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Regulation of Mitogen-Activated Protein Kinase Phosphatase-1 in Vascular Smooth Muscle Cells Dirk Bokemeyer, Marion Lindemann and Herbert J. Kramer Hypertension. 1998;32:661-667 doi: 10.1161/01.HYP.32.4.661 Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1998 American Heart Association, Inc. All rights reserved. Print ISSN: 0194-911X. Online ISSN: 1524-4563
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Regulation of Mitogen-Activated Protein Kinase Phosphatase-1 in Vascular Smooth Muscle Cells Dirk Bokemeyer, Marion Lindemann, Herbert J. Kramer Abstract—Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dualspecificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth–promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs. (Hypertension. 1998;32:661-667.) Key Words: MAP kinase n ERK n SAPK n p38 MAP kinase n MKP-1
M
itogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduce extracellular cues to intracellular responses. A common feature for activation of all MAP kinase isoforms is the requirement for phosphorylation of both a threonine and a neighboring tyrosine regulatory site by a specific upstream protein kinase for activation. Extracellular signal-regulated kinase (ERK) remains the best characterized mammalian MAP kinase.1,2 Binding of extracellular stimuli to their cell membrane receptors induces a sequence of protein kinase reaction, leading to phosphorylation and activation of MEK (MAP kinase/ERK kinase).3 MEK, the specific activator of ERK, is a dual-specificity protein kinase that phosphorylates both threonine and tyrosine regulatory sites in ERK.4 Phosphorylated and activated ERK migrates to the nucleus, where it phosphorylates several transcription factors.3,5,6 In vitro studies3,5,6 and more recently in vivo studies7 have established the pivotal role of the highly conserved MEK-ERK module in the control of cellular proliferation and hypertrophy. In contrast to ERK, more recently described MAP kinases such as stress-activated protein kinase (SAPK), also referred to as c-Jun N-terminal kinase (JNK), and p38 MAP kinase are
suggested to inhibit cellular proliferation and to induce apoptosis.8,9 Interestingly, the mechanism involved in the activation of SAPK and p38 MAP kinase is similar to that involved in the activation of ERK. Thus, highly specific protein kinase cascades lead to dual phosphorylation of tyrosine and threonine residues on these MAP kinases, inducing their full activation.6 In general, the extent of protein phosphorylation is balanced by an antagonism of kinases and phosphatases. Therefore, recently cloned dual-specificity protein tyrosine phosphatases (PTPases), which exhibit dual-catalytic activity toward phosphotyrosine and phosphothreonine in substrate proteins, may play a pivotal role in the regulation of MAP kinase–signaling pathways. The vaccina H-1 gene product (VH-1) was the first phosphatase shown to effectively hydrolyze both phosphotyrosine and phosphoserine/phosphothreonine.10 Recently, MAP kinase phosphatase-1 (MKP-1), a mammalian VH-1–like dual-specificity PTPase, has been isolated. MKP-1 (the human homologue is called CL100 [97% identity]) was demonstrated to dephosphorylate and inactivate not only ERK11–14 but also SAPK and p38 MAP kinase.15,16 Furthermore, the kinetics of gene expression and the cellular localization are consistent with a role for MKP-1
Received March 2, 1998; first decision March 25, 1998; revision accepted June 2, 1998. From the Medical Policlinic/Department of Medicine, Division of Nephrology, University of Bonn, Germany. Correspondence to Dirk Bokemeyer, MD, Medizinische Poliklinik, University of Bonn, Wilhelmstr 35-37, 53111 Bonn, Germany. E-mail [email protected] © 1998 American Heart Association, Inc.
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in the compensatory inactivation of stimulated MAP kinase– signaling pathways. However, the intracellular mechanisms involved in the induction of MKP-1, which is principally regulated at the transcriptional level,17,18 remain to be determined. Previously, we described a cell line–specific regulation of MKP-1 by ERK. In mesangial cells, activation of ERK was shown to induce MKP-1 expression, thereby providing a potential mechanism of feedback inhibition in the control of ERK activity.19 In contrast, activation of ERK suppressed MKP-1 expression in fibroblasts.20 To elucidate the intracellular mechanisms controlling the activity of MKP-1 in vascular smooth muscle cells (VSMCs), in the present study we examined the roles of ERK-, SAPK-, and p38 MAP kinase– signaling pathways in the regulation of MKP-1 expression. Using a selective antagonist of MEK, as well as extracellular stimuli that selectively activate the ERK rather than the SAPK or p38 MAP kinase cascade, we demonstrated the induction of MKP-1 expression by activation of the MEKERK module in VSMCs, suggesting a feedback inhibition of ERK to control its activity. In addition, MKP-1 was inducible in response to activation of stress-response pathways such as the SAPK or p38 MAP kinase cascade. This mode of MKP-1 induction may play an important role in the stress response of VSMCs after activation of the SAPK- or p38 MAP kinase– signaling pathways. These data provide new insights into the regulation of MKP-1 and thereby into the mechanisms involved in the downregulation of MAP kinases in VSMCs.
Methods
RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform procedure.23 The quality of RNA was assessed by measuring the ratio of 28S rRNA to 18S rRNA (2:1) in ethidium-stained denaturing agarose gels. For Northern blot analysis, RNA was denatured by heating for 15 minutes at 65°C in 0.02 mol/L 3-[N-morpholino]propanesulfonic acid, 6.6% formaldehyde, and 50% formamide and then fractionated by electrophoresis in a 1.2% agarose gel. The RNA was transferred to nitrocellulose and hybridized with MKP-1 cDNA (a generous gift of S.M. Keyse, Dundee, UK) labeled by nick translation with [32P]dCTP. After hybridization, the membranes were washed twice with 23 SSPE– 0.1% SDS at 20°C for 10 minutes and twice with 0.13 SSPE– 0.1% SDS at 42°C for 20 minutes. Cellulose membranes were exposed to Fuji RX films with intensifying screens. Each blot was rehybridized with GAPDH cDNA probe.
Western Blot Analysis Confluent VSMCs were washed with ice-cold PBS and lysed in 400 mL Triton X-100 lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mmol/L PMSF) at 4°C. After 5 minutes, the cells were scraped and centrifuged at 4°C for 15 minutes at 10 000g. The soluble cell lysates were mixed 1:4 with 53 Laemmli buffer and heated for 5 minutes at 95°C. Lysates (80 mg) were loaded per lane and separated by SDS–polyacrylamide gel electrophoresis (PAGE). Protein was transferred to nitrocellulose (pore size, 0.45 mm; Schleicher and Schuell) and probed with polyclonal antibodies against MKP-1 (Reference 19; see above ), p42 ERK,21 p46 SAPK, or phosphorylated p38 MAP kinase. The primary antibodies were detected using horseradish peroxidase– conjugated protein A, visualized with the Amersham ECL system after intensive washing of the membranes.
Immunoprecipitation
The minimum essential medium (MEM) cell culture media was from Gibco-BRL. Protein A–sepharose was obtained from Pharmacia Biotech. Cycloheximide was purchased from Calbiochem and [32P]dCTP was purchased from New England Nuclear. Plateletderived growth factor (PDGF), 12-O-tetradecanoylphorbol 13acetate (TPA), anisomycin, angiotensin II (Ang II), and all other reagents were obtained from Sigma Chemical Co.
Confluent VSMCs were washed with ice-cold PBS and lysed in Triton X-100 lysis buffer (as described above) for 5 minutes at 4°C. Insoluble material was removed by centrifugation. Protein (500 mg) of cell lysates was incubated for 2 hours with 1 mL polyclonal anti–MKP-1 antibody.19 Immunocomplexes were adsorbed to protein A–Sepharose and washed 3 times with lysis buffer. The proteins were resolubilized by the addition of an equal volume of 23 Laemmli buffer and were detected by Western blot analysis as described above.
Antibodies
ERK Activity Assay
Materials
Polyclonal antibodies against ERK and SAPK were raised against the C-terminal peptide of either p42 ERK21 (a generous gift from Dr M.J. Dunn, Medical College of Wisconsin, Milwaukee) or p46 SAPK (Santa Cruz Biotechnology). The anti-CL100 antibody was produced by immunizing rabbits with a synthetic peptide corresponding to the C terminus of the human homologue of MKP-1, as described previously.19 Rabbit polyclonal anti–phospho-p38 MAP kinase antibody was purchased from New England Biolabs.
Cell Cultures Rat VSMCs were isolated as described previously.22 Briefly, for preparation of smooth muscle cells, thoracic aortas of male SpragueDawley rats were isolated. After a short incubation in MEM containing collagenase, elastase, and trypsin inhibitor, the adventitia was stripped off. Afterward, the aortas were minced and incubated a second time in MEM containing the same supplements as used before until a single-cell suspension was achieved. VSMCs were cultured in MEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. VSMCs were serum-starved for 20 hours in serum-free MEM before stimulation. Experiments were conducted with cells between the 3rd and 10th passage.
Soluble cell lysates (500 mg; as described above) were incubated for 90 minutes with 10 mL polyclonal anti–p42 ERK antibody (Santa Cruz Biotechnology). Immunocomplexes were adsorbed to protein A–Sepharose, washed twice with lysis buffer and twice with kinase buffer (10 mmol/L MgCl2, 20 mmol/L HEPES, pH 7.4, containing 200 mmol/L Na-orthovanadate), and resuspended in 60 mL kinase buffer containing 50 mmol/L ATP and 5 mCi [g-32P]ATP. The final reaction buffer also contained 15 mg myelin basic protein. The reaction was initiated by incubation at 30°C for 15 minutes. Afterward, 20 mL of 43 Laemmli buffer was added to terminate the reaction. The samples were subjected to SDS-PAGE and autoradiography.
Nonradioactive ERK Activity Assay ERK immunocomplexes were adsorbed to protein A–Sepharose, washed (as described for the radioactive ERK assay), and resuspended in 60 mL kinase buffer containing 50 mmol/L ATP and 2 mg GST-Elk1 fusion protein. The reaction was initiated by incubation at 30°C for 45 minutes. To terminate the reaction, 20 mL of 43 Laemmli buffer was added, and samples were subjected to a 10% SDS-PAGE. Proteins were then analyzed by Western blot analysis, as described above, with a polyclonal anti–phospho-Elk1 antiserum recognizing only the in position 383 phosphorylated form of Elk1.
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Figure 1. Time curve of ERK activation after stimulation of quiescent VSMCs with 10% FBS alone (2Cx) or together with 20 mg/mL cycloheximide (1Cx). Upper panel shows a Western blot analysis detecting ERK2 in whole cell lysates. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). The lower panel shows ERK activity assayed by the ability of immunoprecipitated ERK to phosphorylate the GST-fusion protein of the transcription factor Elk1. The kinase reaction was followed by Western blot analysis (10% SDS-PAGE) with a polyclonal anti–phospho-Elk1 antiserum recognizing only the in position 383 (site of phosphorylation by ERK) phosphorylated form of Elk1. The results shown here were reproduced in 2 independent experiments.
MKP-1 Phosphatase Activity Assay Soluble cell lysates (500 mg; as described above) were incubated for 90 minutes with 1 mL polyclonal anti–MKP-1 antibody. Immunocomplexes were adsorbed to protein A–Sepharose, washed, resuspended in kinase buffer containing 25 U phosphorylated active ERK (New England Biolabs Inc), and incubated for 1 hour at 30°C. Thereafter, 50 mmol/L ATP and 2 mg GST-Elk1 fusion protein were added. After an additional incubation at 30°C for 45 minutes, the reaction was terminated and Elk1 phosphorylation was analyzed as described above.
Results To investigate the relevance of inducible genes such as MKP-1 in the downregulation of ERK in VSMCs, we examined the time curve of ERK2 activity after mitogenic stimulation in the presence and absence of the protein synthesis inhibitor cycloheximide. ERK activity was measured by Western blot analysis of crude cellular lysates detecting ERK2 and by the ability of immunoprecipitated ERK2 to phosphorylate the GST-fusion protein of the transcription factor Elk1. In general, the 2 isoforms of ERK, ERK1 and ERK2, are expected to be activated by the same mechanisms and to be functionally redundant.2,5,6 As shown in Figure 1, FBS induced a rapid activation of ERK followed by a slow inactivation over 6 hours, whereas in the presence of cycloheximide, FBS induced a more sustained activation of ERK. Thus, the synthesis of new protein, presumably of a dual-specificity PTPase such as MKP-1, is required to dephosphorylate and inactivate ERK in VSMCs. As shown in Figure 1, ERK activity begins to decline 30 minutes after stimulation with FBS. To correlate MKP-1 expression with the described ERK activity, we examined the time curve of MKP-1 protein expression in VSMCs stimulated with FBS using polyclonal rabbit antiserum raised against a peptide corresponding to the C terminus of MKP1.19 After immunoprecipitation and immunoblotting with anti–MKP-1 antibody, we detected an inducible band of 39 kDa, corresponding to the expected size of MKP-1, as early as 30 minutes after stimulation (Figure 2). Therefore, the time
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Figure 2. Time curve of MKP-1 protein expression in response to FBS. Quiescent VSMCs were stimulated with 10% FBS for the indicated periods of time. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDS-PAGE and Western blot analysis using anti– MKP-1 antiserum and the ECL detection system. The band correlating to the expected size of 39 kDa of the MKP-1 protein is indicated. The dominant band of '53 kDa, as indicated by the star, correlates to the heavy chain of the antibodies used for immunoprecipitation.
curve of MKP-1 protein expression correlated closely with ERK dephosphorylation and inactivation, suggesting that MKP-1 might be responsible for ERK inactivation in VSMCs. Recently, we demonstrated in NIH3T3 cells that the SAPK cascade, rather than the ERK cascade, induces MKP-1 expression in NIH3T3 cells.18 However, in human mesangial cells, the ERK cascade itself induced MKP-1 expression.19 To examine the role of MAP kinase–signaling pathways in the regulation of MKP-1 in VSMCs, we stimulated quiescent cells with extracellular stimuli that activate either the ERK or the SAPK/p38 MAP kinase pathways. Stimulation with PDGF, TPA, or Ang II induced a rapid and strong activation of ERK that was detected by the electrophoretic retardation, indicating phosphorylated and activated protein forms (Figure 3A). However, anisomycin induced only a weak and transient activation of ERK (Figure 3A). In contrast, anisomycin potently activated SAPK and p38 MAP kinase, whereas PDGF, TPA, and Ang II had no detectable effect on SAPK activity and only weak stimuli of p38 MAP kinase (Figure 3B and 3C). TPA and Ang II, like FBS, potently induced the expression of MKP-1 protein (Figure 4A), suggesting that the ERK cascade induces the expression of MKP-1 in VSMCs. In accordance with this finding, PDGF stimulated the expression of MKP-1 mRNA (Figure 5) and MKP-1 protein (Figure 6B). To examine the functional significance of MKP-1 expression, we measured its catalytic activity toward recombinant phosphorylated and active ERK. Figure 4B demonstrates that the expression of MKP-1 after stimulation with FBS, Ang II, and PDGF correlates with MKP-1 catalytic activity. Anisomycin potently induced the expression of MKP-1 mRNA, 2.2 kb in length24 (Figure 5A). Because anisomycin is an inhibitor of protein synthesis, only MKP-1 mRNA levels are available. These data suggest that the expression of MKP-1 is inducible by multiple intracellular signaling pathways. Because anisomycin activated p38 MAP kinase (Figure 3C), we examined the effect of the p38 MAP kinase antagonist SB 203580 (a generous gift from Dr J.C. Lee25) on the
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Figure 3. Effect of extracellular agonists on the activity of ERK, SAPK, and p38 MAP kinase. A, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis detecting ERK2. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). B, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis detecting p46 SAPK. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). C, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis using an antibody that detects only the phosphorylated form of p38 MAP kinase (MAPK). The intensity of bands correlates to the extent of phosphorylation at the regulatory sites of p38 MAPK.
MKP-1 gene expression to define the role of p38 MAP kinase in anisomycin-induced MKP-1 induction. The antagonist did not affect the PDGF-induced gene expression of MKP-1 (Figure 5A), suggesting that the PDGF-induced expression of MKP-1 is due to an intracellular pathway distinct from the p38 MAP kinase cascade. However, the anisomycin-induced expression of MKP-1 was dose-dependently inhibited after preincubation with SB 203580 (Figure 5B). To examine the role of the ERK cascade in the regulation of the dual-specificity phosphatase MKP-1 in more detail, we performed experiments using the MEK antagonist PD 098059 (a generous gift from Dr A.R. Saltiel26). PD 098059 significantly reduced the PDGF-induced ERK activation in VSMCs as detected by the reduction of the PDGF-induced band shift of ERK and the inhibition of ERK activity assayed in an immunocomplex kinase assay (Figure 6A). Quantification of myelin basic protein phosphorylation by scintillation counting demonstrated a 50% reduction of ERK activity in the presence of PD 098059. As shown in Figure 6B, inhibition of
Figure 4. Effect of ERK-activating stimuli on MKP-1 protein expression and activity. A, Quiescent VSMCs were stimulated with FBS (10%), TPA (100 nmol/L), or Ang II (1027 mol/L) for 30 and 60 minutes. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDSPAGE and Western blot analysis using anti–MKP-1 antiserum and the ECL detection system. The MKP-1 band with an expected size of 39 kDa is indicated. B, Quiescent VSMCs were stimulated with FBS (10%), Ang II (1027 mol/L), or PDGF (20 ng/mL) for 1 hour. MKP-1 immunoprecipitates were incubated with recombinant phosphorylated and active ERK2 (New England Biolabs). ERK activity was assayed by phosphorylation of the GST-fusion protein of Elk1 as described above. The dominant band of '53 kDa (star) correlates to the heavy chain of the antibodies used for immunoprecipitation of MKP-1.
ERK activation by PD 098059 significantly reduced the PDGF-induced gene expression of MKP-1. Based on the effect of the MEK inhibitor and on the effect of cellular stimulation with PDGF, TPA, and Ang II, our data suggest that the ERK cascade induces the expression of MKP-1 in VSMCs. Because MKP-1 is known to dephosphorylate and inactivate ERK, this mode of regulation illustrates a potential mechanism to maintain balanced cell growth through feedback inhibition of ERK in VSMCs.
Discussion A diverse array of extracellular signals utilize MAP kinase signaling cascades to initiate a variety of cell signaling outcomes. The pleiotropic potential of MAP kinases emphasizes the importance of a tight control of their activation. In chromaffin cells (PC12) it was demonstrated that the duration of ERK activation by extracellular stimuli is critical for cell signaling outcomes, because transient activation of MAP kinase induced mitogenesis, whereas sustained activation of MAP kinase induced cell differentiation.27,28 However, in mesangial cells and fibroblasts, only potent mitogens seem to be capable of inducing a sustained phase of ERK activation.21,29 These data emphasize the importance of mechanisms to terminate and control the duration of ERK activity. We19,20 and others11 have demonstrated previously that the downregulation of ERK in NIH3T3 cells and in mesangial cells is dependent on the production of new protein, presumably of the expression of a transcriptionally regulated dual-
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Figure 5. Effect of p38 MAP kinase inhibition on MKP-1 gene expression. A, Quiescent VSMCs were pretreated with the p38 MAP kinase antagonist SB 203580 (10 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with TPA (100 nmol/L), PDGF (20 ng/mL), or anisomycin (500 nmol/L) for the indicated periods. Total RNA was isolated and examined by Northern blot analysis. The blots were probed with MKP-1 cDNA. As a loading control, the same filters were stripped and rehybridized with the GAPDH probe. A representative blot of 3 independent experiments is shown. B, Quiescent VSMCs were pretreated with different concentrations of SB 203580 for 60 minutes before stimulation with anisomycin (500 nmol/L) for 60 minutes. Total RNA was analyzed by Northern blot analysis.
specificity PTPase such as MKP-1. However, this may not be the case in all cell systems. The inactivation of ERK after mitogenic stimulation of PC12 cells, adipose cells (3T3-L1), or endothelial cells (PAE) occurs normally when protein synthesis is inhibited.30,31 We demonstrated in the present study a decline of ERK activity after stimulation with FBS over a period of 6 hours (beginning 30 minutes after stimulation), indicating the action of phosphatase activity toward ERK during this period. Furthermore, FBS induced within 30 minutes a sustained expression of the dual-specificity PTPase MKP-1. Therefore, it is likely that MKP-1, which is known to exhibit catalytic activity toward both regulatory sites on ERK, is involved in ERK downregulation. Moreover, we showed in the present study a prolonged ERK activation in the presence of the protein synthesis inhibitor cycloheximide, emphasizing
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Figure 6. Effect of the MEK antagonist PD 098059 on the PDGF-induced expression of the dual-specificity PTPase MKP-1 in VSMCs. A, Quiescent cells were preincubated with PD 098059 (20 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with PDGF (20 ng/mL) for the indicated periods. The upper panel shows a Western blot analysis detecting p42 ERK (ERK2) as described above (star indicates phosphorylated protein forms), and the lower panel shows ERK activity assayed by the ability of immunoprecipitated ERK to phosphorylate myelin basic protein (MBP). B, Quiescent cells were preincubated with PD 098059 (20 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with PDGF for the indicated periods. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDS-PAGE and Western blot analysis using anti–MKP-1 antiserum and the ECL detection system. The band of MKP-1 protein (39 kDa) is indicated. The dominant band of '53 kDa correlates to the heavy chain of the antibodies used for immunoprecipitation. A representative blot of 3 independent experiments is shown.
the necessity of transcriptionally regulated phosphatases, presumably the dual-specificity PTPase MKP-1, for ERK inactivation in VSMCs. These data are consistent with a study in VSMCs in which MKP-1 antisense oligonucleotides were used.14 In this study, a prolonged activation of ERK with no effect on MEK activity after suppression of MKP-1 protein expression was demonstrated.14 Previously we20 have shown that in NIH3T3 fibroblasts, activation of the SAPK cascade, rather than the ERK cascade, induces the expression of MKP-1. Because MKP-1 is known to dephosphorylate and inactivate ERK,11,13–15 this cross-talk of MAP kinase–signaling cascades may contribute to the inhibition of cell growth after activation of SAPK.8,9,32,33 In contrast to NIH3T3 fibroblasts, activation of the ERK cascade in mesangial cells19 and lung fibroblasts34 has been demonstrated to induce the expression of MKP-1, thereby providing a potential mechanism of feedback inhibition.
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Based on these obvious cell line–specific differences in the regulation of MKP-1 expression, we examined the role of multiple MAP kinase–signaling cascades in the regulation of MKP-1 in VSMCs. With the use of extracellular stimuli that activate the ERK rather than the SAPK or p38 MAP kinase cascade, as well as a synthetic inhibitor of MEK activity, our data suggest that activation of the ERK signaling cascade induces MKP-1 expression in VSMCs. This mode of MKP-1 induction might be responsible for the observed decline in ERK activity 30 minutes after stimulation of quiescent VSMCs. Furthermore, we demonstrated the induction of MKP-1 gene expression after activation of stress-responsive MAP kinase pathways such as the SAPK or p38 MAP kinase cascade. These data correlate with findings that the human homologue of MKP-1 was cloned from a cDNA library from human skin fibroblasts treated with hydrogen peroxide and that this gene was inducible by oxidative stress.24 MKP-1 is known to effectively dephosphorylate ERK.13,15 It is therefore reasonable to speculate that VSMC activation of the SAPK/p38 MAP kinase pathways in response to cellular stress induces the expression of the dual-specificity PTPase MKP-1, thereby inhibiting the stimulation of ERK. This cross-talk between these independent signal transduction pathways would be a logical cellular response to agonists activating the SAPK/p38 MAP kinase pathways, since activation of SAPK and p38 MAP kinase inhibits cell growth and induces apoptosis.8,9 Therefore, inhibition of the growth-stimulatory and anti-apoptotic ERK pathway6,9 would contribute to the effects of activated SAPK/p38 MAP kinase pathways in VSMCs. In addition to its catalytic activity toward ERK, MKP-1 was also shown to hydrolyze phosphotyrosine and phosphothreonine on SAPK and p38 MAP kinase in hela cells and in NIH3T3 fibroblasts.15,16 A recent study in leukemic cells suggested MKP-1 to be even more active toward SAPK and p38 MAP kinase than toward ERK.35 The induction of MKP-1 in response to cellular stress in VSMCs may therefore be important for the deactivation of SAPK and p38 MAP kinase as a negative feedback mechanism. However, further studies will be needed to define the substrate specificity of MKP-1 toward different MAP kinases. Interestingly, other dual-specificity PTPases have also been suggested to exhibit distinct specificities toward multiple MAP kinases (eg, PAC1, which failed to inactivate SAPK in hela and NIH3T3 cells despite its activity toward ERK and p38 MAP kinase,15 and Pyst1, which preferentially dephosphorylates ERK36). Therefore, it would not be surprising if unique members of the group of dual-specificity PTPases, such as MKP-1, PAC1, Pyst1, B23,37 MKP-2,38,39 or MKP-3,40,41 were shown to exhibit selective activities toward distinct MAP kinases, thereby introducing further tiers of control of the regulatory networks. A similar selectivity has already been demonstrated for distinct dual-specificity kinases, each phosphorylating a single MAP kinase.
Acknowledgments This work was supported by a grant from the Deutsche Forschungsgemeinschaft BO 1288/3–1 (Dr Bokemeyer) and in part by a BONFOR research grant from the Faculty of Medicine, University of
Bonn. We would like to thank Stephen M. Keyse (Dundee, UK) for the CL100 cDNA, John C. Lee (King of Prussia, Pa) for the p38 MAP kinase antagonist, and Alan R. Saltiel (Ann Arbor, Mich) for the MEK antagonist.
References 1. Hill CS, Treisman R. Transcriptional regulation by extracellular signals: mechanisms and specificity. Cell. 1995;80:199 –211. 2. Cobb MH, Boulton TG, Robbins DJ. Extracellular signal-regulated kinases: ERKs in progress. Cell Regul. 1991;2:965–978. 3. Johnson GL, Vaillancourt RR. Sequential protein kinase reactions controlling cell growth and differentiation. Curr Opin Cell Biol. 1994;6: 230 –238. 4. Payne DM, Rossomando HJ, Martino P, Erickson AK, Her J-H, Schbanowitz J, Hunt DF, Weber MJ, Sturgill TW. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991;10:885–892. 5. Seger R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995;9: 726 –735. 6. Bokemeyer D, Sorokin A, Dunn MJ. Multiple intracellular MAP kinase signaling cascades. Kidney Int. 1996;49:1187–1198. 7. Bokemeyer D, Guglielmi KE, McGinty A, Sorokin A, Lianos EA, Dunn MJ. Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats. J Clin Invest. 1997;100:582–588. 8. Yan M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, Templeton DJ. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature. 1994;372:798 – 800. 9. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995; 270:1326 –1331. 10. Guan K, Broyles SS, Dixon JE. A Tyr/Ser protein phosphatase encoded by vaccina virus. Nature. 1991;350:359 –362. 11. Sun H, Charles CH, Lau LF, Tonks NK. MKP-1 (3CH134), an immediate early gene product, is a dual specificity phosphatase that dephosphorylates MAP kinase in vivo. Cell. 1993;75:487– 493. 12. Ward Y, Gupta S, Jensen P, Wartmann M, Davis RJ, Kelly K. Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1. Nature. 1994;367:651– 654. 13. Sun H, Tonks NK, Bar-Sagi D. Inhibition of Ras-induced DNA synthesis by expression of the phosphatase MKP-1. Science. 1994;266:285–288. 14. Duff JL, Monia BP, Berk BC. Mitogen-activated protein (MAP) kinase is regulated by the MAP kinase phosphatase (MKP-1) in vascular smooth muscle cells. J Biol Chem. 1995;270:7161–7166. 15. Chu Y, Solski PA, Khosravi-Far R, Der CJ, Kelly K. The mitogen-activated protein kinase phosphatases PAC1, MKP-1, and MKP-2 have unique substrate specificities and reduced activity in vivo toward the ERK2 sevenmaker mutation. J Biol Chem. 1996;271:6497– 6501. 16. Liu Y, Gorospe M, Yang C, Holbrook NJ. Role of mitogen-activated protein kinase phosphatase during the cellular response to genotoxic stress. J Biol Chem. 1995;270:8377– 8380. 17. Sun H, Tonks NK. The coordinated action of protein tyrosine phosphatases and kinases in cell signaling. Trends Biochem Sci. 1994;19: 480 – 485. 18. Charles CH, Abler AS, Lau LF. cDNA sequence of growth factorinducible immediate early gene and characterization of its encoded protein. Oncogene. 1992;7:187–190. 19. Bokemeyer D, Sorokin A, Dunn MJ. Differential regulation of the dualspecificity protein-tyrosine phosphatases CL100, B23, and PAC1 in mesangial cells. J Am Soc Nephrol. 1997;8:40 –50. 20. Bokemeyer D, Sorokin A, Yan M, Ahn NG, Templeton DJ, Dunn MJ. Induction of mitogen-activated protein kinase phosphatase 1 by the stress-activated protein kinase signaling pathway but not by extracellular signal-regulated kinase in fibroblasts. J Biol Chem. 1996;271:639 – 642. 21. Wang Y, Simonson MS, Pouysse´gur J, Dunn MJ. Endothelin rapidly stimulates mitogen-activated protein kinase activity in rat mesangial cells. Biochem J. 1992;287:589 –594. 22. Bokemeyer D, Kramer HJ, Meyer-Lehnert H. Atrial natriuretic peptide blunts the effects of cyclosporine A on Ca21 mobilization in vascular smooth muscle cells. Hypertension. 1993;21:166 –172. 23. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156 –159. 24. Keyse SM, Emslie EA. Oxidative stress and heat shock induce a gene encoding a protein-tyrosine phosphatase. Nature. 1992;359:644 – 647.
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Bokemeyer et al 25. Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stress and interleukin-1. FEBS Lett. 1995;364:229 –233. 26. Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995;270:27489 –27494. 27. Marshall CJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179 –185. 28. Cowley S, Paterson H, Kemp P, Marshall CJ. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell. 1994;77:841– 852. 29. Meloche S, Seuwen K, Page`s G, Pouysse´gur J. Biphasic and synergistic activation of p44 MAPK (ERK1) by growth factors: correlation between late phase activation and mitogenicity. Mol Endocrinol. 1992;638: 845– 854. 30. Wu J, Lau LF, Sturgill TW. Rapid deactivation of MAP kinase in PC12 cells occurs independently of induction of phosphatase MKP-1. FEBS Lett. 1994;353:9 –12. 31. Alessi DR, Gomez N, Moorhead G, Lewis T, Keyse SM, Cohen P. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol. 1995;5:283–295. 32. Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci. 1995;20:117–122. 33. Sanchez I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J, Kyriakis JM, Zon LI. Role of SAPK/ERK kinase-1 in stress-activated pathway regulating transcription factor c-Jun. Nature. 1994;372:794–798.
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34. Brondello JM, Brunet A, Pouysse´gur J, McKenzie FR. The dual specificity mitogen-activated protein kinase phosphatase-1 and -2 are induced by the p42/p44 MAPK cascade. J Biol Chem. 1997;272: 1368 –1376. 35. Franklin CC, Kraft AS. Conditional expression of the mitogen-activated protein kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated protein kinase in U937 cells. J Biol Chem. 1997;272:16917–19623. 36. Groom LA, Sneddon AA, Alessi DR, Dowd S, Keyse SM. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase. EMBO J. 1996;15:3621–3632. 37. Ishibashi T, Bottaro DP, Michieli P, Kelley CK, Aaronson SA. A novel dual specificity phosphatase induced by serum stimulation and heat shock. J Biol Chem. 1994;269:29897–29902. 38. Guan K-L, Butch E. Isolation and characterization of a novel dual specific phosphatase, HVH2, which selectively dephosphorylates the mitogen-activated protein kinase. J Biol Chem. 1995;270:7197–7203. 39. Misra-Press A, Rim CS, Yao H, Roberson MS, Stork PJS. A novel mitogen-activated protein kinase phosphatase. J Biol Chem. 1995;270: 14587–14596. 40. Muda M, Boschert U, Dickinson R, Martinou J-C, Martinou M, Camps M, Schlegel W, Arkinstall S. MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies a new class of mitogen-activated protein kinase phosphatase. J Biol Chem. 1996;271:4319 – 4326. 41. Mourey RJ, Vega QC, Campbell JS, Wenderoth MP, Hauschka SD, Krebs EG, Dixon JE. A novel cytoplasmatic dual specificity protein tyrosine phosphatase implicated in muscle and neuronal differentiation. J Biol Chem. 1996;271:3795–3802.
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Regulation of Mitogen-Activated Protein Kinase Phosphatase-1 in Vascular Smooth Muscle Cells Dirk Bokemeyer, Marion Lindemann, Herbert J. Kramer Abstract—Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dualspecificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth–promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs. (Hypertension. 1998;32:661-667.) Key Words: MAP kinase n ERK n SAPK n p38 MAP kinase n MKP-1
M
itogen-activated protein (MAP) kinases are important mediators involved in the intracellular network of interacting proteins that transduce extracellular cues to intracellular responses. A common feature for activation of all MAP kinase isoforms is the requirement for phosphorylation of both a threonine and a neighboring tyrosine regulatory site by a specific upstream protein kinase for activation. Extracellular signal-regulated kinase (ERK) remains the best characterized mammalian MAP kinase.1,2 Binding of extracellular stimuli to their cell membrane receptors induces a sequence of protein kinase reaction, leading to phosphorylation and activation of MEK (MAP kinase/ERK kinase).3 MEK, the specific activator of ERK, is a dual-specificity protein kinase that phosphorylates both threonine and tyrosine regulatory sites in ERK.4 Phosphorylated and activated ERK migrates to the nucleus, where it phosphorylates several transcription factors.3,5,6 In vitro studies3,5,6 and more recently in vivo studies7 have established the pivotal role of the highly conserved MEK-ERK module in the control of cellular proliferation and hypertrophy. In contrast to ERK, more recently described MAP kinases such as stress-activated protein kinase (SAPK), also referred to as c-Jun N-terminal kinase (JNK), and p38 MAP kinase are
suggested to inhibit cellular proliferation and to induce apoptosis.8,9 Interestingly, the mechanism involved in the activation of SAPK and p38 MAP kinase is similar to that involved in the activation of ERK. Thus, highly specific protein kinase cascades lead to dual phosphorylation of tyrosine and threonine residues on these MAP kinases, inducing their full activation.6 In general, the extent of protein phosphorylation is balanced by an antagonism of kinases and phosphatases. Therefore, recently cloned dual-specificity protein tyrosine phosphatases (PTPases), which exhibit dual-catalytic activity toward phosphotyrosine and phosphothreonine in substrate proteins, may play a pivotal role in the regulation of MAP kinase–signaling pathways. The vaccina H-1 gene product (VH-1) was the first phosphatase shown to effectively hydrolyze both phosphotyrosine and phosphoserine/phosphothreonine.10 Recently, MAP kinase phosphatase-1 (MKP-1), a mammalian VH-1–like dual-specificity PTPase, has been isolated. MKP-1 (the human homologue is called CL100 [97% identity]) was demonstrated to dephosphorylate and inactivate not only ERK11–14 but also SAPK and p38 MAP kinase.15,16 Furthermore, the kinetics of gene expression and the cellular localization are consistent with a role for MKP-1
Received March 2, 1998; first decision March 25, 1998; revision accepted June 2, 1998. From the Medical Policlinic/Department of Medicine, Division of Nephrology, University of Bonn, Germany. Correspondence to Dirk Bokemeyer, MD, Medizinische Poliklinik, University of Bonn, Wilhelmstr 35-37, 53111 Bonn, Germany. E-mail [email protected] © 1998 American Heart Association, Inc.
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in the compensatory inactivation of stimulated MAP kinase– signaling pathways. However, the intracellular mechanisms involved in the induction of MKP-1, which is principally regulated at the transcriptional level,17,18 remain to be determined. Previously, we described a cell line–specific regulation of MKP-1 by ERK. In mesangial cells, activation of ERK was shown to induce MKP-1 expression, thereby providing a potential mechanism of feedback inhibition in the control of ERK activity.19 In contrast, activation of ERK suppressed MKP-1 expression in fibroblasts.20 To elucidate the intracellular mechanisms controlling the activity of MKP-1 in vascular smooth muscle cells (VSMCs), in the present study we examined the roles of ERK-, SAPK-, and p38 MAP kinase– signaling pathways in the regulation of MKP-1 expression. Using a selective antagonist of MEK, as well as extracellular stimuli that selectively activate the ERK rather than the SAPK or p38 MAP kinase cascade, we demonstrated the induction of MKP-1 expression by activation of the MEKERK module in VSMCs, suggesting a feedback inhibition of ERK to control its activity. In addition, MKP-1 was inducible in response to activation of stress-response pathways such as the SAPK or p38 MAP kinase cascade. This mode of MKP-1 induction may play an important role in the stress response of VSMCs after activation of the SAPK- or p38 MAP kinase– signaling pathways. These data provide new insights into the regulation of MKP-1 and thereby into the mechanisms involved in the downregulation of MAP kinases in VSMCs.
Methods
RNA Extraction and Northern Blot Analysis Total cellular RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform procedure.23 The quality of RNA was assessed by measuring the ratio of 28S rRNA to 18S rRNA (2:1) in ethidium-stained denaturing agarose gels. For Northern blot analysis, RNA was denatured by heating for 15 minutes at 65°C in 0.02 mol/L 3-[N-morpholino]propanesulfonic acid, 6.6% formaldehyde, and 50% formamide and then fractionated by electrophoresis in a 1.2% agarose gel. The RNA was transferred to nitrocellulose and hybridized with MKP-1 cDNA (a generous gift of S.M. Keyse, Dundee, UK) labeled by nick translation with [32P]dCTP. After hybridization, the membranes were washed twice with 23 SSPE– 0.1% SDS at 20°C for 10 minutes and twice with 0.13 SSPE– 0.1% SDS at 42°C for 20 minutes. Cellulose membranes were exposed to Fuji RX films with intensifying screens. Each blot was rehybridized with GAPDH cDNA probe.
Western Blot Analysis Confluent VSMCs were washed with ice-cold PBS and lysed in 400 mL Triton X-100 lysis buffer (50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 10% glycerol, 1% Triton X-100, 1 mg/mL aprotinin, 1 mg/mL leupeptin, 1 mmol/L PMSF) at 4°C. After 5 minutes, the cells were scraped and centrifuged at 4°C for 15 minutes at 10 000g. The soluble cell lysates were mixed 1:4 with 53 Laemmli buffer and heated for 5 minutes at 95°C. Lysates (80 mg) were loaded per lane and separated by SDS–polyacrylamide gel electrophoresis (PAGE). Protein was transferred to nitrocellulose (pore size, 0.45 mm; Schleicher and Schuell) and probed with polyclonal antibodies against MKP-1 (Reference 19; see above ), p42 ERK,21 p46 SAPK, or phosphorylated p38 MAP kinase. The primary antibodies were detected using horseradish peroxidase– conjugated protein A, visualized with the Amersham ECL system after intensive washing of the membranes.
Immunoprecipitation
The minimum essential medium (MEM) cell culture media was from Gibco-BRL. Protein A–sepharose was obtained from Pharmacia Biotech. Cycloheximide was purchased from Calbiochem and [32P]dCTP was purchased from New England Nuclear. Plateletderived growth factor (PDGF), 12-O-tetradecanoylphorbol 13acetate (TPA), anisomycin, angiotensin II (Ang II), and all other reagents were obtained from Sigma Chemical Co.
Confluent VSMCs were washed with ice-cold PBS and lysed in Triton X-100 lysis buffer (as described above) for 5 minutes at 4°C. Insoluble material was removed by centrifugation. Protein (500 mg) of cell lysates was incubated for 2 hours with 1 mL polyclonal anti–MKP-1 antibody.19 Immunocomplexes were adsorbed to protein A–Sepharose and washed 3 times with lysis buffer. The proteins were resolubilized by the addition of an equal volume of 23 Laemmli buffer and were detected by Western blot analysis as described above.
Antibodies
ERK Activity Assay
Materials
Polyclonal antibodies against ERK and SAPK were raised against the C-terminal peptide of either p42 ERK21 (a generous gift from Dr M.J. Dunn, Medical College of Wisconsin, Milwaukee) or p46 SAPK (Santa Cruz Biotechnology). The anti-CL100 antibody was produced by immunizing rabbits with a synthetic peptide corresponding to the C terminus of the human homologue of MKP-1, as described previously.19 Rabbit polyclonal anti–phospho-p38 MAP kinase antibody was purchased from New England Biolabs.
Cell Cultures Rat VSMCs were isolated as described previously.22 Briefly, for preparation of smooth muscle cells, thoracic aortas of male SpragueDawley rats were isolated. After a short incubation in MEM containing collagenase, elastase, and trypsin inhibitor, the adventitia was stripped off. Afterward, the aortas were minced and incubated a second time in MEM containing the same supplements as used before until a single-cell suspension was achieved. VSMCs were cultured in MEM supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. VSMCs were serum-starved for 20 hours in serum-free MEM before stimulation. Experiments were conducted with cells between the 3rd and 10th passage.
Soluble cell lysates (500 mg; as described above) were incubated for 90 minutes with 10 mL polyclonal anti–p42 ERK antibody (Santa Cruz Biotechnology). Immunocomplexes were adsorbed to protein A–Sepharose, washed twice with lysis buffer and twice with kinase buffer (10 mmol/L MgCl2, 20 mmol/L HEPES, pH 7.4, containing 200 mmol/L Na-orthovanadate), and resuspended in 60 mL kinase buffer containing 50 mmol/L ATP and 5 mCi [g-32P]ATP. The final reaction buffer also contained 15 mg myelin basic protein. The reaction was initiated by incubation at 30°C for 15 minutes. Afterward, 20 mL of 43 Laemmli buffer was added to terminate the reaction. The samples were subjected to SDS-PAGE and autoradiography.
Nonradioactive ERK Activity Assay ERK immunocomplexes were adsorbed to protein A–Sepharose, washed (as described for the radioactive ERK assay), and resuspended in 60 mL kinase buffer containing 50 mmol/L ATP and 2 mg GST-Elk1 fusion protein. The reaction was initiated by incubation at 30°C for 45 minutes. To terminate the reaction, 20 mL of 43 Laemmli buffer was added, and samples were subjected to a 10% SDS-PAGE. Proteins were then analyzed by Western blot analysis, as described above, with a polyclonal anti–phospho-Elk1 antiserum recognizing only the in position 383 phosphorylated form of Elk1.
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Figure 1. Time curve of ERK activation after stimulation of quiescent VSMCs with 10% FBS alone (2Cx) or together with 20 mg/mL cycloheximide (1Cx). Upper panel shows a Western blot analysis detecting ERK2 in whole cell lysates. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). The lower panel shows ERK activity assayed by the ability of immunoprecipitated ERK to phosphorylate the GST-fusion protein of the transcription factor Elk1. The kinase reaction was followed by Western blot analysis (10% SDS-PAGE) with a polyclonal anti–phospho-Elk1 antiserum recognizing only the in position 383 (site of phosphorylation by ERK) phosphorylated form of Elk1. The results shown here were reproduced in 2 independent experiments.
MKP-1 Phosphatase Activity Assay Soluble cell lysates (500 mg; as described above) were incubated for 90 minutes with 1 mL polyclonal anti–MKP-1 antibody. Immunocomplexes were adsorbed to protein A–Sepharose, washed, resuspended in kinase buffer containing 25 U phosphorylated active ERK (New England Biolabs Inc), and incubated for 1 hour at 30°C. Thereafter, 50 mmol/L ATP and 2 mg GST-Elk1 fusion protein were added. After an additional incubation at 30°C for 45 minutes, the reaction was terminated and Elk1 phosphorylation was analyzed as described above.
Results To investigate the relevance of inducible genes such as MKP-1 in the downregulation of ERK in VSMCs, we examined the time curve of ERK2 activity after mitogenic stimulation in the presence and absence of the protein synthesis inhibitor cycloheximide. ERK activity was measured by Western blot analysis of crude cellular lysates detecting ERK2 and by the ability of immunoprecipitated ERK2 to phosphorylate the GST-fusion protein of the transcription factor Elk1. In general, the 2 isoforms of ERK, ERK1 and ERK2, are expected to be activated by the same mechanisms and to be functionally redundant.2,5,6 As shown in Figure 1, FBS induced a rapid activation of ERK followed by a slow inactivation over 6 hours, whereas in the presence of cycloheximide, FBS induced a more sustained activation of ERK. Thus, the synthesis of new protein, presumably of a dual-specificity PTPase such as MKP-1, is required to dephosphorylate and inactivate ERK in VSMCs. As shown in Figure 1, ERK activity begins to decline 30 minutes after stimulation with FBS. To correlate MKP-1 expression with the described ERK activity, we examined the time curve of MKP-1 protein expression in VSMCs stimulated with FBS using polyclonal rabbit antiserum raised against a peptide corresponding to the C terminus of MKP1.19 After immunoprecipitation and immunoblotting with anti–MKP-1 antibody, we detected an inducible band of 39 kDa, corresponding to the expected size of MKP-1, as early as 30 minutes after stimulation (Figure 2). Therefore, the time
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Figure 2. Time curve of MKP-1 protein expression in response to FBS. Quiescent VSMCs were stimulated with 10% FBS for the indicated periods of time. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDS-PAGE and Western blot analysis using anti– MKP-1 antiserum and the ECL detection system. The band correlating to the expected size of 39 kDa of the MKP-1 protein is indicated. The dominant band of '53 kDa, as indicated by the star, correlates to the heavy chain of the antibodies used for immunoprecipitation.
curve of MKP-1 protein expression correlated closely with ERK dephosphorylation and inactivation, suggesting that MKP-1 might be responsible for ERK inactivation in VSMCs. Recently, we demonstrated in NIH3T3 cells that the SAPK cascade, rather than the ERK cascade, induces MKP-1 expression in NIH3T3 cells.18 However, in human mesangial cells, the ERK cascade itself induced MKP-1 expression.19 To examine the role of MAP kinase–signaling pathways in the regulation of MKP-1 in VSMCs, we stimulated quiescent cells with extracellular stimuli that activate either the ERK or the SAPK/p38 MAP kinase pathways. Stimulation with PDGF, TPA, or Ang II induced a rapid and strong activation of ERK that was detected by the electrophoretic retardation, indicating phosphorylated and activated protein forms (Figure 3A). However, anisomycin induced only a weak and transient activation of ERK (Figure 3A). In contrast, anisomycin potently activated SAPK and p38 MAP kinase, whereas PDGF, TPA, and Ang II had no detectable effect on SAPK activity and only weak stimuli of p38 MAP kinase (Figure 3B and 3C). TPA and Ang II, like FBS, potently induced the expression of MKP-1 protein (Figure 4A), suggesting that the ERK cascade induces the expression of MKP-1 in VSMCs. In accordance with this finding, PDGF stimulated the expression of MKP-1 mRNA (Figure 5) and MKP-1 protein (Figure 6B). To examine the functional significance of MKP-1 expression, we measured its catalytic activity toward recombinant phosphorylated and active ERK. Figure 4B demonstrates that the expression of MKP-1 after stimulation with FBS, Ang II, and PDGF correlates with MKP-1 catalytic activity. Anisomycin potently induced the expression of MKP-1 mRNA, 2.2 kb in length24 (Figure 5A). Because anisomycin is an inhibitor of protein synthesis, only MKP-1 mRNA levels are available. These data suggest that the expression of MKP-1 is inducible by multiple intracellular signaling pathways. Because anisomycin activated p38 MAP kinase (Figure 3C), we examined the effect of the p38 MAP kinase antagonist SB 203580 (a generous gift from Dr J.C. Lee25) on the
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Figure 3. Effect of extracellular agonists on the activity of ERK, SAPK, and p38 MAP kinase. A, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis detecting ERK2. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). B, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis detecting p46 SAPK. Activation is identifiable by the appearance of bands with delayed mobility, indicating phosphorylated protein forms (indicated by star). C, Quiescent VSMCs were stimulated with FBS (10%), PDGF (20 ng/mL), TPA (100 nmol/L), anisomycin (500 nmol/L), or Ang II (1027 mol/L) for 10 and 30 minutes. Whole cell lysates were examined by Western blot analysis using an antibody that detects only the phosphorylated form of p38 MAP kinase (MAPK). The intensity of bands correlates to the extent of phosphorylation at the regulatory sites of p38 MAPK.
MKP-1 gene expression to define the role of p38 MAP kinase in anisomycin-induced MKP-1 induction. The antagonist did not affect the PDGF-induced gene expression of MKP-1 (Figure 5A), suggesting that the PDGF-induced expression of MKP-1 is due to an intracellular pathway distinct from the p38 MAP kinase cascade. However, the anisomycin-induced expression of MKP-1 was dose-dependently inhibited after preincubation with SB 203580 (Figure 5B). To examine the role of the ERK cascade in the regulation of the dual-specificity phosphatase MKP-1 in more detail, we performed experiments using the MEK antagonist PD 098059 (a generous gift from Dr A.R. Saltiel26). PD 098059 significantly reduced the PDGF-induced ERK activation in VSMCs as detected by the reduction of the PDGF-induced band shift of ERK and the inhibition of ERK activity assayed in an immunocomplex kinase assay (Figure 6A). Quantification of myelin basic protein phosphorylation by scintillation counting demonstrated a 50% reduction of ERK activity in the presence of PD 098059. As shown in Figure 6B, inhibition of
Figure 4. Effect of ERK-activating stimuli on MKP-1 protein expression and activity. A, Quiescent VSMCs were stimulated with FBS (10%), TPA (100 nmol/L), or Ang II (1027 mol/L) for 30 and 60 minutes. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDSPAGE and Western blot analysis using anti–MKP-1 antiserum and the ECL detection system. The MKP-1 band with an expected size of 39 kDa is indicated. B, Quiescent VSMCs were stimulated with FBS (10%), Ang II (1027 mol/L), or PDGF (20 ng/mL) for 1 hour. MKP-1 immunoprecipitates were incubated with recombinant phosphorylated and active ERK2 (New England Biolabs). ERK activity was assayed by phosphorylation of the GST-fusion protein of Elk1 as described above. The dominant band of '53 kDa (star) correlates to the heavy chain of the antibodies used for immunoprecipitation of MKP-1.
ERK activation by PD 098059 significantly reduced the PDGF-induced gene expression of MKP-1. Based on the effect of the MEK inhibitor and on the effect of cellular stimulation with PDGF, TPA, and Ang II, our data suggest that the ERK cascade induces the expression of MKP-1 in VSMCs. Because MKP-1 is known to dephosphorylate and inactivate ERK, this mode of regulation illustrates a potential mechanism to maintain balanced cell growth through feedback inhibition of ERK in VSMCs.
Discussion A diverse array of extracellular signals utilize MAP kinase signaling cascades to initiate a variety of cell signaling outcomes. The pleiotropic potential of MAP kinases emphasizes the importance of a tight control of their activation. In chromaffin cells (PC12) it was demonstrated that the duration of ERK activation by extracellular stimuli is critical for cell signaling outcomes, because transient activation of MAP kinase induced mitogenesis, whereas sustained activation of MAP kinase induced cell differentiation.27,28 However, in mesangial cells and fibroblasts, only potent mitogens seem to be capable of inducing a sustained phase of ERK activation.21,29 These data emphasize the importance of mechanisms to terminate and control the duration of ERK activity. We19,20 and others11 have demonstrated previously that the downregulation of ERK in NIH3T3 cells and in mesangial cells is dependent on the production of new protein, presumably of the expression of a transcriptionally regulated dual-
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Figure 5. Effect of p38 MAP kinase inhibition on MKP-1 gene expression. A, Quiescent VSMCs were pretreated with the p38 MAP kinase antagonist SB 203580 (10 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with TPA (100 nmol/L), PDGF (20 ng/mL), or anisomycin (500 nmol/L) for the indicated periods. Total RNA was isolated and examined by Northern blot analysis. The blots were probed with MKP-1 cDNA. As a loading control, the same filters were stripped and rehybridized with the GAPDH probe. A representative blot of 3 independent experiments is shown. B, Quiescent VSMCs were pretreated with different concentrations of SB 203580 for 60 minutes before stimulation with anisomycin (500 nmol/L) for 60 minutes. Total RNA was analyzed by Northern blot analysis.
specificity PTPase such as MKP-1. However, this may not be the case in all cell systems. The inactivation of ERK after mitogenic stimulation of PC12 cells, adipose cells (3T3-L1), or endothelial cells (PAE) occurs normally when protein synthesis is inhibited.30,31 We demonstrated in the present study a decline of ERK activity after stimulation with FBS over a period of 6 hours (beginning 30 minutes after stimulation), indicating the action of phosphatase activity toward ERK during this period. Furthermore, FBS induced within 30 minutes a sustained expression of the dual-specificity PTPase MKP-1. Therefore, it is likely that MKP-1, which is known to exhibit catalytic activity toward both regulatory sites on ERK, is involved in ERK downregulation. Moreover, we showed in the present study a prolonged ERK activation in the presence of the protein synthesis inhibitor cycloheximide, emphasizing
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Figure 6. Effect of the MEK antagonist PD 098059 on the PDGF-induced expression of the dual-specificity PTPase MKP-1 in VSMCs. A, Quiescent cells were preincubated with PD 098059 (20 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with PDGF (20 ng/mL) for the indicated periods. The upper panel shows a Western blot analysis detecting p42 ERK (ERK2) as described above (star indicates phosphorylated protein forms), and the lower panel shows ERK activity assayed by the ability of immunoprecipitated ERK to phosphorylate myelin basic protein (MBP). B, Quiescent cells were preincubated with PD 098059 (20 mmol/L) for 60 minutes (1) or untreated (2) before stimulation with PDGF for the indicated periods. Cell lysates were analyzed by immunoprecipitation with anti–MKP-1 polyclonal antibody followed by SDS-PAGE and Western blot analysis using anti–MKP-1 antiserum and the ECL detection system. The band of MKP-1 protein (39 kDa) is indicated. The dominant band of '53 kDa correlates to the heavy chain of the antibodies used for immunoprecipitation. A representative blot of 3 independent experiments is shown.
the necessity of transcriptionally regulated phosphatases, presumably the dual-specificity PTPase MKP-1, for ERK inactivation in VSMCs. These data are consistent with a study in VSMCs in which MKP-1 antisense oligonucleotides were used.14 In this study, a prolonged activation of ERK with no effect on MEK activity after suppression of MKP-1 protein expression was demonstrated.14 Previously we20 have shown that in NIH3T3 fibroblasts, activation of the SAPK cascade, rather than the ERK cascade, induces the expression of MKP-1. Because MKP-1 is known to dephosphorylate and inactivate ERK,11,13–15 this cross-talk of MAP kinase–signaling cascades may contribute to the inhibition of cell growth after activation of SAPK.8,9,32,33 In contrast to NIH3T3 fibroblasts, activation of the ERK cascade in mesangial cells19 and lung fibroblasts34 has been demonstrated to induce the expression of MKP-1, thereby providing a potential mechanism of feedback inhibition.
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MAP Kinases in Induction of MKP-1
Based on these obvious cell line–specific differences in the regulation of MKP-1 expression, we examined the role of multiple MAP kinase–signaling cascades in the regulation of MKP-1 in VSMCs. With the use of extracellular stimuli that activate the ERK rather than the SAPK or p38 MAP kinase cascade, as well as a synthetic inhibitor of MEK activity, our data suggest that activation of the ERK signaling cascade induces MKP-1 expression in VSMCs. This mode of MKP-1 induction might be responsible for the observed decline in ERK activity 30 minutes after stimulation of quiescent VSMCs. Furthermore, we demonstrated the induction of MKP-1 gene expression after activation of stress-responsive MAP kinase pathways such as the SAPK or p38 MAP kinase cascade. These data correlate with findings that the human homologue of MKP-1 was cloned from a cDNA library from human skin fibroblasts treated with hydrogen peroxide and that this gene was inducible by oxidative stress.24 MKP-1 is known to effectively dephosphorylate ERK.13,15 It is therefore reasonable to speculate that VSMC activation of the SAPK/p38 MAP kinase pathways in response to cellular stress induces the expression of the dual-specificity PTPase MKP-1, thereby inhibiting the stimulation of ERK. This cross-talk between these independent signal transduction pathways would be a logical cellular response to agonists activating the SAPK/p38 MAP kinase pathways, since activation of SAPK and p38 MAP kinase inhibits cell growth and induces apoptosis.8,9 Therefore, inhibition of the growth-stimulatory and anti-apoptotic ERK pathway6,9 would contribute to the effects of activated SAPK/p38 MAP kinase pathways in VSMCs. In addition to its catalytic activity toward ERK, MKP-1 was also shown to hydrolyze phosphotyrosine and phosphothreonine on SAPK and p38 MAP kinase in hela cells and in NIH3T3 fibroblasts.15,16 A recent study in leukemic cells suggested MKP-1 to be even more active toward SAPK and p38 MAP kinase than toward ERK.35 The induction of MKP-1 in response to cellular stress in VSMCs may therefore be important for the deactivation of SAPK and p38 MAP kinase as a negative feedback mechanism. However, further studies will be needed to define the substrate specificity of MKP-1 toward different MAP kinases. Interestingly, other dual-specificity PTPases have also been suggested to exhibit distinct specificities toward multiple MAP kinases (eg, PAC1, which failed to inactivate SAPK in hela and NIH3T3 cells despite its activity toward ERK and p38 MAP kinase,15 and Pyst1, which preferentially dephosphorylates ERK36). Therefore, it would not be surprising if unique members of the group of dual-specificity PTPases, such as MKP-1, PAC1, Pyst1, B23,37 MKP-2,38,39 or MKP-3,40,41 were shown to exhibit selective activities toward distinct MAP kinases, thereby introducing further tiers of control of the regulatory networks. A similar selectivity has already been demonstrated for distinct dual-specificity kinases, each phosphorylating a single MAP kinase.
Acknowledgments This work was supported by a grant from the Deutsche Forschungsgemeinschaft BO 1288/3–1 (Dr Bokemeyer) and in part by a BONFOR research grant from the Faculty of Medicine, University of
Bonn. We would like to thank Stephen M. Keyse (Dundee, UK) for the CL100 cDNA, John C. Lee (King of Prussia, Pa) for the p38 MAP kinase antagonist, and Alan R. Saltiel (Ann Arbor, Mich) for the MEK antagonist.
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