Neutralizing the EGF receptor in glioblastoma cells stimulates cell migration by activating uPAR-initiated cell signaling.

In glioblastoma (GBM), the EGF receptor (EGFR) and Src family kinases (SFKs) contribute to an aggressive phenotype. EGFR may be targeted therapeutically; however, resistance to EGFR-targeting drugs such as Erlotinib and Gefitinib develops quickly. In many GBMs, a truncated form of the EGFR (EGFRvIII) is expressed. Although EGFRvIII is constitutively active and promotes cancer progression, its activity is attenuated compared with EGF-ligated wild-type EGFR, suggesting that EGFRvIII may function together with other signaling receptors in cancer cells to induce an aggressive phenotype. In this study, we demonstrate that in EGFRvIII-expressing GBM cells, the urokinase receptor (uPAR) functions as a major activator of SFKs, controlling phosphorylation of downstream targets, such as p130Cas and Tyr-845 in the EGFR in vitro and in vivo. When EGFRvIII expression in GBM cells was neutralized, either genetically or by treating the cells with Gefitinib, paradoxically, the cells demonstrated increased cell migration. The increase in cell migration was explained by a compensatory increase in expression of urokinase-type plasminogen activator, which activates uPAR-dependent cell signaling. GBM cells that were selected for their ability to grow in vivo in the absence of EGFRvIII also demonstrated increased cell migration, due to activation of the uPAR signaling system. The increase in GBM cell migration, induced by genetic or pharmacologic targeting of the EGFR, was blocked by Dasatinib, highlighting the central role of SFKs in uPAR-promoted cell migration. These results suggest that compensatory activation of uPAR-dependent cell signaling, in GBM cells treated with targeted therapeutics, may adversely affect the course of the disease by promoting cell migration, which may be associated with tumor progression.

Urokinase receptor primes cells to proliferate in response to epidermal growth factor.

Epidermal growth factor (EGF) expresses mitogenic activity by a mechanism that requires the EGF receptor (EGFR). We report that murine embryonic fibroblasts (MEFs) proliferate in response to EGF only when these cells express the urokinase receptor (uPAR). EGFR expression was equivalent in uPAR-/- and uPAR+/+ MEFs. In response to EGF, these cells demonstrated equivalent overall EGFR tyrosine phosphorylation and ERK/MAP kinase activation; however, phosphorylation of Tyr-845 in the EGFR, which has been implicated in cell growth, was substantially decreased in uPAR-/- MEFs. STAT5b activation also was decreased. As Tyr-845 is a c-Src target, we overexpressed c-Src in uPAR-/- MEFs and rescued EGF mitogenic activity. Rescue also was achieved by expressing murine but not human uPAR, suggesting a role for autocrine uPAR cell-signaling. In MDA-MB 231 breast cancer cells, EGF mitogenic activity was blocked by uPAR gene silencing, with antibodies that block uPA-binding to uPAR, and with a synthetic peptide that disrupts uPAR-dependent cell signaling. Again, c-Src overexpression rescued the mitogenic activity of EGF. We conclude that uPAR-dependent cell-signaling may prime cells to proliferate in response to EGF by promoting Tyr-845 phosphorylation and STAT5b activation. The importance of this pathway depends on the c-Src level in the cell.

Cytokeratin 8 functions as a major plasminogen receptor in select epithelial and carcinoma cells.

Cytokeratin 8 (K8) is a member of the intermediate filament (IF) gene family expressed by simple epithelial cells and by some carcinoma cells. The majority of the cellular K8 is assembled with its partner, K18, into highly insoluble 10 nm filaments that extend from the nucleus to the internal leaflet of the plasma membrane. At desmosomes and hemidesmosomes, K8, K18, and other IF proteins are bridged to proteins with transmembrane domains by a family of proteins called plakins. K8 does not have a signal peptide or a well-defined transmembrane domain; however, there is substantial evidence that this protein is available to bind plasminogen and K8-specific antibodies on the surfaces of certain epithelial cells in culture, including hepatocytes, hepatocellular carcinoma cells, and various breast cancer cell lines. This may reflect a novel mechanism of protein penetration through the plasma membrane or binding of secreted K8 to other cell-surface molecules. Cancer cells are known to secrete K8-containing protein complexes in vitro and in vivo. These complexes bind plasminogen as well. The plasminogen-binding activity of K8 is unique amongst IF proteins, probably because its sequence includes a carboxyl-terminal Lys residue. However, a K8 mutant that lacks the C-terminal Lys still binds plasminogen, albeit with decreased affinity. K18 does not bind plasminogen; however, K8 and K18 bind tissue-type plasminogen activator (tPA) equivalently. tPA-binding to K18 may be important in the mechanism whereby K8-K18 complexes promote plasminogen activation by tPA. Numerous studies have demonstrated correlations between high levels of K8 expression and increased migration and invasion of certain cancer cells. These correlations are most easily explained by the function of IF proteins in determining the rigidity of the cytoskeleton; however, the function of cell-surface K8 as a plasminogen receptor merits consideration. We have demonstrated that certain aggressive breast cancer cell lines, which have highly activated endogenous urokinase type-plasminogen activator (uPA)-uPA receptor (uPAR) systems, do not express high levels of cell-surface K8. The membrane macromolecule that is responsible for plasminogen-binding and for supporting activation of plasminogen by uPA on the surfaces of these cell types remains to be determined. This review focuses on the function of K8 as a plasminogen receptor and its potential role in cancer.

Endogenously produced urokinase-type plasminogen activator is a major determinant of the basal level of activated ERK/MAP kinase and prevents apoptosis in MDA-MB-231 breast cancer cells.

Urokinase-type plasminogen activator (uPA) binds to the uPA receptor (uPAR) and activates the Ras-extracellular signal-regulated kinase (ERK) signaling pathway in many different cell types. In this study, we demonstrated that endogenously produced uPA functions as a major determinant of the basal level of activated ERK in MDA-MB-231 breast cancer cells. When these cells were cultured in the presence of antibodies that block the binding of uPA to uPAR, the level of phosphorylated ERK decreased substantially. Furthermore, conditioned medium from MDA-MB-231 cells activated ERK in MCF-7 cells and this response was blocked by uPA-specific antibody. The mitogen-activated protein kinase kinase inhibitor, PD098059, decreased expression of uPA and uPAR in MDA-MB-231 cells. Thus, uPA and the uPAR-ERK signaling pathway form a positive feedback loop in these cells. When this feedback loop was disrupted with uPA- or uPAR-specific antibody, uPA mRNA-specific antisense oligodeoxynucleotides or PD098059, cell growth was inhibited and apoptosis was promoted, as determined by the increase in cytoplasmic nucleosomes and caspase-3 activity. Treating the cells simultaneously with PD098059 and uPA- or uPAR-specific antibody did not further promote apoptosis, compared with either reagent added separately, supporting the hypothesis that uPAR and ERK are components of the same cell growth/survival-regulatory pathway. The ability of uPA to signal through uPAR, maintain an elevated basal level of activated ERK and inhibit apoptosis represents a novel mechanism whereby the uPA-uPAR system may affect breast cancer progression in vivo.

Plasminogen activator inhibitor 1 functions as a urokinase response modifier at the level of cell signaling and thereby promotes MCF-7 cell growth.

Plasminogen activator inhibitor 1 (PAI-1) is a major inhibitor of urokinase-type plasminogen activator (uPA). In this study, we explored the role of PAI-1 in cell signaling. In MCF-7 cells, PAI-1 did not directly activate the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2, but instead altered the response to uPA so that ERK phosphorylation was sustained. This effect required the cooperative function of uPAR and the very low density lipoprotein receptor (VLDLr). When MCF-7 cells were treated with uPA-PAI-1 complex in the presence of the VLDLr antagonist, receptor-associated protein, or with uPA-PAI-1(R76E) complex, which binds to the VLDLr with greatly decreased affinity, transient ERK phosphorylation (<5 min) was observed, mimicking the uPA response. ERK phosphorylation was not induced by tissue-type plasminogen activator-PAI-1 complex or by uPA-PAI-1 complex in the presence of antibodies that block uPA binding to uPAR. uPA-PAI-1 complex induced tyrosine phosphorylation of focal adhesion kinase and Shc and sustained association of Sos with Shc, whereas uPA caused transient association of Sos with Shc. By sustaining ERK phosphorylation, PAI-1 converted uPA into an MCF-7 cell mitogen. This activity was blocked by receptor-associated protein and not observed with uPA-PAI-1(R76E) complex, demonstrating the importance of the VLDLr. uPA promoted the growth of other cells in which ERK phosphorylation was sustained, including beta3 integrin overexpressing MCF-7 cells and HT 1080 cells. The MEK inhibitor, PD098059, blocked the growth-promoting activity of uPA and uPA-PAI-1 complex in these cells. Our results demonstrate that PAI-1 may regulate uPA-initiated cell signaling by a mechanism that requires VLDLr recruitment. The kinetics of ERK phosphorylation in response to uPAR ligation determine the function of uPA and uPA-PAI-1 complex as growth promoters.

A 16-amino acid peptide from human alpha2-macroglobulin binds transforming growth factor-beta and platelet-derived growth factor-BB.

Alpha2-macroglobulin (alpha2M) is a major carrier of transforming growth factor-beta (TGF-beta) in vitro and in vivo. By screening glutathione S-transferase (GST) fusion proteins with overlapping sequences, we localized the TGFbeta-binding site to aa 700-738 of the mature human alpha2M subunit. In separate experiments, we screened overlapping synthetic peptides corresponding to aa 696-777 of alpha2M and identified a single 16-mer (718-733) that binds TGF-beta1. Platelet-derived growth factor-BB (PDGF-BB) bound to the same peptide, even though TGF-beta and PDGF-BB share almost no sequence identity. The sequence of the growth factor-binding peptide, WDLVVVNSAGVAEVGV, included a high proportion of hydrophobic amino acids. The analogous peptide from murinoglobulin, a human alpha2M homologue that does not bind growth factors, contained only three nonconservative amino acid substitutions; however, the MUG peptide failed to bind TGF-beta1 and PDGF-BB. These results demonstrate that a distinct and highly-restricted site in alpha2M, positioned near the C-terminal flank of the bait region, mediates growth factor binding. At least part of the growth factor-binding site is encoded by exon 18 of the alpha2M gene, which is notable for a 5' splice site polymorphism that has been implicated in Alzheimer's Disease.

Urokinase-type plasminogen activator stimulates the Ras/Extracellular signal-regulated kinase (ERK) signaling pathway and MCF-7 cell migration by a mechanism that requires focal adhesion kinase, Src, and Shc. Rapid dissociation of GRB2/Sps-Shc complex is associated with the transient phosphorylation of ERK in urokinase-treated cells.

Urokinase-type plasminogen activator (uPA) stimulates MCF-7 cell migration by binding to the UPA receptor and activating the Ras-extracellular signal-regulated kinase (Ras-ERK) signaling pathway. Studies presented here show that soluble uPA receptor and a peptide derived from the linker region between domains 1 and 2 of the uPA receptor also stimulate cellular migration via a mitogen-activated protein kinase/ERK kinase (MEK)-dependent pathway. Signaling proteins that function upstream of Ras in uPA- stimulated cells remain undefined. To address this problem, we transfected MCF-7 cells to express the noncatalytic carboxylterminal domain of focal adhesion kinase (FAK), FAK(Y397F), kinase-defective c-Src, or Shc FFF, all of which express dominant-negative activity. In each case, ERK phosphorylation and cellular migration in response to uPA were blocked. Both activities were rescued by co-transfecting the cells to express constitutively active MEK1, indicating that FAK, c-Src, and Shc are upstream of MEK. Shc was tyrosine-phosphorylated in uPA-treated cells. The level of phosphorylated Shc was increased within 1 min and remained increased for at least 30 min. Sos co-immunoprecipitated with Shc in cells that were treated with uPA for 1-2.5 min, probably reflecting the formation of Shc-Grb2/Sos complex; however, by 10 min, co-immunoprecipitation of Sos with Shc was no longer observed. Rapid dissociation of Sos from Shc represents a possible mechanism for the transient phosphorylation of ERK in uPA-treated MCF-7 cells.

Interleukin-4 and IL-10 bind covalently to activated human alpha2-macroglobulin by a mechanism that requires Cys949.

Alpha2-macroglobulin (alpha2M) functions as an extracellular carrier of diverse cytokines, including transforming growth factor-beta1 (TGF-beta1), that expresses anti-inflammatory activities. The results presented here demonstrate that interleukin-10 (IL-10) and IL-4, which also regulate the inflammatory response, bind to alpha2M. Unlike TGF-beta, IL-4 and IL-10 bind almost exclusively to the receptor-recognized, or activated, form of alpha2M. Purified IL-4-alpha2M complexes were predominantly covalent due to thiol disulfide exchange involving Cys949 in the alpha2M subunit. Blocking Cys949 with iodoacetamide significantly inhibited IL-4- and IL-10 binding. Bovine serum albumin (BSA), which possesses a free Cys residue and undergoes thiol disulfide exchange reactions, did not compete with alpha2M for the binding of IL-4 or IL-10. These results suggest a model in which IL-4 and IL-10 associate with activated alpha2M to form complexes that are initially noncovalent but unstable. In these complexes, Cys949 is properly aligned to undergo thiol disulfide exchange and generate stable, covalent IL-4-alpha2M and IL-10-alpha2M complexes.

Identical or overlapping sequences in the primary structure of human alpha(2)-macroglobulin are responsible for the binding of nerve growth factor-beta, platelet-derived growth factor-BB, and transforming growth factor-beta.

alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.

Extracellular signal-regulated kinase functions in the urokinase receptor-dependent pathway by which neutralization of low density lipoprotein receptor-related protein promotes fibrosarcoma cell migration and matrigel invasion.

The low density lipoprotein receptor-related protein (LRP) has been reported to regulate cellular migration. In this study, an antisense RNA expression strategy was used to reduce LRP to undetectable levels in HT 1080 fibrosarcoma cells. The LRP-deficient cells demonstrated increased levels of cell-surface uPAR, higher levels of uPA in conditioned medium, increased migration on vitronectin-coated surfaces, and increased invasion of Matrigel. LRP-deficient cells also demonstrated increased levels of phosphorylated extracellular signal-regulated kinase (ERK) in the absence of exogenous stimulants. Antibodies which block binding of endogenously produced uPA to uPAR reduced ERK phosphorylation and migration of LRP-deficient cells to the levels observed with control cells. Inhibitors of ERK activation, including PD098059 and dominant-negative MEK1, also decreased the migration of LRP-deficient but not control cells. By contrast, constitutively active MEK1 stimulated the migration of control but not LRP-deficient cells. Although Matrigel invasion by LRP-deficient cells was inhibited by the proteinase inhibitor, aprotinin, PD098059 in combination with aprotinin was necessary for an optimal effect. Expression of the VLDL receptor in LRP-deficient cells reversed the changes in cellular migration and invasion. These studies demonstrate that binding of endogenously produced uPA to uPAR may serve as a major determinant of basal levels of activated ERK and, by this mechanism, regulate cellular migration and invasion. By regulating the uPA/uPAR system, LRP may also regulate ERK activation, cellular migration, and invasion.

Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

Stable antisense RNA expression neutralizes the activity of low-density lipoprotein receptor-related protein and promotes urokinase accumulation in the medium of an astrocytic tumor cell line.

Low-density lipoprotein receptor-related protein (LRP) binds and internalizes multiple ligands that are structurally and functionally diverse. However, the effects of LRP on cellular phenotype remain unclear. To study LRP in human astrocytic tumor cells, we designed LRP antisense RNA expression constructs in which the antisense cDNA fragment was expressed under the control of the cytomegalovirus (CMV) promoter. U-1242 MG astrocytic tumor cells were transfected with the antisense constructs and cloned from single cells to yield multiple cell lines with decreased LRP expression. Further studies were performed with two cell lines in which LRP antigen was completely eliminated (L(alpha)42) or substantially decreased (Lalpha47), as determined by Western blot analysis. Untransfected U-1242 MG cells and cells that were stably transfected with empty vector (pBK-CMV) bound activated alpha2-macroglobulin (alpha2M) in a specific and saturable manner. The Bmax was about 5000 receptors/cell. Lalpha42 cells did not bind alpha2M, and binding was decreased by >60% in Lalpha47 cells. Lalpha42 and Lalpha47 cells also demonstrated reduced susceptibility to the cytotoxin, Pseudomonas exotoxin A, and accumulated greatly increased levels of urokinase-type plasminogen activator (uPA) in conditioned medium. The accumulation of uPA demonstrates a major role for LRP in the catabolism of this protein in astrocytic tumor cells. The LRP-deficient cell lines, developed using antisense technology, represent a new model system for studying LRP function in astrocytes.

The very low density lipoprotein receptor regulates urokinase receptor catabolism and breast cancer cell motility in vitro.

The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA) and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged with uPA.PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the catabolism of [35S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t(1)/(2) was 8.2 h. uPA.PAI-1 complex accelerated uPAR catabolism (t(1)/(2) to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t(1)/(2) of 7.8 h) and absence (t(1)/(2) of 16.9 h) of uPA.PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 +/- 0.1-fold and by 4.4 +/- 0.3-fold when 1.0 nM uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures. These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular motility.

Epidermal growth factor differentially regulates low density lipoprotein receptor-related protein gene expression in neoplastic and fetal human astrocytes.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytotic receptor that may modify the biological activity of reactive astrocytes in neuroplasticity and neurodegeneration and of malignant astrocytes in brain invasion. In this study, the regulation of LRP by epidermal growth factor receptor (EGFR) ligands in both cultured human fetal astrocytes and astrocytic tumor cell lines (U-251 MG and U-1242 MG) was investigated. All astrocytic cell types expressed LRP, as determined by the binding of activated alpha2-macroglobulin (alpha2M*) on intact cells and by Western and Northern blot analyses of cell extracts. Primary cultured astrocytes expressed the highest levels of alpha2M*-binding capacity (Bmax = 30 fmol/mg protein). This was twofold higher than for the U-1242 MG astrocytoma cells (Bmax = 15 fmol/mg protein) and fourfold greater than for the glioblastoma U-251 MG cells (7.0 fmol/mg protein). Receptor affinity (K(D)) ranged from 0.25 to 0.6 nM in all the astroglial cell types. Functional LRP at the surface was down-regulated by EGF, compared with controls, as indicated by a reduction of both Bmax and LRP-mediated endocytosis by approximately 50% and 60%, respectively. In comparison, EGF treatment of primary astrocytes did not down-regulate LRP expression or LRP-mediated endocytosis. Treatment of the tumor cells with EGF or TGFalpha (25 ng/ml) significantly down-regulated total cellular LRP. Receptor-associated protein (RAP) mRNA expression was not affected by EGF in either tumor cells or primary astrocytes. The reduction of LRP in the tumor cells resulted from a specific decrease in LRP mRNA transcription, as determined by Northern blot and nuclear run-on experiments. These data suggest that EGF mediates a functional down-regulation of LRP endocytotic activity in astrocytic tumor cells and that LRP expression is differentially regulated in neoplastic and non-neoplastic astrocytes.

A modified human alpha 2-macroglobulin derivative that binds tumor necrosis factor-alpha and interleukin-1 beta with high affinity in vitro and reverses lipopolysaccharide toxicity in vivo in mice.

The plasma protein alpha 2-macroglobulin (alpha 2M) has been reported to bind the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta), which play a central role in the pathogenesis of chronic inflammatory disorders, including Crohn's disease and rheumatoid arthritis. In this study, we chemically modified alpha 2M to stabilize a conformation of the protein (termed MAC, Macroglobulin Activated for Cytokine binding) with greatly increased TNF-alpha- and IL-1 beta-binding activity. The equilibrium dissociation constant (KD) for the binding of TNF-alpha to MAC was 80 +/- 20 nM, reflecting a 100-fold increase in affinity compared with native alpha 2M. To test the ability of MAC to neutralize proinflammatory cytokines in vivo, we treated mice with lipopolysaccharide (LPS) by intravenous injection. When MAC (2.5 mg) was administered by intraperitoneal injection 1 hour before the LPS, 12 of 12 mice survived and were without signs of toxicity at 5 days. None of the mice survived in the untreated control group (0/26) or in the group treated with 2.5 mg of unmodified alpha 2M (0/4). MAC also prevented the large increase in expression of inducible nitric oxide synthase in the liver, kidneys, and heart of LPS-treated mice. A novel property of MAC, compared with previously studied anticytokine agents, was its ability to reverse LPS toxicity in 12 of 24 mice when administered after the plasma level of TNF-alpha was elevated. These studies demonstrate that a naturally occurring protein, alpha 2M, can be modified so that it acquires the properties of clinically active monoclonal antibodies. Thus, MAC may have therapeutic potential in the control of chronic inflammatory disorders.

Binding of urokinase-type plasminogen activator to its receptor in MCF-7 cells activates extracellular signal-regulated kinase 1 and 2 which is required for increased cellular motility.

Binding of urokinase-type plasminogen activator (uPA) to its receptor, uPAR, regulates cellular adhesion, migration, and tumor cell invasion. Some of these activities may reflect the ability of uPAR to initiate signal transduction even though this receptor is linked to the plasma membrane only by a glycosylphosphatidylinositol anchor. In this study, we demonstrated that single-chain uPA activates extracellular signal-regulated kinase 1 (ERK1) and ERK2 in MCF-7 breast cancer cells. Phosphorylation of ERK1 and ERK2 was increased 1 min after adding uPA and returned to baseline levels by 5 min. The amino-terminal fragment (ATF) of uPA, which binds to uPAR but lacks proteinase activity, also activated ERK1 and ERK2. Responses to uPA and ATF were eliminated when the cells were pretreated with PD098059, an inhibitor of mitogen-activated protein kinase kinase. uPA and ATF promoted the migration of MCF-7 cells across serum-coated Transwell membranes in vitro. Migration was increased 2.1 +/- 0.4-fold when uPA was added to the top chamber, 4. 8 +/- 0.8-fold when uPA was added to the bottom chamber, and 7.7 +/- 1.0-fold when uPA was added to both chambers. MCF-7 cells that were pulse-exposed to uPA for 30 min, and then washed to remove unbound ligand, demonstrated increased motility even though migration was allowed to occur for 24 h. PD098059 completely neutralized the effects of uPA on MCF-7 cellular motility, irrespective of whether the uPA was present for the entire motility assay or administered by pulse-exposure. These results demonstrate a novel, receptor-dependent signaling activity which is required for uPA-stimulated breast cancer cell migration.

Localization of the binding site for transforming growth factor-beta in human alpha2-macroglobulin to a 20-kDa peptide that also contains the bait region.

alpha2-Macroglobulin (alpha2M) functions as a major carrier of transforming growth factor-beta (TGF-beta) in vivo. The goal of this investigation was to characterize the TGF-beta-binding site in alpha2M. Human alpha2M, which was reduced and denatured to generate 180-kDa subunits, bound TGF-beta1, TGF-beta2, and NGF-beta in ligand blotting experiments. Cytokine binding was not detected with bovine serum albumin that had been reduced and alkylated, and only minimal binding was detected with purified murinoglobulin. To localize the TGF-beta-binding site in alpha2M, five cDNA fragments, collectively encoding amino acids 122-1302, were expressed as glutathione S-transferase (GST) fusion proteins. In ligand blotting experiments, TGF-beta2 bound only to the fusion protein (FP3) that includes amino acids 614-797. FP3 bound 125I-TGF-beta1 and 125I-TGF-beta2 in solution, preventing the binding of these growth factors to immobilized alpha2M-methylamine (alpha2M-MA). The IC50 values were 33 +/- 5 and 26 +/- 6 nM for TGF-beta1 and TGF-beta2, respectively; these values were comparable with or lower than those determined with native alpha2M or alpha2M-MA. A GST fusion protein that includes amino acids 798-1082 of alpha2M (FP4) and purified GST did not inhibit the binding of TGF-beta to immobilized alpha2M-MA. FP3 (0.2 microM) neutralized the activity of TGF-beta1 and TGF-beta2 in fetal bovine heart endothelial (FBHE) cell proliferation assays; FP4 was inactive in this assay. FP3 also increased NO synthesis by RAW 264.7 cells, mimicking an alpha2M activity that has been attributed to the neutralization of endogenously synthesized TGF-beta. Thus, we have isolated a peptide corresponding to 13% of the alpha2M sequence that binds TGF-beta and neutralizes the activity of TGF-beta in two separate biological assays.

Activated alpha 2-macroglobulin reverses the immunosuppressive activity in human breast cancer cell-conditioned medium by selectively neutralizing transforming growth factor-beta in the presence of interleukin-2.

The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). A human breast cancer cell line derived from malignant ascites (BRC 173) secreted TGF-beta, but not IL-10, into tissue culture supernatant (TCS). BRC 173 TCS suppressed natural killer (NK) and lymphokine-activated killer (LAK) cell activity and also blocked the generation of HLA-A*0201-restricted tumor-reactive cytotoxic T-lymphocyte (CTL) lines in vitro. Human alpha 2-macroglobulin (alpha 2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-beta family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. alpha 2M was converted to its activated conformation by reaction with methylamine (alpha 2M-MA) and then incubated with normal human peripheral blood lymphocytes (PBL) in the presence of IL-2 and BRC 173 TCS. Lysis of NK targets (K562) and LAK cell targets (DM6 melanoma) by the PBL was examined after 6 days of culture. PBL cultured in IL-2, without TCS or alpha 2M-MA, were lytic for both target cells. BRC 173 TCS substantially suppressed the lytic activity of the PBL in the presence of IL-2. When TGF-beta-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. alpha 2M-MA (280 nM) neutralized almost all of the immunosuppressive activity in the TCS, restoring 80-100% of the lytic activity without any apparent effect on the activity of IL-2. The ability of alpha 2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate the activated alpha 2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.

Characterization of the binding sites for plasminogen and tissue-type plasminogen activator in cytokeratin 8 and cytokeratin 18.

Cytokeratin 8 (CK8) is an intermediate filament protein that penetrates to the external surfaces of breast cancer cells and is released from cells in the form of soluble heteropolymers. CK8 binds plasminogen and tissue-type plasminogen activator (t-PA) and accelerates plasminogen activation on cancer cell surfaces. The plasminogen-binding site is located at the C-terminus of CK8. In this study, we prepared GST-fusion proteins which contained either 174 amino acids from the C-terminus of CK8 (CK8f) or 134 amino acids from the C-terminus of CK18 (CK18f). A third GST-CK fusion protein was identical to CK8fexcept that the C-terminal lysine was mutated to glutamine (CK8fK483Q). CK8f bound plasminogen; the K(D) was 0.5 microM. Binding was completely inhibited by epsilonACA. CK8fK483Q also bound plasminogen, albeit with decreased affinity (K(D) approximately 1.5 microM). CK18f did not bind plasminogen at all. All three fusion proteins bound t-PA equivalently, providing the first evidence that CK18 may function as a t-PA receptor, t-PA and plasminogen cross-competed for binding to CK8f. Thus, t-PA and plasminogen cannot bind to the same CK8f monomer simultaneously. Nevertheless, CK8f still promoted plasminogen activation, probably reflecting the fact that CK8f was purified in dimeric or tetrameric form. These studies demonstrate that CK8 may promote plasminogen activation by t-PA only when present in an oligomerized state. CK18 may participate in the oligomer, together with CK8, based on its ability to bind t-PA.

LDL receptor family-dependent and -independent pathways for the internalization and digestion of lipoprotein lipase-associated beta-VLDL by rat vascular smooth muscle cells.

Lipoprotein lipase (LPL) promotes the binding and internalization of beta-VLDL (very low density lipoprotein) by many cell types. We examined the function of receptors in the LDL receptor family (LRF) and heparan sulfate proteoglycans (HSPG) in the metabolism of LPL-associated beta-VLDLa by rat vascular smooth muscle cells (VSMCs) in culture. These cells express LDL receptor-related protein and the VLDL receptor, but not the LDL receptor. LPL greatly increased the binding of 125I-labeled beta-VLDL to VSMCs at 4 degrees C. Binding was almost entirely inhibited by heparin, but essentially unaffected by the potent LRF-antagonist, receptor-associated protein (RAP), indicating that LRFs do not contribute significantly to the VSMC binding capacity for LPL-associated beta-VLDL. At 37 degrees C, RAP inhibited the rapid internalization of LPL-associated 125I-labeled beta-VLDL and the digestion of the beta-VLDL into trichloroacetic acid soluble radioactivity; these processes still occurred, but at a decreased rate. RAP did not inhibit the ability of beta-VLDL-LPL complex to stimulate VSMC ACAT activity. Furthermore, in Oil red-O histochemistry experiments, which model foam cell transformation in vitro, RAP paradoxically increased cholesteryl ester storage in VSMCs treated with beta-VLDL and LPL under specific cell culture conditions. These results support a model in which the internalization of LPL-associated beta-VLDL by VSMCs is mediated by two pathways, one involving LRFs and a second that is independent of LRFs, probably involving direct uptake by HSPG. The LRF-dependent pathway leads to less cellular storage of cholesteryl ester and thus may be antiatherogenic under certain conditions.