MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells

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1 MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells ROBERT H. WEISS, 1,2,3 ELIZABETH A. MAGA, 1 AND AL RAMIREZ 1 1 Division of Nephrology, Department of Internal Medicine, and 2 Cell and Developmental Biology Graduate Group, University of California, Davis 95616; and 3 Department of Veterans Affairs Northern California Health Care System, Pleasant Hill, California Weiss, Robert H., Elizabeth A. Maga, and Al Ramirez. MEK inhibition augments Raf activity, but has variable effects on mitogenesis, in vascular smooth muscle cells. Am. J. Physiol. 274 (Cell Physiol. 43): C1521 C1529, Proteins comprising the mitogen-activated protein (MAP) kinase signaling cascade are activated by a variety of growth factors, but the precise role of this series of kinase reactions, especially Raf kinase and MAP kinase kinase (MEK), in vascular smooth muscle (VSM) cell mitogenesis is not known. In this study, a specific and selective inhibitor of MEK, PD-98059, was used to examine the role of MEK in DNA synthesis and Raf-1 activity in VSM cells stimulated with serum as well as with growth factors encompassing both tyrosine kinase and G protein-coupled receptor classes. Although significant increases in DNA synthesis are seen after stimulation of VSM cells with either 10% serum, plateletderived growth factor (PDGF)-BB, or -thrombin, preincubation of the cells with 50 µm PD for 1 h inhibits stimulation by PDGF and thrombin, but not by serum. There is a dose-dependent inhibition of the mitogenic effect by PD in all cases; these results are not affected when PD is added at times ranging from 4 h before to 2 h after growth factor addition (times at which PD exerts its inhibitory effect). In the presence of PD-98059, stimulated MAP kinase activity is attenuated when growth factors are added between 10 min and 4 h, times which correspond to both early and sustained phases of MAP kinase activity. In addition, Raf-1 activity is markedly increased by incubation of the cells with PD-98059, despite attenuation of hyperphosphorylation of this kinase. Thus growth factors coupled to both tyrosine kinase and G protein receptors require components of the MAP kinase cascade (MEK and/or MAP kinase) for VSM cell mitogenesis, whereas serum is capable of stimulatory effects in the absence of active MEK and MAP kinase. Furthermore, there exists a functional feedback stimulatory effect of inhibited MEK on its upstream activator Raf-1 in the case of serum as well as growth factors coupled to tyrosine kinase and G protein receptors. mitogen-activated protein kinase; -thrombin; plateletderived growth factor-bb; PD GROWTH FACTORS TRANSMIT their signals from the cell surface to the nucleus by utilizing a series of sequentially activated kinase cascades, which are integrated into a complex network known as the mitogen-activated protein (MAP)/extracellular signal-regulated kinase pathways (reviewed in Ref. 16). Although elements of this cascade have been shown to be involved in the regulation of the proliferative response of cells to both tyrosine kinase and G protein-coupled receptor growth factors, the actual sequence of molecular events that occurs to transmit the mitogenic response from the cell surface receptor to the nucleus is not completely understood. Upon activation of many different cell types by a variety of growth factor receptors, the MAP kinase pathway becomes activated initially by the formation of Ras-GTP. The ensuing recruitment of Raf to the plasma membrane by Ras causes its activation, by unknown factors, and transmission of the signal through the cascade reactions then occurs (11, 23). Raf-1 phosphorylates and activates MAP kinase kinase (MEK), which in turn phosphorylates and activates MAP kinase in a biphasic fashion (17, 18). Subsequently, active MAP kinase is translocated to the nucleus where it phosphorylates transcription factors, such as Elk-1, which leads to transcription of c-fos (9, 25). Although the elements of the MAP kinase pathways function to propagate their signals from the cell membrane to the nucleus, there is evidence that feedback loops also occur (22, 31). Although it had been inferred that hyperphosphorylation of Raf was associated with its activation, we (2) and others (8, 24) recently showed that hyperphosphorylation of Raf is not required for its catalytic activity. Other investigators have demonstrated that hyperphosphorylation of Raf-1 is associated with a decrease in its affinity with the cell membrane and inactivation of this kinase, at least in cells stimulated with serum (28), whereas still others have shown that Raf hyperphosphorylation is the consequence of events occurring downstream of MAP kinase (22). However, although the proliferative response of the receptor tyrosine kinases has been thought to be conferred by the Ras/Raf/ MAP kinase pathway, there is also evidence of Rasindependent signaling by the platelet-derived growth factor (PDGF) receptor (4). The recent discovery of PD has allowed detailed investigation of the means by which the mitogenic signal is transmitted through Raf to its downstream effectors. PD has been shown to be a specific inhibitor of MEK, acting by preventing phosphorylation and thus activation of both MEK-1 and MEK-2 (1, 7, 15, 20). Because activation of the MAP kinase pathway is associated with many mitogenic growth factors, we asked what effect MEK inhibition has on DNA synthesis in vascular smooth muscle (VSM) cells stimulated by serum as well as by growth factors coupled to tyrosine kinase or G protein-coupled receptors. We now show that, although PDGF-BB and -thrombin-mediated mitogenesis is significantly inhibited by PD-98059, this inhibitor allowed 10% serum to remain stimulatory. Furthermore, in the case of cells stimulated by PDGF-BB, -thrombin, or 10% serum, /98 $5.00 Copyright 1998 the American Physiological Society C1521

2 C1522 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS PD caused inhibition of MAP kinase but stimulation of Raf-1 catalytic activity, despite loss of Raf-1 hyperphosphorylation. MATERIALS AND METHODS Materials. Cultures of A10 embryonic thoracic aorta, smooth muscle, DB1X rat cells were obtained from the American Type Culture Collection. -Thrombin was kindly supplied to us by Dr. J. Fenton (Albany, NY). PD-98059, Elk-1 fusion protein, LumiGLO (Phototope-HRP Western Detection System), phospho-map kinase, and phospo-elk-1 antibodies were obtained from New England Biolabs (Beverly, MA). Human recombinant PDGF-BB, sheep polyclonal antihuman c-raf kinase COOH terminal, inactive glutathione S-transferase (GST)-MAP kinase kinase 1 (MEK-1), and inactive GST-p42 MAP kinase (MAPK) were obtained from UBI (Lake Placid, NY). Sheep polyclonal anti-human c-raf kinase COOH terminal was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit horseradish peroxidase (HRP)-linked IgG was obtained from Bio-Rad (Richmond, CA). Components for the enhanced chemiluminescence (ECL) system and [ 3 H]thymidine were obtained from Amersham (Arlington Heights, IL). All other reagents were from Sigma (St. Louis, MO). Cell culture. A10 cells were maintained in DMEM with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 U/ml) in a humidified atmosphere of 5% CO 2-95% air at 37 C. Culture medium was changed every other day until the cells were confluent. The cells were growth-arrested by placing them in quiescence medium containing DMEM, 20 mm HEPES (ph 7.4), 5 mg/ml transferrin, 0.5 mg/ml BSA, 50 U/ml penicillin, and 50 U/ml streptomycin. Quiescence medium was changed daily for 1 2 days before each experiment. Cells were harvested with 5 ml trypsin and subcultured at a 1:10 dilution weekly. DNA synthesis. Cultured A10 cells were used at passages Before each experiment, the cells were harvested, plated onto 24-well plates, grown to confluence, and then serum starved for 24 h. For dose-response studies, 10, 20, 30, 40, 50, or 60 µm of the MEK-1/2 inhibitor PD was added 1 h before growth factor addition. For time course studies, 50 µm PD was added 4, 2, and 1 h before, at the same time as, and 1 and 2 h after growth factor addition. [ 3 H]thymidine incorporation was assessed as previously described (30). MAP kinase activity assay. A10 cells were grown to confluence in 6-cm dishes and serum starved for 48 h. Cells were treated with 50 µm PD for 1 h. Control cells were treated with the same quantity of DMSO. The cells were stimulated for 10 min with either 10% serum, PDGF-BB (30 ng/ml), or -thrombin (1 U/ml). The plates were washed one time with ice-cold PBS, and 0.5 ml lysis buffer [20 mm Tris, ph 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm -glycerolphosphate, 1 mm Na 3 VO 4, 1 µg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride (PMSF)] was added to each plate. The plates were incubated on ice for 5 min, and then the cells were scraped off the plate and transferred to an Eppendorf tube. The lysate was passed through a 25-gauge needle four times and then centrifuged at 14,000 rpm for 10 min at 4 C. Active MAP kinase was immunoprecipitated from each lysate by placing normalized amounts of lysate (as determined spectrophotometrically) in a tube with phospho-map kinase antibody in a 1:50 dilution lysate-antibody and incubated overnight at 4 C with gentle shaking. Protein A-Sepharose beads (50 µl) were added to each tube and were placed for1hat4 C with shaking. Samples were centrifuged at 14,000 rpm for 30 s, and the pellet was washed twice with 500 µl lysis buffer and twice with 500 µl kinase buffer [25 mm Tris, ph 7.5, 5 mm -glycerolphosphate, 2 mm dithiothreitol (DTT), 0.1 mm Na 3 VO 4, and 10 mm MgCl 2 ]. The immunoprecipitate was resuspended in 50 µl kinase buffer containing 100 µm ATP and 1 µg Elk-1 fusion protein, a MAP kinase substrate. Samples were incubated at 30 C for 30 min, and the reaction was terminated by adding 25 µl 3 SDS sample buffer. Samples were boiled for 5 min, vortexed lightly, and centrifuged at 14,000 rpm for 2 min. Thirty microliters of each sample were electrophoresed on a 12.5% SDS-PAGE gel, and the proteins were electrophoretically transferred to a nitrocellulose membrane. MAP kinase activity was determined by Western blotting with an antibody specific for phosphorylated Elk-1. Membranes were blocked for 1 h with 5% nonfat dry milk-tris-buffered saline (TBS)-0.1% Tween at room temperature. Membranes were incubated with a 1:1,000 dilution of phospho-elk-1 antibody in 5% BSA-TBS-0.05% Tween overnight at 4 C with gentle shaking. The membranes were washed three times for 5 min with TBS-0.1% Tween and incubated for 1 h atroom temperature with a 1:2,000 dilution of goat-anti rabbit HRP in 5% nonfat dry milk-tbs-0.1% Tween. After three washes of 5 min with TBS-0.1% Tween, the membranes were placed in 10 ml of 1 LumiGLO for 1 min, exposed to film for various times, and developed. Western blotting. A10 cells were grown to confluence on 6-cm culture dishes and serum deprived for 48 h. Cells were treated with 50 µm PD or DMSO for 1 h. After stimulation for 10 min with either 10% serum, PDGF-BB (30 ng/ml), or -thrombin (1 U/ml), the cells were washed three times with ice-cold PBS and lysed in place by the addition of ice-cold RIPA-A buffer [50 mm Tris-Cl, ph 7.4, 150 mm NaCl, 1 mm EGTA, 1 mm sodium orthovanadate, 1 mm sodium fluoride, 1 mm PMSF (freshly prepared), 1% Nonidet P-40, and 1 µg/ml aprotinin]. After 10-min incubation at 4 C, the lysate was scraped off with a rubber spatula and centrifuged at 14,000 rpm at 4 C for 10 min. The supernatant was saved, and the protein concentration was determined spectrophotometrically at 595-nm absorbance with the Bio-Rad protein concentration reagent. Aliquots containing identical amounts of protein for each lysate were heated with SDS-PAGE gel loading buffer in a boiling water bath for 5 min, loaded onto a 7.5% polyacrylamide gel with 5% stacking gel, and electrophoresed at 100 V for 2 h. Molecular weight standards were electrophoresed on the same gel. Subsequently, the gel was electrophoretically transferred onto a nitrocellulose filter in transfer buffer (193 mm glycine, 25 mm Tris, and 20% methanol, ph 8.3). The nitrocellulose filters were blocked for 1 h with 5% nonfat dry milk in TBS-T (20 mm Tris, 137 mm NaCl, ph 7.5, 0.1% Tween 20), washed in TBS-T, and incubated at room temperature for1hina1:1,000 dilution of Raf-1 antibody in TBS-T. The filter was washed again in TBS-T and incubated for 1 h with a 1:15,000 dilution of goat anti-rabbit HRP-coupled IgG in TBS-T. The final washes were with TBS-T for 15 min and 4 5 min. The ECL system was used for detection. Raf activity assay. Raf activity was determined by an in vitro kinase cascade reaction. Briefly, immunoprecipitated active Raf will activate an inactive MEK, which can then activate an inactive MAP kinase. Any Raf-activated MAP kinase then phosphorylates the MAP kinase substrate Elk-1. The amount of Raf activity is related to the amount of phosphorylated Elk-1 by a phospho-specific Elk-1 antibody. A10 cells were grown to confluence in 6-cm dishes and serum starved for 48 h. Cells were treated with 50 µm PD for 1 h. Control cells were treated with DMSO. The

3 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS C1523 cells were then stimulated for 10 min with either serum, PDGF-BB (30 ng/ml), or -thrombin (1 U/ml). All dishes were washed three times with ice-cold PBS and placed in 500 µl buffer A (50 mm Tris, ph 7.5, 1 mm EDTA, 1 mm EGTA, 0.1% -mercaptoethanol, 1% Triton X-100, 50 mm sodium fluoride, 5 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 0.5 mm Na 3 VO 4, 0.1 mm PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) on ice for 10 min. Cells were scraped into an Eppendorf tube, passed through a 25-gauge needle four times, and centrifuged at 14,000 rpm for 10 min at 4 C. Lysates were normalized for protein amount (as determined spectrophotometrically) and immunoprecipitated overnight with 2 µg sheep polyclonal anti-human c-raf kinase COOH terminal at 4 C with shaking. Protein G agarose beads (100 µl) that had been washed with PBS were added to each tube and incubated for 1 h at 4 C. The immunoprecipitates were washed one time with buffer A plus 0.5 M NaCl followed by one wash with buffer A. Samples were resuspended in 70 µl assay dilution buffer (20 mm MOPS, ph 7.2, 25 mm glycerolphosphate, 5 mm EGTA, 1 mm Na 3 VO 4,and1mM DTT), and 10 µl of a 75 mm MgCl 2 /500 µm ATP mix were added to each tube. Components of the c-raf kinase cascade were added as follows: 0.4 µg of inactive GST-MAP kinase kinase 1 (MEK-1) and 1.0 µg of inactive GST-p42 MAP kinase. Samples were incubated at 30 C for 30 min. Four microliters of this mixture were then added to a new tube containing 46 µl kinase buffer plus 100 µm ATP and 1 µg Elk-1 fusion protein. Samples were incubated for 30 min at 30 C. Detection of Elk-1 phosphorylation was carried out as described above for the MAP kinase assay. RESULTS PD inhibits PDGF-BB- and -thrombin-, but not serum-induced, mitogenesis. To determine the effect of PD on mitogenesis induced by both tyrosine kinase and G protein-coupled receptors in VSM cells, we first examined the effect of these growth factors, as well as serum, on VSM cell mitogenesis. In the absence of PD-98059, stimulation of the cells by serum, PDGF- BB, and -thrombin results in a significant increase (6.75-, 6.08-, and 2.01-fold control, respectively) in Fig. 1. Vascular smooth muscle (VSM) cell mitogenesis induced by serum and growth factors. Confluent, serum-starved VSM cells were stimulated with 10% serum, -thrombin (1 U/ml), or platelet-derived growth factor (PDGF)-BB (30 ng/ml) for 24 h. [ 3 H]thymidine (1 µci/well) was added for the last 6 h of incubation, and TCAprecipitable radioactivity was assessed by liquid scintillation. Data are expressed as means SE of 8 14 wells per data point. *P 0.05 compared with control. Fig. 2. Effect of PD on VSM cell mitogenesis. Confluent, serum-starved VSM cells were incubated with PD (50 µm; hatched bars) for 1 h before addition of 10% serum, -thrombin (1 U/ml), or PDGF-BB (30 ng/ml) for an additional 24 h. Control cells (solid bars) were incubated with same volume of DMSO solvent. [ 3 H]thymidine (1 µci/well) was added for last 6hofincubation, and TCA-precipitable radioactivity was assessed by liquid scintillation. Data are expressed as means SE of 3 wells per data point and are representative of 3 separate experiments. *P 0.05 ( )PD compared with ( )PD [ 3 H]thymidine incorporation (Fig. 1). The relatively small increase in [ 3 H]thymidine incorporation after -thrombin stimulation is consistent with previous reports in these cells (19). In subsequent experiments, cells were preincubated with 50 µm of the MEK inhibitor PD for 1 h before stimulation with growth factor or 10% serum; preincubation with PD under these conditions causes significant inhibition of MAP kinase activity (see below). After 18 h of growth factor incubation, [ 3 H]thymidine was added for 6 h before TCA precipitation. In all cells except those treated with serum, DNA synthesis was significantly decreased by preincubation with PD (Fig. 2), suggesting that serum, but not PDGF-BB or thrombin, utilizes a pathway for mitogenesis that is independent of MEK. To assess the growth response of VSM cells to varying concentrations of PD-98059, we next performed doseresponse experiments using these growth factors with the MEK inhibitor. For clarity of comparison among the different experiments, [ 3 H]thymidine incorporation is graphed as percent change relative to cells that were appropriately stimulated but were not preincubated with PD The effect of incubation of the cells with 10% serum (squares in Fig. 3A) is consistently stimulatory when compared with control cells that were not stimulated (triangles in Fig. 3A) at concentrations from 10 to 50 µm PD However, incubation of these cells with PD abolished the stimulatory effect of both PDGF-BB (circles in Fig. 3B) and -thrombin (diamonds in Fig. 3B) at these same concentrations. This is consistent with the data above (Fig. 2) showing inhibition of growth by PDGF-BB or -thrombin, but not by 10% serum, in cells incubated with PD

4 C1524 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS addition, although this stimulation only reached statistical significance when PD was added before serum. Mitogenesis induced by both PDGF-BB (circles in Fig. 4B) and -thrombin (diamonds in Fig. 4B) is completely inhibited by PD at these same times. PD decreases MAP kinase activity. When activated by MEK, MAP kinase translocates to the nucleus to stimulate such transcriptional activators as Elk-1 (9). Because MEK is positioned in the phosphorylation Fig. 3. Dose-dependent inhibition of VSM cell mitogenesis with PD Confluent, serum-starved VSM cells were incubated with PD at concentrations indicated for 1 h before addition of 10% serum (A) or -thrombin (1 U/ml) or PDGF-BB (30 ng/ml) (B) for an additional 24 h. [ 3 H]thymidine (1 µci/well) was added for last 6hof incubation, and TCA-precipitable radioactivity was assessed by liquid scintillation. Control cells were incubated with PD at concentrations indicated but were not stimulated. Data are expressed as percent change compared with cells that did not receive PD and are means SE of 3 wells per data point. *P 0.05 compared with control cells that were incubated with same concentrations of PD but not stimulated. Experiment shown is pooled from 3 experiments. To explore the dependence of activation of MEK on phase of the cell cycle at which it is inhibited, we next examined the effect of time of addition of the inhibitor relative to growth factor on synchronized, serumstarved cells. As above, [ 3 H]thymidine is graphed as percent change relative to cells that were appropriately stimulated but were not preincubated with PD Serum is stimulatory in VSM cells exposed to 50 µm PD (squares in Fig. 4A) as compared with control cells that were not stimulated (triangles in Fig. 4A), regardless of whether the MEK inhibitor is added at times ranging from 4 h before to 2 h after serum Fig. 4. Time of PD addition does not affect growth trend. Confluent, serum-starved VSM cells were incubated with PD (50 µm) at indicated time relative to 10% serum (A) or -thrombin (1 U/ml) or PDGF-BB (30 ng/ml) (B) addition. For example, 2 represents an experiment in which PD was added 2 h before growth factor addition. Cells were incubated for 24 h after growth factor addition. [ 3 H]thymidine (1 µci/well) was added for last 6 h of incubation, and TCA-precipitable radioactivity was assessed by liquid scintillation. Control cells were incubated with PD at concentrations indicated but were not stimulated. Data are expressed as percent change compared with control cells that did not receive PD and are means SE of 3 wells per data point. *P 0.05 compared with control cells that were incubated with same concentrations of PD but not stimulated. Experiment shown is pooled from 3 experiments.

5 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS C1525 Fig. 5. Mitogen-activated protein (MAP) kinase activity is decreased by PD Confluent, serum-starved VSM cells were incubated, where indicated with an asterisk, with PD (50 µm) for 1 h. Subsequently, cells were either unstimulated (C) or stimulated with 10% serum (S), -thrombin (T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for 10 min and lysed. Lysate was immunoprecipitated with MAP kinase antibody, and assessment of phosphorylation of Elk-1 by MAP kinase was determined as described in MATERIALS AND METHODS. Experiment shown is representative of 3 experiments. cascade such that it activates MAP kinase, we next examined several time courses of catalytic activity of this downstream molecule as measured by phosphorylation of Elk-1 by immunoprecipitated MAP kinase. In initial experiments, VSM cells were treated with 50 µm PD for 1 h and then stimulated with either 10% serum, PDGF-BB, or -thrombin. In all cases, the presence of the MEK inhibitor attenuates MAP kinase activity as seen by decreased phosphorylation of the MAP kinase-specific Elk-1 substrate (Fig. 5). To determine whether PD is inhibitory toward MEK (as measured by MAP kinase activity) at times that would be required for early events in the cell cycle to take place, we examined MAP kinase activity in cells that had been incubated with PD for various times before stimulation with PDGF-BB and serum. MAP kinase activity was inhibited by PD when the cells were incubated with PD for from 1 to 2 h before serum (Fig. 6A) or PDGF (Fig. 6B) stimulation, but by 4 h, the inhibition was much less pronounced or absent. It is significant that the maximal inhibition of MAP kinase occurred when PD was added 1 h before serum stimulation (Fig. 6A); under these conditions, serum was maximally stimulatory ( 1 in Fig. 4A), further supporting the independence of serumstimulated growth from MAP kinase activation. The activation of MAP by a variety of growth factors has been shown to be biphasic in nature, with a rapid phase ending by 30 min and a sustained phase beginning at 30 min and lasting up to 4 h (5, 10, 17). We next asked whether PD is inhibitory during both phases of activation. Serum-deprived VSM cells were incubated with PDGF-BB concomitantly with PD for from 10 min to 24 h before lysis and determination of MAP kinase activity. Although significant inhibition of MAP kinase activity was seen with 10 min and1hofpdgf and PD incubation, times of 4 h and later did not show consistent inhibition by PD (Fig. 7). These data demonstrate that PD inhibits both the early phase (10 min after stimulation) and at least part of the sustained phase (1 h after stimulation) of MAP kinase activation and are consistent with the data in Fig. 6 showing that there is minimal effect of PD (at least toward MAP kinase inhibition) by 4 h of incubation in the media. PD inhibits hyperphosphorylation but increases catalytic activity of Raf-1. Although Raf lies upstream of MEK in the MAP kinase signaling cascade, there are reports that the activity of this kinase is affected by more distal signaling molecules such as MAP kinase (27, 28, 31) or molecules downstream of MAP kinase (22). Raf activity had traditionally been assessed either by examination of its phosphorylation state, based on its migration on an SDS gel, or by determination of its catalytic activity, but recent data from several laboratories, including our own, have indicated that these two assays are not equivalent (1, 2, 8). Hyperphosphorylation is indicated by the shift of the band to a higher molecular weight as opposed to nonstimulated cells; a slower migrating band indicates phosphorylation of Raf-1. The state of phosphorylation of Raf-1 was examined by Western blotting of growth factor-exposed cell lysates that had been pretreated with 50 µm PD In the absence of PD-98059, the characteristic hyperphosphorylation of Raf was seen when cells were treated with serum, PDGF-BB, or -thrombin (Fig. 8). In the presence of PD-98059, the mobility of Raf-1 is faster (i.e., the band migrates further down the gel at a lower molecular weight) than without PD in the case of serum, -thrombin, Fig. 6. MAP kinase activity is inhibited by PD for up to 4 h. Confluent, serum-starved VSM cells were incubated, where indicated with an asterisk, with PD (50 µm) for 1, 2, or 4 h before addition of 10% serum (A) or PDGF-BB (30 ng/ml) (B) for 10 min. Cells not exposed to inhibitor were incubated in the same volume of DMSO. Control cells (C) were not stimulated and were lysed at 1 h. Lysates were immunoprecipitated with MAP kinase antibody, and assessment of phosphorylation of Elk-1 by MAP kinase was determined as described in MATERIALS AND METHODS.

6 C1526 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS Fig. 7. MAP kinase is inhibited by PD when added with growth factor at times 4 h. Confluent, serum-starved VSM cells were incubated concurrently with PDGF-BB (30 ng/ml) and, where indicated with an asterisk, PD (50 µm). Cells not exposed to inhibitor were incubated with the same volume of DMSO. After 10 min or 1, 4, 8, or 24 h, cells were lysed. Control cells (C) were not stimulated and were lysed at 1 h. Lysates were immunoprecipitated with MAP kinase antibody, and assessment of phosphorylation of Elk-1 by MAP kinase was determined as described in MATERIALS AND METHODS. and PDGF, indicating that the hyperphosphorylation of Raf-1 in all three cases is reduced by the MEK inhibitor. We next examined the catalytic activity of Raf-1 in VSM cells treated with the MEK inhibitor. Raf-1 was immunoprecipitated from stimulated cells and subjected to a cascade reaction ultimately leading to phosphorylation of Elk-1 (see MATERIALS AND METHODS). Elk-1 phosphorylation was assessed by Western blotting against a phospho-elk-1 antibody. In the absence of PD-98059, Raf activity of cells stimulated with 10% serum, -thrombin, or PDGF-BB was unchanged from that of control cells after 10 min of growth factor stimulation (Fig. 9A). In the presence of PD-98059, Raf-1 activity was greatly increased over that of control cells for 10% serum, -thrombin, or PDGF-BB, indicating that, despite abolition of hyperphosphorylation by PD-98059, the catalytic activity of Raf-1 was increased by the MEK inhibitor. To control for quantity of immunoprecipitated Raf-1 in the Raf activity assay, we electrophoresed an aliquot of the immunoprecipitated lysate used in the assay and performed Western blotting with Raf-1 antibody. All of the lanes show an equivalent quantity of Raf-1 (Fig. 9B), despite significant changes in catalytic activity (Fig. 9A). DISCUSSION The role of the MAP kinase cascade in mitogenic signaling is an area of intense interest, since growth factors are involved in a variety of disease processes ranging from atherosclerosis to cancer to kidney disease. Although mitogenic signals from the tyrosine kinase-coupled growth factors have been clearly linked, through Grb2, Sos, and Ras, to the MAP kinase cascade, the mechanism by which the G protein-coupled receptors activate Ras and the distal MAP kinase cascade effectors is not known. There is, however, clear evidence that this cascade is activated by G proteincoupled receptors such as -thrombin (2) and angiotensin II (2, 3). The MEK inhibitor PD is a highly specific inhibitor of activation of MEK-1 and MEK-2 and thus of activation of MAP kinase (1, 7, 15, 20). PD binds to inactive forms of MEK and prevents its activation by upstream components such as c-raf (1). Inhibition of MEK by PD is not due to competition for ATP or substrate (MAP kinase) binding (7, 15) and is most likely due to an allosteric effect (7). Because activation of components of the MAP kinase cascade results from stimulation of cells by a variety of growth factors, it has been assumed that transmission of the signal along this pathway is essential for the induction of mitogenesis by these growth factors. In support of this assumption, inhibition of MEK with PD has been associated with a decrease not only in PDGF-stimulated [ 3 H]thymidine incorporation in 3T3 cells (7), but also with a block in nerve growth factor (NGF)-induced differentiation in PC-12 cells (20). We now show for the first time that attenuation of MEK activity has a dose-dependent inhibitory effect on DNA synthesis in VSM cells stimulated by both tyrosine kinase and G protein-coupled receptors as well as by 10% serum. However, although serum retains a stimulatory effect at all concentrations of PD Fig. 8. Hyperphosphorylation of Raf is inhibited by PD Confluent, serum-starved VSM cells were incubated, where indicated with an asterisk, with PD (50 µm) for 1 h. Subsequently, cells were either unstimulated (C) or stimulated with 10% serum (S), -thrombin (T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for 10 min, lysed, and Western blotted with Raf-1 antibody. Arrowheads indicate control phosphorylation state of Raf-1. Multiple control lysates were run on same gel as indicators of mobility of basal unstimulated Raf-1. Experiment shown is representative of 3 experiments. Fig. 9. Raf catalytic activity is increased by PD Confluent, serum-starved VSM cells were incubated, where indicated with an asterisk, with PD (50 µm) for 1 h. Subsequently, cells were either unstimulated (C) or stimulated with 10% serum (S), thrombin (T; 1 U/ml), or PDGF-BB (P; 30 ng/ml) for 10 min and lysed. A: lysate was immunoprecipitated with Raf-1 antibody, and assessment of phosphorylation of Elk-1 in an in vitro cascade reaction was determined as described in MATERIALS AND METHODS. B: above immunoprecipitated lysate was Western blotted with Raf-1 antibody. Experiments shown are representative of 3 separate experiments.

7 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS C1527 tested, stimulation of these cells by PDGF-BB and -thrombin is significantly inhibited at these same concentrations. PD inhibits MEK for up to 4 h; the time at which PD is added relative to growth factor has no effect on the overall trend of growth, consistent with data in Swiss 3T3 cells (1) and showing that it is not significant whether MEK is inhibited early or late in the cell cycle (13, 29) for it to demonstrate the effects observed. Control cells exhibited an attenuated growth response to PD-98059, as has been previously reported (21). We have shown that serum does not require MEK or MAP kinase for its mitogenic effects. That there exist pathways alternative to the Ras/Raf/MEK/MAPK cascade leading from receptor tyrosine kinases to the proliferative machinery has been suggested by other investigators. Using dominant inhibitory constructs in NIH/3T3 cells, Barone and Courtneidge (4) showed the existence of a signaling pathway from the PDGF receptor to the early oncogenes, which is dependent on Src but which bypasses Ras. Others have shown that dominant negative Ras does not abolish MAP kinase activation due to NGF (32). These authors further showed that tyrosine phosphorylation of phospholipase C- is not affected by the inhibitory Ras construct, an experiment which suggests another alternative pathway of receptor tyrosine kinase activation of mitogenic machinery. Although cellular differentiation is often associated with cessation of growth, it can also be induced by prolonged activation of the MAP kinase pathway in neuronal cells by NGF (6). However, in this mode of MAP kinase activation, there is evidence that Raf may utilize signaling pathways independent of MEK and MAP kinase (14), consistent with our finding that Raf activity is increased (by PD-98059) despite inhibition of MAP kinase activity. There is also evidence of MEK-dependent, but MAP kinase-independent, activity in response to some growth factors. The finding that insulin stimulates SOS phosphorylation through MEK (leading to dissociation of the Grb2-SOS complex), despite complete inhibition of MAP kinase (12), suggests that there also exist alternative signaling pathways between MEK and MAP kinase. Conversely, there exist MEK-independent pathways for MAP kinase activation. One such pathway, mediating the prolonged phase of MAP kinase activation, has been demonstrated in Swiss 3T3 cells in response to PDGF and was thought to be mediated through phosphatidylinositol 3-kinase or protein kinase C (10). Our finding, that PD inhibits both the early and at least part of the sustained phase (5, 10, 17) of MAP kinase activation, is consistent with a previously published report that sustained MAP kinase activation is linked to the mitogenic potential of MAP kinase when activated by serum growth factors (18). Whether MAP kinase recovers from inhibition by 4 h or PD is degraded in the media is not known; the latter explanation is unlikely given that others have shown activity of PD in culture media for up to 16 h (1). The differences observed among the growth stimulators tested is likely due to the fact that serum contains other cytokines and growth factors that act on DNA synthesis through pathways independent of the MAP kinase cascade. The precise pathway by which serum stimulates growth in these studies, independent of MEK and MAP kinase, was not determined. On the other hand, it has been shown that strong activators of growth, such as epidermal growth factor in 3T3 cells, may still retain enough residual MEK activity so as to be able to activate the MAP kinase cascade despite MEK inhibition by PD (1). Our finding that MEK is not inhibited after prolonged times (after 4 h) of PD incubation may be significant, since it is possible that serum could utilize this pathway whereas the specific growth factors examined may require earlier MEK activation (18). The likely existence of feedback loops in the MAP kinase cascade has already been suggested. This possibility has been strengthened recently by the findings of Wartmann and Davis (27), who showed that Raf hyperphosphorylation occurred after serum stimulation despite a reduction of Raf catalytic activity. These authors later showed that inhibition of MEK prevented Raf hyperphosphorylation while restoring levels of membrane-bound Raf (28). Data from another laboratory were consistent with the possibility that MAP kinase feeds back to phosphorylate the T-669 site of the epidermal growth factor receptor (31). In light of these findings, we examined the effect of MEK inhibition on activation of MAP kinase cascade component activation in VSM cells activated by a variety of growth factors. That PD decreased MAP kinase activity was expected since this molecule is the substrate for activated MEK, but our finding that Raf activity was increased in the case of all three growth-stimulatory conditions despite loss of Raf hyperphosphorylation is consistent with previous work in other cells (1, 27, 28) and extends those results to several classes of growth factor receptor. Whether this increase in Raf activity with PD is due to a feedback stimulatory effect of poorly activated MAP kinase on Raf, or whether it is due to an accumulation of activated Raf which cannot be phosphatased before activation of its downstream effector, remains to be determined. Indeed, while the growth of serum-stimulated cells is not greatly inhibited by PD-98059, the MAP kinase activity is reduced by 62% (by laser densitometry measurement of the protein band on the gel), demonstrating that MEK is being strongly inhibited by PD and, furthermore, that the growth seen with serum is due to activation of other than the MAP kinase pathway. We now show that the presence of PD alters the state of PDGF-induced Raf-1 hyperphosphorylation in VSM cells, consistent with data from another laboratory using D-609 to stimulate Chinese hamster ovary cells overexpressing Raf-1 (28). In the absence of PD-98059, Raf is hyperphosphorylated after serum, PDGF-BB, and -thrombin stimulation, whereas the magnitude of Raf-1 hyperphosphorylation is diminished after incubating the cells with PD Active

8 C1528 MEK INHIBITION IN VASCULAR SMOOTH MUSCLE CELLS MEK has been shown to interact with Raf-1 in NIH/3T3 cells and to cause the mobility shift (hyperphosphorylation) of a COOH-terminal fragment of Raf-1 (8). In that study, cells possessing active MAP kinase had hyperphosphorylated Raf-1, whereas those with the MAP kinase activity inactivated by PD had Raf-1 that was not hyperphosphorylated. The data in this study are also consistent with our previous report that angiotensin II causes catalytic activation of Raf-1 despite the fact that hyperphosphorylation was not observed (2). It is possible that hyperphosphorylation occurs before activation (28) and that at the point when our cells were lysed in that study (10 min after stimulation), angiotensin II-induced hyperphosphorylation had already occurred. However, because we did observe concurrent hyperphosphorylation and catalytic activation when the cells were stimulated with thrombin and 10% serum (2), it is further possible that different growth stimulants display different kinetics toward these discrete events. Employing an in vitro cascade reaction with MEK and MAP kinase to measure specific Raf-1 catalytic activity, we observed an increase in Raf-1 activity in stimulated cells in the presence of PD This could be due to accumulation of Raf activity, since its substrate MEK is made unavailable. However, this scenario seems unlikely due to the fact that MEK inhibition also affects the phosphorylation state of Raf-1. Active MEK has been shown to be capable of causing hyperphosphorylation of Raf-1, and inactivated MEK abolished the hyperphosphorylated state of Raf-1, indicating that active MEK is responsible for keeping Raf-1 in the hyperphosphorylated and inactive state (8). We believe that these data support the theory of a negative-feedback model of Raf activity (26, 28, 31). PD has been shown to inhibit the hyperphosphorylation of Sos (15), and it has been suggested that this would prevent the inactivation of Ras, thereby allowing Raf-1 to remain active (1). The resulting Raf-1 activity due to the inhibition of MAP kinase activity may also be a continued negative feedback of the entire pathway, as active Raf-1 may continue the signal of no growth to signaling molecules further upstream. This work was supported by the Research Service of the United States Department of Veterans Affairs and by a gift from Dialysis Clinics, Inc. Address for reprint requests: R. H. Weiss, Div. of Nephrology, TB 136, Dept. of Internal Medicine, Univ. of California, Davis, CA Received 9 December 1997; accepted in final form 25 February REFERENCES 1. Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, and A. R. Saltiel. 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