Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes

doi:10.1074/jbc.273.24.15249

Multiple Signal Transduction Pathways Link Na⁺/K⁺-ATPase to Growth-related Genes in Cardiac Myocytes

THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES*

From the ^‡Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43614 and the ^§Division of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215

Abstract

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na⁺/K⁺-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes,i.e. those for c-Fos, skeletal α-actin, atrial natriuretic factor, and the α₃-subunit of Na⁺/K⁺-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal α-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) α₃ repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca²⁺, and were attenuated by a Ca²⁺/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na⁺/K⁺-ATPase share early segments involving Ca²⁺ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca²⁺ through different mechanisms.

Footnotes

↵* This work was supported by National Institutes of Health Grant HL-36573 awarded by the National Heart, Lung and Blood Institute, United States Public Health Service, Department of Health and Human Services; by a grant-in-aid from the American Heart Association; and by funds contributed in part by the American Heart Association, Ohio-West Virginia Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: Dept. of Pharmacology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4182; Fax: 419-383-2871; E-mail:xie{at}opus.mco.edu.
↵1 The abbreviations and trivial names used are: MAPK, mitogen-activated protein kinase; ANF, atrial natriuretic factor; ERK, extracellular signal-regulated protein kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpi-perazine dihydrochloride; HA1004,N-(2-guanidinoethyl)-5-iso-quinolinesulfonamide hydrochloride; MEK, MAPK kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; skACT, skeletal α-actin; Raf, Raf-1 kinase; W-7,N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride.
↵2 P. Kometiani, J. Li, A. Askari, and Z. Xie, unpublished observations.
- Received February 6, 1998.
- Revision received March 30, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.