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.

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.

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.

Catabolic pathways for streptokinase, plasmin, and streptokinase activator complex in mice. In vivo reaction of plasminogen activator with alpha 2-macroglobulin.

The catabolic pathways of streptokinase, plasmin, and activator complex prepared with human plasminogen were studied in mice. (125)I-streptokinase clearance occurred in the liver and was 50% complete in 15 min. Incubation with mouse plasma had no effect on the streptokinase clearance rate. Complexes of plasmin and alpha(2)-plasmin inhibitor were eliminated from the plasma by a specific and saturable pathway. Competition experiments demonstrated that this pathway is responsible for the clearance of injected plasmin. Streptokinase-plasminogen activator complex formed with either (125)I-plasminogen or (125)I-streptokinase cleared in the liver at a significantly faster rate than either of the uncomplexed proteins (50% clearance in <3 min). Streptokinase incubated with human plasma also demonstrated this accelerated clearance. p-Nitrophenyl-p'-guanidinobenzoate-HCl or pancreatic trypsin inhibitor-treated complex cleared slowly compared with untreated complex independent of which protein was radiolabeled. Significant competition for clearance was demonstrated between alpha(2)-macroglobulin-trypsin and activator complex only when the plasmin(ogen) was the radiolabeled moiety. Large molar excesses of alpha(2)-plasmin inhibitor-plasmin failed to retard the clearance of activator complex. Hepatic binding of streptokinase-plasmin, in liver perfusion experiments, was dependent upon prior incubation with plasma (8-10% uptake compared to a background of approximately 2.5%). Substitution of human alpha(2)-macroglobulin for plasma also resulted in binding when the incubation was performed for 10 min at 37 degrees C (7.5%). Electrophoresis experiments confirmed the transfer of 0.8 mol plasmin/mol alpha(2)-macroglobulin when activator complex was incubated at 37 degrees C with alpha(2)-macroglobulin for 40 min. Streptokinase transfer from activator complex to alpha(2)-macroglobulin was negligible. The in vivo clearance of activator complex is proposed to involve active attack of the complex on the alpha(2)-macroglobulin "bait region," resulting in facilitated plasmin transfer. Dissociated streptokinase is rapidly bound and cleared by sites in the liver.

A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function.

A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.

Regulation of macrophage alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein by lipopolysaccharide and interferon-gamma.

alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a broad specificity receptor that may function in lipoprotein metabolism, proteinase regulation, and growth factor regulation. In this study, we demonstrated that alpha 2M-R/LRP expression in macrophages can be markedly decreased by LPS and by IFN-gamma. Regulation of alpha 2M-R/LRP in RAW 264.7 cells was demonstrated at the mRNA, antigen, and receptor-function levels. In receptor-function studies, the decrease in alpha 2M-R/LRP expression was detected as a 90% decrease in the Bmax or maximum receptor binding capacity for activated alpha 2M after treatment with LPS or IFN-gamma. Western blot analysis of whole cell lysates demonstrated significant loss of alpha 2M-R/LRP heavy-chain. Northern blot analysis of poly(A)+ RNA revealed a marked decrease in alpha 2M-R/LRP mRNA after treatment with LPS (79% decrease) or IFN-gamma (70% decrease). Other cytokines, including tumor necrosis factor-alpha, transforming growth factor-beta-1, and interleukin-6 did not regulate alpha 2M-R/LRP. The ability of LPS and IFN-gamma to regulate alpha 2M-R/LRP was confirmed in experiments with primary cultures of murine bone marrow macrophages. These studies demonstrate that macrophage alpha 2M-R/LRP is subject to significant downregulation by physiologically significant cytokines and signaling macromolecules.

An alpha 2-macroglobulin receptor-dependent mechanism for the plasma clearance of transforming growth factor-beta 1 in mice.

Radioiodinated transforming growth factor-beta 1 (TGF-beta 1) bound to the plasma proteinase inhibitor, alpha 2-macroglobulin (alpha 2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When alpha 2M conformational change was induced with methylamine, 125I-TGF-beta 1 binding significantly increased. Intravenously injected 125I-TGF-beta 1 cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-beta 1-alpha 2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t1/2 of 4 min. Greater than 90% of the radioactivity was recovered in the liver. The clearance and distribution of 125I-TGF-beta 1-alpha 2M-methylamine were equivalent to those observed with 125I-alpha 2M-methylamine and 125I-alpha 2M-trypsin. The latter two radioligands clear via specific alpha 2M receptors in the liver. Large molar excesses of alpha 2M-trypsin or alpha 2M-methylamine competed with 125I-TGF-beta 1-alpha 2M-methylamine for plasma clearance. Native alpha 2M, which does not bind to the alpha 2M receptor, did not compete. The receptor binding domain of alpha 2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting alpha 2M preparations still bound 125I-TGF-beta 1; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that alpha 2M can mediate the plasma clearance of a growth factor via the alpha 2M receptor system. We propose that alpha 2M, the alpha 2M receptor, and proteinases may function as a concerted system to regulate TGF-beta 1 activity and the activity of related factors in vivo.

Clearance and distribution of recombinant murine gamma-interferon in mice.

Recombinant murine gamma-interferon (rIFN-gamma) was radiolabeled by a novel procedure which does not require the use of preiodinated Bolton-Hunter reagent (specific activities of 0.5-3.0 microCi/micrograms). Gel filtration chromatography of the radiolabeled preparation yielded two peaks. The early eluting peak contained disulfide stabilized aggregates with minimal interferon antiviral activity. The second peak contained activity that was consistently greater than or equal to that of the nonradiolabeled rIFN-gamma. Two bands with apparent molecular weights of 17,000 and 34,000 were observed when the second peak was analyzed by SDS gel electrophoresis. Fractions comprising each of the two chromatography peaks were pooled separately and subjected to gel filtration again on identical columns 24 h after completion of the first column run. The elution volumes of each peak remained unchanged suggesting that the two forms are not in rapid equilibrium. The plasma clearance rates of [125I]rIFN-gamma before and after purification by chromatography were initially rapid but multiphasic. The slower phases of clearance did not result from stable association of the rIFN-gamma with plasma proteins. In organ distribution studies, the liver and spleen sequestered significant amounts of [125I]rIFN-gamma; however, the highest concentration of rIFN-gamma was recovered in the kidneys. A functional nephrectomy procedure was used to further study the role of the kidneys in rIFN-gamma clearance. Eliminating the kidneys significantly increased the amount of rIFN-gamma retained in the circulation, particularly at later times when the vascular [125I]rIFN-gamma levels were approximately threefold higher than in nonnephrectomized mice.

Effects of diethyldithiocarbamate and nine other nucleophiles on the intersubunit protein cross-linking and inactivation of purified human alpha 2-macroglobulin by cis-diamminedichloroplatinum(II).

The cross-linking and inactivation of the plasma protein alpha 2-macroglobulin by cis-diamminedichloroplatinum(II) (cisplatin; Gonias, S. L., and Pizzo, S. V. J. Biol. Chem., 256: 12478-12484, 1981) was used to study platinum(II)-protein binding in the presence of compounds of therapeutic or biochemical significance. Diethyldithiocarbamate, potassium cyanide (KCN), sodium thiocyanate, L-methionine, N-acetyl-L-cysteine, 2-aminoethanethiol, L-cysteine, L-lysine, L-histidine, and L-arginine demonstrated variable capacity to inhibit reaction of cisplatin with protein and to reverse bidentate platinum(II)-protein binding in the in vitro model system. alpha 2-Macroglobulin lost 90% of its activity and was completely cross-linked, as determined with polyacrylamide gel electrophoresis, after reaction with cisplatin (0.6 to 1.0 mM). When diethyldithiocarbamate (4 to 15 mM) was incubated with alpha 2-macroglobulin and cisplatin, protein inactivation and cross-linking were totally prevented. In experiments with alpha 2-macroglobulin-platinum(II) complex, purified by gel filtration chromatography, 1.0 mM diethyldithiocarbamate completely reactivated the protein and eliminated nearly all of the intersubunit cross-links. Only KCN was comparably effective as an inhibitor of the reaction of cisplatin with alpha 2-macroglobulin; however, KCN was significantly less reactive with preformed platinum(II)-protein bonds than was diethyldithiocarbamate. N-Acetyl-L-cysteine, 2-aminoethanethiol, and L-cysteine were moderately reactive with free cisplatin. This group of compounds also demonstrated a low level of reactivity with the purified alpha 2-macroglobulin-platinum(II) complex. L-Methionine inhibited reaction of cisplatin with the protein, but was ineffective at reversing the reaction in the concentration range studied. The remaining compounds had little or no effect on the reaction of cisplatin with alpha 2-macroglobulin. The ability of diethyldithiocarbamate to displace nucleophilic protein groups from highly stable bonds with platinum(II) may be critical in its function as a rescue agent, limiting cisplatin toxicity towards nontumor cells.

Altered clearance of human alpha 2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II).

Clearance studies were performed in mice using alpha 2-macroglobulin (alpha 2M), alpha 2M-trypsin complex and alpha 2M-CH3NH2 complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 microM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native alpha 2M was unchanged following incubation with cis-DDPt. alpha 2M-trypsin and alpha 2M-CH3NH2 cleared rapidly from the circulation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and alpha 2M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of alpha 2M subunits was obtained; however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of alpha 2M did not duplicate the effect of cis-DDPt on alpha 2M clearance, nor did it inhibit the effect of cis-DPPt on alpha 2M clearance. Plasma elimination of alpha 2M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.

Inactivation of alpha 2-antiplasmin by limited reaction with cis-dichlorodiammineplatinum (II)

alpha 2-Antiplasmin (alpha 2AP), a serpin proteinase inhibitor with two methionine residues in its reactive center, was treated with cis-dichlorodiammineplatinum (II) (cis-DDP). This compound has been utilized previously to specifically modify methionine residues. After reaction, the alpha 2AP demonstrated decreased inhibitory activity against plasmin, miniplasmin, trypsin and chymotrypsin. The reduction in activity depended on the concentration of cis-DDP; however, the amount of activity retained by the treated alpha 2AP was equivalent with each of the four proteinases. alpha 2AP that was incubated with 1.0 mM cis-DDP for 3 h at 37 degrees C was 90% inactivated. These same conditions resulted in the binding of only 1.0-1.5 mol of platinum per mol of inhibitor. In experiments with lower concentrations of cis-DDP, the amount of incorporated platinum directly correlated with the amount of inactivated alpha 2AP (1:1 stoichiometry). Reactions and functions of alpha 2AP that do not result in proteinase inhibition were not affected by cis-DDP. Cleavage of alpha 2AP by elastase, which occurs near the proteinase inhibition site, was unaffected. In addition, the affinity of alpha 2AP for the K1-3 region of plasminogen remained unchanged after treatment. These data strongly suggest that the reaction of alpha 2AP with cis-DDP involves principally a single site on the inhibitor and that this site is critical for proteinase inhibitory activity. The most likely candidate is the P'1 methionine which is adjacent to the peptide bonds cleaved in the proteinase inhibitory reactions but not in the elastase reaction.

Physical properties of human alpha 2-macroglobulin following reaction with methylamine and trypsin.

Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin complex and alpha 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native alpha 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between alpha 2-macroglobulin-methylamine and native alpha 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in alpha 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with alpha 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (S0(20),W) of 20.5 was determined for alpha 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted alpha 2-macroglobulin. This increase in sedimentation velocity is attributed to a 10% decrease in alpha 2-macroglobulin Stokes radius. alpha 2-Macroglobulin-trypsin complex prepared by reaction of the protease at a 2-fold molar excess with the inhibitor was a S0(20),W of 20.3. Although this sedimentation coefficient does reflect compacting of the alpha 2-macroglobulin structure compared to native alpha 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from pockets in the alpha 2-macroglobulin structure.

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.

Transformation of hairy cell leukemia to EBV genome-containing aggressive B cell lymphoma.

Hairy cell leukemia is a preplasmacytic B cell leukemia which is not EBV associated, although elevated titers of Epstein-Barr virus (EBV) antibodies have been seen in this leukemia and chronic lymphocytic leukemia. Hairy cells are not readily susceptible to EBV infection in vitro, even though they are EBV receptor-positive B cells. We have observed a 59-year-old patient who after 9 years of hairy cell leukemia developed a well-differentiated IgG-kappa monoclonal B cell lymphoma without further evidence of hairy cell leukemia. Pathologically, the lymphoma showed plasmacytic differentiation, and in the patient's serum, a 2 g/dl monoclonal IgG-kappa component was present. DNA extracted from the lymphomatous lymph node hybridized with DNA fragments of a reiterated sequence of EBV, IR1. The transformation, with no chemotherapy involved, from a preplasmacytic leukemia into a lymphoplasmacytic lymphoma with monoclonal gammopathy may be related to the entry of EBV into these cells. Studies at the molecular level may help understand mechanisms of malignant transformation or interconversion in lymphoproliferative disorders of the B cell type.

Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line.

Low density lipoprotein receptor-related protein (LRP) is a major receptor for multiple ligands, including chylomicron and VLDL remnants, bacterial toxins, viruses, proteinases, lipoprotein lipase, and activated alpha2-macroglobulin (alpha2M). In this study, we used Northern blot analyses and nuclear run-on experiments to demonstrate that interferon-gamma (IFN-gamma) causes a concentration-dependent decrease in steady-state LRP mRNA expression and gene transcription rate in RAW 264.7 cells. IFN-gamma also markedly increased expression of inducible nitric oxide synthase (NOS), as expected; however, the increase in nitric oxide was not responsible for the down-regulation of LRP expression since the NOS inhibitor, N(G)-monomethyl-L-arginine, did not preserve LRP expression in IFN-gamma-treated cells. Transforming growth factor-beta1 (TGF-beta1; 2.5 ng/mL) had no independent effect on LRP expression and did not modify the response to IFN-gamma when the two cytokines were added simultaneously to cultures. When TGF-beta1 was added 24 h prior to IFN-gamma, the extent of LRP down-regulation was significantly reduced. Specific binding of the LRP ligand, activated (125)I-alpha2M, was decreased by 76 +/- 5% in cells treated with 100 U/mL IFN-gamma, but only by 45 +/- 7% in cells treated with 100 U/mL IFN-gamma after TGF-beta1-pretreatment. The antagonistic activity of TGF-beta1 on the IFN-gamma response in RAW 264.7 cells did not result from a change in LRP mRNA stability or IFN-gamma receptor expression, as determined by Northern blot analyses and (125)I-IFN-gamma binding experiments. The studies presented here suggest that the balance between IFN-gamma and TGF-beta1 may be critical in determining LRP expression at sites of infection and inflammation.

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.

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.

Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex.

Activin A is a member of the transforming growth factor-beta family of growth factors and a potent regulator of cellular activity. A number of binding proteins for activin A have been identified, including alpha 2-macroglobulin (alpha 2M). Alpha 2M has several conformational states that are known to have different growth factor-binding properties. The effect of alpha 2M conformation on activin A binding has not been characterized. The aims of this study were to determine 1) whether activin A binds preferentially to the native (alpha 2M-N) or "activated" (alpha 2M*) conformation of alpha 2M, 2) the affinity of different alpha 2M conformations for activin A, and 3) the fate of activin A complexed with alpha 2M-N or alpha 2M* in vivo. [125I]Activin A associated with alpha 2M in plasma and follicular fluid and with purified alpha 2Ms. In this qualitative assay, more activin A was associated with alpha 2M* than with alpha 2M-N. The affinity of the activin A-alpha 2M interaction was determined. The Kd values for activin A-alpha 2M* and activin A-alpha 2M-N were 190 +/- 30 and 510 +/- 60 nM, respectively. The plasma clearance profiles and tissue distribution of uncomplexed activin A and purified alpha 2M*-activin A complex were determined. Radiolabeled activin A cleared in a biphasic manner, with rapid clearance over the initial 10 min and substantially slower clearance over the subsequent 20 min. During the slow phase of clearance, activin A formed a complex with circulating alpha 2M-N. In contrast, radiolabeled activin A-alpha 2M* complexes were rapidly cleared from plasma with a half-life of approximately 5 min and were specifically targeted to alpha 2M receptors in vivo. These studies reveal that alpha 2M can maintain activin A in the circulation or rapidly target the hormone for plasma clearance depending on the conformational state of the carrier protein in vivo.

Chemical modification of alpha2-macroglobulin to generate derivatives that bind transforming growth factor-beta with increased affinity.

alpha2-Macroglobulin (alpha2M) binds a number of cytokines, including transforming growth factor-beta1 (TGF-beta1) and TGF-beta2. The affinity of these interactions depends on the alpha2M conformation. In this investigation, we treated human alpha2M with cis-dichlorodiammineplatinum (II) (cis-Pt), a crosslinking reagent that partially 'locks' the alpha2M conformation, and then with methylamine to generate a preparation (alpha2M-P/M) consisting of stable alpha2M conformational intermediates. alpha2M-P/M bound TGF-beta1 and TGF-beta2 with higher affinity than any other form of alpha2M studied to date. The equilibrium dissociation constants were 14 and 2 nM for TGF-beta1 and TGF-beta2, respectively. alpha2M-P/M, at 100 nM, neutralized the activity of TGF-beta1 by about 75% in an endothelial cell proliferation assay. The equivalent concentration of native alpha2M or methylamine-modified alpha2M had no effect. These studies demonstrate that the potential of alpha2M as a cytokine carrier and neutralizer may not be fully realized in either the native or completely activated conformations.

Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein is increased in reactive and neoplastic glial cells.

alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a multi-functional cell-surface receptor that has been implicated in diverse physiologic processes. In normal human brain, alpha 2M-R/LRP is expressed principally by pyramidal neurons with localization to cell bodies and proximal processes. By contrast, alpha 2M-R/LRP is not present in either the cell bodies or processes of most normal macroglia (including astrocytes). In this investigation, we studied the expression of alpha 2M-R/LRP in the brain, following tissue injury or neoplastic transformation, by immunohistochemistry. Significantly increased alpha 2M-R/LRP immunoreactivity was identified in reactive astrocytes, indicating the expression of this receptor is regulated in vivo in response to brain injury. alpha 2M-R/LRP immunoreactivity was also detected in glial cell tumors; this finding is novel since malignant transformation is typically thought to turn off expression of this receptor.