(Received for publication, October 3, 1994; and in revised form, November 28, 1994)
From the
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.
Vascular smooth muscle cell (VSMC) ()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.
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).
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).
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).
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
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.
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 -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.