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Selective Inhibition of Platelet-Derived Growth Factor (PDGF) Receptor Autophosphorylation and PDGF-Mediated Cellular Events by a Quinoline Derivative
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
234, 285–292 (1997)
EX973616
Selective Inhibition of Platelet-Derived Growth Factor (PDGF) Receptor Autophosphorylation and PDGF-Mediated Cellular Events by a Quinoline Derivative Mikio Yagi,1 Shinichiro Kato, Yoshiko Kobayashi, Kazuo Kubo, Shinichi Oyama, Toshiyuki Shimizu, Tsuyoshi Nishitoba, Toshiyuki Isoe, Kazuhide Nakamura, Hideya Ohashi, Nami Kobayashi, Noriko Iinuma, Tatsushi Osawa, Rie Onose,* and Hiroyuki Osada* Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., Takasaki, Japan; and *The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan
This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF b-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC50 0.1 mM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 mM. In a cell-free kinase experiment, it showed selective inhibition of PDGF b-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sistransformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases. q 1997 Academic Press
INTRODUCTION
Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells, such as fibroblasts, vascu1 To whom reprint requests should be addressed at Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-12, Japan. Fax: /81-273-47-5280. E-mail: [email protected].
lar smooth muscle cells, glomerular mesangial cells, and capillary endothelial cells [1, 2]. It consists of a dimer of two polypeptides chains, A and B, and is found either as a homodimer, AA or BB, or as a heterodimer, AB [2]. The receptors for PDGF consist of two subunits, a [3] and b [4], and each subunit dimerizes after ligand binding. Both types of PDGF receptor have protein tyrosine kinase activity in their intracellular domains [5], which phosphorylates their tyrosine residues after ligand binding and receptor dimerization. This autophosphorylation is essential for transmission of the signals of cell growth [6, 7]. A similar phenomenon is also known to occur in other growth factor receptors bearing tyrosine kinase activity, such as epidermal growth factor (EGF) receptor [8], basic fibroblast growth factor (bFGF) receptor [9], insulin b-receptor [10], and stem cell factor (SCF) receptor (c-kit) [11]. PDGF and its receptor are known to participate in various physiological processes such as embryonal development and wound healing [12]. However, their overexpression is involved in a number of pathophysiological processes, including several forms of neoplasia, atherosclerosis, glomerulonephritis, and tissue fibrosis [13]. For instance, abnormal proliferation of glomerular mesangial cells (MC) is observed in chronic glomerulonephritis experimental animal models [14–17] and also in human cases [18, 19], in which up-regulation of PDGF and/or its receptor is suggested to be a major determinant of disease progression. In vitro study also revealed that PDGF strongly stimulates the growth of cultured MC [20, 21]. Much effort has been expended to block the action of PDGF. Recently, several compounds that inhibit PDGF receptor autophosphorylation were reported [22–26]. We have also synthesized a novel series of quinoline derivatives that strongly inhibit PDGF receptor autophosphorylation of cultured MC [27]. Among them, we discovered that Ki6783 showed the most potent and selective inhibition. We studied the inhibitory effect of Ki6783 on autophosphorylation of the PDGF receptor under several conditions. To de-
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FIG. 1. Chemical structure of Ki6783.
termine its selectivity, we investigated the inhibitory effect of Ki6783 on autophosphorylation of other receptors and protein kinases. We also studied various biological activities of this compound, such as inhibition of MC proliferation, recovery of the abnormal cell shape in v-sis-transformed NIH3T3 fibroblast cells, and modulation of intracellular events. MATERIALS AND METHODS Chemicals. Ki6783 was chemically synthesized according to the procedure described previously [27]. Its chemical structure is shown in Fig. 1. For the experiments in this study, it was dissolved in dimethyl sulfoxide (DMSO) and diluted at various concentrations with the solvent. The sample solution was added to the culture medium in which the final concentration of DMSO was adjusted to 0.5%. Cell culture. MC were cultured from isolated rat glomeruli according to the method previously described [28]. Briefly, glomeruli were obtained from the renal cortex of male Wistar–Kyoto rats (8– 12 weeks of age, Charles River, Japan) by sequential sieving. The glomeruli were suspended in RPMI 1640 medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal bovine serum (FBS, Summit Biotechnology), supplemented with 50 mg/ml of streptomycin sulfate (Wako Pure Chemical Industry, Osaka, Japan) and 18 mg/ml of penicillin G potassium (Wako Pure Chemical Industry). Glomeruli were plated onto 75-cm2 plastic tissue culture flasks (Nunc, Rockilde, Denmark) and cultured at 377C. On the 21st day of culture, MC grown from cultured glomeruli were trypsinized and subcultured after glomeruli were removed with nylon mesh. After five subculturing passages, the MC were seeded onto 24-well or 96-well plates (Nunc) and used for tyrosine phosphorylation assay or growth assay. NIH3T3 cells (American Tissue Culture Collection (ATCC) Rockville, MD), v-sis-transformed NIH3T3 cells (which were a kind gift from Dr. S. A. Aaronson, Mt. Sinai Medical Center, NY), and HepG2 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and used for each experiment. RPMI 1640 containing 10% FBS was used for the culture of A431 cells (ATCC). Human aortic smooth muscle cells (hAOSMC) were obtained from KURABO (Osaka, Japan) and cultured in S-GM medium containing 5% FBS. K17 cells used for the stem cell factor (SCF) receptor (c-kit) autophosphorylation assay were constructed as one of a series of c-kit transformants [29] by Dr. H. Ohashi in our laboratory (Kirin Brewery Co., Ltd.) and were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Gibco Laboratories) containing 10% FBS. Receptor autophosphorylation assay in intact cells. MC, NIH3T3 cells, and hAOSMC were used for the assay of PDGF b-receptor autophosphorylation as follows. Cells which grew to near confluency in 24-well plates were growth arrested by cultivation in RPMI 1640 medium containing 0.5% FBS for 3 days. Then the medium was removed and 0.25 ml of the medium containing 0.1% BSA and various concentrations of Ki6783 were added into each well. After incubation at 377C for 1 h, recombinant human PDGF-BB (Upstate Biotechnology Inc. (UBI), New York, NY) dissolved in the medium containing
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0.1% BSA was added to each well, which was adjusted to 50 ng/ml as a final concentration. Cells were incubated at 377C for 10 min for autophosphorylation of the receptor. After removal of the medium, cells were washed with phosphate-buffered saline (PBS, Nissui Seiyaku Co.), and then 50 ml of ice-cold Tris-buffered saline (TBS) containing 1% Triton X-100, 2 mM sodium orthovanadate, and 1 mM EDTA was added in order to lyse cells by subsequent incubation at 47C for 30 min. An equal amount of TBS containing 1% sodium lauryl sulfate was added to the lysate and mixed well. Electrophoresis was carried out using 7.5% polyacrylamide gel to separate solubilized proteins. The proteins in the gel were transferred to Immobilon PVDF transfer membrane (Daiichi Pure Chemicals, Tokyo, Japan). Western blot analysis was performed with anti-phosphotyrosine monoclonal antibody (4G10, UBI, 1 mg/10 ml). Bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham) at a 1:2000 dilution as the second antibody and with the enhanced chemiluminescence (ECL) detection system (Amersham, UK). The density of each protein band was quantitated using a VIDAS Plus image analyzer (Carl Zeiss, Oberkochen, Germany). After analysis of tyrosine phosphorylation, bound antibodies on the blotting membrane were removed by incubation with stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris–HCl) at 707C for 30 min. PDGF b-receptor on the membrane was detected by Western blot with rabbit anti-PDGF b-receptor antiserum (UBI) at a 1:1000 dilution and confirmed the corresponding bands in the Western blot of phosphorylated tyrosine. Tyrosine phosphorylation assay of other receptors was carried out by the same method as that for the PDGF receptor. A431 cells, NIH3T3 cells, and HepG2 cells were used to study autophosphorylation of EGF receptor, bFGF receptor, and insulin b-receptor induced by EGF (UBI), bFGF (UBI), and insulin (Sigma Chemical Co., St. Louis, MO), respectively. The autophosphorylation assay of c-kit in K17 cells induced by recombinant murine SCF which was provided by Dr. H. Tsumura (Kirin Brewery Co., Ltd.) was carried out as described previously (29). Cell-free receptor tyrosine kinase assay. At first, lysate of quiescent NIH3T3 cells after cultivation in DMEM containing 0.5% FBS for 2 days was prepared by treatment of the detergent buffer (50 mM Hepes (pH 7.5), 1.5 mM MgCl2 , 150 mM NaCl, 1 mM EGTA, 10% glycerol, and 1% Triton X-100). The lysate was reacted with PDGF-BB (50 ng/ml) at 47C for 30 min, and then PDGF b-receptor in the lysate was immunoprecipitated with rabbit anti-PDGF b-receptor antiserum at a 1:100 dilution as a final concentration and with protein A–Sepharose beads (Pharmacia Biotech, Sweden). The immunoprecipitates were washed first with HNTG buffer (20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10 mM MgCl2) followed by 10 mM Tris–HCl (pH 7.5) and suspended in ice-cold reaction buffer (50 mM Tris–HCl (pH 7.5) and 10 mM MnCl2). The suspension was incubated with Ki6783 at 47C for 10 min. The tyrosine kinase reaction was initiated by the addition of 20 mM ATP (disodium salt, Sigma) to the samples, followed by incubation at 47C for 10 min. The reaction was terminated by the addition of SDS sample buffer (0.14 M Tris–HCl (pH 7.5), 22.4% glycerol, 6% SDS, 0.02% bromophenol blue, 10% 2-mercaptoethanol), and the samples were separated by SDS–PAGE on a 7.5% gel. After transfer of the protein in the gel to the membrane, tyrosine phosphorylation of PDGF b-receptor was analyzed by Western blot using anti-phosphotyrosine antibodies and detected by ECL fluorography as described above. The tyrosine phosphorylation assay of EGF receptor was carried out as previously described [30] in a manner similar to that used for the PDGF receptor. A431 cells were used to prepare the lysate. Kinetic study for determining the mode of inhibition. The mode of inhibition of Ki6783 with respect to ATP was examined by essentially the same protocol as in the cell-free PDGF receptor autophosphorylation experiment described above. The velocity of the enzymatic reaction and the inhibitory mode of Ki6783 were determined
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AN INHIBITOR OF PDGF RECEPTOR AUTOPHOSPHORYLATION by Lineweaver–Burk plot. The Ki value of the compound was estimated by Dixon plot. Assay for other kinases. The effect of Ki6783 on the activity of protein kinase C (PKC) was examined as described [31]. The effect of Ki6783 on the activity of protein kinase A (PKA) was examined with the Pierce assay system (Pierce). The effect of Ki6783 on the activity of MAP kinase (MAPK) was examined by the use of myelin basic protein (MBP) as a substrate and purified MAP kinase derived from Xenopus laevis as described previously [32]. The activity of MAP kinase kinase (MAPKK) was studied with kinase-negative MAPK as a substrate and purified MAPKK [32]. MAP kinase activation assay in intact cells. Cultured NIH3T3 cells which grew to subconfluence on dishes (9 cm diameter) were washed twice with PBS and incubated at 377C for 12 h after the addition of DMEM. Ki6783 was added to the medium and incubated for 30 min. Thereafter, 100 ng/ml of PDGF-BB was added and incubated for 5 min. The medium was removed and cells were washed with PBS. Then the lysate buffer (10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) was added, and cells were incubated for 10 min on ice. After cells were scraped off the dishes, the soluble fraction of the lysate was used for the MAP kinase assay by the use of the BIOTRAK p42/p44 MAP kinase assay system (Amersham). Assay of DNA synthesis. MC were seeded onto 96-well plates (Nunc) and cultured in RPMI 1640 containing 10% FBS just before they grew to near confluency. The medium was then changed to that containing 0.5% FBS, and cell growth was arrested by subsequent cultivation for 3 days. Thereafter, the medium was removed, and cells were washed twice with the medium. The medium was changed to RPMI 1640 containing 0.1% BSA, and 50 ng/ml of PDGF-BB, 155 kBq/ml of [methyl-3H]thymidine (New England Nuclear, Boston, MA) and various concentrations of Ki6783 were added into each well. After cultivation for 24 h, cells were harvested and collected on the glass filter with the cell harvester (Wallac, Turku, Finland). The radioactivity of [3H]thymidine incorporated into the cells was determined by the liquid scintillation counting system (b Plate, Wallac). Cell proliferation assay. Growth of MC was arrested by the same method as that used in the DNA synthesis study. Thereafter the medium containing 30 ng/ml of PDGF-BB, 2% FBS, 0.1% BSA, and Ki6783 (0.01–100 mM) was added to each well. After cultivation for 48 or 72 h, intact cell number was counted in a hemocytometer. Observation of morphology of sis-transformed NIH3T3 cells. vsis-transformed NIH3T3 cells were seeded onto 96-well plates and cultured. After 2 days, media were changed to DMEM containing 10% FBS and Ki6783, and cells were cultured subsequently for 48 h, for observation of cell shape. NIH3T3 cells were also cultured as a control.
RESULTS
Inhibition of receptor autophosphorylation by Ki6783 in intact cells. In the first series of experiments, the effect of Ki6783 on receptor autophosphorylation was investigated in intact cells. Quiescent MC were pretreated with various concentrations of Ki6783 for 1 h and then exposed to 50 ng/ml of PDGF-BB for 10 min to obtain the maximum autophosphorylation of PDGF b-receptor. Antiphosphotyrosine immunoblot analysis of Triton X-100 soluble proteins of PDGF-stimulated MC showed the major tyrosine phosphorylated protein band at 180 kDa (Fig. 2A). This protein was confirmed as PDGF b-receptor by the subsequent reprobing of
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FIG. 2. Selective effect of Ki6783 on autophosphorylation of PDGF b-receptor (A) versus EGF receptor (B) in cultured rat mesangial cells (PDGF b-receptor) and A431 cells (EGF receptor), respectively. Quiescent cells were incubated with various concentrations of Ki6783 for 1 h and subsequently incubated in the presence of 50 ng/ml of recombinant human PDGF-BB (/) or 500 ng/ml of EGF or in the absence of these factors (0). Cell lysates were electrophoresed in a 7.5% polyacrylamide gel, and Western blot analysis using antiphosphotyrosine (pY) antibodies was carried out as described under Materials and Methods. Western blot analysis using anti-PDGF breceptor antibodies (PDGF b-R; in A) or anti-EGF receptor antibodies was also carried out in the same membranes to confirm corresponding bands. Fluorography was performed with the ECL system.
the same immunoblot membrane with anti-PDGF breceptor antibody. Ki6783 decreased the amount of phosphorylated tyrosine of the PDGF b-receptor in a dose-dependent manner (Fig. 2A). Quantitation of the fluorogram by densitometry indicated that the concentration of 50% inhibition (IC50) of PDGF b-receptor autophosphorylation by Ki6783 is 0.13 mM (Table 1). Inhibitory activity of Ki6783 to PDGF b-receptor autophosphorylation in different species was also examined by the use of mouse NIH3T3 fibroblast cells and human aortic smooth muscle cells (hAOSMC). As shown in Table 1, this compound inhibited phosphorylation of this receptor with a strength almost equal to that observed in MC (IC50 0.07 mM in NIH3T3 and 0.15 mM in hAOSMC). To test the selectivity of inhibition, EGF (500 ng/ml), bFGF (20 ng/ml), and insulin (5 mg/ml) were added to the culture media of quiescent A431 cells, NIH3T3 cells, and HepG2 cells, respectively. Tyrosine phosphorylation of EGF receptor (Fig. 2B, 150 kDa), bFGF receptor (130 kDa), and insulin b-receptor (95 kDa) was detected by the antiphosphotyrosine immunoblot of the cell lysates. Ki6783 did not inhibit phosphorylation of these receptors even at 100 mM (Table 1). We also tested the effect of Ki6783 on autophosphorylation of stem cell factor (SCF) receptor (c-kit), which belongs to the PDGF receptor superfamily. Stable c-kit transformant K17 cells [29] were stimulated by recombinant murine SCF (100 nM) to obtain auto-
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TABLE 1 Selective Inhibition of Ki6783 on PDGF b-Receptor Tyrosine Kinase among Various Receptor Tyrosine Kinases and Protein Kinases in Intact Cells or in the Cell-Free Experiments Intact cells assay PDGF b-R
EGF-R bFGF-R Insulin b-R c-kit
Rat MC NIH3T3 hAOSMC A431 NIH3T3 HepG2 K17
IC50 (mM)
Cell-free assay
IC50 (mM)
0.13 0.07 0.15 ú100 ú100 ú100 õ1.0
PDGF b-R EGF-R PKA PKC MAPK MAPKK
0.025 ú10 ú10 ú10 ú10 ú10
Note. IC50 value of each assay is represented in the table. Experimental procedures and abbreviations are described under Materials and Methods.
phosphorylation of c-kit. Pretreatment of K17 with 1 mM Ki6783 decreased the amount of phosphorylation of c-kit to less than 50% of the control level (Table 1). Inhibition of protein kinases by Ki6783 under the cellfree conditions. In the next series of experiments, the effect of Ki6783 on several protein kinases was tested. Immunoprecipitates of PDGF b-receptor of NIH3T3 cell lysates were used for the assay of PDGF b-receptor tyrosine kinase. Ki6783 inhibited the tyrosine kinase activity in a dose-dependent manner (Fig. 3). The IC50 of the compound was 0.025 mM. In contrast, Ki6783 had no effect on the activities of EGF receptor tyrosine kinase, PKA, PKC, MAPK, or MAPKK even at 10 mM. The mode of autophosphorylation inhibition. The kinetics of the inhibitory action of Ki6783 on PDGF b-receptor tyrosine kinase was analyzed with various concentrations of Ki6783 and ATP. The Lineweaver–
FIG. 3. Effect of Ki6783 on tyrosine phosphorylation of immunoprecipitated PDGF b-receptor (PDGF b-R). Lysates of the quiescent NIH3T3 cells were immunoprecipitated with anti-PDGF b-receptor after treatment with PDGF-BB (50 ng/ml) for 30 min. Ki6783 was added to the immunoprecipitates for 10 min and the phosphorylation reaction was carried out for 10 min by the addition of ATP (20 mM). SDS–PAGE, Western blot analysis, and fluorography using antiphosphotyrosine (pY) antibodies and anti-PDGF b-R antibodies were carried out by the same method as that of the autophosphorylation assay using intact cells.
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FIG. 4. Lineweaver–Burk plot of the inhibition study of Ki6783. Phosphorylation reaction of PDGF b-receptor immunoprecipitates was performed in the presence of five concentrations of ATP and five concentrations of Ki6783. The vertical axis represents the inverse of the velocity of the enzymatic reaction and the horizontal axis represents the inverse of the concentration of added ATP.
Burk plot of the kinase assay is shown in Fig. 4. Regression lines at various concentrations of Ki6783 crossed at one point at an ATP concentration equal to 0 mM; the mode of inhibition of Ki6783 was estimated to be competitive action against ATP. The Km value of the assay and the Ki value of Ki6783 were calculated as 73.0 mM and 58.1 nM, respectively, from the plot. Inhibition of MAP kinase activation by Ki6783. To test whether Ki6783 actually blocks intracellular signal transduction next to PDGF receptor autophosphorylation in intact cells, the effect on MAP kinase activation was examined (Fig. 5). After incubation with PDGF-BB (100 ng/ml) for 5 min, MAP kinase activity in NIH3T3 cells was increased about fivefold more than
FIG. 5. Effect of Ki6783 on MAP kinase activation in NIH3T3 cells. Quiescent NIH3T3 cells were incubated with Ki6783 for 30 min and incubated in the presence of 100 ng/ml of PDGF-BB for 5 min. Cell lysates were prepared and a MAP kinase assay was carried out as described under Materials and Methods. The vertical axis represents the relative MAP kinase activity in which the mean value of the negative control (PDGF(0) and Ki6783(0)) is 1. Each value is expressed by the mean { SEM of three experiments.
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DISCUSSION
FIG. 6. Inhibitory effect of Ki6783 on DNA synthesis in cultured rat mesangial cells. Quiescent mesangial cells were treated with 50 ng/ml of PDGF-BB and various concentrations of Ki6783 and [3H]thymidine (155 kBq/ml), and incubated for 24 h. Radioactivity incorporated into the cell was determined as described under Materials and Methods. The vertical axis is relative DNA synthesis, in which the mean radioactivity in the absence of Ki6783 is 100%, was expressed. Values are expressed as the means { SEM of four experiments.
that of nontreated cells. Ki6783 (1 and 10 mM) decreased MAP kinase activation induced by PDGF. Effects of Ki6783 on DNA synthesis and proliferation of mesangial cells. In order to test biological activities of Ki6783 due to the inhibition of PDGF, the effect of the compound on cell growth was examined using cultured rat MC. In the study of DNA synthesis, [3H]thymidine incorporation of MC for 24 h was increased about 10-fold by the treatment of PDGF-BB (50 ng/ml). Ki6783 decreased the amount of incorporated [3H]thymidine in a dose-dependent manner as shown in Fig. 6. The IC50 was about 0.1 mM. The number of intact MC increased after the addition of PDGF-BB (30 ng/ml) and FBS (2%) to the culture medium for 48 and 72 h. Ki6783 inhibited the increase in cell number at both incubation times in a dose-dependent manner (Fig. 7). No cytotoxicity was observed 72 h after the addition of 100 mM Ki6783 to the culture medium containing PDGF and FBS. Normalization of the morphology of sis-transformed NIH3T3 cells by Ki6783. Sis codes DNA of PDGF-B chain, and v-sis is known as a variant of the sis gene from simian sarcoma virus [33]. v-sis-transfected NIH3T3 cells acquire the transformed phenotype characterized by the loss of density-dependent growth arrest and spindle-shaped morphology due to autocrine growth stimulation via the PDGF receptor. The effect of Ki6783 on the transformed phenotype was tested. When sis-transformed cells were treated with Ki6783 for 48 h, normalization of the morphology was observed, such as the change from a non-contact-inhibited phenotype to a flattened, contact-inhibited morphology similar to that of nontransformed NIH3T3 cells (Fig. 8).
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In the present study, we showed that our original quinoline derivative, Ki6783, strongly inhibits tyrosine phosphorylation of PDGF b-receptor in intact cells as well as in the cell-free experiment and that this activity is much more selective for this receptor than for other growth factor receptors. We previously reported that several 4-substituted quinoline derivatives possess this activity and that Ki6783 is the strongest compound among them [27]. This is the first report describing the characterization of the inhibitory action of a 4-substituted quinoline derivative, although other kinds of compounds have been reported as inhibitors of PDGF receptor autophosphorylation, such as staurosporine derivatives [23] and 3substituted quinoline derivatives [26, 34]. Ki6783 is thought to have one of the more potent activities among these agents. Inhibitory activity of PDGF b-receptor phosphorylation in intact cells was about five times weaker than that in the cell-free experiment based on IC50 values. This discrepancy might be explained by the slow uptake into the cells. Ki6783 also inhibited PDGF b-receptor autophosphorylation in several types of cultured cells, such as rat mesangial cells, mouse NIH3T3 cells, and human aortic smooth muscle cells, with almost the same strength. From these results, it is estimated that the association site of the receptor with the compound is highly homologous among different species. In this study, we did not examine the effect of the compound on the PDGF a-receptor because the amount of the receptor was too small to detect autophosphorylation compared to the b-type receptor (data not shown). Another interesting characteristic of this compound is its high selectivity for the PDGF receptor compared
FIG. 7. Inhibition of mesangial cell proliferation by Ki6783 on intact cell number of mesangial cells. Quiescent mesangial cells were treated with 30 ng/ml of PDGF-BB, FBS (2%), and various concentrations of Ki6783 and incubated for 48 and 72 h. The cells were harvested by trypsinization, and intact cell number was counted. Values are means { SEM of six to eight experiments in each group.
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FIG. 8. Effect of Ki6783 on the morphology of v-sis-transformed NIH3T3 cells. v-sis-transformed NIH3T3 cells were cultivated in the absence (A) or in the presence of Ki6783 (1 mM) (B) for 48 h. No transfected NIH3T3 cells were cultivated for the same period (C). Microphotographs were taken with a phase-contrast microscope (1200).
to other growth factor receptors. Ki6783 did not inhibit autophosphorylation of the EGF receptor, bFGF receptor, or insulin b-receptor even at 100 mM. These results indicate that the selectivity for the PDGF b-receptor is about 1000 times greater than that of the others. Such high selectivity was also demonstrated in the cell-free experiment, in which Ki6783 did not inhibit EGF receptor tyrosine kinase or other serine–threonine-type protein kinases such as PKA, PKC, MAPK, and MAPKK even at 10 mM. These results indicate that PDGF receptor has a characteristic binding site for Ki6783 which affects its tyrosine kinase activity. However, the kinetic analysis indicated its mode of action as a competitive inhibition to ATP. Since all protein kinases have an ATP binding site, the question arises why and how Ki6783 can recognize and inhibit PDGF receptor so selectively. As piceatannol, a protein kinase inhibitor, was reported to inhibit competitively the peptide substrate as well as ATP [35], it may be that Ki6783 also possesses such multiple modes of action. Otherwise, we estimate that Ki6783 may selectively decrease the
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catalytic efficiency of PDGF receptor tyrosine kinase by an unknown mechanism. A highly selective inhibitor of EGF receptor autophosphorylation, PD153035, was recently reported [36]. Interestingly, its chemical structure (4,6,7-trisubstituted quinazoline) resembles that of Ki6783. However, the reason each receptor can precisely distinguish these similar chemical structures is unknown. Recently, the catalytic specificity of various tyrosine kinases of growth factor receptors was examined [37, 38]. Although the actual catalytic site has not been determined completely, such a study will elucidate the mechanism of inhibition by these compounds. Ki6783 strongly inhibited autophosphorylation of the SCF receptor (c-kit). As this receptor is known to be closely related to the PDGF receptor with respect to their protein structures [11], this compound may be able to recognize it. Further study of the effect of the compound on the phosphorylation of other receptors in the PDGF receptor superfamily such as KDR [39], flt1 [40], and fms [41] remains to be done. After the ligand binding and autophosphorylation of
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PDGF receptor, the signal is transduced by the sequential pathway in the cytoplasm, and at last it reaches the nucleus to stimulate DNA synthesis or other cellular events [42, 43]. Activation of MAP kinase is well known to take place during this signal transduction [42, 43]. Ki6783 inhibited MAPK activation in NIH3T3 stimulated by PDGF-BB, although it did not inhibit MAPK activity under the cell-free condition. Thus, it is suggested that the compound can actually block the intracellular signal transduction after PDGF receptor autophosphorylation. About 40% of MAPK to the control was activated in the presence of 1 mM Ki6783, whereas complete inhibition of autophosphorylation of PDGF breceptor was observed at that dose. This may be due to a difference in concentration of PDGF-BB, incubation time, or cell type between these two experiments. Although NIH3T3 cells were used in this study because of convenience of cultivation, further experiments using mesangial cells are necessary. A second central finding of this study is that Ki6783 has inhibitory activity against growth and phenotypic change of cells bearing the PDGF receptor. PDGF is considered a ‘‘competence factor’’ for cell growth, which shifts the stage in the cell cycle from G1Q phase to G1 phase (44). In particular, PDGF-BB and -AB are known to have strong growth stimulating activity via the PDGF-b receptor in several types of cells (2). In this study, we also found that PDGF-BB and -AB stimulated DNA synthesis and proliferation in mesangial cells in contrast to no effect of PDGF-AA (data not shown). Ki6783 decreased PDGF-BB-stimulated thymidine incorporation into the cell, in which dose dependency was almost the same as that of the inhibition of receptor autophosphorylation. Furthermore, we tested the effect of the compound on cell proliferation. Ki6783 inhibited the increase in intact cell number after the incubation with PDGF-BB and 2% FBS as a growth progression factor. In this experiment, the compound showed weaker activity than that in the DNA synthesis study. This may be explained by contribution of other growth factors in FBS to cell growth. Interestingly, Ki6783 did not show any cytotoxicity even at 100 mM after 72 h incubation. This suggests that Ki6783 has a strong cytostatic but not cytotoxic effect on the growth of mesangial cells. Several studies have been reported using protein tyrosine kinase inhibitors, which supported or opposed the correlation between the activities of enzyme inhibition and growth inhibition [25, 45, 46]. Our study indicated that the inhibitory activity of Ki6783 on receptor autophosphorylation was well correlated with that on cell growth. Finally, Ki6783 was shown to clearly reverse the morphological change of v-sis-transformed NIH3T3 fibroblast cells from the characteristic spindle shape to the normal flattened shape usually observed in non-
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transfected NIH3T3 cells. Some tyrosine kinase inhibitor was also reported to have the same activity at 50 mM (25). We could observe such an effect by the addition of only 1 mM Ki6783. These results suggest that Ki6783 has the potent activity to block the phenotypic change triggered by overexpression of PDGF. PDGF and its receptor system are known to be involved in various pathophysiological processes, including several forms of neoplasia, atherosclerosis, glomerulonephritis, and tissue fibrosis [13]. In particular, participation of PDGF in excessive proliferation of glomerular mesangial cells has been well recognized in the progression of chronic glomerular diseases [14–21, 47]. The therapeutic importance of blocking PDGF action in these diseases has also been discussed [16]. From these points of view, Ki6783 may have a possible use in inhibiting disease progression as a therapeutic agent. Furthermore, highly selective inhibition of PDGF action may be beneficial with respect to fewer adverse effects. Our present study is the first attempt to examine the effect of a selective PDGF receptor autophosphorylation inhibitor on mesangial cell growth. In vivo study is now under way. We are grateful to Dr. Tadashi Yamamoto (Niigata University) for helpful suggestions regarding the culture of mesangial cells, and to Dr. Eisuke Nishida (Kyoto University) for providing us with recombinant MAPK and MAPKK.
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24. Bryckart, M. C., Eldor, A., Fontanay, M., Gazit, A., Osherov, N., Gilon, C., Levitzki, A., and Tobelem, G. (1992) Exp. Cell Res. 199, 255–261. 25. Kovalenko, M., Gazit, A., Bo¨hmer, A., Rorsman, C., Ro¨nnstrand, L., Heldin, C-H., Waltenberger, J., Bo¨hmer, F-D., and Levitzki, A. (1994) Cancer Res. 54, 6106–6114. 26. Maguire, M. P., Sheets, K. R., McVety, K., Spada, A. P., and Zilberstein, A. (1994) J. Med. Chem. 37, 2129–2137. 27. Kubo, K., Shimizu, T., Ohyama, S., Murooka, H., Nishitoba, T., Kato, S., Kobayashi, Y., Yagi, M., Isoe, T., Nakamura, K., Osawa, T., and Izawa, T., manuscript in preparation. 28. Kreisberg, J. I., and Karnovsky, M. J. (1983) Kidney Int. 23, 439–447. 29. Ohashi, H., Kameda, R., Nishikawa, M., Kawagishi, M., and Liu, Y-C. (1994) Cytotechnology 16, 27–35. 30. Carpenter, G., King, L., Jr., and Cohen, S. (1978) Nature 276, 409–410.
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Received January 31, 1997 Revised version received April 1, 1997
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Selective Inhibition of Platelet-Derived Growth Factor (PDGF) Receptor Autophosphorylation and PDGF-Mediated Cellular Events by a Quinoline Derivative Mikio Yagi,1 Shinichiro Kato, Yoshiko Kobayashi, Kazuo Kubo, Shinichi Oyama, Toshiyuki Shimizu, Tsuyoshi Nishitoba, Toshiyuki Isoe, Kazuhide Nakamura, Hideya Ohashi, Nami Kobayashi, Noriko Iinuma, Tatsushi Osawa, Rie Onose,* and Hiroyuki Osada* Pharmaceutical Research Laboratory, Kirin Brewery Company, Ltd., Takasaki, Japan; and *The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama, Japan
This report describes the biological effects of our original compound, Ki6783 ((3,4-dimethoxy)-4-phenoxy-6,7-dimethoxyquinoline), a potent and selective inhibitor of platelet-derived growth factor (PDGF) receptor autophosphorylation. This compound strongly inhibited autophosphorylation of the PDGF b-receptor in cultured rat glomerular mesangial cells (MC) bearing this receptor (IC50 0.1 mM), although it did not inhibit autophosphorylation of other growth factor receptors even at 100 mM. In a cell-free kinase experiment, it showed selective inhibition of PDGF b-receptor tyrosine kinase. A kinetic study of the compound to this tyrosine kinase revealed a competitive mode of action to ATP. [3H]Thymidine incorporation and cell proliferation of MC were inhibited by Ki6783 in a dose-dependent manner after Ki6783 and PDGF-BB were added to the culture medium. Furthermore, this compound normalized the fibrotic cell shape of v-sistransformed NIH3T3 cells, which grow in an autocrine manner via the PDGF receptor. These effects could be explained by the inhibition of intracellular signal transduction triggered by PDGF receptor autophosphorylation, in which activation of mitogen-activated protein kinase occurs. These results suggest that Ki6783 is one of the more potent and selective inhibitors of PDGF receptor autophosphorylation and that it may be useful in ameliorating cell abnormalities due to excess action of PDGF and its receptor systems in several diseases. q 1997 Academic Press
INTRODUCTION
Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells, such as fibroblasts, vascu1 To whom reprint requests should be addressed at Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki-shi, Gunma 370-12, Japan. Fax: /81-273-47-5280. E-mail: [email protected].
lar smooth muscle cells, glomerular mesangial cells, and capillary endothelial cells [1, 2]. It consists of a dimer of two polypeptides chains, A and B, and is found either as a homodimer, AA or BB, or as a heterodimer, AB [2]. The receptors for PDGF consist of two subunits, a [3] and b [4], and each subunit dimerizes after ligand binding. Both types of PDGF receptor have protein tyrosine kinase activity in their intracellular domains [5], which phosphorylates their tyrosine residues after ligand binding and receptor dimerization. This autophosphorylation is essential for transmission of the signals of cell growth [6, 7]. A similar phenomenon is also known to occur in other growth factor receptors bearing tyrosine kinase activity, such as epidermal growth factor (EGF) receptor [8], basic fibroblast growth factor (bFGF) receptor [9], insulin b-receptor [10], and stem cell factor (SCF) receptor (c-kit) [11]. PDGF and its receptor are known to participate in various physiological processes such as embryonal development and wound healing [12]. However, their overexpression is involved in a number of pathophysiological processes, including several forms of neoplasia, atherosclerosis, glomerulonephritis, and tissue fibrosis [13]. For instance, abnormal proliferation of glomerular mesangial cells (MC) is observed in chronic glomerulonephritis experimental animal models [14–17] and also in human cases [18, 19], in which up-regulation of PDGF and/or its receptor is suggested to be a major determinant of disease progression. In vitro study also revealed that PDGF strongly stimulates the growth of cultured MC [20, 21]. Much effort has been expended to block the action of PDGF. Recently, several compounds that inhibit PDGF receptor autophosphorylation were reported [22–26]. We have also synthesized a novel series of quinoline derivatives that strongly inhibit PDGF receptor autophosphorylation of cultured MC [27]. Among them, we discovered that Ki6783 showed the most potent and selective inhibition. We studied the inhibitory effect of Ki6783 on autophosphorylation of the PDGF receptor under several conditions. To de-
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FIG. 1. Chemical structure of Ki6783.
termine its selectivity, we investigated the inhibitory effect of Ki6783 on autophosphorylation of other receptors and protein kinases. We also studied various biological activities of this compound, such as inhibition of MC proliferation, recovery of the abnormal cell shape in v-sis-transformed NIH3T3 fibroblast cells, and modulation of intracellular events. MATERIALS AND METHODS Chemicals. Ki6783 was chemically synthesized according to the procedure described previously [27]. Its chemical structure is shown in Fig. 1. For the experiments in this study, it was dissolved in dimethyl sulfoxide (DMSO) and diluted at various concentrations with the solvent. The sample solution was added to the culture medium in which the final concentration of DMSO was adjusted to 0.5%. Cell culture. MC were cultured from isolated rat glomeruli according to the method previously described [28]. Briefly, glomeruli were obtained from the renal cortex of male Wistar–Kyoto rats (8– 12 weeks of age, Charles River, Japan) by sequential sieving. The glomeruli were suspended in RPMI 1640 medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal bovine serum (FBS, Summit Biotechnology), supplemented with 50 mg/ml of streptomycin sulfate (Wako Pure Chemical Industry, Osaka, Japan) and 18 mg/ml of penicillin G potassium (Wako Pure Chemical Industry). Glomeruli were plated onto 75-cm2 plastic tissue culture flasks (Nunc, Rockilde, Denmark) and cultured at 377C. On the 21st day of culture, MC grown from cultured glomeruli were trypsinized and subcultured after glomeruli were removed with nylon mesh. After five subculturing passages, the MC were seeded onto 24-well or 96-well plates (Nunc) and used for tyrosine phosphorylation assay or growth assay. NIH3T3 cells (American Tissue Culture Collection (ATCC) Rockville, MD), v-sis-transformed NIH3T3 cells (which were a kind gift from Dr. S. A. Aaronson, Mt. Sinai Medical Center, NY), and HepG2 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and used for each experiment. RPMI 1640 containing 10% FBS was used for the culture of A431 cells (ATCC). Human aortic smooth muscle cells (hAOSMC) were obtained from KURABO (Osaka, Japan) and cultured in S-GM medium containing 5% FBS. K17 cells used for the stem cell factor (SCF) receptor (c-kit) autophosphorylation assay were constructed as one of a series of c-kit transformants [29] by Dr. H. Ohashi in our laboratory (Kirin Brewery Co., Ltd.) and were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Gibco Laboratories) containing 10% FBS. Receptor autophosphorylation assay in intact cells. MC, NIH3T3 cells, and hAOSMC were used for the assay of PDGF b-receptor autophosphorylation as follows. Cells which grew to near confluency in 24-well plates were growth arrested by cultivation in RPMI 1640 medium containing 0.5% FBS for 3 days. Then the medium was removed and 0.25 ml of the medium containing 0.1% BSA and various concentrations of Ki6783 were added into each well. After incubation at 377C for 1 h, recombinant human PDGF-BB (Upstate Biotechnology Inc. (UBI), New York, NY) dissolved in the medium containing
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0.1% BSA was added to each well, which was adjusted to 50 ng/ml as a final concentration. Cells were incubated at 377C for 10 min for autophosphorylation of the receptor. After removal of the medium, cells were washed with phosphate-buffered saline (PBS, Nissui Seiyaku Co.), and then 50 ml of ice-cold Tris-buffered saline (TBS) containing 1% Triton X-100, 2 mM sodium orthovanadate, and 1 mM EDTA was added in order to lyse cells by subsequent incubation at 47C for 30 min. An equal amount of TBS containing 1% sodium lauryl sulfate was added to the lysate and mixed well. Electrophoresis was carried out using 7.5% polyacrylamide gel to separate solubilized proteins. The proteins in the gel were transferred to Immobilon PVDF transfer membrane (Daiichi Pure Chemicals, Tokyo, Japan). Western blot analysis was performed with anti-phosphotyrosine monoclonal antibody (4G10, UBI, 1 mg/10 ml). Bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham) at a 1:2000 dilution as the second antibody and with the enhanced chemiluminescence (ECL) detection system (Amersham, UK). The density of each protein band was quantitated using a VIDAS Plus image analyzer (Carl Zeiss, Oberkochen, Germany). After analysis of tyrosine phosphorylation, bound antibodies on the blotting membrane were removed by incubation with stripping buffer (100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, and 62.5 mM Tris–HCl) at 707C for 30 min. PDGF b-receptor on the membrane was detected by Western blot with rabbit anti-PDGF b-receptor antiserum (UBI) at a 1:1000 dilution and confirmed the corresponding bands in the Western blot of phosphorylated tyrosine. Tyrosine phosphorylation assay of other receptors was carried out by the same method as that for the PDGF receptor. A431 cells, NIH3T3 cells, and HepG2 cells were used to study autophosphorylation of EGF receptor, bFGF receptor, and insulin b-receptor induced by EGF (UBI), bFGF (UBI), and insulin (Sigma Chemical Co., St. Louis, MO), respectively. The autophosphorylation assay of c-kit in K17 cells induced by recombinant murine SCF which was provided by Dr. H. Tsumura (Kirin Brewery Co., Ltd.) was carried out as described previously (29). Cell-free receptor tyrosine kinase assay. At first, lysate of quiescent NIH3T3 cells after cultivation in DMEM containing 0.5% FBS for 2 days was prepared by treatment of the detergent buffer (50 mM Hepes (pH 7.5), 1.5 mM MgCl2 , 150 mM NaCl, 1 mM EGTA, 10% glycerol, and 1% Triton X-100). The lysate was reacted with PDGF-BB (50 ng/ml) at 47C for 30 min, and then PDGF b-receptor in the lysate was immunoprecipitated with rabbit anti-PDGF b-receptor antiserum at a 1:100 dilution as a final concentration and with protein A–Sepharose beads (Pharmacia Biotech, Sweden). The immunoprecipitates were washed first with HNTG buffer (20 mM Hepes (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10 mM MgCl2) followed by 10 mM Tris–HCl (pH 7.5) and suspended in ice-cold reaction buffer (50 mM Tris–HCl (pH 7.5) and 10 mM MnCl2). The suspension was incubated with Ki6783 at 47C for 10 min. The tyrosine kinase reaction was initiated by the addition of 20 mM ATP (disodium salt, Sigma) to the samples, followed by incubation at 47C for 10 min. The reaction was terminated by the addition of SDS sample buffer (0.14 M Tris–HCl (pH 7.5), 22.4% glycerol, 6% SDS, 0.02% bromophenol blue, 10% 2-mercaptoethanol), and the samples were separated by SDS–PAGE on a 7.5% gel. After transfer of the protein in the gel to the membrane, tyrosine phosphorylation of PDGF b-receptor was analyzed by Western blot using anti-phosphotyrosine antibodies and detected by ECL fluorography as described above. The tyrosine phosphorylation assay of EGF receptor was carried out as previously described [30] in a manner similar to that used for the PDGF receptor. A431 cells were used to prepare the lysate. Kinetic study for determining the mode of inhibition. The mode of inhibition of Ki6783 with respect to ATP was examined by essentially the same protocol as in the cell-free PDGF receptor autophosphorylation experiment described above. The velocity of the enzymatic reaction and the inhibitory mode of Ki6783 were determined
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AN INHIBITOR OF PDGF RECEPTOR AUTOPHOSPHORYLATION by Lineweaver–Burk plot. The Ki value of the compound was estimated by Dixon plot. Assay for other kinases. The effect of Ki6783 on the activity of protein kinase C (PKC) was examined as described [31]. The effect of Ki6783 on the activity of protein kinase A (PKA) was examined with the Pierce assay system (Pierce). The effect of Ki6783 on the activity of MAP kinase (MAPK) was examined by the use of myelin basic protein (MBP) as a substrate and purified MAP kinase derived from Xenopus laevis as described previously [32]. The activity of MAP kinase kinase (MAPKK) was studied with kinase-negative MAPK as a substrate and purified MAPKK [32]. MAP kinase activation assay in intact cells. Cultured NIH3T3 cells which grew to subconfluence on dishes (9 cm diameter) were washed twice with PBS and incubated at 377C for 12 h after the addition of DMEM. Ki6783 was added to the medium and incubated for 30 min. Thereafter, 100 ng/ml of PDGF-BB was added and incubated for 5 min. The medium was removed and cells were washed with PBS. Then the lysate buffer (10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) was added, and cells were incubated for 10 min on ice. After cells were scraped off the dishes, the soluble fraction of the lysate was used for the MAP kinase assay by the use of the BIOTRAK p42/p44 MAP kinase assay system (Amersham). Assay of DNA synthesis. MC were seeded onto 96-well plates (Nunc) and cultured in RPMI 1640 containing 10% FBS just before they grew to near confluency. The medium was then changed to that containing 0.5% FBS, and cell growth was arrested by subsequent cultivation for 3 days. Thereafter, the medium was removed, and cells were washed twice with the medium. The medium was changed to RPMI 1640 containing 0.1% BSA, and 50 ng/ml of PDGF-BB, 155 kBq/ml of [methyl-3H]thymidine (New England Nuclear, Boston, MA) and various concentrations of Ki6783 were added into each well. After cultivation for 24 h, cells were harvested and collected on the glass filter with the cell harvester (Wallac, Turku, Finland). The radioactivity of [3H]thymidine incorporated into the cells was determined by the liquid scintillation counting system (b Plate, Wallac). Cell proliferation assay. Growth of MC was arrested by the same method as that used in the DNA synthesis study. Thereafter the medium containing 30 ng/ml of PDGF-BB, 2% FBS, 0.1% BSA, and Ki6783 (0.01–100 mM) was added to each well. After cultivation for 48 or 72 h, intact cell number was counted in a hemocytometer. Observation of morphology of sis-transformed NIH3T3 cells. vsis-transformed NIH3T3 cells were seeded onto 96-well plates and cultured. After 2 days, media were changed to DMEM containing 10% FBS and Ki6783, and cells were cultured subsequently for 48 h, for observation of cell shape. NIH3T3 cells were also cultured as a control.
RESULTS
Inhibition of receptor autophosphorylation by Ki6783 in intact cells. In the first series of experiments, the effect of Ki6783 on receptor autophosphorylation was investigated in intact cells. Quiescent MC were pretreated with various concentrations of Ki6783 for 1 h and then exposed to 50 ng/ml of PDGF-BB for 10 min to obtain the maximum autophosphorylation of PDGF b-receptor. Antiphosphotyrosine immunoblot analysis of Triton X-100 soluble proteins of PDGF-stimulated MC showed the major tyrosine phosphorylated protein band at 180 kDa (Fig. 2A). This protein was confirmed as PDGF b-receptor by the subsequent reprobing of
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FIG. 2. Selective effect of Ki6783 on autophosphorylation of PDGF b-receptor (A) versus EGF receptor (B) in cultured rat mesangial cells (PDGF b-receptor) and A431 cells (EGF receptor), respectively. Quiescent cells were incubated with various concentrations of Ki6783 for 1 h and subsequently incubated in the presence of 50 ng/ml of recombinant human PDGF-BB (/) or 500 ng/ml of EGF or in the absence of these factors (0). Cell lysates were electrophoresed in a 7.5% polyacrylamide gel, and Western blot analysis using antiphosphotyrosine (pY) antibodies was carried out as described under Materials and Methods. Western blot analysis using anti-PDGF breceptor antibodies (PDGF b-R; in A) or anti-EGF receptor antibodies was also carried out in the same membranes to confirm corresponding bands. Fluorography was performed with the ECL system.
the same immunoblot membrane with anti-PDGF breceptor antibody. Ki6783 decreased the amount of phosphorylated tyrosine of the PDGF b-receptor in a dose-dependent manner (Fig. 2A). Quantitation of the fluorogram by densitometry indicated that the concentration of 50% inhibition (IC50) of PDGF b-receptor autophosphorylation by Ki6783 is 0.13 mM (Table 1). Inhibitory activity of Ki6783 to PDGF b-receptor autophosphorylation in different species was also examined by the use of mouse NIH3T3 fibroblast cells and human aortic smooth muscle cells (hAOSMC). As shown in Table 1, this compound inhibited phosphorylation of this receptor with a strength almost equal to that observed in MC (IC50 0.07 mM in NIH3T3 and 0.15 mM in hAOSMC). To test the selectivity of inhibition, EGF (500 ng/ml), bFGF (20 ng/ml), and insulin (5 mg/ml) were added to the culture media of quiescent A431 cells, NIH3T3 cells, and HepG2 cells, respectively. Tyrosine phosphorylation of EGF receptor (Fig. 2B, 150 kDa), bFGF receptor (130 kDa), and insulin b-receptor (95 kDa) was detected by the antiphosphotyrosine immunoblot of the cell lysates. Ki6783 did not inhibit phosphorylation of these receptors even at 100 mM (Table 1). We also tested the effect of Ki6783 on autophosphorylation of stem cell factor (SCF) receptor (c-kit), which belongs to the PDGF receptor superfamily. Stable c-kit transformant K17 cells [29] were stimulated by recombinant murine SCF (100 nM) to obtain auto-
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TABLE 1 Selective Inhibition of Ki6783 on PDGF b-Receptor Tyrosine Kinase among Various Receptor Tyrosine Kinases and Protein Kinases in Intact Cells or in the Cell-Free Experiments Intact cells assay PDGF b-R
EGF-R bFGF-R Insulin b-R c-kit
Rat MC NIH3T3 hAOSMC A431 NIH3T3 HepG2 K17
IC50 (mM)
Cell-free assay
IC50 (mM)
0.13 0.07 0.15 ú100 ú100 ú100 õ1.0
PDGF b-R EGF-R PKA PKC MAPK MAPKK
0.025 ú10 ú10 ú10 ú10 ú10
Note. IC50 value of each assay is represented in the table. Experimental procedures and abbreviations are described under Materials and Methods.
phosphorylation of c-kit. Pretreatment of K17 with 1 mM Ki6783 decreased the amount of phosphorylation of c-kit to less than 50% of the control level (Table 1). Inhibition of protein kinases by Ki6783 under the cellfree conditions. In the next series of experiments, the effect of Ki6783 on several protein kinases was tested. Immunoprecipitates of PDGF b-receptor of NIH3T3 cell lysates were used for the assay of PDGF b-receptor tyrosine kinase. Ki6783 inhibited the tyrosine kinase activity in a dose-dependent manner (Fig. 3). The IC50 of the compound was 0.025 mM. In contrast, Ki6783 had no effect on the activities of EGF receptor tyrosine kinase, PKA, PKC, MAPK, or MAPKK even at 10 mM. The mode of autophosphorylation inhibition. The kinetics of the inhibitory action of Ki6783 on PDGF b-receptor tyrosine kinase was analyzed with various concentrations of Ki6783 and ATP. The Lineweaver–
FIG. 3. Effect of Ki6783 on tyrosine phosphorylation of immunoprecipitated PDGF b-receptor (PDGF b-R). Lysates of the quiescent NIH3T3 cells were immunoprecipitated with anti-PDGF b-receptor after treatment with PDGF-BB (50 ng/ml) for 30 min. Ki6783 was added to the immunoprecipitates for 10 min and the phosphorylation reaction was carried out for 10 min by the addition of ATP (20 mM). SDS–PAGE, Western blot analysis, and fluorography using antiphosphotyrosine (pY) antibodies and anti-PDGF b-R antibodies were carried out by the same method as that of the autophosphorylation assay using intact cells.
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FIG. 4. Lineweaver–Burk plot of the inhibition study of Ki6783. Phosphorylation reaction of PDGF b-receptor immunoprecipitates was performed in the presence of five concentrations of ATP and five concentrations of Ki6783. The vertical axis represents the inverse of the velocity of the enzymatic reaction and the horizontal axis represents the inverse of the concentration of added ATP.
Burk plot of the kinase assay is shown in Fig. 4. Regression lines at various concentrations of Ki6783 crossed at one point at an ATP concentration equal to 0 mM; the mode of inhibition of Ki6783 was estimated to be competitive action against ATP. The Km value of the assay and the Ki value of Ki6783 were calculated as 73.0 mM and 58.1 nM, respectively, from the plot. Inhibition of MAP kinase activation by Ki6783. To test whether Ki6783 actually blocks intracellular signal transduction next to PDGF receptor autophosphorylation in intact cells, the effect on MAP kinase activation was examined (Fig. 5). After incubation with PDGF-BB (100 ng/ml) for 5 min, MAP kinase activity in NIH3T3 cells was increased about fivefold more than
FIG. 5. Effect of Ki6783 on MAP kinase activation in NIH3T3 cells. Quiescent NIH3T3 cells were incubated with Ki6783 for 30 min and incubated in the presence of 100 ng/ml of PDGF-BB for 5 min. Cell lysates were prepared and a MAP kinase assay was carried out as described under Materials and Methods. The vertical axis represents the relative MAP kinase activity in which the mean value of the negative control (PDGF(0) and Ki6783(0)) is 1. Each value is expressed by the mean { SEM of three experiments.
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FIG. 6. Inhibitory effect of Ki6783 on DNA synthesis in cultured rat mesangial cells. Quiescent mesangial cells were treated with 50 ng/ml of PDGF-BB and various concentrations of Ki6783 and [3H]thymidine (155 kBq/ml), and incubated for 24 h. Radioactivity incorporated into the cell was determined as described under Materials and Methods. The vertical axis is relative DNA synthesis, in which the mean radioactivity in the absence of Ki6783 is 100%, was expressed. Values are expressed as the means { SEM of four experiments.
that of nontreated cells. Ki6783 (1 and 10 mM) decreased MAP kinase activation induced by PDGF. Effects of Ki6783 on DNA synthesis and proliferation of mesangial cells. In order to test biological activities of Ki6783 due to the inhibition of PDGF, the effect of the compound on cell growth was examined using cultured rat MC. In the study of DNA synthesis, [3H]thymidine incorporation of MC for 24 h was increased about 10-fold by the treatment of PDGF-BB (50 ng/ml). Ki6783 decreased the amount of incorporated [3H]thymidine in a dose-dependent manner as shown in Fig. 6. The IC50 was about 0.1 mM. The number of intact MC increased after the addition of PDGF-BB (30 ng/ml) and FBS (2%) to the culture medium for 48 and 72 h. Ki6783 inhibited the increase in cell number at both incubation times in a dose-dependent manner (Fig. 7). No cytotoxicity was observed 72 h after the addition of 100 mM Ki6783 to the culture medium containing PDGF and FBS. Normalization of the morphology of sis-transformed NIH3T3 cells by Ki6783. Sis codes DNA of PDGF-B chain, and v-sis is known as a variant of the sis gene from simian sarcoma virus [33]. v-sis-transfected NIH3T3 cells acquire the transformed phenotype characterized by the loss of density-dependent growth arrest and spindle-shaped morphology due to autocrine growth stimulation via the PDGF receptor. The effect of Ki6783 on the transformed phenotype was tested. When sis-transformed cells were treated with Ki6783 for 48 h, normalization of the morphology was observed, such as the change from a non-contact-inhibited phenotype to a flattened, contact-inhibited morphology similar to that of nontransformed NIH3T3 cells (Fig. 8).
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In the present study, we showed that our original quinoline derivative, Ki6783, strongly inhibits tyrosine phosphorylation of PDGF b-receptor in intact cells as well as in the cell-free experiment and that this activity is much more selective for this receptor than for other growth factor receptors. We previously reported that several 4-substituted quinoline derivatives possess this activity and that Ki6783 is the strongest compound among them [27]. This is the first report describing the characterization of the inhibitory action of a 4-substituted quinoline derivative, although other kinds of compounds have been reported as inhibitors of PDGF receptor autophosphorylation, such as staurosporine derivatives [23] and 3substituted quinoline derivatives [26, 34]. Ki6783 is thought to have one of the more potent activities among these agents. Inhibitory activity of PDGF b-receptor phosphorylation in intact cells was about five times weaker than that in the cell-free experiment based on IC50 values. This discrepancy might be explained by the slow uptake into the cells. Ki6783 also inhibited PDGF b-receptor autophosphorylation in several types of cultured cells, such as rat mesangial cells, mouse NIH3T3 cells, and human aortic smooth muscle cells, with almost the same strength. From these results, it is estimated that the association site of the receptor with the compound is highly homologous among different species. In this study, we did not examine the effect of the compound on the PDGF a-receptor because the amount of the receptor was too small to detect autophosphorylation compared to the b-type receptor (data not shown). Another interesting characteristic of this compound is its high selectivity for the PDGF receptor compared
FIG. 7. Inhibition of mesangial cell proliferation by Ki6783 on intact cell number of mesangial cells. Quiescent mesangial cells were treated with 30 ng/ml of PDGF-BB, FBS (2%), and various concentrations of Ki6783 and incubated for 48 and 72 h. The cells were harvested by trypsinization, and intact cell number was counted. Values are means { SEM of six to eight experiments in each group.
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FIG. 8. Effect of Ki6783 on the morphology of v-sis-transformed NIH3T3 cells. v-sis-transformed NIH3T3 cells were cultivated in the absence (A) or in the presence of Ki6783 (1 mM) (B) for 48 h. No transfected NIH3T3 cells were cultivated for the same period (C). Microphotographs were taken with a phase-contrast microscope (1200).
to other growth factor receptors. Ki6783 did not inhibit autophosphorylation of the EGF receptor, bFGF receptor, or insulin b-receptor even at 100 mM. These results indicate that the selectivity for the PDGF b-receptor is about 1000 times greater than that of the others. Such high selectivity was also demonstrated in the cell-free experiment, in which Ki6783 did not inhibit EGF receptor tyrosine kinase or other serine–threonine-type protein kinases such as PKA, PKC, MAPK, and MAPKK even at 10 mM. These results indicate that PDGF receptor has a characteristic binding site for Ki6783 which affects its tyrosine kinase activity. However, the kinetic analysis indicated its mode of action as a competitive inhibition to ATP. Since all protein kinases have an ATP binding site, the question arises why and how Ki6783 can recognize and inhibit PDGF receptor so selectively. As piceatannol, a protein kinase inhibitor, was reported to inhibit competitively the peptide substrate as well as ATP [35], it may be that Ki6783 also possesses such multiple modes of action. Otherwise, we estimate that Ki6783 may selectively decrease the
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catalytic efficiency of PDGF receptor tyrosine kinase by an unknown mechanism. A highly selective inhibitor of EGF receptor autophosphorylation, PD153035, was recently reported [36]. Interestingly, its chemical structure (4,6,7-trisubstituted quinazoline) resembles that of Ki6783. However, the reason each receptor can precisely distinguish these similar chemical structures is unknown. Recently, the catalytic specificity of various tyrosine kinases of growth factor receptors was examined [37, 38]. Although the actual catalytic site has not been determined completely, such a study will elucidate the mechanism of inhibition by these compounds. Ki6783 strongly inhibited autophosphorylation of the SCF receptor (c-kit). As this receptor is known to be closely related to the PDGF receptor with respect to their protein structures [11], this compound may be able to recognize it. Further study of the effect of the compound on the phosphorylation of other receptors in the PDGF receptor superfamily such as KDR [39], flt1 [40], and fms [41] remains to be done. After the ligand binding and autophosphorylation of
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PDGF receptor, the signal is transduced by the sequential pathway in the cytoplasm, and at last it reaches the nucleus to stimulate DNA synthesis or other cellular events [42, 43]. Activation of MAP kinase is well known to take place during this signal transduction [42, 43]. Ki6783 inhibited MAPK activation in NIH3T3 stimulated by PDGF-BB, although it did not inhibit MAPK activity under the cell-free condition. Thus, it is suggested that the compound can actually block the intracellular signal transduction after PDGF receptor autophosphorylation. About 40% of MAPK to the control was activated in the presence of 1 mM Ki6783, whereas complete inhibition of autophosphorylation of PDGF breceptor was observed at that dose. This may be due to a difference in concentration of PDGF-BB, incubation time, or cell type between these two experiments. Although NIH3T3 cells were used in this study because of convenience of cultivation, further experiments using mesangial cells are necessary. A second central finding of this study is that Ki6783 has inhibitory activity against growth and phenotypic change of cells bearing the PDGF receptor. PDGF is considered a ‘‘competence factor’’ for cell growth, which shifts the stage in the cell cycle from G1Q phase to G1 phase (44). In particular, PDGF-BB and -AB are known to have strong growth stimulating activity via the PDGF-b receptor in several types of cells (2). In this study, we also found that PDGF-BB and -AB stimulated DNA synthesis and proliferation in mesangial cells in contrast to no effect of PDGF-AA (data not shown). Ki6783 decreased PDGF-BB-stimulated thymidine incorporation into the cell, in which dose dependency was almost the same as that of the inhibition of receptor autophosphorylation. Furthermore, we tested the effect of the compound on cell proliferation. Ki6783 inhibited the increase in intact cell number after the incubation with PDGF-BB and 2% FBS as a growth progression factor. In this experiment, the compound showed weaker activity than that in the DNA synthesis study. This may be explained by contribution of other growth factors in FBS to cell growth. Interestingly, Ki6783 did not show any cytotoxicity even at 100 mM after 72 h incubation. This suggests that Ki6783 has a strong cytostatic but not cytotoxic effect on the growth of mesangial cells. Several studies have been reported using protein tyrosine kinase inhibitors, which supported or opposed the correlation between the activities of enzyme inhibition and growth inhibition [25, 45, 46]. Our study indicated that the inhibitory activity of Ki6783 on receptor autophosphorylation was well correlated with that on cell growth. Finally, Ki6783 was shown to clearly reverse the morphological change of v-sis-transformed NIH3T3 fibroblast cells from the characteristic spindle shape to the normal flattened shape usually observed in non-
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transfected NIH3T3 cells. Some tyrosine kinase inhibitor was also reported to have the same activity at 50 mM (25). We could observe such an effect by the addition of only 1 mM Ki6783. These results suggest that Ki6783 has the potent activity to block the phenotypic change triggered by overexpression of PDGF. PDGF and its receptor system are known to be involved in various pathophysiological processes, including several forms of neoplasia, atherosclerosis, glomerulonephritis, and tissue fibrosis [13]. In particular, participation of PDGF in excessive proliferation of glomerular mesangial cells has been well recognized in the progression of chronic glomerular diseases [14–21, 47]. The therapeutic importance of blocking PDGF action in these diseases has also been discussed [16]. From these points of view, Ki6783 may have a possible use in inhibiting disease progression as a therapeutic agent. Furthermore, highly selective inhibition of PDGF action may be beneficial with respect to fewer adverse effects. Our present study is the first attempt to examine the effect of a selective PDGF receptor autophosphorylation inhibitor on mesangial cell growth. In vivo study is now under way. We are grateful to Dr. Tadashi Yamamoto (Niigata University) for helpful suggestions regarding the culture of mesangial cells, and to Dr. Eisuke Nishida (Kyoto University) for providing us with recombinant MAPK and MAPKK.
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Received January 31, 1997 Revised version received April 1, 1997
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