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

Jennifer L Duff,Brett P Monia,Bradford C Berk

doi:10.1074/jbc.270.13.7161

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 micrograms/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.

Highlights

From the tDepartment of Biochemistry, Emory University, Atlanta, Georgia 30322, the fJDepartment of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, California 92008, and the §Division of Cardiology, Department of Medicine, University of Washington, Seattle, Washington 98195
Using actinomycin D and antisense oligonucleotides targeted to MAP kinase phosphatase-l (MKP-l), we demonstrate that MKP-l regulates mitogen-activated protein (MAP) kinase in vascular smooth muscle cells (VSMC)
MKP-l antisense oligonucleotides did not affect the activity of MEK indicating that sustained activation of MAP kinase was due to inhibition of MKP·l expression

Summary

To whomcorrespondenceand reprint requests should be addressed

University of Washington, Division of Cardiology, Mail Stop RG-22, Seattle, WA 98195. 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]. Our laboratory recently showed that angiotensin II stimulates rapid expression ofMKP-l in VSMC [23] with a time course that corresponds with the inactivation of MAP kinase. These data suggest that MKP-l may regulate the transient activation of MAP kinases by angiotensin II in vivo. We show that a ntisense oligonucleotides complementary to MKP-l inhibit MKP-l expression and caused a prolonged activation of MAP kinase sugges ting th at MKP -l is t he domina nt MAP kinase phosphatase in vivo

MATERIALS AND METHODS

10 JL10I mM Na

RESULTS

A Co nt ro l