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(Circulation Research. 2003;93:1026.)
© 2003 American Heart Association, Inc.

Reports

Role of 14-3-3-Mediated p38 Mitogen-Activated Protein Kinase Inhibition in Cardiac Myocyte Survival

Shaosong Zhang, Jie Ren, Cindy E. Zhang, Ilya Treskov, Yibin Wang, Anthony J. Muslin

From the Center for Cardiovascular Research, Departments of Medicine (S.Z., J.R., C.E.Z., I.T., A.J.M.) and Cell Biology and Physiology (S.Z., J.R., C.E.Z., A.J.M.), Washington University School of Medicine, St Louis, Mo; Department of Anesthesiology and Medicine (Y.W.), David Geffen School of Medicine, UCLA, Los Angeles, Calif.

Correspondence to Anthony J. Muslin, Center for Cardiovascular Research, Box 8086, Washington University School of Medicine, 660 S Euclid Ave, St Louis, MO 63110. E-mail amuslin{at}im.wustl.edu

Abstract

14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic, and checkpoint control pathways. Targeted expression of dominant-negative 14-3-3 (DN-14-3-3) to murine postnatal cardiac tissue potentiates Ask1, c-jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) activation. DN-14-3-3 mice are unable to compensate for pressure overload, which results in increased mortality, dilated cardiomyopathy, and cardiac myocyte apoptosis. To evaluate the relative role of p38 MAPK activity in the DN-14-3-3 phenotype, we inhibited cardiac p38 MAPK activity by pharmacological and genetic methods. Intraperitoneal injection of SB202190, an inhibitor of p38 and p38ß MAPK activity, markedly increased the ability of DN-14-3-3 mice to compensate for pressure overload, with decreased mortality. DN-14-3-3 mice were bred with transgenic mice in which dominant-negative p38 (DN-p38) or dominant-negative p38ß (DN-p38ß) MAPK expression was targeted to the heart. Compound transgenic DN-14-3-3/DN-p38ß mice, and to a lesser extent compound transgenic DN-14-3-3/DN-p38 mice, exhibited reduced mortality and cardiac myocyte apoptosis in response to pressure overload, demonstrating that DN-14-3-3 promotes cardiac apoptosis due to stimulation of p38 MAPK activity.

Key Words: signal transduction • apoptosis • cardiac • 14-3-3 protein • p38 mitogen-activated protein kinase

14-3-3 proteins are intracellular phosphoserine-binding adapter molecules.^1,2 14-3-3 proteins modulate several important signal transduction, apoptotic, and checkpoint control pathways by binding to many signaling proteins, including the protein kinases Ask1³ and Raf-1.⁴ Ask1 is a mitogen-activated protein kinase kinase kinase (MAPKKK) that participates in the c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) pathways.⁵ 14-3-3 binding to Ask1 inhibits its enzymatic activity.^3,6

Previously, we investigated the biological effects of 14-3-3 proteins in cultured cells and in transgenic animals by use of dominant-negative forms of 14-3-3 (DN14-3-3).⁶ DN-14-3-3-transfected fibroblasts exhibited increased Ask1, p38 MAPK, and JNK activities. Inhibition of 14-3-3 activity also caused cultured cells to become sensitized to proapoptotic stimuli, including UV irradiation and serum deprivation. The proapoptotic effect of DN-14-3-3 in fibroblasts was blocked by treatment with the p38 MAPK inhibitor SB202190.

Transgenic mice were generated with cardiac-specific overexpression of DN-14-3-3.⁶ These mice appeared normal at baseline but were unable to compensate for pressure overload induced by transverse aortic constriction (TAC), a mild stimulus of cardiac myocyte apoptosis. DN-14-3-3 mice tolerated TAC poorly and most animals died from overwhelming cardiac dysfunction. TUNEL studies revealed that there was massive cardiac myocyte apoptosis in DN-14-3-3 ventricular tissue but not in nontransgenic cardiac tissue after TAC. To evaluate the importance of p38 MAPK activation in the DN-14-3-3 phenotype, we inhibited cardiac p38 MAPK activity by pharmacological and genetic methods in the present work.

Materials and Methods

Transgenic mice with cardiac-specific expression of DN-14-3-3 were evaluated in this work.⁶ DN-14-3-3 mice were subjected to pressure overload by TAC in the presence or absence of intraperitoneal administration of SB202190, 5 mg · kg^-1 · d^-1, a chemical inhibitor of p38 and p38ß MAPK. Next, DN-14-3-3 mice were bred with transgenic mice in the same strain that had cardiac-specific expression of dominant-negative forms of p38 MAPK (DN-p38) or p38ß MAPK (DN-p38ß) to generate compound transgenic DN-14-3-3/DN-p38 or DN-14-3-3/DN-p38ß mice.⁷ Compound transgenic mice were also subjected to pressure overload by TAC, and survival, MAPK activation, and apoptosis were assayed by conventional methods.

An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.

Results

An experimental protocol to administer SB202190, a specific inhibitor of p38 and p38ß MAPK, was established in DN-14-3-3 transgenic mice. In this method, SB202190 was delivered daily by intraperitoneal injection, beginning 3 days before TAC. After 10 days of administration (7 days after TAC), ventricular tissue was isolated and cytosolic protein lysates were generated. Anti-p38 MAPK immunoprecipitates from these lysates were produced with an antibody that recognizes both p38 and p38ß MAPK. Immunoprecipitations were assayed for p38 MAPK enzymatic activity by in vitro kinase assay with recombinant ATF-2 protein used as a substrate. In vitro kinase assays demonstrated that daily administration of 5 mg/kg of SB202190 inhibited p38 MAPK activity in cardiac tissue (Figure 1A). In addition, analysis of protein lysates by anti-phospho-HSP27 immunoblotting demonstrated that SB202190 treatment blocked cardiac phosphorylation of HSP27, a well-defined substrate of p38 MAPK (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of SB202190 administration on signaling and survival after TAC. A, Systemic administration of SB202190 inhibits cardiac p38 kinase activity. Mice were treated with SB202190, 5 mg · kg-1 · d-1, for 10 days by intraperitoneal injection. Anti-p38 MAPK immunoprecipitates obtained from ventricular lysates were evaluated by in vitro kinase assay with recombinant ATF-2 protein used as a substrate (top). Parallel samples of ventricular lysates were analyzed by anti-pan p38 MAPK immunoblotting to demonstrate that equal amounts of protein were used to generate the immunoprecipitates. B, Kaplan-Meier survival curves for DN-14-3-3 transgenic mice subjected to TAC and treated with SB202190 or vehicle control. Daily intraperitoneal injections of SB202190 (or vehicle control) were administered to DN-14-3-3 mice for 3 days before TAC and were continued for 7 days after TAC.

To determine whether inhibition of p38 MAPK activity could block the DN-14-3-3 phenotype, we performed a series of TAC experiments. DN-14-3-3 mice that were injected with control buffer did not tolerate TAC and only 21% of animals survived (6 of 29) for 7 days after the surgical procedure. In contrast, 82% of animals (9 of 11) treated with SB202190 for 3 days before TAC survived for at least 7 days after surgery (Figure 1B). The difference in survival observed in SB202190-treated DN-14-3-3 mice was significant by the Kaplan-Meier log-rank test (²=11.0, P=0.0009).

To specifically determine which p38 MAPK isoform was important for the abnormal response of DN-14-3-3 mice to TAC, transgenic mice with cardiac-specific expression of DN-p38 and DN-p38ß were bred with DN-14-3-3 mice.⁷ DN-14-3-3 protein levels were not altered in compound transgenic DN-14-3-3/DN-p38 and DN-14-3-3/DN-p38ß compared with single transgenic DN-14-3-3 mice (Figure 2). We previously demonstrated that DN-p38 mice exhibit reduced p38 MAPK activation and that DN-p38ß transgenic mice exhibit reduced p38ß MAPK activation in cardiac tissue.⁷

View larger version (43K): [in this window] [in a new window]   Figure 2. A, DN-14-3-3 protein levels in cardiac tissue from single transgenic DN-14-3-3 mice or from compound transgenic DN-14-3-3/DN-p38 or DN-14-3-3/DN-p38ß mice. The 14-3-3 transgenic construct contained a myc-1 epitope tag. Cytosolic lysates derived from left ventricular tissue were analyzed by immunoblotting with an anti-myc-1 epitope primary antibody (top) and an anti-pan-14-3-3 primary antibody (middle). Bottom, anti-ERK immunoblot to control for protein loading. B, Kaplan-Meier survival curves for DN-14-3-3 single transgenic, DN-14-3-3/DN-p38 double transgenic, or DN-14-3-3/DN-p38ß double transgenic mice subjected to TAC.

TAC experiments were performed on transgenic mice and, in each case, compound transgenic mice were compared with single transgenic DN-14-3-3 littermates and nontransgenic littermates. As expected, single transgenic DN-14-3-3 mice were intolerant to pressure overload and only 6% of mice (1 of 17) survived for 7 days after TAC. In contrast, DN-14-3-3/DN-p38ß mice were completely resistant to TAC-induced death and 100% of animals (10 of 10) survived. The difference in survival observed in compound transgenic DN-14-3-3/DN-p38ß mice compared with single transgenic DN-14-3-3 mice was significant by the log-rank test (²=16.6, P<0.0001). DN-14-3-3/DN-p38 mice were partially resistant to TAC-induced death and 60% of animals (9 of 15) mice survived (Figure 2). The difference in survival observed in compound transgenic DN-14-3-3/DN-p38 mice compared with DN-14-3-3 mice was significant by analysis with the log-rank test (²=7.1, P=0.008).

p38 MAPK promotes apoptosis in certain cell types and this may be due to direct phosphorylation of p53 or other effectors. In response to pressure overload, we previously demonstrated that DN-14-3-3 mice develop overwhelming cardiac myocyte death.⁶ Apoptosis in cardiac tissue was assessed in the present study by caspase-3 cleavage assays that confirmed that both DN-14-3-3/DN-p38ß and DN-14-3-3/DN-p38 cardiac tissue had reduced apoptosis 7 days after TAC compared with DN-14-3-3 single transgenic mice (Figure 3). The percentage of left ventricular cardiac myocytes that were immunoreactive for cleaved caspase-3 7 days after TAC was 4.2±0.93% for DN-14-3-3 mice, 0.049±0.012% for DN-14-3-3/DN-p38ß mice, and 0.19±0.067% for DN-14-3-3/DN-p38 mice, and 0.045±0.015% for nontransgenic mice.

View larger version (79K): [in this window] [in a new window]   Figure 3. p38 MAPK activity required for caspase-3 activation after pressure overload. Nontransgenic, single transgenic DN-14-3-3, compound transgenic DN-14-3-3/DN-p38, or compound transgenic DN-14-3-3/DN-p38ß mice were subjected to TAC and survivors were killed after 7 days. Ventricular sections were analyzed by immunohistochemistry with a primary antibody directed against cleaved (activated) caspase-3. A, Prominent anti-activated caspase-3 immunoreactivity, demarcated by brown nuclear staining, in left ventricular tissue from DN-14-3-3 mice. B, Minimal anti-activated caspase-3 immunostaining in ventricular tissue from compound transgenic DN-14-3-3/DN-p38 mice. Minimal anti-activated caspase-3 immunostaining was also observed in DN-14-3-3/DN-p38ß mice (data not shown).

Discussion

14-3-3 proteins bind to a variety of signal transduction, checkpoint control, and apoptotic pathway proteins.^1,2 14-3-3 binding to BAD and FKHRL1 inhibits the proapoptotic activity of these proteins, thereby promoting cell survival.^8,9 14-3-3 proteins also bind to and inhibit the activity of Ask1, a MAPKKK in the p38 MAPK and JNK pathways.³ We previously demonstrated that inhibition of 14-3-3 activity in cultured fibroblasts promoted apoptosis in a p38 MAPK-dependent fashion.⁶ We also showed that dominant-negative 14-3-3 increased the sensitivity of cardiac tissue to proapoptotic stimuli.⁶

In the present study, we addressed the role of p38 MAPK in the phenotype observed in DN-14-3-3 transgenic mice by both pharmacological and genetic means. Our results show that 14-3-3-mediated p38 MAPK inhibition is a critical factor to promote cardiac myocyte survival and suggest that p38ß MAPK plays a more important role than p38 MAPK in the apoptosis response. But, we cannot rule out the possibility that each DN-p38 MAPK protein inhibits the other isoform to some degree. Several other studies demonstrated that p38 MAPK activity leads to cardiac myocyte apoptosis. For example, one group found that doxorubicin- and tumor necrosis factor- (TNF-)-dependent murine cardiac myocyte apoptosis was dependent on p38 MAPK activation.^10,11 Another group showed that hypoxia-, angiotensin II-, and norepinephrine-mediated canine cardiac myocyte apoptosis is dependent on p38 MAPK activity.¹²

The specific targets of p38 MAPK that are important for a proapoptotic response are not completely known but may include the transcription factor p53. It is not clear whether p38 MAPK-mediated p53 phosphorylation is important in cardiac myocyte apoptosis, but our results suggest that inhibition of p38 MAPK in human patients may be a useful method to ameliorate cardiac myocyte death in response to provocative stimulation such as ischemia or pressure overload.

Acknowledgments

Acknowledgments

This work was supported by grants from the NIH (GM54620, HL61567), the American Heart Association, the Washington University/Pfizer Corporation Biomedical Research Program, and the Burroughs Wellcome Fund (A.J.M.). S.Z. is a recipient of an American Heart Association Heartland Affiliate Beginning Grant-in-Aid.

Footnotes

Original received July 28, 2003; resubmission received September 23, 2003; revised resubmission received October 17, 2003; accepted October 20, 2003.

References

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This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 93/11/1026    most recent 01.RES.0000104084.88317.91v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhang, S. Articles by Muslin, A. J. Search for Related Content PubMed PubMed Citation Articles by Zhang, S. Articles by Muslin, A. J. Related Collections Apoptosis Cell signalling/signal transduction Genetically altered mice