©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitogen-activated Protein (MAP) Kinase Is Regulated by the MAP Kinase Phosphatase (MKP-1) in Vascular Smooth Muscle Cells
EFFECT OF ACTINOMYCIN D AND ANTISENSE OLIGONUCLEOTIDES (*)

(Received for publication, October 3, 1994; and in revised form, November 28, 1994)

Jennifer L. Duff (1) (3) Brett P. Monia (2) Bradford C. Berk (3)(§)

From the  (1)Department of Biochemistry, Emory University, Atlanta, Georgia 30322, the (2)Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008, and the (3)Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Angiotensin II stimulates hypertrophic growth of vascular smooth muscle cells (VSMC) and activates many growth-promoting kinases such as mitogen-activated protein (MAP) kinase. A novel transcriptionally regulated phosphatase, MAP kinase phosphatase-1 (MKP-1), is induced by angiotensin II in VSMC and selectively dephosphorylates MAP kinase in vitro. Using actinomycin D and antisense oligonucleotides targeted to MKP-1, we demonstrate that MKP-1 regulates MAP kinase in VSMC. Both actinomycin D and MKP-1 antisense oligonucleotides inhibited MKP-1 mRNA expression and caused prolonged activation of the p42 and p44 MAP kinases as measured by in-gel-kinase assays and Western blot. For example, MAP kinase activity 120 min after angiotensin II treatment was 30% (range 25-35%), 79%, and 74% of maximum in control, actinomycin D-treated (3 µg/ml, 30 min), and antisense oligonucleotide-treated (300 nM, 6 h) cells, respectively. A sense oligonucleotide was without effect (34%). MKP-1 antisense oligonucleotides did not affect the activity of MEK indicating that sustained activation of MAP kinase was due to inhibition of MKP-1 expression. These findings demonstrate that inactivation of MAP kinase by angiotensin II is mediated predominantly by MKP-1, suggesting an important role for MKP-1 and other related phosphatases in the regulation of MAP kinases in VSMC.


INTRODUCTION

Vascular smooth muscle cell (VSMC) (^1)growth is an important event in the development of atherosclerosis and restenosis following balloon angioplasty. Several data suggest a role for the potent vasoconstrictor angiotensin II in restenosis and VSMC growth. Angiotensin II stimulates hypertrophic growth of cultured VSMC (1) , as well as hyperplastic growth of VSMC from injured vessels (2) and genetically hypertensive rats(3) . Inhibiting angiotensin II effects by blocking production with angiotensin-converting enzyme inhibitors or by receptor antagonism with angiotensin II receptor antagonists prevents neointimal growth in the rat carotid artery injury model(4, 5) . In addition, angiotensin II stimulates many of the same signal transduction events as growth factors including protein tyrosine phosphorylation(6) , stimulation of c-fos(7) , and activation of mitogen-activated protein (MAP) kinases(8, 9) .

MAP kinases are a family of serine/threonine protein kinases activated as an early response to a variety of stimuli involved in cellular growth, transformation, and differentiation (for review see (10) ). Stimulation of the p42 and p44 isoforms of MAP kinase (Erk2 and Erk1, respectively) requires dual phosphorylation on Thr-183 and Tyr-185 residues. A dual specificity protein kinase, MAP kinase kinase or MEK, catalyzes the phosphorylation of MAP kinase (11) and is itself regulated by serine phosphorylation by MEK kinase and/or Raf kinase (12, 13) . Both Raf kinase (6, 14) and a MAP kinase activating activity (MEK) (15) are activated by angiotensin II and may play a role in regulating MAP kinase activity in VSMC. However, angiotensin II stimulation of MAP kinase phosphorylation and activation is a transient event with peak activation seen at 5 min and return to baseline by 60-120 min(8, 9) . Therefore, an additional level of MAP kinase regulation must also occur through dephosphorylation and inactivation by a protein phosphatase.

A novel dual specificity protein phosphatase, 3CH134, was found to dephosphorylate tyrosine-phosphorylated MAP kinase selectively in vitro, 15-200-fold more rapidly than other tyrosine-phosphorylated substrates examined(16) . 3CH134 was expressed in COS cells and shown to selectively dephosphorylate and inactivate MAP kinase stimulated by serum, Ras, or Raf(17) . 3CH134 was, therefore, renamed MKP-1 for MAP kinase phosphatase-1 (17) and belongs to a family of vaccinia virus-like phosphatases such as CL100(18) , PAC-1(19) , erp(20) , and hVH1(21) . CL100 (22) and hVH1 (21) were also shown to dephosphorylate and inactivate MAP kinases that had been activated in vitro with MEK. Constitutive expression of the immediate early gene erp, which has a sequence identical with MKP-1, has a negative effect on fibroblast proliferation(20) . However, under normal conditions, MKP-1 appears to be absent and is regulated by transcription with transient induction of gene expression. Our laboratory recently showed that angiotensin II stimulates rapid expression of MKP-1 in VSMC (23) with a time course that corresponds with the inactivation of MAP kinase. These data suggest that MKP-1 may regulate the transient activation of MAP kinases by angiotensin II in vivo.

To determine if MKP-1 is the dominant MAP kinase phosphatase in vivo, we used antisense oligonucleotides to block MKP-1 expression in VSMC. Antisense oligonucleotides have proven to be a powerful tool to investigate the function of several signal transduction proteins such as Ras(24) , protein kinase C(25) , and c-Myc(26) . Due to the transcriptional regulation and the short half-life of MKP-1 mRNA and protein, MKP-1 is an ideal target molecule for antisense strategies. In the present study, we show that antisense oligonucleotides complementary to MKP-1 inhibit MKP-1 expression and caused a prolonged activation of MAP kinase suggesting that MKP-1 is the dominant MAP kinase phosphatase in vivo.


MATERIALS AND METHODS

Cell Culture

VSMC were isolated from 200-250-g male Sprague-Dawley rats and maintained in 10% calf serum/Dulbecco's modified Eagle's medium (DMEM) as described previously(23) . Passage 5 to 13 VSMC at 70-80% confluence were growth-arrested by incubation in 0.1% calf serum/DMEM for 48 h prior to use.

Oligonucleotide Synthesis

Phosphorothioate oligonucleotides were synthesized and purified as described previously(24, 27) .

Oligonucleotide Treatment

Oligonucleotides were added to the cells at a concentration of 100-1000 nM in Opti-MEM containing DOTMA/DOPE solution (Lipofectin^R) (Life Technologies, Inc.) at a concentration of 2.5 µg/ml when 100 nM DNA was added. After 6 h, the medium was removed, 0.1% calf serum/DMEM was added, and cells were allowed to recover for 30 min prior to stimulation by angiotensin II.

Northern Blot Analysis

Total RNA was extracted from VSMC by the guanidine isothiocyanate-cesium chloride gradient procedure, and Northern blot analysis was performed as described previously(23) . Radiolabeling of the probes, MKP-1 or glyceraldehyde-3-phosphate dehydrogenase, and hybridization on Nytran membranes were also performed as described previously(23) . Densitometric analysis of autoradiograms exposed in the linear range of film density was made on a LaCie scanner using NIH image software.

MAP Kinase Activity Assay

Quiescent VSMC treated with oligonucleotides were stimulated with or without 100 nM angiotensin II for various time periods then harvested for MAP kinase activity using an in-gel-kinase assay as described previously(28) . Cells were lysed in buffer containing 50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na(3)VO(4), 10 mM HEPES, pH 7.4, 0.1% Triton X-100, 500 µM phenylmethanesulfonyl fluoride, and 10 µg/ml leupeptin, then flash-frozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells were scraped off the dish and centrifuged at 14,000 rpm (4 °C for 30 min), and protein concentrations were determined using the Bradford protein assay (Bio-Rad). Equal amounts of protein (5-10 µg) were separated by SDS-polyacrylamide gel electrophoresis through a gel containing 0.4 mg/ml myelin basic protein (MBP). The gel was then incubated twice in buffer A (50 mM HEPES, pH 7.4, and 5 mM beta-mercaptoethanol) containing 20% isopropyl alcohol for 30 min, once in buffer A for 1 h, twice in buffer A containing 6 M guanidine HCl for 30 min, twice in buffer A containing 0.04% Tween 20 at 4 °C for 16 h and 2 h, once in buffer A containing 100 µM Na(3)VO(4) and 10 mM MgCl(2) at 30 °C for 30 min, and once in buffer A containing 100 µM Na(3)VO(4), 10 mM MgCl(2), 50 µM ATP, and 50 µCi of [-P]ATP for 1 h at 30 °C. The reaction was terminated by washing the gel 5-8 times in fixative solution containing 10 mM sodium pyrophosphate and 5% trichloroacetic acid for 15 min. The gel was dried and subjected to autoradiography.

MAP Kinase Western Blot Analysis

Quiescent VSMC treated with oligonucleotides were stimulated with or without 100 nM angiotensin II for various time periods and then harvested as described above for the MAP kinase activity assay. Equal amounts of protein (20 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes, then Western blot analysis was performed using polyclonal Erk1 and Erk2 antibodies (Santa Cruz Biotechnology). After incubation with horseradish peroxidase-conjugated secondary antibody, the blots were developed using enhanced chemiluminescence (Amersham).

MEK Activity Assay

MEK activity was assayed according to modifications of procedures previously described by Ohmichi et al.(29) and Pang et al.(30) . Growth-arrested VSMC were treated with or without oligonucleotides for 6 h as indicated above and then treated with angiotensin II for various times. Cells were washed in ice-cold phosphate-buffered saline, lysed in 400 µl of buffer containing 10 mM Tris, pH 7.5, 70 mM NaCl, 1% Triton X-100, 50 mM beta-glycerophosphate, 100 µM Na(3)VO(4), and 4 µg/ml leupeptin, then immediately flash-frozen in a dry ice/ethanol bath. The cell extracts were thawed on ice, scraped, sonicated for 5 s, then centrifuged at 14,000 rpm at 4 °C for 30 min. Extracts were loaded onto a DE52 ion exchange column (500 µl bed volume) equilibrated in buffer containing 10 mM Tris, pH 7.5, 70 mM NaCl, and 50 mM beta-glycerophosphate. The flow-through fraction containing MEK was collected and bound proteins, which include MAP kinase, were eluted by addition of 400 µl of equilibration buffer containing 500 mM NaCl. The flow-through was assayed for its ability to phosphorylate an agarose-bound glutathione S-transferase fusion protein containing a catalytically inactive MAP kinase (GST-(K71A)Erk1) (Kinetek Biotechnology Corp.). Lysate protein (20 µg) was incubated in a total volume of 150 µl containing 50 mM Tris, pH 7.5, 2 mM EGTA, 10 mM MgCl(2), 40 µM ATP, 2 µCi of [-P]ATP, and 5 µg of GST-(K71A)Erk1 for 20 min at 30 °C. The reaction was stopped by the addition of 200 µl ice-cold buffer containing 50 mM Tris, pH 7.5, 2 mM EGTA, and 10 mM MgCl(2), and the beads were washed and pelleted twice by brief centrifugation at 2,000 rpm. 6 times SDS sample buffer was added, and the samples were separated by 10% SDS-polyacrylamide gel electrophoresis. The gel was then dried and subjected to autoradiography.


RESULTS

Actinomycin D Inhibits MKP-1 Expression and Sustains MAP Kinase Activity

We have previously demonstrated that angiotensin II rapidly induces MKP-1 mRNA in VSMC with peak expression at 30 min(23) . Induction of MKP-1 mRNA expression is a transient event with a return to baseline 2 h after angiotensin II treatment. Actinomycin D was used to inhibit angiotensin II-induced transcription and block the production of MKP-1 mRNA. Pretreatment of cells with 3 µg/ml actinomycin D completely blocked the angiotensin II-induced expression of MKP-1 mRNA (Fig. 1). Inhibition of transcription with actinomycin D also caused sustained activation of angiotensin II-stimulated MAP kinases as detected by in-gel-kinase assays (Fig. 2A). Because the activities of the p42 and p44 isoforms of MAP kinase were prolonged to the same extent, we combined the values of both p42 and p44 and determined the percent of maximum based on the peak activity at 5 min (Fig. 2B). The activity of the p42 and p44 MAP kinases 120 min after angiotensin II treatment returned to 25% of maximum in the control cells (Fig. 2B), whereas treatment with actinomycin D sustained the activity of MAP kinases after 120 min to 79% of maximum, suggesting that a transcriptionally regulated protein or proteins are involved in MAP kinase inactivation. To examine the phosphorylation state of MAP kinase after actinomycin D treatment, Western blot analysis was performed which detects a slower migrating form of MAP kinase that is due to phosphorylation. Phosphorylation of the p42 and p44 MAP kinases was prolonged when VSMC were treated with actinomycin D as detected by the persistence of the ``band shift'' 240 min after angiotensin II stimulation (Fig. 2C).


Figure 1: Effect of actinomycin D on MKP-1 expression. VSMC were pretreated with or without 3 µg/ml actinomycin D for 30 min, then stimulated with 100 nM angiotensin II for the indicated time points. Total RNA was prepared, and 5 µg was examined by Northern blot analysis. Nytran membranes were hybridized with a P-labeled MKP-1 probe and then rehybridized with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe.




Figure 2: Effect of actinomycin D on MAP kinase activity and phosphorylation. A, activity. VSMC were pretreated with 3 µg/ml actinomycin D for 30 min then stimulated with 100 nM angiotensin II for the indicated time points. Cells were harvested in lysis buffer as described under ``Materials and Methods,'' and in-gel-kinase assays were performed using myelin basic protein incorporated in the gel. B, MAP kinase assays (n = 2) as described in A were averaged and calculated as the percent maximum based on the peak activation at 5 min. C, phosphorylation. VSMC were treated as in A. Cells were harvested in lysis buffer as described under ``Materials and Methods,'' and Western blot analysis was performed using Erk1 and Erk2 antibodies (Santa Cruz).



Inhibition of MKP-1 mRNA Expression by Antisense Oligonucleotides

To specifically inhibit the expression of MKP-1, several antisense oligonucleotides corresponding to different regions of the MKP-1 cDNA were synthesized (Table 1). The oligonucleotides were made as phosphorothioate derivatives which have been shown to enhance nuclease resistance and support RNase H cleavage of hybridizing RNA(27) . Using the program OLIGO(25) , we designed 11 oligonucleotides, each 20 bases in length, that had the highest melting temperature (T(m)) possible without being self-complementary. Each oligonucleotide was then tested for its ability to inhibit the expression of MKP-1 mRNA after angiotensin II treatment. VSMC were pretreated for 3 h with oligonucleotides at a concentration of 200 nM in serum-free Opti-MEM containing the cationic lipid Lipofectin. Lipofectin has previously been shown to improve antisense oligonucleotide uptake and prevent degradation in cultured cells(31) . After oligonucleotide uptake, cells were stimulated for 30 min with angiotensin II, and Northern blot analysis was performed to measure the steady state levels of MKP-1 mRNA. As shown in Fig. 3, the ability of the oligonucleotides to inhibit MKP-1 mRNA expression varied greatly. Oligonucleotides ISIS 8149 and ISIS 8153-8159 were very effective at inhibiting MKP-1 mRNA expression, yet varied in potency with ISIS 8155 being the most potent at 90 ± 9% inhibition. Oligonucleotides ISIS 8150, ISIS 8151, and ISIS 8152 were of minimal or no efficacy; showing only -15-20% inhibition. These results agree with several other investigators who found that the 3`-untranslated region is an effective region to target for antisense strategies(25, 26, 27) . Lipofectin alone had no effect on angiotensin II induction of MKP-1 mRNA (data not shown).




Figure 3: Effect of antisense oligonucleotides on MKP-1 expression. VSMC were pretreated for 3 h with 200 nM oligonucleotide in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min then stimulated with 100 nM angiotensin II for 30 min. Total RNA was prepared, and 5 µg was examined by Northern blot analysis. Nytran membranes were hybridized with a P-labeled MKP-1 probe and then rehybridized with a glyceraldehyde-3-phosphate dehydrogenase probe. As a correction for differences in loading, the signal intensity of each RNA sample hybridized to the MKP-1 probe was divided by that hybridized to glyceraldehyde-3-phosphate dehydrogenase probe. Values are expressed as the percent inhibition based on the positive control (angiotensin II stimulation for 30 min without oligonucleotide treatment).



We next examined the concentration response relationship of ISIS 8149 and the most effective oligonucleotide, ISIS 8155. As shown in Fig. 4, A and B, both oligonucleotides ISIS 8149 and ISIS 8155 caused a concentration-dependent inhibition of MKP-1 mRNA expression. The time course for the inhibition of MKP-1 mRNA expression is shown in Fig. 4C. Antisense oligonucleotide ISIS 8155 completely blocked the induction of MKP-1 mRNA for up to 120 min after angiotensin II addition. To determine the time during which antisense oligonucleotide inhibition persisted, cells were allowed to recover from oligonucleotide and Lipofectin treatment for 18, 24, or 48 h, then angiotensin II was added for 30 min, and mRNA was harvested for Northern blot analysis. As shown in Fig. 4D, antisense oligonucleotide ISIS 8155 was still effective at inhibiting MKP-1 mRNA expression after 24 h; however, by 48 h, ISIS 8155 was no longer effective at inhibiting MKP-1 expression in response to angiotensin II.


Figure 4: Dose response and time course of antisense oligonucleotide inhibition of MKP-1 mRNA expression. A, dose response. VSMC were pretreated for 6 h with or without the indicated concentrations of oligonucleotides ISIS 8155 or ISIS 8149 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min then stimulated with 100 nM angiotensin II for 30 min. Total RNA was prepared, and 5 µg was examined by Northern blot analysis as in Fig. 3. B, normalization of dose response. As a correction for differences in loading, the signal intensity of each RNA sample hybridized to the MKP-1 probe was divided by that hybridized to the glyceraldehyde-3-phosphate dehydrogenase probe. C, time course. VSMC were pretreated for 6 h with or without 300 nM oligonucleotide ISIS 8155 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min, then stimulated with 100 nM angiotensin II for the indicated time points. Total RNA was prepared, and 5 µg was examined by Northern blot analysis as in Fig. 3. D, recovery time from antisense. VSMC were pretreated for 6 h with or without 300 nM oligonucleotide ISIS 8155 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 18, 24, or 48 h, then stimulated with 100 nM angiotensin II for 30 min. Total RNA was prepared, and 5 µg was examined by Northern blot analysis as in Fig. 3.



A sense oligonucleotide (ISIS 9245) and a scrambled (ISIS 9244) oligonucleotide corresponding to ISIS 8155 were also synthesized and tested for their ability to inhibit MKP-1 expression. MKP-1 mRNA expression was partially inhibited by the sense oligonucleotide ISIS 9245 (50 ± 27% inhibition at 200 nM); however, comparison of the sequence of the sense oligonucleotide to the MKP-1 cDNA sequence showed a region where 9 of 11 bases were complementary, suggesting that ISIS 9245 may have been binding as an antisense oligonucleotide to MKP-1 mRNA. In contrast, the scrambled oligonucleotide, ISIS 9244, had no significant effect (28 ± 13% inhibition).

Sustained Activation and Phosphorylation of MAP Kinases by MKP-1 Antisense Oligonucleotides

To examine the effect of inhibiting MKP-1 expression on MAP kinase activity, in-gel-kinase assays were performed on lysates from control and oligonucleotide-treated VSMC. Angiotensin II stimulated the activity of p42 and p44 MAP kinases in control cells with the time course shown in Fig. 2A(8) . Peak activity was seen at 5 min with a return to near baseline by 120 min. Antisense oligonucleotide ISIS 8155 caused a sustained activation of the p42 and p44 MAP kinases (Fig. 5A). MAP kinase activity 120 min after angiotensin II stimulation (normalized to the maximum activity 5 min after angiotensin II stimulation) was 74 ± 5% of maximum in antisense oligonucleotide-treated cells and 35 ± 7% of maximum in control cells (Fig. 5B). The sense oligonucleotide ISIS 9245 did not significantly sustain MAP kinase activity which was 34 ± 2% of the maximum 120 min after angiotensin II treatment (Fig. 5C).


Figure 5: Inhibition of MKP-1 expression sustains MAP kinase activity. A, VSMC were pretreated for 6 h with 300 nM antisense oligonucleotide ISIS 8155 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min, then stimulated with 100 nM angiotensin II for the indicated time points. Cells were harvested in lysis buffer as described under ``Materials and Methods,'' and in-gel-kinase assay was performed using myelin basic protein (MBP) incorporated in the gel. B, MAP kinase assays (n = 3-6) as described above were averaged and calculated as the percent maximum based on the peak activation at 5 min. C, VSMC were pretreated for 6 h with 300 nM antisense oligonucleotide ISIS 8155 or sense oligonucleotide 9245 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min, then stimulated with 100 nM angiotensin II for 120 min. In-gel-kinase assay was performed as described in A. Assays (n = 3) were averaged and calculated as the percent maximum based on the peak activation at 5 min.



The specificity of p42 and p44 MAP kinases as substrates for MKP-1 was shown by the observation that two unregulated kinases at 58 kDa and 110 kDa were not affected by antisense oligonucleotide treatment (Fig. 5A). However, two other MBP kinases that are regulated by angiotensin II, at 85 kDa and 65 kDa, were also sustained by MKP-1 antisense oligonucleotides (Fig. 5A and after prolonged exposure for 3 h).

To examine the phosphorylation state of MAP kinase after antisense oligonucleotide treatment, Western blot analysis was performed. Antisense oligonucleotide ISIS 8155 sustained MAP kinase phosphorylation as shown by persistence of the band shift still present 60 min after angiotensin II treatment (data not shown).

Effect of Sustained Activation of MAP Kinase on MEK

To determine if there was sustained activation of the upstream kinase, MEK, that contributed to the sustained activation of MAP kinase, MEK activity was studied. To assay MEK activity, MEK was partially purified from control and antisense oligonucleotide-treated cells and, activity was measured by phosphorylation of catalytically inactive MAP kinase (GST-(K71A)Erk1). Angiotensin II rapidly and transiently stimulated the activity of MEK in control cells with peak activation seen at 5 min and return to baseline by 20 min (Fig. 6, A and B). Antisense oligonucleotide treatment with ISIS 8155 caused a small decrease in peak MEK activity that was not significant, indicating that the sustained activation of MAP kinase was primarily due to the loss of MKP-1.


Figure 6: Effect of MKP-1 inhibition on MEK activity. A, VSMC were pretreated for 6 h with or without 300 nM antisense oligonucleotide ISIS 8155 in Opti-MEM containing Lipofectin. Cells were allowed to recover in 0.1% calf serum/DMEM for 30 min, then stimulated with 100 nM angiotensin II for the indicated time points. Cells were harvested in lysis buffer as described under ``Materials and Methods,'' and endogenous MAP kinase was removed by binding to a DE52 ion exchange column. MEK activity was measured by incubating lysates with a catalytically inactive GST-(K71A)Erk1 fusion protein in the presence of [-P]ATP. After 20 min, the reaction was terminated with 6 times SDS sample buffer, and phosphorylated GST-(K71A)Erk1 was resolved by SDS-polyacrylamide gel electrophoresis. B, MEK assays (n = 3) as described above were averaged and calculated as the percent maximum based on the peak activation at 5 min.




DISCUSSION

MKP-1 is a dual specificity phosphatase that selectively dephosphorylates MAP kinases in vitro(16, 17, 21, 22) . In the present study, we have demonstrated that MKP-1 regulates the dephosphorylation and inactivation of MAP kinases in angiotensin II-stimulated VSMC. Data that support an essential role for MKP-1 in VSMC include the following. 1) The time course for angiotensin II-mediated induction of MKP-1 mRNA expression correlates with the time course for the inactivation of angiotensin II-stimulated MAP kinases (23) . 2) Inhibiting transcription with actinomycin D causes prolonged activation of the p42 and p44 MAP kinases, suggesting that inactivation of MAP kinase is mediated by a transcriptionally regulated protein phosphatase(s). 3) Specific inhibition of MKP-1 with antisense oligonucleotides also causes prolonged activation of the p42 and p44 MAP kinases, while a control sense oligonucleotide showed no effect on MAP kinase activity. 4) Prolonged activation of MAP kinase was primarily due to inhibition of MKP-1 rather than stimulation of MEK, because MEK activity was not changed in VSMC treated with MKP-1 antisense oligonucleotides.

Regulation of MAP kinases in VSMC, therefore, not only occurs at the level of phosphorylation but also at the level of transcription since inactivation requires new gene expression. Many of the kinases involved in signal transduction are primarily regulated by phosphorylation cascades. However, there are several examples which suggest that kinases may also be regulated at the level of transcription. For example, Webster et al.(32) showed that the activity of a novel kinase, Sgk, is regulated transcriptionally by glucocorticoids and serum in fibroblasts. On the other hand, protein phosphatases, which are known to be regulated by post-translational events such as phosphorylation or binding to regulatory molecules(33) , may also be regulated by transcription. The family of phosphatases that include MKP-1 are all regulated at the level of transcription. Recently, another dual specificity phosphatase named PAC1, which exhibits significant amino acid sequence similarity with MKP-1 and CL100, was shown to have stringent substrate specificity toward MAP kinase in vitro(19, 34) . Thus, other more distantly related members of the MKP-1 phosphatase family, that are also transcriptionally regulated, may regulate MAP kinase and its related kinase family members such as p38 (35) and stress-activated protein kinases(36) . Several candidate MKP-1 homologues have recently been described that appear to be widely expressed suggesting that transcriptional regulation of protein kinases is important in MAP kinase signal transduction(37) .

The present study also suggests that other kinases besides p42 and p44 MAP kinase may be regulated by MKP-1 transcription. Two MBP kinases (65 kDa and 85 kDa) showed prolonged activity in cell lysates treated with MKP-1 antisense oligonucleotides (Fig. 5A). The 85-kDa MBP kinase was also activated in actinomycin D-treated VSMC lysates, further indicating that this kinase is regulated by transcription. The high substrate specificity of MKP-1, CL100, and hVH1 toward p42 and p44 MAP kinases was shown in vitro using several different phosphorylated substrates(16, 21, 22) . Transfection of MKP-1 into COS cells causes a specific decrease in p42 MAP kinase tyrosine phosphorylation as detected by Western blot(17) . However, because all phosphoproteins may not be detected by antiphosphotyrosine antibodies (38) , the MKP-1-regulated proteins detected in this study may also serve as substrates for MKP-1. Future work will be required to determine the extent to which these kinases are regulated by induction of MKP-1.

Several insights regarding the role of MEK activity in regulating MAP kinase may be inferred from the present study. 1) Increases in MEK activity correlate temporally with the activation of MAP kinase in angiotensin II-stimulated VSMC. 2) Decreases in MEK activity also correlate temporally with inactivation of MAP kinase suggesting that the MEK phosphatase and MKP-1 are regulated in parallel. MEK activity is regulated by serine phosphorylation by upstream kinases such as Raf-1 or MEK kinase(12, 13) . The in vivo MEK phosphatase is unknown; however, the serine/threonine protein phosphatase 2A has been shown to dephosphorylate and inactivate MEK in vitro(39) . 3) Finally, our results indicate that the regulation of MAP kinase activity is tightly controlled by both the upstream kinase, MEK, and the downstream phosphatase, MKP-1.

In conclusion, this study demonstrates that MKP-1 regulates the activity of the p42 and p44 MAP kinases in VSMC after angiotensin II stimulation. MAP kinases are known to be regulated by post-translational phosphorylation events via MEK; however, our results indicate that MAP kinases are also regulated by transcription events involving a MAP kinase specific phosphatase (MKP-1). Transcription of MKP-1 may be important in determining the effect of angiotensin II on growth of VSMC which is characterized by cell hypertrophy rather than cell hyperplasia(1) . The rapid and transient activation of MAP kinase in angiotensin II-stimulated VSMC correlates with the results of several other investigators who reported that nonmitogenic stimuli, such as nerve growth factor and thrombin-mimicking peptide, cause transient activation of MAP kinase, while mitogenic stimuli, such as epidermal growth factor and alpha-thrombin, cause sustained, biphasic activation of MAP kinase(40, 41) . Thus, the induction of MKP-1 may represent a new mechanism by which angiotensin II regulates VSMC growth.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: University of Washington, Division of Cardiology, Mail Stop RG-22, Seattle, WA 98195. Tel.: 206-685-6960; Fax: 206-616-1580 or 206-543-3169.

(^1)
The abbreviations used are: VSMC, vascular smooth muscle cell; MAP kinase, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; MEK, MAP kinase/Erk kinase; MKP-1, mitogen-activated protein kinase phosphatase; Lipofectin, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE); MOPS, 3-(N-morpholino)propanesulfonic acid; MBP, myelin basic protein; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We thank Lester F. Lau (Dept. of Genetics, University of Illinois) for the MKP-1 cDNA, Steven L. Pelech and David L. Charest (Kinetek Biotechnology Corp., University of British Columbia) for the GST-(K71A)Erk1 fusion protein, Jordan E. Wilson for assistance with the Western blots, and members of the laboratory for critical reading of the manuscript.


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