Role of 14-3-3-Mediated p38 Mitogen-Activated Protein Kinase Inhibition in Cardiac Myocyte Survival
14-3-3 family members are dimeric phosphoserine-binding proteins that regulate signal transduction, apoptotic, and checkpoint control pathways. Targeted expression of dominant-negative 14-3-3η (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 p38α and p38β 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 p38α (DN-p38α) or dominant-negative p38β (DN-p38β) MAPK expression was targeted to the heart. Compound transgenic DN-14-3-3/DN-p38β mice, and to a lesser extent compound transgenic DN-14-3-3/DN-p38α 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.
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-14-3-3η.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-14-3-3η 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 p38α and p38β 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 p38α MAPK (DN-p38α) or p38β MAPK (DN-p38β) to generate compound transgenic DN-14-3-3/DN-p38α or DN-14-3-3/DN-p38β 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.
An experimental protocol to administer SB202190, a specific inhibitor of p38α and p38β 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 p38α and p38β 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).
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 (χ2=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-p38α and DN-p38β 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-p38α and DN-14-3-3/DN-p38β compared with single transgenic DN-14-3-3 mice (Figure 2). We previously demonstrated that DN-p38α mice exhibit reduced p38α MAPK activation and that DN-p38β transgenic mice exhibit reduced p38β MAPK activation in cardiac tissue.7
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-p38β 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-p38β mice compared with single transgenic DN-14-3-3 mice was significant by the log-rank test (χ2=16.6, P<0.0001). DN-14-3-3/DN-p38α 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-p38α mice compared with DN-14-3-3 mice was significant by analysis with the log-rank test (χ2=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-p38β and DN-14-3-3/DN-p38α 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-p38β mice, and 0.19±0.067% for DN-14-3-3/DN-p38α mice, and 0.045±0.015% for nontransgenic mice.
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 14-3-3η 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 p38β MAPK plays a more important role than p38α 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-α (TNF-α)-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.
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.
Original received July 28, 2003; resubmission received September 23, 2003; revised resubmission received October 17, 2003; accepted October 20, 2003.
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