TNF-␣ signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF-␣ receptor GIANLUIGI CONDORELLI,*,‡,†,1 CARMINE MORISCO,**,2 MICHAEL V. G. LATRONICO,*,†,2 PIER PAOLO CLAUDIO,†† PAUL DENT,# PHILIP TSICHLIS,* GEROLAMA CONDORELLI,¶ GIACOMO FRATI,† ALESSANDRA DRUSCO,* CARLO M. CROCE,* AND CLAUDIO NAPOLI**,° *Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; ‡II Faculty of Medicine, University “La Sapienza”, 00161, Rome, Italy; †I.R.C.C.S. Neuromed, Pozzilli (IS), Italy; **Department of Medicine, University of Naples, 80131, Naples, Italy; #Department of Radiation Oncology, Massey Cancer Center, Medical College of Virginia, Richmond, Virginia, USA; ¶Centro di Endocrinologia ed Endocrinologia Sperimentale, C.N.R., 80131, Naples, Italy; and ††Biotechnology Department, College of Science and Technology, Sbarro Institute for Cancer Research, Temple University, Philadelphia, PA 19122 Cardiomyocyte hypertrophy and apoptosis have been implicated in the loss of contractile function during heart failure (HF). Moreover, patients with HF have been shown to exhibit increased levels of tumor necrosis factor ␣ (TNF-␣) in the myocardium. However, the multiple signal transduction pathways generating from the TNF-␣ receptor in cardiomyocytes and leading preferentially to apoptosis or hypertrophy are still unknown. Here we demonstrate in neonatal rat cardiomyocytes that 1) TNF-␣ induces phosphorylation of AKT, activation of NF-B, and the phosphorylation of JUN kinase; 2) blocking AKT activity prevents NF-B activation, suggesting a role for AKT in regulating NF-B function; 3) AKT and JUN are both critical for the hypertrophic effects of TNF-␣, since dominantnegative mutants of these genes are capable of inhibiting TNF-␣-induced ANF-promoter up-regulation and increase in cardiomyocyte cell size, and 4) blocking NF-B, AKT, or JUN alone or in combination does not sensitize cardiomyocytes to the proapoptotic effects of TNF-␣, in contrast to other cell types, suggesting a cardiac-specific pathway regulating the anti-apoptotic events induced by TNF-␣. Altogether, the data presented evidence the role of AKT and JUN in TNF-␣induced cardiomyocyte hypertrophy and apoptosis.— Condorelli, G., Morisco, C., Latronico, M., Claudio, P. P., Dent, P., Tsichlis, P., Condorelli, G., Frati, G., Drusco, A., Croce, C. M., Napoli, C. TNF-␣ signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF-␣ receptor. FASEB J. 16, 1732–1737 (2002) ABSTRACT
Key Words: cardiomyocyte apoptosis 䡠 hypertrophy 䡠 AKT 䡠 heart failure 䡠 tumor necrosis factor ␣
The cardiovascular system is constantly exposed to the bloodstream and its components. Of these, various 1732
cytokines may play an important role in heart failure (HF) (1). Indeed, clinical and experimental studies have revealed an increase in serum concentration of tumor necrosis factor ␣ (TNF-␣), as well as other cytokines, in HF patients. Recently the potential clinical use of anti-TNF-␣ targeted drugs has spurred an even greater interest toward the elucidation of the cardiac effects of TNF-␣ (2). The interaction between TNF-␣ and its receptor (TNFR) can activate different signal transduction cascades involved in several pathophysiological conditions (3, 4). NF-B, AKT, and JUN have been shown to be activated by TNF-␣ (3). In hepatocytes (5) or endothelial cells (6) and other cell types, the block of NF-B by I-B sensitizes cells to the proapoptotic effect of TNF-␣. Similarly, JUN-AP1 induction by TNF-␣-dependent JUN kinase (JNK) activation is anti-apoptotic (7). In cardiomyocytes, TNF-␣ can induce hypertrophy (8, 9) or apoptosis, the latter in the presence of an inhibitor of protein synthesis (cycloheximide) (10). Mice lacking TNFR show an increased number of apoptotic myocytes after ischemia-reperfusion injury, thus implying a protective effect of antiapoptotic pathways generating from the TNFR also in vivo (11). Moreover, recent evidence has suggested that TNF-␣ induces cardiomyocyte protection due to upregulation of interleukin 6 (IL-6), which, once secreted, would act as an autocrine mediator, inducing IL-6 receptor activation and protection from serum starved apoptosis through gp130-dependent anti-apoptotic signaling (12). The molecular pathways mediating apoptosis or hypertrophy in cardiac myocytes have begun to be unraveled (13). Nonetheless, the exact sequence of signal 1 Correspondence: Kimmel Cancer Center, 233 S. 10th St., Rm. 1006, Philadelphia PA 19107, USA. E-mail: gianluigi.
[email protected] 2 These authors contributed equally to this manuscript.
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transduction events generating from TNFR and their relative role in triggering either cardiomyocyte hypertrophy or apoptosis has not yet been clarified. To gain a more complete appreciation of the extent and biological relevance of TNF-␣ in cardiac apoptosis and hypertrophy, we have sought to investigate the main signal transduction pathways deriving from the TNFR in neonatal rat cardiomyocytes.
MATERIALS AND METHODS Materials All reagents were purchased from Sigma (St. Louis, MO) unless stated otherwise and were of the highest grade available. Rat recombinant TNF-␣ was from BioSource International (Camarillo, CA); LY294002 was from Calbiochem (San Diego, CA). Antibodies for phosphorylated and phosphorylation state-independent molecules were from New England Biolabs (Beverly, MA), luciferase assay kits from Promega (Madison, WI); ELISA apoptotic kits were from Roche Diagnostics (Nutley, NJ). Adenoviruses Construction of adenovirus Ad5sIB (expressing the mutant form S32A/S36A) (5), Ad5LacZ (containing the Escherichia coli -galactosidase gene), and TAM 67 JUN amino-terminal mutant (14) have been described. The dominant-negative AKT (dn-AKT) mutant used in this study is the T308A/S473A form (15). Ad5LacZ, used in control experiments, was from Quantum Biotechnology (San Jose, CA). All are E1-deleted forms of recombinant adenovirus type 5. Ad5 were propagated, harvested, titered, and purified as previously reported (16). Expression of the transgenes was checked by western blots.
Lipofectamine (GIBCO BRL) in 1 mL DMEM medium/well. To determine the activity of NF-B transcription factor, a plasmid (1 g/mL) containing a 5⫻ repeated NF-B binding site linked to firefly luciferase (a kind gift from Dr. Srinivasan Srinivasula, Thomas Jefferson University) was used in conventional luciferase assays. Twenty-four hours after transfection, the culture medium was changed to the cardiac myocyte culture medium without serum. Myocytes were cultured in the presence or absence of TNF-␣ (25 ng/mL) for an additional 24 h. For some experiments, transfected myocytes were infected with 10 or 50 MOI of sI-B adenovirus. Myocytes were then lysed with the reporter lysis buffer (Promega) and luciferase activities were measured. An SV 40 promoter-driven -galactosidase construct (SV 40 -gal, 0.5 g/mL) was cotransfected and -gal activity was determined by using Lumi-Gal 530 (Lumigen, Southfield, MI). Luciferase values were divided by the -gal values to correct for differences in the transfection efficiency. Luciferase activity was evaluated in the presence or absence of LY294002 at a final concentration of 10 M. Similarly, the activation of ANF gene expression was studied by a luciferase promoter assay using a ⫺1050⫹ANF promoter region hooked to the luciferase gene (17). Immunoblotting Cardiac myocytes were grown in 6-well plates (1⫻106 cells per well). After infection with adenovirus and stimulation with TNF-␣ for 10 min, myocytes were lysed with 100 L of lysis buffer (50 mM HEPES (pH 7.6), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM -glycerophosphate, 1 mM Na3VO4, 10 mM NaF, 30 mM Na pyrophosphate, 2 mM DTT, 1 mM AEBSF). Samples were subjected to SDS-PAGE, transferred onto PVDF membranes, and probed with anti-phospho-AKT (Ser 473), antiphospho-I-B (Ser 32), or anti-phospho-JNK (Thr183/Tyr185) antibodies according to the manufacturer’s instructions. Phosphorylation state-independent antibodies were used as a control for sample variations. Horseradish peroxidase-conjugated goatanti-rabbit was used as the secondary antibody. The bound secondary was detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL).
Cardiomyocyte preparation Immunofluorescence All procedures involving animals were performed in accordance with institutional guidelines for the care and use of laboratory animals. Hearts removed from decapitated 1- to 3-day-old neonatal Wistar rats with the atria dissected away were minced and digested with a mixture of 108 U/mL collagenase type II (Worthington) and 0.9 mg/mL pancreatin (Life Technologies, Grand Island, NY) to obtain free cells. Myocytes were enriched on a Percoll gradient and plated on gelatin-coated dishes overnight in DMEM/medium 199 (4:1) supplemented with 10% horse serum, 5% fetal calf serum, 200 M l-glutamine, and penicillin/streptomycin (all from Life Technologies) at a density of 1 ⫻ 105 cells/cm2. The next day cells were rinsed three times and the plating medium was replaced with serum-free medium consisting only of DMEM/medium 199 (4:1), l-glutamine, and penicillin/streptomycin; 10 M cytosine--d-furanoarabinoside was added to stop proliferation of noncardiomyocytes and cultures contained ⬎95% cardiac myocytes. All cultures were serum starved for 24 h before starting the experiments.
For visualization of NF-B translocation, an anti-NF-B antibody directed against the p65 subunit (Boehringer Mannheim, Mannheim, Germany) was used. Plates with and without sI-B pretreatment were stimulated with TNF-␣, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and nonspecific sites were blocked with 3% BSA. The primary Ab was used at a dilution of 1:50 and incubated at room temperature for 1 h. The bound Ab was then detected with a biotinylated anti-mouse IgG, followed by decoration with Cy3-conjugated streptavidin (18). Determination of protein/DNA content To determine effects on cell size, the ratio between cellular protein and DNA content was measured as previously reported (17). Quantitation of apoptosis
Transient transfection and reporter gene assays For transient transfections, myocytes were plated at a density of 1 ⫻ 106 per well in 6-well plates (17). Twenty-four hours after plating, the media was changed to DMEM/F12 without supplement. Transfections were carried out using 10 L/mL of TNF-␣ SIGNAL TRANSDUCTION IN NEONATAL CARDIOCYTES
Cardiomyocytes were infected with adenovirus and treated with TNF-␣ for 24 h, after which cells were harvested by trypsinization, counted, resuspended in PBS, and snap-frozen in liquid N2 until use. Aliquots containing 105 cells were used for detection of apoptosis with an ELISA based kit. As a 1733
control, MCF-7 cells were seeded in 35 mm plates, grown in DMEM supplemented with 10% serum, and treated as for cardiomyocytes. Statistical analysis Results are expressed as mean ⫾ sd. Statistical analysis was performed with Student’s t test and values of P ⬍ 0.05 were considered to be significant. All experiments were repeated at least three times.
RESULTS Activation of NF-B by TNF-␣ To determine whether TNF-␣ induces NF-B activity in cardiomyocytes, a luciferase assay was conducted. Cells transfected with NF-B reporter gene showed a significant increase of luciferase activity when stimulated with TNF-␣ (Fig. 1A). This effect was significantly decreased when cells were preinfected for 24 h with a nonphosphorylatable, constitutive active mutant adenovirus of I-B, called I-B super-repressor (sI-B) (5). This mutant cannot be phosphorylated by TNF-␣-dependent IK kinases, thus impeding its degradation; as a consequence, NF-B cannot be released from I-B inhibition and is retained in the cytoplasm. Therefore, TNF-␣ efficiently trans-activates NF-B and sI-B blocks NF-B activation in cardiomyocytes. Figure 1B shows that NF-B fluorescence is nuclear after 40 min of TNF-␣ challenge, whereas NF-B is retained in the cytoplasm of cardiomyocytes pretreated with sI-B (Fig. 1C).
Figure 1. A) Effects of sI-B on TNF-␣-induced NF-B transactivation. A synthetic promoter containing a 5⫻ NF-B binding site was used. sI-B adenovirus (IKB) induced a dose-related inhibition of TNF-␣-induced NF-B trans-activation. (10⫽10 MOI; 50⫽50 MOI). B, C) TNF-␣ induces nuclear translocation of NF-B, which is prevented by sI-B. Cells were treated for 40 min with TNF-␣ in the absence (B) or presence (C) of sI-B adenovirus (IB). NF-B was detected by immunofluorescence. 1734
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Figure 2. A) Activation of NF-B luciferase promoter is dependent on PI3K. Inhibition of PI3K with LY294002 inhibits TNF-␣-dependent NF-B trans-activation significantly. B) TNF-␣ (T␣) induces AKT phosphorylation, which is prevented by inhibitors of PI3K activation. C) TNF-␣-dependent I-B phosphorylation is prevented by inhibitors of PI3K. Ly ⫽ LY94002; WM ⫽ wortmannin.
Activation of AKT by TNF-␣ and control of NF-B activity by AKT AKT phosphorylation can be induced by TNF-␣, suggesting that AKT activation can account for at least some anti-apoptotic effects of TNF-␣ (19, 20). These results prompted us to determine whether AKT was activated by TNF-␣ in cardiomyocytes. Cells were therefore subjected to TNF-␣ challenge and AKT phosphorylation was measured by Western blot analysis with phospho-AKT antiserum. Figure 2B shows that AKT is phosphorylated after 10 min of stimulation with TNF-␣. Moreover, AKT phosphorylation is effectively blocked by pretreatment with inhibitors of phosphoinositide 3-Kinase (PI3K), namely, LY294002 (LY) and wortmannin (WM), thus demonstrating the causal role of PI3K in TNF-␣-dependent AKT activation. Since I-B can be phosphorylated by TNF-␣ through AKT in other cell types, Western blot analyses were performed on extracts from cardiomyocytes treated with TNF-␣ in the presence or absence of LY or WM. Data show that whereas TNF-␣ clearly induces I-B phosphorylation in the absence of PI3K inhibitors, both LY and WM blocked such phosphorylation, demonstrating the PI3K/AKT dependence of I-B phosphorylation in cardiomyocytes (Fig. 2C). Moreover, the block of AKT phosphorylation significantly decreased TNF-␣ induced NF-B activation, as evaluated by luciferase activity, demonstrating the functional consequences of PI3K inhibition on NF-B-dependent transcription (Fig. 2A). The effect of AKT on NF-B activity was further assessed after transduction of cardiomyocytes with a dominant-negative mutant of AKT, since reagents used to block PI3K are not AKT specific.
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Figure 3. AKT-dependent NF-B trans-activation. A) dn-AKT prevents NF-B-luciferase promoter activation after TNF-␣ challenge. B) dn-AKT prevents TNF-␣-induced phosphorylation of GSK-3.
Results show that dn-AKT prevents TNF-␣ induced NF-B activation (Fig. 3A). The dn-AKT used in this study was also able to inhibit the phosphorylation of GSK-3 by endogenous AKT after stimulation with TNF-␣ (Fig. 3B). JNK is activated by TNF-␣ in cardiomyocytes The JNK pathway can be activated by TNF-␣ in a variety of cell lines (3). To investigate whether TNF-␣ activates the JNK pathway in cardiomyocytes, protein extracts from cells treated with TNF-␣ were subjected to Western blot analysis using a phospho-JNK antiserum (Fig. 4A). Data show that TNF-␣ does induce JNK phosphorylation. JNK phosphorylation was not sensitive to PI3K inhibitors since LY had no effect on the state of JNK phosphorylation (not shown). Similarly, the transcription factor substrate of JNK, JUN (14), is phosphorylated at its amino-terminal after stimulation with TNF-␣ in cardiomyocytes; its phosphorylation is also independent of PI3K activity since it is not prevented by the PI3K-blocking agent LY (Fig. 4B).
expressing a dominant-negative, amino-terminal deleted mutant form of JUN, TAM 67, decreased but did not prevent the hypertrophic effects of TNF-␣ on cardiomyocytes. Only the coincubation of TAM 67 and LY significantly inhibited the TNF-␣ induced hypertrophy on cardiomyocytes. The block of PI3K and of JUN both induced a decrease in cell size in serum-starved cardiomyocytes. Moreover, the hypertrophic effect of TNF-␣ was prevented by dn-AKT gene transduction (Fig. 5B). These data demonstrate that TNF-␣ induced hypertrophy is dependent on both AKT and JNK-JUN pathways. One marker of cardiomyocyte hypertrophy is ANF, whose transcription is activated by hypertrophic-inducing stimuli. Thus, luciferase assays using the ANF promoter were performed in order to determine whether TNF-␣ induces ANF gene activation and whether AKT and JUN control this process. Results demonstrate that dominant-negative AKT and JUN gene transductions both prevented the TNF-␣-mediated increase in ANF gene expression (Fig. 5C). It had earlier been shown that coincubation of cardiomyocytes with TNF-␣ and cycloheximide, an inhibitor of protein synthesis, is a robust apoptotic stimulus (10). Since blocking NF-B or JUN sensitizes other cell types toward the proapoptotic effects of TNF-␣, we determined whether such phenomena can also take place in cardiomyocytes. NF-B, JUN, and AKT were blocked with adenoviruses expressing sI-B, dn-AKT, and TAM 67, respectively, alone or in combination. As a control, lacZ-expressing adenovirus was used. Blocking NF-B, AKT, or JUN alone (Fig. 6) or in combination (not shown) did not sensitize cardiomyocytes toward the proapoptotic effect of TNF-␣. In contrast, sI-B sensitized MCF-7 cells to the proapoptotic effects of TNF-␣.
Biological consequences of blocking NF-B, AKT, and JUN in cardiomyocytes The biological consequences of TNF-␣ on cardiomyocytes were evaluated as induction of hypertrophy or apoptosis. Cardiomyocytes were subjected to TNF-␣ stimulation for 48 h and hypertrophy was evaluated as the change in the ratio between DNA and protein content (Fig. 5). TNF-␣ induces a significant increase in protein/DNA content after 48 h of treatment. This increase is less marked but still significant in the presence of the PI3K inhibitor LY (Fig. 5A). Preincubation of cardiomyocytes with an adenovirus TNF-␣ SIGNAL TRANSDUCTION IN NEONATAL CARDIOCYTES
Figure 4. TNF-␣⫺dependent phosphorylation of JNK kinase and JUN. A) JUN kinase (JNK) is phosphorylated after TNF-␣ stimulation. A Western blot of JNK with phospho-JNK-specific antibodies is shown. B) c-JUN phosphorylation at Ser 63 after stimulation with TNF-␣. LY 29 ⫽ LY294002. 1735
Figure 5. TNF-␣ stimulation induces a hypertrophic response that is dependent on AKT and JUN. A) The ratio between protein and DNA content was measured after incubation with TNF-␣ for 48 h with or without an inhibitor of PI3K (LY294002) or dominant-negative JUN (TAM 67). The TNF-␣-induced increase in cardiomyocyte size was efficiently inhibited by preventing PI3K and JUN activities. B) dn-AKT prevents TNF-␣-induced cardiomyocyte hypertrophy. C) TAM 67 and dn-AKT prevent TNFdependent ANF promoter activation.
DISCUSSION In the present study, we simultaneously analyzed multiple signal transduction events generating from the TNF-␣ receptor in cardiomyocytes and defined the hypertrophic pathways. Our results provide new insights into the role of TNF-␣ signaling and are consistent with the concept that cardiac hypertrophy seems to be dependent on both JUN and AKT. In fact, TAM 67 and dn-AKT were both capable of reducing the hypertrophic effect of TNF-␣. We found that AKT also controls I-B phosphorylation and NF-B activity in cardiomyocytes. However, we determined that blocking either NF-B or AKT could not sensitize cardiomyocytes to the proapoptotic effects of TNF-␣. This is in contrast to events taking place in other cell types such as MCF-7. Indeed, in endothelial cells and hepatocytes, blocking NF-B triggers caspase-dependent apoptosis (5, 6). In other cell lines it has been proved that
TNF-␣-dependent NF-B activation induces the transcription of anti-apoptotic genes such as IAPs (21). With regard to JUN, the consequences of its inhibition are cell type and stimulus specific. In fact, JUN can be either proapoptotic or anti-apoptotic. It has been reported that JUN has an early anti-apoptotic effect on TNF-␣ stimulation (7). On the other hand, the block of JUN in some cell types protects from apoptosis induced by oxidative stress (4, 22). In neonatal rat cardiomyocytes, we have now shown that blocking JUN, while decreasing the hypertrophic effects of TNFR stimulation, does not sensitize the cells to the proapoptotic effect of TNF-␣. Note that the combination of the block of AKT, JUN, and NF-B did not predispose cardiomyocytes to TNF-␣-mediated apoptosis either. This phenomenon may be explained by at least two lines of reasoning. One possibility is that an anti-apoptotic pathway exists in cardiomyocytes further upstream of PI3K, AKT, NF-B, or JUN, and therefore was not blocked by the reagents used in our experiments. The other is that the block of each signal transduction pathway can render cells prone to the proapoptotic effect of TNF-␣ in metabolic conditions different from our experimental setting. Further studies are needed in order to clarify this issue. The JUN/AKT/TNF-␣-dependent framework may constitute a target of new drugs for cardiac hypertrophy and apoptosis. This research was possible thanks to grants from Terapia dei Tumori Italy-USA, “Fondi 1% SSN”, Italy, A.I.R.C., from AHA to G.C.; and National Institutes of Health grant R01DK52825 to P.D. We thank members of the scientific community for reagent availability.
Figure 6. Block of NF-B, AKT, or JUN, alone or in combination, does not sensitize cardiomyocytes to the proapoptotic effects of TNF-␣. Apoptosis was measured using an ELISAbased method in cardiomyocytes (CMC) and MCF-7 cells, used as a control. The increase in OD relative to Lac Z-only-treated cells is shown of a representative experiment. 1736
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