Eur J Biochem 269, 3945–3957 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03068.x Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade Hiroshi Kobayashi1, Mika Suzuki1, Naohiro Kanayama1, Takashi Nishida2, Masaharu Takigawa2 and Toshihiko Terao1 Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan; 2Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan Our laboratory showed that bikunin, a Kunitz-type protease inhibitor, suppresses 4b-phorbol 12-myristate 13-acetate (PMA)- or tumor necrosis factor-alpha (TNFa)-induced urokinase-type plasminogen activator (uPA) expression in different cell types In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS-2/8, and two human ovarian cancer cell lines, HOC-I and HRA The present study showed that (a) bikunin suppresses the expression of constitutive and PMA-induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal-regulated kinase (ERK) activation system is necessary for the PMA-induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA-induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression We conclude that bikunin downregulates constitutive and PMA-stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK-dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene Tumor cell invasiveness is a complex, multistep process that involves cell attachment, the proteolysis of matrix components, and the migration of cells through the disrupted matrix [1] Activation of receptor-bound uPA on the cell surface appears to play an important role in cancer cell invasion and metastasis [2] In a number of cancers, the expression of the uPA and uPAR is required for the invasive phenotype [3,4] uPAR binds uPA with high affinity with a Kd of  0.5 nM [5,6] It has been shown previously that increased levels of uPA and uPAR correlate well with higher invasive phenotype [7] Most uPAR protein is concentrated at invasive foci [8]; it accelerates plasmin formation at the cell surface Overexpression of a human uPAR cDNA increased the ability of tumor cells to penetrate a barrier of reconstituted basement membrane For colon cancer, a high uPAR expression was strongly correlated with the poor prognosis in colon cancer [9] In contrast, exposure to anti-uPAR Ig [10], soluble uPAR [11,12], or stable transfection with antisense uPAR cDNA [13] rescues the invasiveness of tumor cells The uPAR protein is inducible by epidermal growth factor (EGF), transforming growth factor-beta (TGF-b) [14,15], hepatocyte growth factor (HGF) [16], vascular endothelial growth factor (VEGF) [17], interferon gamma (IFN-c), tumor necrosis factor-alpha (TNF-a) [18], and by the tumor promoter, phorbol ester [5,19] Activation of the protein kinase C pathway by PMA has been reported to increase uPAR mRNA in certain cell types [20] An increase in mRNA stability in response to PMA have been also reported [21] Bikunin [also known as urinary trypsin inhibitor (UTI)], a light chain of the interalpha-inhibitor (IaI) family, is a Correspondence to H Kobayashi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan Fax: + 81 53 435 2309, Tel.: + 81 53 435 2308, E-mail: hirokoba@hama-med.ac.jp Abbreviations: ATF, N-terminal fragment of uPA; DIP, diisopropyl fluorophosphate; DMEM, Dulbecco’s minimum Eagle’s medium; EGF, epidermal growth factor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; GraPDH, glyceroaldehyde-3-phosphate dehydrogenase; HGF, hepatocyte growth factor; HI-8, a C-terminal domain of bikunin; IaI, interalphainhibitor; IFN-c, interferon-gamma; PMA, 4b-phorbol 12-myristate 13-acetate; TGFb, tumor growth factor-beta; TNFa, tumor necrosis factor-alpha; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; UTI, urinary trypsin inhibitor; VEGF, vascular endothelial cell growth factor (Received 20 December 2001, revised 20 May 2002, accepted 21 June 2002) Keywords: bikunin; extracellular signal-regulated kinase; urokinase-type plasminogen activator; uPA receptor; tumor invasion Ó FEBS 2002 3946 H Kobayashi et al (Eur J Biochem 269) Kunitz-type protease inhibitor [22] and an effective inhibitor of calcium influx in cell transporter system [23] Bikunin also inhibits TNF-a-mediated translocation and activation of PKC [24] as well as PMA-dependent activation of PKC and MAP kinase cascade [25] We reported that it inhibits tumor invasion and metastasis possibly through suppression of cell-associated plasmin activity and expression of uPA mRNA and protein However, little is known concerning the potential role of bikunin in the regulation of uPAR mRNA and its protein In this paper, the positive modulation of uPAR mRNA and protein by PMA and the negative regulatory effects by bikunin on uPAR gene expression in human cancer cells (ovarian cancer cell lines HOC-I and HRA as well as chondrosarcoma cell line HCS-2/8) are reported We undertook the present study to determine the role of exogenously added bikunin on regulation of extracellular signal-regulated kinase (ERK)-dependent uPAR expression Further, bikunin transfection experiments were carried out to determine whether endogenously produced bikunin is necessary for inhibition of uPAR expression possibly through suppression of an upstream target(s) of ERK phosphorylation MATERIALS AND METHODS Materials 4b-Phorbol 12-myristate 13-acetate (PMA), calphostin C and staurosporin were purchased from Sigma, St Louis, MO, USA Human urokinase (high molecular mass twochain uPA) was a gift from Yoshitomi Pharmaceutical, Osaka, Japan Two-chain uPA was inactivated with diisopropyl fluorophosphates (DIP) to form DIP-uPA, as described previously [26] The N-terminal fragment of uPA (ATF) was purified, as described previously [27] a1-antitrypsin was from Kaketsuken, Kumamoto, Japan Polyclonal anti-(phospho-ERK1/2) Ig and anti-(c-Jun) Ig were from Santa Cruz Biotechnology Polyclonal antiMEK1/2 Ig was from Transduction Laboratories Total ERK was detected using an antibody from Zymed (San Francisco, CA, USA) A uPA-specific antibody, which recognizes the N-terminus of uPA (#3471), polyclonal rabbit anti-uPAR Ig (399R) and a specific ELISA for uPAR (IMUBIND 893) were obtained from American Diagnostica (Greenwich, CT, USA) Nonimmune anti(mouse/rabbit IgG) Ig and anti-(mouse/rabbit IgG) Ig conjugated with horseradish peroxidase were from Dako (Copenhagen, Denmark) Purified bikunin and recombinant C-terminal Kunitz domain II, HI-8 (Thr78-Asn147), were provided by E Morishita (Mochida Pharmaceutical Co., Gotenba, Shizuoka) Bikunin derivatives [O-glycosidelinked N-terminal glycopeptide (bikunin-m1; Ala1-Lys21), N-glycoside-linked C-terminal tandem Kunitz-domains (bikunin-m2; Lys22-Leu143), bikunin klacking O-glycoside (bikunin-c; Ala1–Leu143), asialo bikunin (bikunin-a; Ala1–Leu143), bikunin lacking N-glycoside (bikunin-n; Ala1–Leu143)] were prepared as described previously [28] The MEK inhibitors, PD098059 and U0126, were purchased from Calbiochem [32P]dATP random prime labeling Mega Prime kit was purchased from Amersham Japan All reagents used were of analytical grade Cell culture The human ovarian cancer cell lines HOC-I [29] and HRA [30], as well as the human chondrosarcoma cell line HCS-2/8 [31], have been described previously The HRA cells were provided by Y Kikuch and the HCS-2/8 cells were a gift from M Takigawa, both at Okayama University, Okayama, Japan All cell lines stained negative for mycoplasma contamination Cells were maintained in RPMI-1640 medium (HOC-I and HRA) or Dulbecco’s minimum Eagle’s medium (DMEM) (HCS-2/8) supplemented with penicillin (100 mL)1), streptomycin (100 lgỈmL)1) and 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.; Rockville, MD, USA) at 37 °C in 5%CO2/air atmosphere Before stimulation, cells were washed three times with NaCl/Pi and incubated overnight in complete medium containing 1% fetal bovine serum The test drugs were added and incubation was continued for different time lapses After culture, medium was aspirated and cells were harvested and washed extensively Immediately before harvest, cell viability was consistently found to be > 90% The ELISA procedure we propose measures native IaI and bikunin in human plasma (H Kobayashi, M Suzuki, Y Hirashima & T Terao, unpublished data) EDTA-plasma from healthy individuals revealed a mean level of  250 and  10 lgỈmL)1 of IaI and bikunin, respectively Therefore, the bikunin concentration of culture medium containing 1% fetal bovine serum may correspond to about  0.1 lgỈmL)1 Plasmid construction and transfection into the HRA cell line The a1 microglobulin-bikunin cDNA was cloned by PCR using the human liver cDNA library (Clontech, Palo Alto, CA, USA) as a template with primers (P1: 5¢-ACCG AGCCTCGAGGATATACCAAGGCAGAGGAGC-3¢, P2: 5¢-ACTTGCAGAGCGGCCGCTTGTCAGTTGGA GAAGC-3¢) Cloned cDNA was inserted into the XhoI– NotI site of the pCIneo vector (Clontech) In order to remove the internal sequence of 500 bases from this vector, an inverted PCR was performed with primers (P3: 5¢-GAGAG TCAGCGCTGCTGTGCTACCCCAAGA-3¢, P4: 5¢-ACT TGGATGTTGTCGGGCGGCGTTGGCACA-3¢) The resulting PCR product was digested with Eco47III and selfligated Finally, using the vector as a template, bikunin cDNA was cloned by PCR with primers (P1, P2), and inserted into the SmaI site of pCMV (cytomegalovirus promoter)-IRES (internal ribosome entry site)-bsr (blasticidin S hydrochloride resistant gene) vector [32] The bikunin expression vector pCMV-bikunin-IRES-bsr and the control vector pCMV-luciferase-IRES-bsr [32] encoding LUC (luciferase) were transfected into HRA cells by the standard calcium phosphate precipitation method [33] The cells were selected in the presence of 10 mgỈmL)1 blasticidin S hydrochloride (Funakoshi Co Ltd, Tokyo, Japan) Resistant clones were obtained after four weeks, and bik+ clones and luc+ clones were obtained The cells were subsequently maintained in the presence of 10 mgỈmL)1 blasticidin S hydrochloride The initial bik+ mass cultures were subjected to at least two rounds of subcloning in order to obtain stable bik+ clones DNA sequencing verified the correct insertion of the bik cDNA Finally, HRA bik+ tumor cell clones with bik Ó FEBS 2002 overexpression were confirmed by immunocytochemical staining and Western blot analysis [34] cDNA synthesized from luciferase transfected tumor cells (luc+ clones) was used as a control Northern blot analysis Northern blot analysis was performed using standard methods Ten micrograms of RNA were separated in 1.2% agarose gels and blotted onto Hybond N+ membranes Prehybridization and hybridization were performed in 50% formamide at 42 °C with · 106 c.p.m.ỈmL uPAR cDNA probe, as described previously [35], and filters were reprobed with the cDNA for glyceraldehyde-3-phosphate dehydrogenase to correct for the amount of RNA loaded onto the filters A 1.1 kb XbaI–EcoRI fragment of uPAR cDNA [36] was radiolabeled with [32P]dATP via random hexamer primer extension and used as hybridization probe After each hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at )70 °C Filters were quantitated by scanning densitometry using a Bio-Rad model 620Video Densitometer with a ID ANALYST software package for Macintosh Western blot uPAR, phosphorylated and total ERK were detected by immunoblot analysis To detect uPAR protein, conditioned media were individually harvested and the remaining monolayers were scraped and lysed in 50 mM Hepes, 0.5 M NaCl, 0.05% Tween-20, 1% Triton X-100, mM phenylmethanesulfonyl fluoride, 10 lgỈmL)1 E-64, 10 lgỈmL)1 leupeptin to prepare cell lysates For the detection of ERK proteins, the cells were extracted with 1.0% Nobidet P-40, 50 mM Hepes, 100 mM NaCl, mM EDTA, lgỈmL)1 leupeptin, 0.4 mgỈmL)1 sodium vanadate, 0.4 mgỈmL)1 sodium fluoride, mgỈmL)1 dithiothreitol, pH 7.4 Samples were stored at )20 °C and used only once after thawing Equal amount of cellular protein (50 or 20 lg per lane) were subjected to 12% SDS/PAGE and transferred to poly(vinylidene difluoride) membranes Filters were probed with primary antibodies and revealed with a biotinylated anti(rabbit/mouse IgG) Ig and avidin-peroxidase Peroxidase was detected by enhanced chemiluminescence uPAR protein assay: measurement of ligand binding DIP-uPA was radioiodinated with carrier-free Na-125I, as described previously [37] DIP-uPA was labeled with 125I, resulting in a specific radioactivity of 4900 c.p.m.Ỉng)1, without loss of latent protease activity Binding assays were performed at °C as described previously [37] In brief, confluent monolayers of HRA were grown in 24-well plate wells using complete medium supplemented with 10% fetal bovine serum The cells were washed twice each with medium supplemented with 1% fetal bovine serum The cells were maintained overnight medium supplemented with 1% fetal bovine serum Monolayers were cooled to °C and washed twice with Tyrode’s-Hepes solution containing 1% fetal bovine serum (washing buffers) Prior to performing binding studies, confluent cultures were subjected to a mild acid wash (50 mM glycine-HCl, pH 3.0, 0.1 M NaCl) to dissociate uPAR-associated ligands The cells were then Suppression of uPAR by bikunin (Eur J Biochem 269) 3947 incubated at °C for h in binding buffer (150 mM NaCl, 10 mM Hepes, mM CaCl2, mM MgCl2, 1% fetal bovine serum) supplemented with 10 nM [125I]DIP-uPA and washed four times To determine nonspecific binding, 50-fold higher concentrations of unlabeled DIP-uPA were added to the incubate Unbound [125I]DIP-uPA was removed and the contents of the wells were removed using M NaOH and radioactivity of the cell lysates was measured using a gamma-counter The cells bound [125I]DIP-uPA in a specific and saturable manner at 10 nM [125I]DIP-uPA Each experimental point was performed in at least triplicate wells The nonspecific binding, determined as the percent of input counts bound in the presence of 0.5 lM unlabeled uPA, was approximately 9% and was subtracted from all raw data to give the specific bound counts Invasion assay Invasion assays were performed essentially as described previously [25] The effects of agents that alter the activity of uPA/uPAR expression, including neutralizing monoclonal antibodies against uPA and uPAR, on the invasiveness of HRA cells were determined by measuring the ability of cells treated with these agents to pass through a layer of the extracellular matrix extract Matrigel coating a filter using chemoinvasion chambers Statistical analysis Data are presented as mean ± SD All statistical analysis was performed using STATVIEW for Macintosh The Mann– Whitney U-test was used for the comparisons between different groups P < 0.05 was considered significant RESULTS Induction of uPAR mRNA by PMA in HRA cells Unstimulated cells (HRA, HOC-I and HCS-2/8) expressed different levels of 1.1 kb uPAR transcripts (Fig 1A) uPAR mRNA in HRA cells incubated with PMA (100 nM) for h was increased 7.5-fold as compared with the unstimulated cells, which appeared at h, peaked at h and declined at 24 h (Fig 1B) The effect of PMA on uPAR expression was dose-dependent at PMA concentrations of 10–100 nM, with a maximum increase seen after treatment of HRA cells with 100 nM of PMA (Fig 1C) Similar PMA effects on uPAR mRNA were found in the two other cancer cell lines (data not shown) Suppression by bikunin of uPAR mRNA accumulation by PMA We showed previously that bikunin plays an important role in signaling of PMA [24] and TNFa [25] It is of interest to determine whether bikunin also affects the expression of PMA-induced uPAR mRNA (Fig 2) When concentrations of bikunin (0.1–1 lM) known to inhibit the uPA production [25] were added in the presence of 100 nM PMA (data not shown) to HRA cells, there was a dose-dependent inhibition of  50 and  70% of the uPAR mRNA level at concentrations of 0.1 and lM, Ó FEBS 2002 3948 H Kobayashi et al (Eur J Biochem 269) Fig Induction of uPAR mRNA by PMA in tumor cells (A) Relative levels of uPAR mRNA in HRA, HOC-I, and HCS-2/8 cells Cells were grown to 90% confluence Total cellular RNA was extracted and separated on 1.2% agarose/formaldehyde gel and transferred to Hybond N+ membrane Filters were hybridized with 32P-labeled uPAR cDNA or with 32P-labeled GAPDH cDNA probe Top, representative autoradiograms; Bottom, Levels of uPAR mRNA expression as quantified by densitometric scanning (B) Stimulation of uPAR gene expression in HRA cells HRA cells were grown to 90% confluence and then stimulated with PMA (100 nM) for the indicated periods of time C, PMA stimulates uPAR gene expression in a dose-dependent manner HRA cells were incubated for h with different doses of PMA Results are the mean ± SD of four different determinations, with unlike superscripts (a–d) are different (P < 0.05) We examined the capacity of truncated proteins [deglycosylated bikunin (bikunin-c) and HI-8] and a related protein (a1-antitrypsin) to suppress PMA-stimulated uPAR mRNA expression We showed that bikunin might inhibit uPAR expression by mechanisms different from direct protease inhibition HI-8 is the C-terminal domain of bikunin, which is active fragment for protease inhibitor but is not recognized by the cell-associated bikunin binding sites We could not see any effects of deglycosylated bikunin, HI-8, and a1-antitrypsin on suppressing uPAR expression Similar effects on bikunin specificity were found in the other cell lines (data not shown) Suppression by bikunin of unstimulated and PMA-induced uPAR protein expression Fig Bikunin specifically suppresses PMA-stimulated uPAR gene expression HRA cells were incubated for h with or without 100 nM PMA in the presence or absence of bikunin, its truncated proteins [deglycosylated bikunin (bikunin-c) and HI-8] and its related protein (a1-antitrypsin) Total cellular RNA was extracted and analyzed for uPAR mRNA expression by Northern blot analysis and compared with untreated control cells (Ctr) Top, representative autoradiograms; Bottom, Levels of uPAR mRNA expression as quantified by densitometric scanning Results are the mean ± SD of three different determinations, with unlike superscripts (a–e) are different (P < 0.05) respectively, as determined by scanning densitometry After treatment with lM bikunin alone (lane 2), 30% reduction of uPAR mRNA was observed Similar effects of bikunin inhibition were found in the other cell lines (data not shown) We further investigated whether bikunin could inhibit unstimulated and PMA-stimulated [125I]DIP-uPA binding capacity on the cell surface It has been established that PMA treatment resulted in an increase in the number of binding sites and a decrease of the affinity of the uPAR [38,39] The quantification of uPAR expression by measurement of the amount of uPA bound is thus not ideal Actually, it could be underestimating the level of upregulation Notwithstanding these limitations, in this study, uPAR expression was evaluated by measuring cell-associated [125I]DIP-uPA binding capacity The dose-dependent ability of bikunin to inhibit expression of uPAR by cells is clearly demonstrated using the [125I]DIP-uPA binding assay (Fig 3-A) HRA cells bound [125I]DIP-uPA in a specific and saturable manner with a maximum effect at a concentration of 10 nM (data not shown) Equivalent Kd value was determined ( 1.6 nM), which is consistent with the known binding affinity of uPA for uPAR [5] In cells treated with PMA, cell-associated uPA-binding capacity was significantly decreased in the presence of 100 nM bikunin The maximal Ó FEBS 2002 Suppression of uPAR by bikunin (Eur J Biochem 269) 3949 Fig Effects of PMA and bikunin on functional uPAR protein expression in HRA cells measured by the [125I]DIP-uPA binding assay (A) HRA cells pretreated with bikunin (0, 10, 100, 1000, and 5000 nM) or HI-8 (5000 nM) for h were incubated with or without 100 nM PMA for 12 h B, Competitive inhibition of solid-phase [125I]DIP-uPA binding to HRA monolayer cells by unlabeled competitors The PMA-stimulated cells (100 nM, 12 h) were treated with 10 nM [125I]DIP-uPA in the presence of 1000 nM unlabeled competitors [uPA, amino-terminal fragment of uPA (ATF), bikunin, deglycosylated bikunin (bikunin-c) and HI-8] Levels of uPAR expression as quantified by [125I]DIP-uPA binding assay The percent fractions bound on the surface of the cells treated with or without 100 nM PMA correspond to  or  1.4%, respectively, of [125I]DIPuPA added Results are the mean ± SD of three different determinations, with unlike superscripts (a–f) are different (P < 0.05) suppression of PMA-induced uPAR expression was obtained at 1000 nM bikunin Constitutive uPAR expression without stimulation by PMA was also affected by 100– 1000 nM bikunin Contrary to bikunin, HI-8 failed to suppress PMA-stimulated [125I]DIP-uPA binding at concentrations of HI-8 as high as 5000 nM Similar PMA and bikunin effects on uPAR protein were found in the two other cancer cell lines (data not shown) We examined whether bikunin directly inhibits [125I]DIPuPA binding to the cells The stimulated cells were treated with [125I]DIP-uPA in the presence of several unlabeled competitors [uPA, ATF, bikunin, deglycosylated bikunin (bikunin-c), and HI-8] As shown in Fig 3B, we found that bikunin and its derivatives did not directly inhibit [125I]DIPuPA binding to the uPAR on the cell surface In a parallel experiment, we measured the uPAR levels in cells stimulated with or without PMA using a specific ELISA for uPAR (data not shown) The levels of uPAR protein in unstimulated and PMA-stimulated HRA cells were 4.5 ± 0.53 and 18.0 ± 2.48 ng per 106 cells, respectively, demonstrating that, after stimulation, uPAR protein levels increased about fourfold The levels of uPAR protein in PMA-stimulated cells treated with bikunin (100 and 1000 nM) were 12.3 ± 0.89 and 10.0 ± 0.97 ng per 106 cells, respectively Thus, the dose-dependent ability of bikunin to inhibit expression of uPAR protein by cells was also demonstrated using ELISA Results obtained after exposing the cells to bikunin before and after stimulation by PMA are presented in Fig Preincubation of the cells with bikunin during h before 100 nM PMA stimulation results in a concentration-dependent inhibition of the induction of [125I]DIP-uPA binding (which is associated with uPAR protein expression) At concentrations of 100 and 1000 nM, [125I]DIP-uPA binding is inhibited by  35 and  50%, respectively In contrast, no significant decrease of uPAR expression is observed when bikunin is added to the medium h after stimulation by PMA More than 90% of the control value still remains at the highest bikunin concentration (1000 nM) tested Cell viability, monitored by LDH leakage in the culture medium and trypan blue dye exclusion test, is not altered under the different exposure conditions (data not shown) These experiments demonstrated that a marked decrease of uPAR expression is observed when bikunin is added to the medium before stimulation by PMA The effects of bikunin and its derivatives on uPAR protein expression To further determine which domains of bikunin are sufficient to suppress uPAR levels in cells, we determined Fig Effect of exposure of cells to bikunin before and after stimulation by PMA Levels of uPAR expression as quantified by [125I]DIP-uPA binding assay Values represent means ± SD of three experiments (A) 10 nM bikunin; (B) 100 nM bikunin; and (C) 1000 nM bikunin a–f, means ± SD with unlike superscripts are different (P < 0.05) Results are representative of two separate experiments Ó FEBS 2002 3950 H Kobayashi et al (Eur J Biochem 269) Table Dose-dependent suppression of uPAR expression and ERK phosphorylation stimulated by PMA after intact and truncated bikunin treatment of HRA cells The relative amount of PMA-induced cell-associated uPAR expression suppressed in response to increasing concentrations of intact and truncated bikunins is shown The amount of uPAR accumulated in the cells treated without or with 100 nM PMA in the absence of competitor corresponds to 4.5 ± 0.53 and 18.0 ± 2.48 ng per 106 cells, respectively The amount of ERK phosphorylation in cell lysates treated with 100 nM PMA in the absence of competitor corresponds to 100% as judged by a densitometric scan The data of antitryptic activity, cell binding and uPA suppression are from [28,] These data are representative of two independent experiments Bikunin Molecular mass (kDa) Antitryptic activity (IC50; lgỈmL) Cell binding (IC50) nM uPA suppression Cell lysate (IC50; nM) Conditioned medium (IC50; nM) uPAR suppression ERK suppression Bikunin-m1 Bikunin-m2 Bikunin-c Bikunin-a Bikunin-n HI-8 40 1.6 12 10–30 >30 30 21 1.1 350 25 1.4 15 39 1.5 15 35 1.9 0.9 >1000 20 28  200  100 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 30 35  200  100 40  200  100 >1000 >1000 >1000 >1000 whether the downregulation observed with intact bikunin could be induced by bikunin derivatives Dose-dependent suppression of uPAR in cells treated with PMA in response to treatment with intact bikunin or bikunin derivatives is shown in Table The levels of uPAR protein in cell lysates were determined using a specific ELISA for uPAR Bikunin-a and bikunin-n effectively suppressed uPAR levels Bikunin-a and bikunin-n were essentially equipotent to intact bikunin with respect to the inhibition of uPAR levels as judged by ELISA, with ID50 values of  200 nM Other bikunin derivatives had no discernible effect on uPAR levels in cells When HRA cells were preincubated with bikunin for h, we could detect suppression of phosphorylation of ERK1/2 in a dose-dependent manner The results, based on densitometric scanning, show that lM bikunin inhibits constitutive and PMA-triggered phosphorylation of ERK1/2, by  50 and  70%, respectively However, lM HI-8 failed to change significantly the expression of phosphorylated Suppression by bikunin of ERK1/2 activity in the unstimulated and PMA-stimulated HRA cells Recent studies demonstrated that activation of a PMAdependent signal pathway involves a relay of phosphorylation of several proteins making up the MAP kinase pathway [40] uPAR gene expression is modulated by multiple signal transduction pathways EGF and PMA cause increased uPAR transcription [15] An increased level of phosphorylated ERK is associated with increased level of cell-surface uPAR, which results in enhanced invasion [10] To determine the role of bikunin in the regulation of ERK1/2 phosphorylation, the unstimulated and PMA-stimulated HRA cells were analyzed for the phosphorylation of ERK1/2 (Fig 5) Immunoblotting of cell extracts with anti-ERK1/2 Ig indicated the presence of immunoreactive proteins (44 kDa ERK1 and 42 kDa ERK2) The HRA cells express primarily ERK1 and some ERK2, as has been previously reported for HRA cells [24] Anti-(phosphoERK) Ig showed a strong kinase activity in the PMAstimulated cells corresponding in size to ERK1, while unstimulated cells contained low ERK1 activity We found that, in the stimulated cells, phosphorylation of ERK1/2 is modified within 30 of induction by PMA and then returned to the uninduced state after h (data not shown) We further demonstrated that the high level of uPAR expression may have aided in the use of HRA cell line as a model system, since levels of HRA cell ERK1/2 activation were typically high Compared to HRA cells, lower levels of phosphorylated ERK1/2 were found in HOC-I and HCS-2/8 cells (data not shown) Fig PMA-stimulated cells demonstrate increased levels of activated ERK1/2All cells were extracted when 90% confluent Cells pretreated with bikunin or HI-8 for h were incubated with PMA for 30 and then extracted to assess ERK phosphorylation Cells were solubilized in lysis buffer supplemented with protease inhibitors Equal amounts of cellular protein (50 lgỈlane)1) were loaded in each lane, subjected to SDS/PAGE, and electrotransferred to PVDF membrane for detection with antiphospho-ERK1/2 (active) and anti-ERK1/2 Ig (total) Immunoblot analysis was performed to detect phosphorylated and total ERK1/2 in unstimulated and PMA-stimulated cells Results are representative of two separate experiments Top, representative immunoblotting; Bottom, Levels of phosphorylated ERK1 expression as quantified by densitometric scanning Ó FEBS 2002 Suppression of uPAR by bikunin (Eur J Biochem 269) 3951 proteins Bikunin did not reduce total ERK1/2 antigen level Therefore, these results show that bikunin inhibits both constitutive and PMA-induced phosphorylation of ERK1/2 at the concentration (0.1–1 lM) that prevents uPAR expression Again, similar effects on ERK phosphorylation were found in the two other cell lines (data not shown) Domain specificity of bikunin on ERK activation Dose-dependent suppression of ERK phosphorylation in the stimulated cells in response to pretreatment with intact bikunin or bikunin derivatives is shown in Fig and Table Exposure of cells to bikunin, bikunin-a and bikunin-n resulted in decrease in the amount of ERK phosphorylation in cell lysates as judged by immunoblotting, indicating that ID50 was about 200 nM Other bikunin derivatives had no significant effect on suppression of ERK phosphorylation The inhibition curves for bikunin-induced suppression of uPAR expression and ERK activation are shifted towards higher bikunin concentrations in relation to that for bikunin-induced suppression of uPA expression ERK activation is associated with uPAR expression In order to determine whether the ERK activation system is necessary for the increase in uPAR mRNA expression, we cultured these cells in the presence of PD098059 or an alternative MEK1 inhibitor U0126, which prevents the activation of MEK1 [41,42] HRA cells were treated for 12 Fig Dose-dependent inhibition by bikunin and its derivatives of ERK phosphorylation in the PMA-stimulated HRA cells Cells pretreated with bikunin or its derivatives for h were incubated with 100 nM PMA for 30 and then extracted (20 lgỈlane)1) to assess ERK phosphorylation Experiments were performed twice with similar results and h with varying concentrations of the inhibitors and 100 nM PMA, respectively PD098059 (Fig 7A, lanes 2–4) and U0126 (Fig 7C, lane 4) reduced the uPAR mRNA expression in a dose-dependent manner Bikunin showed no additive effect on PD098059-mediated suppression of uPAR expression (not shown) The reduced amount of uPAR mRNA reflected a diminished amount of uPAR protein as determined by the [125I]DIP-uPA binding assay and ELISA Fig Levels of uPAR mRNA expression in unstimulated or PMA-stimulated cells treated with bikunin and/or several inhibitors A, Cells were pretreated with PD098059 (20, 40, or 60 lM), sodium vanadate (0.1 mM), or bikunin (1000 nM) or vehicle for h before the addition of 100 nM PMA After cells were incubated for h, total cellular RNA was extracted and analyzed for uPAR mRNA expression by Northern blot analysis (B) Unstimulated cells were treated with PD098059 (60 lM) or vehicle for 12 h C, Cells were pretreated with staurosporin (50 nM), PD098059 (60 lM), U0126 (20 lM), or with a combination of each agent h before the addition of 100 nM PMA Top, representative autoradiograms; Bottom, Levels of uPAR mRNA expression as quantified by densitometric scanning A, Results are the mean ± SD of three different determinations with unlike superscripts (a–e) are different (P < 0.05) (B) and (C), experiments were performed twice with similar results 3952 H Kobayashi et al (Eur J Biochem 269) (data not shown) The expression of uPAR mRNA in unstimulated cells was slightly inhibited by treatment of the cells with PD098059 (Fig 7B) or U0126 alone (not shown) These results suggest that, in unstimulated cells, MEK1 inhibition in itself does not significantly affect uPAR mRNA levels, although the PMA-stimulated ERK activity downregulation by bikunin is suggested to be the major mechanism through which bikunin works The overexpression of uPAR mRNA in the PMAstimulated cells was also inhibited by treatment of the cells with PKC inhibitors, calphostin C (data not shown) and staurosporin, as measured by Northern blotting (Fig 7C) However, MEK1 inhibitors showed no additive effect on PKC inhibitor-mediated suppression of uPAR overexpression We next examined whether stimulation of ERK1 activity was associated with increased uPAR mRNA expression For this, HRA cells were treated with a protein tyrosine phosphatase inhibitor (sodium vanadate) [43] 0.1 mM sodium vanadate significantly increased uPAR mRNA 2.3-fold (Fig 7A, lane 5) Even in the presence of bikunin, however, the level of ERK phosphorylation was not decreased back to the basal level observed without sodium vanadate, suggesting that bikunin has no ability to inhibit overexpression of uPAR mRNA in HRA cells treated with sodium vanadate We showed that effects of these inhibitors not reflect a decrease in cell viability over the time frame of these experiments (data not shown) cDNA transfection of bikunin In order to determine whether bikunin expression in cells transfected with bikunin gene is necessary for the decrease in ERK phosphorylation, followed by suppression of uPAR mRNA expression, HRA cells were transfected to express bikunin gene As shown in Fig 8, bikunin was not detected in extracts (data not shown) and conditioned media of HRA and luc+ clone, by immunoblot analysis, but was easily identified in conditioned medium of bik+ clone We examined whether cDNA transfection of bikunin does not alter levels of representative signaling proteins in bik+ clone, luc+ clone, and HRA cells Total antigenic levels of MEK1/2, ERK1/2, and c-Jun were equivalent in the different cell types (data not shown), suggesting that bikunin transfection did not nonspecifically inhibit cellular expression of proteins which impact on MAP kinase cascade Down-regulation of uPAR mRNA level in bikunin-transfected cells Figure 9A shows that the intensity of the uPAR mRNA band was much lower in bik+ clone than in HRA and luc+ clone Scanning autoradiograms of the hybridization signals with a laser densitometer and normalization with the glyceraldehyde-3-phosphate signal showed a fourfold decrease in uPAR mRNA in bik+ clone as compared with HRA and luc+ clone Immunoreactive uPAR protein was detected in these cells The bik+ clone expressed significantly low levels of immunoreactive uPAR protein with molecular mass of 50 kDa under nonreducing conditions (Fig 9B) Based on densitometric acanning, the expression of uPAR at the protein level in the parental cells was reduced by 50–60% in bik+ clone Ó FEBS 2002 Fig Expression of bikunin protein in HRA cells transfected with cDNA coding for human bikunin Detection of bikunin protein by Western blot Equal amounts (10 lLỈlane)1) of conditioned media derived from the same number of cells of parental HRA (lane 1), luc+ clone (lane 2), and bik+ clone (lane 3) were applied to SDS/12% polyacrylamide gel electrophoresis followed by Western blot with antibik antibody Expression of the 50–80 kDa bik protein is upregulated in bik+ clones Molecular mass standards are indicated at the left Results are representative of two separate experiments Expression of uPAR in HRA cells is associated with an active ERK1/2 AS we showed in this study that exogenous bikunin suppresses PMA-induced uPAR expression via an inhibition of ERK1/2 phosphorylation, studies were undertaken to determine whether the level of ERK phosphorylation is decreased in bik+ clone in the presence or absence of exogenous PMA stimuli (Fig 10) Anti-phospho-ERK antibody showed a strong kinase activity in the parental and luc+ clone, high uPAR expressor, corresponding in size to ERK1 On the other hand, bik+ clone, low uPAR expressor, contained low ERK1/2 kinase activity It is unlikely that the large difference in ERK activity between the parental and bik+ clone is a consequence of different growth rates, as we were unable to show a reproducible decrease in proliferation of the bik+ clone (data not shown) The results show that not only exogenously applied bikunin (1 lM) but also bikunin gene transfection significantly inhibits constitutive and PMA-triggered phosphorylation of ERK1/2 by about 70% Similar effects of bikunin on suppression of MAP kinase activation were found in the two other cancer cell lines (data not shown) The effect of bikunin and HI-8 as well as neutralizing antibodies against uPA and uPAR on HRA cell invasiveness Our previous experiments showed that treatment with PMA produced a significant stimulation of the invasiveness of HRA cells in a dose-dependent manner, with a maximum stimulation at 100 nM PMA [25] Figure 11 shows the effect Ó FEBS 2002 Suppression of uPAR by bikunin (Eur J Biochem 269) 3953 Fig Downregulation of uPAR level in bikunin-transfected cells (A) Downregulation of uPAR mRNA level in bikunin-transfected cells Ten micrograms of total RNA isolated from HRA cells, bik+ cells, and luc+ cells, shown in the upper panel, was electrophoresed in a 1.2% agarose/ formaldehyde gel and then transferred to Hybond N+ membrane The membrane was then hybridized with a radiolabeled cDNA probe specific for uPAR mRNA The same blot was stripped and hybridized with radiolabeled GAPDH cDNA to check for equality of loading uPAR mRNA levels were measured by scanning autoradiograms with a laser densitometer, and relative hybridization signals were calculated by assigning an arbitrary value of 100 to the highest intense signal seen by Northern blot analysis correlated for mRNA loading inequalities Results are the mean ± SD of three different determinations with unlike superscripts (a and b) are different (P < 0.05) (B) Expression of uPAR protein in bikunin transfected cells Cell lysates of control and bikunin transfected cell clones treated with or without PMA were used to analyze uPAR expression by Western blot Results are representative of two separate experiments of adding increasing concentrations of antibodies or bikunin and HI-8 on the invasiveness of the PMA-stimulated cells (left panel) and bik+ clone (right panel) The PMA-stimulated cell invasion was specifically reversed by concurrent treatment with either neutralizing anti-uPA Ig or anti-uPAR Ig, as well as with bikunin These data support that HRA cells leading to invasion is induced through the upregulation of the uPA/uPAR system Bikunin, but not HI-8, which could induced change in uPA/uPAR expression, could in turn modify the invasive behavior of HRA cells However, bikunin had no additive effect on antibodymediated suppression of cell invasiveness The bik+ clone invasion was also reversed by concurrent treatment with either neutralizing anti-uPA Ig or anti-uPAR Ig, but not with exogenous bikunin DISCUSSION Fig 10 Bikunin gene transfection inhibits constitutive and PMA-triggered phosphorylation of ERK1/2All cells were extracted when 90% confluent Cells were stimulated with PMA for 30 and then extracted to assess ERK1/2 Immunoblot analysis to detect phosphorylated and total ERK1/2 in unstimulated and PMA-stimulated cells Results are representative of two separate experiments Top, representative immunoblotting; Bottom, Levels of phosphorylated ERK1 expression as quantified by densitometric scanning Results are representative of two separate experiments Several authors have reported that the serine protease uPA and its receptor uPAR play a key role in the invasive and metastatic capacity of tumor cells [3,4,44] Phorbol ester and a variety of growth factors including EGF, FGF, and VEGF, which upregulate uPAR synthesis, also stimulate ERK activity [15,45–49] Further, ERK may represent an essential step in the pathway by which cytokine and integrins promote cellular motility [50] It has been established that PMA induces ERK activity in a number of systems [45], however, the signaling mechanism by which PMA modulates uPAR expression is not completely understood The effect of PMA on the expression of other genes has been ascribed to activation of the classical pathway (Ras fi c-Raf1 fi ERK signaling cascade) [51] An alternative pathway consists of the sequential activation of Rac1, MEKK1, c-Jun N-terminal kinase kinase (JNKK) 3954 H Kobayashi et al (Eur J Biochem 269) Ó FEBS 2002 Fig 11 Suppression of invasiveness in PMAstimulated HRA cells and bik+ clone by treatment with antibodies and bikunin HRA cells and bik+ clone (5 · 104 cells) were placed in the top wells of a chemoinvasion chamber apparatus with the neutralizing antibodies against uPA (10 lgỈmL)1) and uPAR (10 lgỈmL)1), bikunin (0.5 lM) and HI-8 (1 lM) in the presence of 100 nM PMA Each point represents the mean of measurements made on two independent wells This is a representative experiment selected from two performed and the c-Jun N-terminal kinase (JNK) subset of MAPK [52] Recent studies also demonstrated that the PMAdependent stimulation of uPAR gene expression requires a JNK1-dependent and -independent signaling modules [53] Tumor dormancy is induced by downregulation of uPAR [54] It has been reported thata  70% reduction in the uPAR level in human carcinoma HEp3 cells induced a protracted state of tumor dormancy in vivo Therefore, treatment of uPAR-rich cells, which maintain high ERK activity in vivo, with reagents interfering with the uPAR signal to ERK activation, mimic the in vivo dormancy induced by downregulation of uPAR With these in mind, we investigated the regulation by bikunin of ERK activation as a signaling molecule in PMA-induced uPAR overexpression in highly invasive human ovarian cancer cell lines and human chondrosarcoma cell line We previously reported that bikunin expression is associated with a less malignant phenotype [24] There is evidence that bikunin significantly prevented pulmonary metastasis of mouse Lewis lung carcinoma 3LL cells [55] Our ongoing study shows that transfection of the highly invasive and metastatic HRA cell line lacking bikunin expression with bikunin-encoding constructs causes a marked decrease in their metastatic ability (Suzuki & Kobayash, unpublished data) We have clearly demonstrated in the recent studies [28,56–59] that bikunin can suppress PMA-stimulated upregulation of uPA mRNA and protein via a specific receptor for bikunin, which results in bikunin-mediated suppression of cell invasiveness The precise mechanism by which expression of bikunin and decrease in the metastatic ability of the bikunin transfectants might be linked to the expression of uPA has been explored in our laboratory However, nothing is known about the mechanism by which bikunin would modulate the uPAR expression The present study showed that (a) the effect of PMA on uPAR expression is time- and dose-dependent; (b) exogenously added bikunin is able to suppress the expression of constitutive and PMA-induced uPAR mRNA and protein; (c) a marked decrease of uPAR expression is observed when bikunin is added to the medium before stimulation by PMA; (d) PMA promotes cellular uPAR expression, at least, by activating an ERK-dependent signaling pathway: the ERK activation system is necessary for the increase in uPAR expression, since PD098059 and U0126 reduce the uPAR expression; (e) bikunin markedly suppresses PMA- induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (f) the inhibition curves for bikunininduced suppression of uPAR expression and ERK activation are shifted towards higher bikunin concentrations in relation to that for bikunin-induced suppression of uPA expression; (g) the inhibition of ERK1/2 by bikunin depends on receptor binding, since HI-8 does not inhibit ERK1/2 phosphorylation: O-glycoside-linked core protein without N-glycoside, that is the active domain for suppression of uPA expression, is required for bikunin-mediated suppression of uPAR production and ERK phosphorylation; (h) stimulation of ERK1 activity by sodium vanadate is associated with increased uPAR expression: bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate, indicating that bikunin is able to suppress uPAR mRNA and protein possibly through inhibition of upstream components of ERK activation in MAP kinase cascade: a set of consecutive signaling molecules which teleologically alter the program of gene expression; (i) bikunin transfection experiments demonstrated that uPAR expression is associated with an active ERK and the bikunin expression is necessary for inhibition of uPAR expression possibly through suppression of ERK phosphorylation; (j) not only exogenous bikunin but also bikunin gene transfection markedly inhibits PMA-triggered phosphorylation of ERK1/2; and (k) the HRA cells leading to invasion is mainly through upregulation of uPA/uPAR expression: suppression by neutralizing antibodies to uPA/ uPAR of cell invasion is almost complete, while inhibition by bikunin alone is partial Bikunin showed no additive effect on neutralizing antibody-mediated suppression of invasion Therefore, the present results clearly show that bikunin (O-glycoside-linked core protein without N-glycoside) specifically inhibits expression of uPAR mRNA and protein possibly through suppression of an upstream target(s) of the ERK-dependent cascade A recent publication demonstrated that binding of uPA to uPAR activates ERK1/2, which is required for increased cellular motility in breast cancer cells [26] Furthermore, uPA [60] and uPAR [49] expression are increased in response to multiple factors that activate ERK1/2 It is thus possible that uPA might regulate expression of uPA itself and its own receptor via ERK activation Our previous and present results demonstrated that bikunin specifically inhibits constitutive and inducible gene expression including Ó FEBS 2002 uPA and uPAR possibly through suppression of upstream targets of ERK Thus, one of the functions of bikunin is the regulation of uPA synthesis and uPA binding by modulating uPAR It is most likely that the PMA-induced uPAR expression shares the characteristics of that of uPA expression, since the increased uPAR expression is affected by PKC inhibitors (calphostin C and staurosporin) and the uPA expression is also affected by ERK inhibition [61] However, the effect of bikunin inhibition of uPAR expression and ERK phosphorylation is less potent than that of uPA suppression, suggesting that PKC and ERK are involved in the expression of the uPA and uPAR proteins possibly through the parallel pathways, but not the same ones Further, total antigenic levels of MEK1/2, ERK1/2, and c-Jun were equivalent in the different cell types, suggesting that exogenous bikunin and bikunin transfection not nonspecifically inhibit cellular expression of proteins which impact on MAP kinase cascade These results show that bikunin may be selective in inhibiting gene expression The effect of bikunin on the expression of other proteins regulated through PKC and ERK could give information on whether bikunin has ubiquitous effects on protein expression We found that the effects of bikunin are not due to protease inhibition as the single Kunitz-type domain HI-8 is not inhibitory with respect to uPA and uPAR mRNA expression, while full-length bikunin is A further characterization of the bikunin domains responsible for the effect on uPAR expression and ERK activation was intensively examined as previously carried out by us with regard to uPA expression The binding and signaling properties of intact bikunin and truncated bikunins are summarized in Table We conclude that O-glycoside-linked core protein without N-glycoside is essential to the bikunin-mediated suppression of uPA/uPAR production and ERK activation These signaling properties of the truncated bikunin indicate that the simultaneous binding of the chondroitin-4-sulfate side chain of bikunin and the N-terminal Kunitz-domain I are required for effective signaling properties The results from a recent report by us on bikunin inhibition of CD44 clustering as a mechanism by which it might inhibit uPA expression will also be investigated with respect to uPAR expression [62] In addition, PMA can enhance uPAR mRNA stability in certain tumor cells [21] However, we found a relatively similar half-life of uPAR mRNA of 10–12 h in the parental cells, luc+ clone and bik+ clone (data not shown) It is unlikely that bikunin reduces uPAR mRNA stability Again, we found that bikunin had no measurable effects on cell viability or on the yield of the total RNA We believe that the present study provides new insight into how bikunin may be involved in the modulation of the metastatic phenotype: suppression of uPA/uPAR-dependent increased cell adhesion, migration and invasion by bikunin, at least, via suppression of an upstream target(s) of ERK 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