, in the presence of plasmin both latency-associated peptide and TGF-beta1 were decreased in the same cell lysates, indicating that L-TGF-beta1 associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-beta1 and CD36, a receptor for TSP-1, there was colocalization of TGF-beta1 with CD36. Because TSP-1 but not TGF-beta1 is a natural ligand for CD36, these findings suggest that the L-TGF-beta1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-beta1 complex with CD36 is necessary to the activation of L-TGF-beta1 because antibodies to CD36 prevent the colocalization of TGF-beta1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-beta1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-beta1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.

Thrombospondin peptides are potent inhibitors of mesangial and glomerular endothelial cell proliferation in vitro and in vivo.

BACKGROUND: Thrombospondin 1 (TSP1), a multifunctional, matricellular glycoprotein, is expressed de novo in many inflammatory disease processes, including glomerular disease. Short peptide fragments derived from the type I properdin repeats of the TSP1 molecule mimic anti-angiogenic and/or transforming growth factor-beta (TGF-beta)-activating properties of the whole TSP1 glycoprotein. We investigated the effects of D-reverse peptides derived from the type I domain of TSP1 in experimental mesangial proliferative glomerulonephritis in the rat (anti-Thy1 model), as well as their effects on cultured mesangial and glomerular endothelial cells. METHODS: Effects of TSP peptides on proliferation of mesangial or glomerular endothelial cells in culture after growth arrest or growth factor stimulation (fibroblast growth factor-2, platelet-derived growth factor-BB, 10% fetal calf serum) were measured by [3H]thymidine incorporation assay. Adhesion of rat mesangial cells (MCs) to a TSP-peptide matrix was assayed using an attachment-hexosaminidase assay. TSP peptides were intraperitoneally injected daily in rats that had received an intravenous injection of polyclonal anti-Thy1 antibody to induce mesangial proliferative glomerulonephritis. On biopsies from days 2, 5, and 8 of anti-Thy1 disease, mesangial and glomerular endothelial proliferation, matrix expansion, mesangial activation, and microaneurysm formation were assessed. Functional parameters such as blood pressure and proteinuria were also measured. RESULTS: An 18-amino acid peptide (type I peptide) with anti-angiogenic and TGF-beta-activating sequences decreased mesangial and glomerular endothelial cell proliferation in vitro and in vivo and reduced microaneurysm formation and proteinuria in experimental glomerulonephritis. Analogues lacking the TGF-beta-activating sequence mimicked most effects of the type I peptide. The mechanism of action of these peptides may include antagonism of fibroblast growth factor-2 and alteration of MC adhesion. The TGF-beta-activating sequence alone did not have significant effects on mesangial or glomerular endothelial cells in vitro or in experimental kidney disease in vivo. CONCLUSION: Peptides from TSP1 may be promising therapeutics in treating glomerular disease with mesangial and endothelial cell injury.

The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta.

One of the primary points of regulation of transforming growth factor-beta (TGF-beta) activity is control of its conversion from the latent precursor to the biologically active form. We have identified thrombospondin-1 as a major physiological regulator of latent TGF-beta activation. Activation is dependent on the interaction of a specific sequence in thrombospondin-1 (K412RFK415) with the latent TGF-beta complex. Platelet thrombospon-din-1 has TGF-beta activity and immunoreactive mature TGF-beta associated with it. We now report that the latency-associated peptide (LAP) of the latent TGF-beta complex also interacts with thrombospondin-1 as part of a biologically active complex. Thrombospondin.LAP complex formation involves the activation sequence of thrombospondin-1 (KRFK) and a sequence (LSKL) near the amino terminus of LAP that is conserved in TGF-beta1-5. The interactions of LAP with thrombospondin-1 through the LSKL and KRFK sequences are important for thrombospondin-mediated activation of latent TGF-beta since LSKL peptides can competitively inhibit latent TGF-beta activation by thrombospondin or KRFK-containing peptides. In addition, the association of LAP with thrombospondin-1 may function to prevent the re-formation of an inactive LAP.TGF-beta complex since thrombospondin-bound LAP no longer confers latency on active TGF-beta. The mechanism of TGF-beta activation by thrombospondin-1 appears to be conserved among TGF-beta isoforms as latent TGF-beta2 can also be activated by thrombospondin-1 or KRFK peptides in a manner that is sensitive to inhibition by LSKL peptides.

Retinoic acid alters the mechanism of attachment of malignant astrocytoma and neuroblastoma cells to thrombospondin-1.

Based on the hypothesis that the attachment of neuroectodermal cells to thrombospondin-1 (TSP-1) may affect tumor spread and play a role in the anti-tumor effects of retinoic acid, we investigated the expression of TSP-1 in these cells in situ and the effect of retinoic acid on the morphology of TSP-1-adherent neuroblastoma (SK-N-SH) and malignant astrocytoma (U-251MG) cells in vitro. TSP-1-adherent SK-N-SH cells demonstrated process outgrowth, with further neuronal differentiation after retinoic acid treatment, consistent with the in situ studies showing that TSP-1 expression occurs in a differentiation-specific manner in neuroblastic tumors. TSP-1-adherent U-251MG cells failed to spread; however, after retinoic acid treatment the cells demonstrated broad lamellipodia containing radial actin fibers and organization of integrins alpha3beta1 and alpha5beta1 in clusters in lamellipodia and filopodia. The attachment of both SK-N-SH and U-251MG cells to TSP-1 was found to be mediated by heparan sulfate proteoglycans, integrins, and the CLESH-1 adhesion domain first identified in CD36. Heparin and heparitinase treatment inhibited TSP-1 attachment. Integrins alpha3beta1 and alpha5beta1 mediated TSP-1 attachment of SK-N-SH cells, and integrins alpha3beta1, alpha5beta1, and alphavbeta3 mediated TSP-1 attachment of U-251MG cells. Attachment was dependent on the RGD sequence which is located in the carboxy-terminus of TSP-1. Treatment with a pharmacologic dosage of retinoic acid altered the TSP-1 cell adhesion mechanism in both cell lines in that neither heparin nor micromolar concentrations of the RGD peptide inhibited attachment; after treatment, attachment was inhibited by the CSVTCG peptide located in the type I repeat domain of TSP-1 and a recombinant adhesion domain (CLESH-1) from CD36. Expression of CD36 was found in the retinoic acid-treated U-251MG cells. These data indicate that neuroectodermally derived cells utilize several mechanisms to attach to TSP-1, and these are differentially modulated by treatment with retinoic acid. These data also suggest that the CSVTCG sequence of TSP-1 modulates or directs cytoskeletal organization in neuroblastoma and astrocytoma cells.

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 microg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 microg/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, gamma-catenin, and p120(Cas). These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.

Signaling of de-adhesion in cellular regulation and motility.

Adhesion is a process that can be divided into three separate stages: (1) cell attachment, (2) cell spreading, and (3) the formation of focal adhesions and stress fibers. With each stage the adhesive strength of the cell increases. De-adhesion can be defined as the process involving the transition of the cell from a strongly adherent state, characterized by focal adhesions and stress fibers, to a state of intermediate adherence, represented by a cell that is spread, but that lacks stress fibers terminating at adhesion plaques. We propose that this modification of the structural link between the actin cytoskeleton and the extracellular matrix results in a more malleable cellular state conducive for dynamic processes such as cytokinesis, mitogenesis, and motility. Anti-adhesive proteins, including thrombospondin, tenascin, and SPARC, rapidly signal de-adhesion, potentially mediating proliferation and migration during development and wound healing. Intracellular signaling molecules involved in the regulation of de-adhesion are only beginning to be identified. Interestingly, many of the same signaling proteins recognized to play important roles during the process of adhesion have also been found to act during de-adhesion. Characterization of the precise mechanisms by which these signals modulate adhesive structures and the cytoskeleton will further our understanding of the regulation of adhesive strength and its function in cellular physiology.

Thrombospondin-1 is a major activator of TGF-beta1 in vivo.

The activity of TGF-beta1 is regulated primarily extracellularly where the secreted latent form must be modified to expose the active molecule. Here we show that thrombospondin-1 is responsible for a significant proportion of the activation of TGF-beta1 in vivo. Histological abnormalities in young TGF-beta1 null and thrombospondin-1 null mice were strikingly similar in nine organ systems. Lung and pancreas pathologies similar to those observed in TGF-beta1 null animals could be induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-beta1 by thrombospondin-1. Although these organs produced little active TGF-beta1 in thrombospondin null mice, when pups were treated with a peptide derived from thrombospondin-1 that could activate TGF-beta1, active cytokine was detected in situ, and the lung and pancreatic abnormalities reverted toward wild type.

EGF receptor regulation of cell motility: EGF induces disassembly of focal adhesions independently of the motility-associated PLCgamma signaling pathway.

A current model of growth factor-induced cell motility invokes integration of diverse biophysical processes required for cell motility, including dynamic formation and disruption of cell/substratum attachments along with extension of membrane protrusions. To define how these biophysical events are actuated by biochemical signaling pathways, we investigate here whether epidermal growth factor (EGF) induces disruption of focal adhesions in fibroblasts. We find that EGF treatment of NR6 fibroblasts presenting full-length WT EGF receptors (EGFR) reduces the fraction of cells presenting focal adhesions from approximately 60% to approximately 30% within 10 minutes. The dose dependency of focal adhesion disassembly mirrors that for EGF-enhanced cell motility, being noted at 0.1 nM EGF. EGFR kinase activity is required as cells expressing two kinase-defective EGFR constructs retain their focal adhesions in the presence of EGF. The short-term (30 minutes) disassembly of focal adhesions is reflected in decreased adhesiveness of EGF-treated cells to substratum. We further examine here known motility-associated pathways to determine whether these contribute to EGF-induced effects. We have previously demonstrated that phospholipase C(gamma) (PLCgamma) activation and mobilization of gelsolin from a plasma membrane-bound state are required for EGFR-mediated cell motility. In contrast, we find here that short-term focal adhesion disassembly is induced by a signaling-restricted truncated EGFR (c'973) which fails to activate PLCgamma or mobilize gelsolin. The PLC inhibitor U73122 has no effect on this process, nor is the actin severing capacity of gelsolin required as EGF treatment reduces focal adhesions in gelsolin-devoid fibroblasts, further supporting the contention that focal adhesion disassembly is signaled by a pathway distinct from that involving PLCgamma. Because both WT and c'973 EGFR activate the erk MAP kinase pathway, we additionally explore here this signaling pathway, not previously associated with growth factor-induced cell motility. Levels of the MEK inhibitor PD98059 that block EGF-induced mitogenesis and MAP kinase phosphorylation also abrogate EGF-induced focal adhesion disassembly and cell motility. In summary, we characterize for the first time the ability of EGFR kinase activity to directly stimulate focal adhesion disassembly and cell/substratum detachment, in relation to its ability to stimulate migration. Furthermore, we propose a model of EGF-induced motogenic cell responses in which the PLCgamma pathway stimulating cell motility is distinct from the MAP kinase-dependent signaling pathway leading to disassembly and reorganization of cell-substratum adhesion.

Cyclic GMP-dependent protein kinase inhibits osteopontin and thrombospondin production in rat aortic smooth muscle cells.

Vascular lesions resulting from injury are characterized by a thickening of the intima brought about in part through the production of increased amounts of extracellular matrix proteins by the vascular smooth muscle cells (VSMCs). In this study, we tested the hypothesis that cGMP-dependent protein kinase (PKG), an important mediator of NO and cGMP signaling in VSMCs, inhibits the production of two extracellular matrix proteins, osteopontin and thrombospondin, which are involved in the formation of the neointima. VSMCs deficient in PKG were stably transfected with cDNAs encoding either the holoenzyme PKG-Ialpha or the constitutively active catalytic domain of PKG-I in order to directly examine the effects of PKG on osteopontin and thrombospondin production. Cells expressing either of the PKG constructs had dramatically reduced levels of osteopontin and thrombospondin-1 protein compared with control-transfected PKG-deficient cells. PKG transfection also altered the morphology of the VSMCs. These results indicate that PKG may be involved in suppressing extracellular matrix protein expression, which is one important characteristic of synthetic secretory VSMCs. Suppression of these matrix proteins may underlie the effects of NO-cGMP signaling to inhibit VSMC migration and phenotypic modulation.

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase.

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.