Biomedical subjects

A Mazar

Publications and source records attributed to A Mazar.

At least 19 recordsLinked to original sources

PubMed2000

A region in domain II of the urokinase receptor required for urokinase binding.

The urokinase receptor is composed of three homologous domains based on disulfide spacing. The contribution of each domain to the binding and activation of single chain urokinase (scuPA) remains poorly understood. In the present paper we examined the role of domain II (DII) in these processes. Repositioning DII to the amino or carboxyl terminus of the molecule abolished binding of scuPA as did deleting the domain entirely. By using alanine-scanning mutagenesis, we identified a 9-amino acid continuous sequence in DII (Arg(137)-Arg(145)) required for both activities. Competition-inhibition and surface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for scuPA approximately 5-fold due to an increase in the "off rate." Mutation of Arg(137), Arg(142), and Arg(145), each to alanine, leads to an approximately 100-fold decrease in affinity attributable to a 10-fold decrease in the apparent "on rate" and a 6-fold increase in off rate. These differences were confirmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A displayed a 50% reduction in scuPA-mediated plasminogen activation activity, whereas the 3-arginine variant was unable to stimulate scuPA activity at all. Mutation of the three arginines did not affect binding of a decamer peptide antagonist of scuPA known to interact with DI and DIII. However, this mutation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild type DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer function but appears to be required for interdomain cooperativity.

Animals↗

PubMed2000

Novel interactions between urokinase and its receptor.

Urokinase-type plasminogen activator (uPA) binds to its receptor (uPAR) with a K(d) of about 1 nm. The catalytic activity of the complex is apparent at uPA concentrations close to K(d). Other functions of the complex, such as signal transduction, are apparent at much higher concentrations (35-60 nm). In the present study, we show that uPA and recombinant soluble uPAR (suPAR), at concentrations that exceed the K(d) and the theoretical saturation levels (10-80 nm), establish novel interactions that lead to a further increase in the activity of the single-chain uPA (scuPA)/suPAR and two-chain uPA (tcuPA)/suPAR complexes. Experiments performed using dynamic light scattering, gel filtration, and electron microscopy techniques indicate that suPAR forms dimers and oligomers. The three techniques provide evidence that the addition of an equimolar concentration of scuPA leads to the dissociation of these dimers and oligomers. Biacore data show that suPAR dimers and oligomers bind scuPA with decreased affinity when compared with monomers. We postulate that uPAR is present in equilibrium between oligomer/dimer/monomer forms. The binding of uPA to suPAR dimers and oligomers occurs with lower affinity than the binding to monomer. These novel interactions regulate the activity of the resultant complexes and may be involved in uPA/uPAR mediated signal transduction.

Fibrinolysis↗

PubMed2000

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo.

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

Amino Acid Sequence↗

PubMed1999

Transcriptional induction of the urokinase receptor gene by a constitutively active Src. Requirement of an upstream motif (-152/-135) bound with Sp1.

Since c-src overexpression increases colonic cell invasiveness and because both Src activity and urokinase receptor protein are elevated in invasive colon cancers, the present study was undertaken: 1) to determine if a constitutively active Src regulates urokinase receptor expression and 2) to identify required cis-elements and trans-acting factors. SW480 colon cancer cells transfected with an expression plasmid (c-srcY527F) encoding a constitutively active Src protein manifested increased urokinase receptor gene expression and Src activity. Treatment of the src transfectants with a Src-inhibitor (PD173955) reduced urokinase receptor protein levels and laminin degradation. Inasmuch as we recently implicated an upstream region of the urokinase receptor promoter (-152/-135) in constitutive urokinase receptor expression, we determined its role for the induction by src. Whereas the activity of a CAT reporter driven by this region was stimulated by c-srcY527F, the u-PAR promoter mutated at the Sp1-binding motif in the -152/-135 region was not. Nuclear extracts from the src transfectants demonstrated increased Sp1 binding to region -152/-135 compared with those from SW480 cells. Finally, endogenous urokinase receptor protein amounts in 10 colon cancers and corresponding normal colon correlated with Src specific activity. These data suggest that urokinase receptor gene expression is regulated by Src partly via increased Sp1 binding.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

Pulmonary alveolar proteinosis in Israel: ethnic clustering.

BACKGROUND: Pulmonary alveolar proteinosis is a rare disease in which a surfactant-like phospholipid-rich protein accumulates in the lungs. The disease is amenable to effective therapy by total lung lavage. OBJECTIVES: To investigate the prevalence, ethnic distribution and course of PAP in Israel. METHODS: A countrywide survey was conducted during which pulmonologists were questioned about patients with PAP. The patients were examined and their charts, radiological images, pathological slides and physiological data were reviewed. RESULTS: The survey yielded 15 patients (8 females) during the period 1976-98 (14 in the last decade), giving a prevalence of 3.7 x 10(6) and an incidence of 0.36 x 10(6)/year. Mean age of the patients was 33 +/- 13 years (range 0.5-46 years). Seven patients were North African (two were siblings), four were from Iraq and two were Arabs; there was only one Ashkenazi Jew (a child). Symptoms at the onset were dyspnea and chest pain. Spontaneous remission occurred in at least 3 patients, and 10 patients required 1-4 bronchoalveolar lavage treatments. The subjective and physiological response was favorable, but there was less consistent radiological improvement. CONCLUSION: The prevalence of PAP in Israel is approximately 3.7 x 10(6). Most cases occurred in Jews who had immigrated from North Africa or Iraq, and two were siblings. The prevalence among the Arab population appears to be similar. This clustering suggests the existence of a genetic predisposition. The course of the disease appears to be similar to that reported elsewhere.

Adolescent↗

PubMed1998

Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules.

The urokinase-type plasminogen activator receptor (u-PAR) has been implicated in tumor progression, and previous studies have shown that the expression of this gene is strongly up-regulated by PMA. Although the signaling mechanism by which PMA modulates u-PAR expression is not known, the effect of this phorbol ester on the expression of other genes has been ascribed to activation of the c-Raf-1-ERK signaling pathway. However, in the current study we examined an alternate possibility that the inductive effect of PMA on u-PAR expression also required a JNK1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer cells, which contain low JNK activities, resulted in a rapid (5 min) increase in JNK activity. Maximal JNK activity (12-fold induction) occurred after 30 min; this preceding the earliest detected rise in u-PAR protein (2 h). Dose-response studies with PMA also indicated that the increased JNK activity was tightly correlated with elevated u-PAR protein levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (-184) and in PMA-treated cells this motif was bound with c-Jun as indicated from mobility shift assays. PMA up-regulated the c-Jun trans acting activity as indicated by the higher activity of a GAL4-regulated luciferase reporter in phorbol-ester-treated cells co-transfected with an expression vector encoding the c-Jun transactivation domain fused to the GAL4 DNA-binding domain. The ability of PMA to stimulate u-PAR promoter activity was effectively titrated out by the co-expression of either a kinase-defective JNK1 or a dominant negative MEKK1 the latter being an upstream activator of JNK1. Conversely, u-PAR promoter activity was stimulated by the co-expression of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid. We also determined the biological significance of the JNK1-dependent signaling cascade in regulating u-PAR promoter activity by c-Ha-ras since this oncogene is activated and/or overexpressed in a variety of tumors including ovarian cancer. Transfection of an activated c-Ha-ras into OVCAR-3 cells stimulated u-PAR promoter activity over 20-fold and this could be countered by the individual expression of dominant negative expression constructs to Rac-1, MEKK1 or JNK1. Taken together, these data suggest that the PMA- or c-Ha-Ras-dependent stimulation of u-PAR gene expression requires a JNK1-dependent signaling module and that, at least for PMA, the concurrent stimulation of a JNK1-independent signaling module is also required. Thus, caution should be exercised in invoking linear signaling modules to account for the regulation of inducible gene expression.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Cells, Cultured↗

PubMed1997

Prevention of breast cancer growth, invasion, and metastasis by antiestrogen tamoxifen alone or in combination with urokinase inhibitor B-428.

Urokinase (urokinase plasminogen activator, uPA) and its cell surface receptor (uPA receptor, uPAR) play an important role in a variety of physiological and pathological processes requiring cell migration and tissue remodeling. Using our syngeneic model of uPAR overexpression by the rat breast cancer cell line Mat B-III, we have examined the ability of the nonsteroidal antiestrogen, tamoxifen (TAM), and of a selective synthetic inhibitor of uPA, 4-iodo benzo[b]thiophene-2-carboxamidine (B-428), to inhibit expression of uPA and uPAR as well as cell growth, invasion, and metastasis of wild-type Mat B-III cells and of cells overexpressing uPAR (Mat B-III-uPAR). Both TAM and B-428 inhibited uPAR gene transcription, mRNA expression, protein production and also decreased the proliferative and invasive capacity of Mat B-III and Mat B-III-uPAR. The effects of TAM and B-428 were more pronounced when these agents were tested in combination. Both control and experimental cells (1 x 10(6) cells) were inoculated orthotopically into the mammary fat pad of syngeneic female Fisher rats, and animals were infused i.p. with either TAM and B-428 alone or in combination for 2 weeks. Control animals receiving vehicle alone developed large tumors and macroscopic metastases to lungs, liver, and lymph nodes. In contrast to this, experimental animals receiving TAM and B-428 showed a significant decrease in primary tumor volume and metastases. Combination therapy had especially marked effects in blocking progression of the primary tumor in experimental animals inoculated with highly aggressive Mat B-III-uPAR cells. These results underscore the utility of anti-proteolytic agents (B-428) in addition to standard hormone therapy (TAM) in advanced breast cancer patients where the uPA/uPAR system plays a key role in tumor progression.

Amidines↗

PubMed1997

Plasminogen activation by pro-urokinase in complex with its receptor--dependence on a tripeptide (Spectrozyme plasmin).

The intrinsic activity of single-chain pro-urinary-type plasminogen activator (pro-uPA) and whether its receptor (uPAR) potentiates this activity remains controversial. In this report, the pro-uPA/uPAR-(1-281)-peptide complex in solution is shown to have equivalent plasminogen-activator activity to that of active two-chain uPA (tc-uPA). However, the activity of the complex was dependent on a synthetic tripeptide, Spectrozyme plasmin (Spl, H-D-2-aminohexanoic acid(Ahx)-hexatyrosyl-lysine-p-nitroanilide), which can also be used as a chromogenic substrate for plasmin. Furthermore, this activity could be completely suppressed by commonly used carrier proteins and detergents. The pro-uPA/uPAR-(1-281)-peptide complex at 1 nM displayed similar activity to that of tc-uPA for either [Glu1]plasminogen or [Lys77]plasminogen in chromogenic assays with Spl present as the plasmin substrate. When assayed with another plasmin substrate, S2251, the pro-uPA/uPAR-(1-281)-peptide complex was unable to activate plasminogen. The pro-uPA/uPAR-(1-281)-peptide complex and tc-uPA also showed a similar extent of plasminogen activation as measured by SDS/PAGE, when incubated with plasminogen and Spl in the presence of 100 micro M aprotinin, and plasminogen activation by pro-uPA alone was also stimulated in the presence of Spl in this assay. Activation of plasminogen by the pro-uPA/uPAR-(1-281)-peptide strictly required the presence of Spl, and pro-uPA remained in single-chain form during these assays. This activity of the pro-uPA/uPAR-(1-281)-peptide complex but not that of tc-uPA was completely inhibited by human serum albumin, bovine serum albumin, Tween-80, Triton X-100, and Pluronic-F68. Taken together, the data indicates that uPAR-(1-281)-peptide itself is not sufficient to augment pro-uPA activity and the presence of an effector molecule (e.g. Spl) is required to elicit the full plasminogen-activator activity of the pro-uPA/uPAR-(1-281)-peptide complex. It remains to be seen whether there is a physiological counterpart to this phenomenon.

Enzyme Activation↗

PubMed1997

Embryonic fibroblasts that are genetically deficient in low density lipoprotein receptor-related protein demonstrate increased activity of the urokinase receptor system and accelerated migration on vitronectin.

Low density lipoprotein receptor-related protein (LRP) mediates the endocytosis of diverse ligands, including urokinase plasminogen activator (uPA) and its receptor, uPAR, which have been implicated in cellular migration. The purpose of this study was to determine whether LRP affects cellular migration. Murine embryonic fibroblasts (MEF) that are LRP-deficient due to targeted gene disruption and exotoxin selection (MEF-2), heterozygous fibroblasts (PEA-10), and wild-type fibroblasts (MEF-1) were compared. When cultures were denuded of cells in a 1-mm-wide strip, all three cell types migrated into the denuded area. The MEF-2 cells migrated nearly twice as rapidly as the MEF-1 cells or PEA-10 cells. The difference in migration velocity was duplicated in culture wells that were precoated with serum or vitronectin and partially duplicated in wells coated with fibronectin but not in wells coated with type I collagen or Matrigel. uPA was detected in MEF-2 conditioned medium (CM) at a concentration of 0.30 +/- 0.02 nM, which was 13-fold higher than the level detected in MEF-1 CM or PEA-10 CM, suggesting one potential mechanism for the enhanced migration of MEF-2 cells. uPAR was also increased on MEF-2 cells by 4-5-fold, as determined by PI-PLC release, and by 2.5-fold, as determined by a uPA/uPAR activity assay. Mannosamine treatment, which down-regulates cell-surface uPAR, decreased MEF-2 migration by 40% without significantly affecting MEF-1 migration. MEF-2 CM, which is uPA-rich, increased the rate of MEF-1 migration, and MEF-1 CM did not. These studies demonstrate alterations in cellular migration and in the activity of the uPA/uPAR system which accompany complete deficiency of LRP expression in fibroblasts. We propose that uPA and uPAR form an autocrine loop for promoting fibroblast migration and that LRP counteracts the activity of this system.

Animals↗

PubMed1997

Soluble human urokinase receptor is composed of two active units.

The mechanism by which single-chain urokinase (scuPA) binds to its receptor (uPAR) is incompletely understood. We report that a fragment comprising the first domain of recombinant soluble uPAR (sDI) as well as a fragment comprising the remaining domains (sDII-DIII) competes with the binding of recombinant full-length soluble uPAR (suPAR) to scuPA with an IC50 = 253 nM and an IC50 = 1569, respectively. sDII-III binds directly to scuPA with Kd = 238 nM. Binding of scuPA to each fragment also induces the expression of plasminogen activator activity. sDI and sDII-DIII (200 nM each) induced activity equal to 66 and 36% of the maximum activity induced by full-length suPAR (5 nM), respectively. Each fragment also stimulates the binding of scuPA to cells lacking endogenous uPAR. Although scuPA binds to sDI and to sDII-DIII through its amino-terminal fragment, the fragments act synergistically to inhibit the binding of suPAR and to stimulate plasminogen activator activity. Furthermore, sDII-DIII retards the velocity and alters the pattern of cleavage of sDI by chymotrypsin. These results suggest that binding of scuPA to more than one epitope in suPAR is required for its optimal activation and association with cell membranes.

Humans↗

PubMed1996

Single-chain urokinase-type plasminogen activator bound to its receptor is relatively resistant to plasminogen activator inhibitor type 1.

Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule to its cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affinity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of scuPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor-bound tcuPA. Binding of PaI-1 to suPAR/scuPA complexes is totally reversible and can be overcome by increasing the concentration of plasminogen, suggesting a competitive mechanism of inhibition (Ki = 18 nmol/L). Binding of scuPA to suPAR also retards its cleavage by plasmin. These results indicates that binding of single-chain urokinase to its receptor promotes its activity, retards its inhibition, and protects it from conversion to a two-chain form of the enzyme, a step that may precede its inactivation and clearance from cell surfaces. These results are consistent with a physiologic role for receptor-bound single-chain urokinase as a cellular plasminogen activator.

Cell Line↗

PubMed1995

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.

Capillaries↗

PubMed1994

Urokinase inhibits HL-60 cell proliferation in vitro.

The binding of urokinase-type plasminogen activator (u-PA) to its receptor (u-PA-R) is required for morphological and functional maturation during monocyte differentiation of the promyelocytic leukaemia line HL-60. This paper reports that monocyte differentiation of HL-60 cells induced by 1,25 dihydroxyvitamin D2 (vitamin D2) results in a marked increase in expression of u-PA and u-PA-R. This increase in u-PA expression is of greater magnitude than is observed after culture with interferon-gamma (IFN gamma), another potent inducer of monocytic differentiation. Dimethyl sulphoxide (DMSO), an agent that induces granulocytic differentiation, also increased expression of u-PA. However, culture with the granulocyte-inducing all-trans retinoic acid (RA) did not induce an increase in surface expression of u-PA or u-PA-R. The vitamin D2-induced increase in cell-surface u-PA was not coincident with an increase in steady-state levels of u-PA mRNA, suggesting that intracellular stores of this protein, translational or post-translational mechanisms of regulation, or some other regulatory mechanism may be responsible for the increase in u-PA during differentiation. To ascertain an association between the increased expression of cell-surface u-PA and reduced proliferation that accompanies differentiation, the effect of u-PA on cellular proliferation of HL-60 cells was measured. Both pro-u-PA (whole molecule) and fragments of u-PA that retained receptor-binding capability caused a marked inhibition of HL-60 proliferation in the absence of vitamin D2.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoreceptors↗

PubMed1994

Expression of urokinase receptors by human trophoblast. A histochemical and ultrastructural analysis.

BACKGROUND: Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors by trophoblast may facilitate these processes by focusing plasminogen activator activity to discrete sites on the cell surface and promoting the activation of cell-bound plasminogen. However, although urokinase receptors are expressed by cultured trophoblast, the expression of these receptors by trophoblast in vivo has not been examined. EXPERIMENTAL DESIGN: Immunohistochemistry and immunoelectron microscopy were used to characterize the expression of urokinase receptors by villous and extravillous trophoblast at several points in gestation. RESULTS: Urokinase receptors were expressed in a polarized fashion at the leading edge of migrating extravillous trophoblast cells. Receptors were also abundantly expressed during the first and second trimesters of gestation by villous trophoblast, where they were located on apical villous projections and within intracellular vacuoles, a subset of which were lysosomes. CONCLUSIONS: The polarized expression of urokinase receptors by invasive extravillous trophoblast cells is consistent with a role for these receptors in mediating the extent and directionality of trophoblast migration. In contrast, the expression of urokinase receptors by villous trophoblast, which are not actively invasive in vivo, may serve to facilitate the generation of plasmin at the interface of these cells with maternal plasma, thereby limiting the deposition of fibrin within the placental intervillous spaces. Diminished urokinase receptor expression by villous trophoblast at term may represent a physiologic adaptation to diminish local fibrinolysis and limit hemorrhage at parturition.

Cell Movement↗

PubMed1993

Effect of tethered peptidylchloromethylketone inhibitors on thermal stability and domain interactions of urokinase and other serine proteases.

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

Amino Acid Chloromethyl Ketones↗

PubMed1993

Regulation of urokinase-type plasminogen activator expression in squamous-cell carcinoma of the oral cavity.

We undertook a study to determine the role and regulation of expression of urokinase-type plasminogen activator in squamous-cell carcinoma of the oral cavity. The contribution of urokinase to the invasive process was clearly demonstrated in experiments in which in vitro invasion by a cultured squamous-cell carcinoma cell line was substantially reduced by a monoclonal antibody directed at the catalytic site of urokinase. The conditioned medium from 2 invasive cell lines contained high levels of urokinase. Examination of resected tumors for urokinase revealed (a) localization of the antigen to the tumor cells and (b) higher levels of the plasminogen activator in tumor tissue than in adjacent non-malignant tissue. These results suggested that elevated expression of urokinase is a common feature of this malignancy. The mRNA half-lives of cell lines expressing high and low levels of urokinase were similar, indicating that elevated levels of mRNA for the plasminogen activator were not a consequence of a more stable transcript. No evidence of u-PA gene amplification was obtained by Southern blotting of DNA derived from the cell lines expressing high urokinase levels. Transfection of squamous-cell carcinoma cells, which express high levels of urokinase, with a construct bearing the chloramphenicol acetyl transferase gene driven by the full-length (2345 bp) urokinase promoter indicated activation of the urokinase promoter in trans. In conclusion, our results suggest that transcriptional activation of the urokinase gene, in squamous-cell carcinomas of the oral cavity, leads to elevated levels of urokinase mRNA/protein which, in turn, can promote the invasive phenotype.

Blotting, Western↗

PubMed1993

Comparative localization of receptors for plasmin and for urokinase on MCF 7 cells.

The receptor for plasmin and the receptor for urokinase-type plasminogen activator were characterized on MCF 7 cells either independently or simultaneously, using fluorescence microscopy and confocal microscopy. The plasmin receptor was visualized, as previously described by Correc et al. (Int. J. Cancer 50, 767, 1992) using biotinylated plasminogen and fluoresceinated streptavidin. The urokinase receptor was revealed by both polyclonal and monoclonal antibodies reacting specifically with this receptor and by binding of urokinase aminoterminal fragment. On unfixed cells, these methods gave the same heterogeneous patterns of surface staining, consisting of contours and grains, localized mainly at the upper nonadherent face of the tumor cells by confocal microscopy. Only a part of the cells was stained. When both receptors were characterized together, their presence was found on the same cells and they gave almost superimposable patterns in many cases, as shown by confocal microscopy. In contrast, when MCF 7 cells were fixed or permeabilized before staining, quite different patterns were observed: almost all the cells were labeled. The staining was mainly cytoplasmic and localized preferentially in the center and close to the upper face of the cells. Similar results were found with antiserum against urokinase receptor and monoclonal antivinculin antibody. It is likely that the receptor for urokinase and the receptor for plasmin have similar localizations on MCF 7 cells, thus resulting in a functional cooperation.

Breast Neoplasms↗