INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

TUMOR NECROSIS FACTOR (TNF)- is a proinflammatory cytokine with pleiotropic biological effects and mediates diverse pathological processes such as cachexia during cancer, shock in infection, and inflammation in autoimmune disease (35, 36). Elevated circulating levels of TNF- in patients with chronic heart failure caused by ischemic heart disease and dilated cardiomyopathy (14, 16) suggest the involvement of TNF- in the pathogenesis of cardiovascular diseases. In the heart, resident macrophages and cardiac myocytes produce TNF-, and TNF- receptors are expressed in cardiac myocytes (13, 17, 34). However, the role of TNF- in the myocardium is controversial. TNF- is regarded as an important factor that can induce hypertrophy and resistance to hypoxic stress of cultured cardiac myocytes (19, 20, 39). Continuous infusion of TNF- leads to a significant increase of left ventricular myocyte cross-sectional areas in rats (1). On the other hand, it has also been demonstrated that TNF- induces myocardial dysfunction and apoptosis of cardiomyocytes (12, 17, 29). Furthermore, little is known about TNF--induced signal transduction pathways in cardiac myocytes.

Phosphatidylinositol (PI) 3-kinase is an enzyme that catalyzes the phosphorylation of the D-3 position of phosphatidylinositides. On ligand binding, several growth factor receptors and cytokine receptors are able to stimulate PI3-kinase activity (27, 33). The lipid products of PI3-kinase interact with protein modules, such as pleckstrin homology (PH) and a Src homology 2 (SH2) domains of effector molecules, and then activate and localize the downstream enzymes as well as their substrates (27, 33). Although the role of PI3-kinase in intracellular signaling is underscored by its implication in a plethora of biological responses, relatively little is known about the downstream elements of PI3-kinase. Recently, Akt/protein kinase B (PKB) was identified as a downstream target of PI3-kinase. Akt/PKB, also named RAC (related to A and C) protein kinase, is a serine-threonine kinase that contains a PH domain in its NH₂-terminal end region and a catalytic domain closely related to both cAMP-dependent protein kinase and protein kinase C (PKC) (2, 5, 6). It is shown that the kinase activity of Akt/PKB is stimulated by growth factors acting through receptor tyrosine kinases, such as platelet-derived growth factor, epidermal growth factor, and insulin receptors, and the activation of Akt/PKB by these growth factors is mediated by PI3-kinase (2, 5, 6). A signaling pathway from PI3-kinase to Akt/PKB is implicated in some cellular responses of PI3-kinase, including protection from apoptosis in various cell types (2, 5, 6) and protein synthesis in skeletal muscle and adipose tissue (10, 37). However, less is known about the participation of the PI3-kinase-Akt/PKB pathway in intracellular signaling pathways and cellular functions of TNF- in cardiac myocytes.

In the present study, we examined the role of TNF- and the involvement of the PI3-kinase-Akt/PKB pathway in cultured cardiac myocytes. We showed here that TNF- induced stimulation of protein synthesis, which coincided with enlargement of cell surface area. Evidence is also provided to demonstrate that the activation of the signaling pathway of PI3-kinase-Akt/PKB is required for the stimulation of protein synthesis induced by TNF-. These results indicate the involvement of the PI3-kinase-Akt/PKB pathway in the TNF--induced hypertrophic response in cardiac myocytes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. Sprague-Dawley rats were purchased from Charles River (Osaka, Japan). The standard culture medium was Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F-12) from Life Technologies (Gaithersburg, MD). Recombinant rat TNF- was from Genzyme (Cambridge, MA). LY-294002 was from Calbiochem (San Diego, CA). The sheep polyclonal antibody against Akt/PKB was from Upstate Biotechnology (Lake Placid, NY). The rabbit polyclonal phosphospecific Akt/PKB antibody, which detects Akt/PKB only when phosphorylated at Ser-473, was from New England Bio Labs (Beverly, MA). Protein G-Sepharose 4 fast flow and protein A-Sepharose CL-4B were from Amersham Pharmacia Biotech (Uppsala, Sweden).

Cell culture. Primary cultured ventricular myocytes were prepared from neonatal rat hearts as described previously (18). Cardiac myocytes were distributed at a density of 5.0 × 10⁴/cm². The culture medium was DMEM/F-12 supplemented with 5% calf serum and penicillin-streptomycin (0.02 U/ml and 0.02 mg/ml, respectively). 5-Bromodeoxyuridine (100 mM) was added during the first 24 h to prevent proliferation of nonmyocytes. The medium was changed 24 h after seeding the cells to serum-free medium, which is DMEM/F-12 containing 0.1% bovine serum albumin and ITS (10 mg/ml insulin, 10 mg/ml transferrin, and 10 ng/ml selenious acid; Becton Dickinson Labware).

Recombinant adenovirus vectors. Adenovirus vectors encoding a dominant negative mutant of PI3-kinase (AxCAp85), a dominant negative mutant of Akt (AxCAAkt-AA), and bacterial -galactosidase (AxCALacZ) were prepared as described previously (10, 24, 32). p85 is a mutant of 85-kDa regulatory subunit of PI3-kinase that lacks the binding site for the 110-kDa catalytic subunit of PI3-kinase (24) and has been widely used as a dominant negative mutant of PI3-kinase (10, 24, 33). Akt-AA is a mutant of Akt/PKB in which Thr-308 and Ser-473 are replaced by alanine (10, 32). Cardiac myocytes were infected with adenovirus vectors at the indicated multiplicity of infection (MOI) 24 h after seeding the cells. The cells were subjected to experiments 48 h after infection.

Cellular morphology and cell surface area. After 48 h in the serum-depleted medium, cardiac myocytes were stimulated with or without 2,000 U/ml of TNF-. The cellular morphology was examined and photographed under light microscopy. The cell surface area of cardiac myocytes was measured by use of image analysis software (NIH image). At least 100 cells/condition were scored for size measurement.

Cell viability assay. Cell viability was assessed by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay according to recommendations of the manufacturer (Boehringer Mannheim). Cardiac myocytes were seeded on 96-well plates at the density of 7,000 cells/well, because our control experiments showed good correlation between absorbance at 450 nm and cell number at a cell concentration between 0.1 and 2 × 10⁴/well. Cells were then stimulated with or without 2,000 U/ml of TNF- for 24 h.

PI3-kinase assay. Lipid kinase activity of PI3-kinase was measured as described previously (24). Cardiac myocytes were stimulated as indicated. Cells were lysed into buffer containing 1% Nonidet-P40, 137 mM NaCl, 20 mM Tris · HCl, pH 8.0, 10% glycerol, 1 mM CaCl₂, 1 mM MgCl₂, 2 mM sodium orthovanadate, 25 mg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride. After removal of insoluble materials by centrifugation at 15,000 rpm for 20 min, protein concentrations in supernatants were normalized with the use of BioRad protein assay. The lysates (500 µg protein) were incubated with 2 µg of anti-phosphotyrosine monoclonal antibody (PY 20; Transduction Laboratories, Lexington, KY) for 2 h at 4°C. The immunocomplexes were immunoprecipitated with 30 µl of a 1:1 slurry of protein A-Sepharose CL-4B for 2 h at 4°C. Immunoprecipitates were washed twice with each of following solutions: 1) phosphate-buffered saline containing 1% NP-40; 2) 100 mM Tris · HCl, pH 7.5, and 500 mM LiCl; and 3) 10 mM Tris · HCl, pH 7.2, 100 mM NaCl, and 1 mM EDTA. After the final wash, immunoprecipitates were incubated with 10 µg of sonicated PI (Avanti Polar Lipids, Alabaster, AL) and [-³²P]ATP (1 µCi/sample) for 15 min at 30°C. The phosphorylation reaction was stopped by addition of 15 µl of 4 N HCl and 130 µl of chloroform-methanol (1:1). The biphasic mixture was microcentrifugated to extract lipids. The bottom organic layer was carefully collected, and ³²P-labeled phospholipids were resolved by thin-layer chromatography (TLC) with the use of Silica gel 60 plates (MCB reagents; Merck, Rahway, NJ) and a chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2) solvent system. The radioactivities in the PI3-monophosphate fraction were determined with the use of a Fujix bioimaging analyzer (BAS-2000).

Akt/PKB kinase assay. Akt/PKB kinase activity was measured as described previously (31). Cardiac myocytes were stimulated as indicated. The cells were washed twice with ice-cold phosphate-buffered saline and lysed into buffer A (50 mM Tris · HCl, pH 7.5, 0.1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 10 mM sodium -glycerophosphate, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 0.1% 2-mercaptoethanol) containing 1 mM microcystin LR (Research Biochemicals International, Natick, MA). After removal of insoluble materials by centrifugation at 15,000 rpm for 20 min, protein concentrations in supernatants were normalized using Bio-Rad protein assay. For immunoprecipitation, after the lysates (500 µg protein) were preabsorbed with 15 µl of a 1:1 slurry of protein G-Sepharose for 30 min at 4°C, the lysates were incubated with sheep polyclonal anti-Akt/PKB antibody (2 µg) for 2 h at 4°C. The immunocomplexes were immunoprecipitated with 30 µl of a 1:1 slurry of protein G-Sepharose for 2 h at 4°C. The immunocomplexes were washed three times with buffer A containing 500 mM NaCl, twice with buffer B (50 mM Tris · HCl, pH 7.5, 0.03% Brij-35, 0.1 mM EGTA, and 0.1% 2-mercaptoethanol), and then once with assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM sodium -glycerophosphate, 1 mM sodium orthovanadate, and 1 mM dithiothreitol). The beads were resuspended in 30 µl of kinase reaction mixture {assay dilution buffer containing 25 mM MgCl₂, 170 mM ATP, 1 µg histone H2B (Boehringer Mannheim), and 1 µCi [-³²P]ATP} and incubated at 30°C for 30 min. Kinase reactions were stopped by addition of 7 µl of 5× sample buffer, after which the samples were boiled for 5 min at 100°C and electrophoresed on 15% SDS-polyacrylamide gels. The gels were dried, and the radioactivities were analyzed using a Fujix bioimaging analyzer (BAS-2000).

Immunoblot analysis. Immunoblot analysis with phosphospecific or total Akt/PKB antibody was carried out as described previously (31). Samples were subjected to 10% SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). Blots were incubated with polyclonal phosphospecific Akt/PKB antibody or total Akt/PKB antibody, and the primary antibodies were detected using horseradish peroxidase-labeled anti-rabbit IgG and anti-sheep IgG, respectively, followed by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Protein synthesis assay. Protein synthesis was measured by [³H]leucine incorporation as described previously (30). Cardiac myocytes were stimulated with 2,000 U/ml of TNF- for 24 h. [³H]leucine (0.5 µCi/ml) was added 4 h before harvest. Cells were washed three times with ice-cold phosphate-buffered saline and incubated with 5% trichloroacetic acid at 4°C for 30 min. Trichloroacetic acid-precipitable materials were washed twice with 5% trichloroacetic acid and solubilized in 0.1 N NaOH at 37°C for 30 min. The radioactivity was measured by liquid scintillation spectrometry.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Enlargement of cell surface area and stimulation of protein synthesis induced by TNF- in cardiac myocytes. Because TNF- has been reported to induce both hypertrophic change and apoptosis in cardiac myocytes (12, 29), we first examined the effect of TNF- on cell surface area, protein synthesis, and viability of cardiac myocytes. TNF- increased cell surface area by 1.5-fold (Fig. 1, A and B), which coincided with stimulation of protein synthesis (see Fig. 5). WST-1 assay revealed that TNF- did not affect the viability of cardiac myocytes (Fig. 1C), indicating that TNF- did not induce apoptosis in our cells. In this study, we examined the signal transduction pathway leading to protein synthesis stimulated by TNF-, which is one of the important characteristics of cardiac myocyte hypertrophy.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Tumor necrosis factor (TNF)- induces enlargement of cardiac myocytes but does not induce death of cardiac myocytes. Cultured cardiac myocytes were treated with or without 2,000 U/ml of TNF- for 24 h. The cellular morphology was examined and photographed under light microscopy (A). Cell surface area was measured by an NIH image analyzer (B). Cell viability was assessed by WST-1 assay (C). Values are means ± SE of 3 independent trials. OD450, optical density at 450 nm.

TNF--stimulated kinase activity of PI3-kinase. To examine the effect of TNF- on kinase activity of PI3-kinase, cultured cardiac myocytes were treated with TNF-, and kinase activity of PI3-kinase was measured by in vitro kinase assay with the use of PI as a substrate. As shown in Fig. 2A, TNF--induced PI3-kinase activation was detected within 2 min after addition of TNF-. TNF- induced an approximately sixfold increase in the activity of PI3-kinase maximally at ~10 min. TNF- increased the kinase activity of PI3-kinase in a concentration-dependent manner (Fig. 2B). Half-maximum and maximum effects were achieved at 100 and 2,000 U/ml of TNF-, respectively. When a PI3-kinase-specific inhibitor, LY-294002, was added to the anti-phosphotyrosine immunoprecipitates, the spot corresponding to PI3-monophosphate completely disappeared (data not shown), confirming the specificity of this activity to PI3-kinase.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   TNF--stimulated kinase activity of phosphatidylinositol (PI) 3-kinase in cardiac myocytes. Cultured cardiac myocytes were treated with 2,000 U/ml of TNF- for various periods of time (A) or for 10 min with various concentrations of TNF- (B). Anti-phosphotyrosine immunoprecipitates were subjected to in vitro kinase assay using PI as a substrate. Radioactivities of phosphorylated PI were quantitated. Values are means ± SE of 3 independent trials and are expressed as multiples of control. The experiments shown represent 1 of 3 independent trials that gave nearly identical results. PI 3-P, PI3-monophosphate.

TNF--stimulated kinase activity of Akt/PKB. Next, we examined the effect of TNF- on kinase activity of Akt/PKB, because Akt/PKB has been reported to be a downstream target of PI3-kinase (2, 5, 6). The effect of TNF- on kinase activity of Akt/PKB was time and concentration dependent (Fig. 3). TNF--stimulated Akt/PKB activation was detected at 5 min after addition of TNF- and reached a maximum at 15 min and then declined (Fig. 3A). Half-maximum and maximum effects were achieved at 100 and 2,000 U/ml of TNF-, respectively (Fig. 3B). Because Akt/PKB was shown to be activated by phospholipid binding and phosphorylation at Thr-308 and Ser-473 (2, 5, 6), we assessed the effect of TNF- on the phosphorylation state of Akt/PKB at Ser-473 by immunoblotting with the phosphospecific Akt/PKB antibody, which recognizes Akt/PKB only when phosphorylated at Ser-473. Stimulation of cardiac myocytes with TNF- caused a marked increase in the phosphorylation at Ser-473, whereas total Akt/PKB proteins were not altered by treatment with TNF- (Fig. 3C). These results indicate that in cardiac myocytes, TNF- stimulates the kinase activity of Akt/PKB, and this activation is coincident with Ser-473 phosphorylation.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   TNF--stimulated kinase activity of Akt/protein kinase B (PKB) in cardiac myocytes. Cultured cardiac myocytes were treated with 2,000 U/ml of TNF- for various periods of time (A) or for 15 min with various concentrations of TNF- (B). Anti-Akt/PKB immunoprecipitates were subjected to in vitro kinase assay using histone H2B as a substrate. Radioactivities of phosphorylated histone H2B were quantitated. Values are means ± SE of 3 independent trials and are expressed as multiples of control. C: cultured cardiac myocytes were treated with (+) or without () 2,000 U/ml of TNF- for 15 min. Whole cell lysates were analyzed by immunoblotting with phosphospecific Akt/PKB antibody (Phospho Akt) or total Akt/PKB antibody (Akt). The experiments shown represent 1 of 3 independent trials that gave nearly identical results.

PI3-kinase-dependent activation of Akt/PKB by TNF-. PI3-kinase is shown to be necessary and sufficient for growth factor-dependent activation of Akt/PKB, although other pathways independent of PI3-kinase for activation of Akt/PKB have been proposed (11, 23). We tested the effects of a PI3-kinase-specific inhibitor, LY-294002, and the expression of a dominant negative mutant of PI3-kinase on TNF--induced Akt/PKB activity. LY-294002 is a specific inhibitor of PI3-kinase but has no inhibitory effect against PI4-kinase or a number of intracellular serine-threonine or tyrosine kinases at 50 µM (27, 38). LY-294002 (50 µM) completely inhibited the TNF--induced activation of Akt/PKB (Fig. 4A). Infection with AxCAp85 increased the expression of p85 in a MOI-dependent manner (Fig. 4B, bottom). In cells infected with AxCAp85, TNF--induced activation of Akt/PKB was inhibited in a MOI-dependent manner (Fig. 4B, middle), which was well correlated with the inhibition of PI3-kinase activity by the mutant of PI3-kinase (Fig. 4B, top). The infection with AxCALacZ did not alter the TNF--induced activation of PI3-kinase and Akt/PKB. These results clearly indicate that TNF--induced activation of Akt/PKB is mediated by PI3-kinase in cardiac myocytes.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   PI3-kinase-dependent activation of Akt/PKB by TNF-. A: cultured cardiac myocytes were incubated with or without 50 µM of LY-294002 for 10 min before stimulation with 2,000 U/ml of TNF- for 10 min. Anti-Akt/PKB immunoprecipitates were subjected to in vitro kinase assay with the use of histone H2B as a substrate. The experiment shown represents 1 of 3 independent trials that gave nearly identical results. B: cardiac myocytes were infected with adenoviruses encoding -galactosidase (AxCALacZ) or a dominant negative mutant of PI3-kinase (AxCAp85) at the indicated multiplicity of infection (MOI) and stimulated with 2,000 U/ml of TNF- for 10 min. Anti-phosphotyrosine immunoprecipitates were subjected to in vitro kinase assay using PI as a substrate (top). Anti-Akt/PKB immunoprecipitates were subjected to in vitro kinase assay using histone H2B as a substrate (middle). Whole cell lysates were analyzed by immunoblotting with anti-PI3-kinase antibody (bottom). The experiments shown represent 1 of 3 independent trials that gave nearly identical results.

PI3-kinase-Akt/PKB pathway-mediated protein synthesis induced by TNF- in cardiac myocytes. Because TNF- has been reported to induce protein synthesis in cultured cardiac myocytes (19, 39), we next investigated whether the activation of the PI3-kinase-Akt/PKB pathway is required for protein synthesis stimulated by TNF- in cardiac myocytes. Treatment with TNF- induced a 1.5-fold increase in [³H]leucine incorporation (Fig. 5). To assess the role of PI3-kinase in protein synthesis by TNF-, the effects of LY-294002 and a dominant negative mutant of PI3-kinase on protein synthesis were tested. LY-294002 completely inhibited the TNF--induced protein synthesis (Fig. 5A, left). Infection of cells with AxCAp85 suppressed TNF--induced protein synthesis in a MOI-dependent manner, whereas the infection with AxCALacZ did not inhibit it (Fig. 5A, right). These results indicated that PI3-kinase activity is necessary for TNF--induced protein synthesis in cardiac myocytes. Next, we investigated the role of Akt/PKB in TNF--induced protein synthesis by use of the adenovirus expressing a dominant negative mutant of Akt/PKB (AxCAAkt-AA). Infection of cells with AxCAAkt-AA also inhibited TNF--induced protein synthesis in a MOI-dependent manner (Fig. 5B). Therefore, the activation of the PI3-kinase-Akt/PKB pathway is required for protein synthesis stimulated by TNF- in cardiac myocytes.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   PI3-kinase-Akt/PKB pathway-mediated protein synthesis induced by TNF- in cardiac myocytes. A: cultured cardiac myocytes were incubated with or without 50 µM of LY-294002 for 10 min before stimulation with 2,000 U/ml of TNF- for 24 h. Protein synthesis was measured by [3H]leucine incorporation (left). Cultured cardiac myocytes were infected with AxCALacZ or AxCAp85 at the indicated MOI. Protein synthesis was measured by [3H]leucine incorporation 24 h after TNF- (2,000 U/ml) stimulation (right). Values are means ± SE of 3 independent trials and are expressed as multiples of control. B: cultured cardiac myocytes were infected with AxCALacZ or AxCAAkt-AA at the indicated MOI. Protein synthesis was measured by [3H]leucine incorporation 24 h after TNF- (2,000 U/ml) stimulation. Values are means ± SE of 3 independent trials and are expressed as multiples of control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, the role and signal transduction pathway of TNF- in cultured neonatal cardiac myocytes were examined. First, we showed that TNF- stimulated protein synthesis and enlargement of cell surface area without affecting cell viability. Next, it was demonstrated that TNF- induced activation of PI3-kinase, which resulted in Akt/PKB activation. These results were in accordance with recent studies performed in other cell types such as HeLa cells or human cervical carcinoma cells (21, 22). Although it is also reported that endotoxin, which is a frequent contaminant of recombinant proteins, can provoke the PI3-kinase/Akt pathway (25), neutralization of the stimulatory effect of TNF- on Akt/PKB phosphorylation with a neutralizing anti-TNF- antibody indicated that the stimulatory effects were attributed to TNF- (data not shown). Evidence was also provided that the activation of the PI3-kinase-Akt/PKB pathway was required for protein synthesis stimulated by TNF- in cardiac myocytes.

A number of enzymes possessing PI3-kinase activity have been identified and are divided into four classes: class 1a, 1b, 2, and 3 (27). Among them, the most characterized is class 1a PI3-kinase, which is a heterodimer composed of a regulatory subunit p85 and a catalytic subunit p110 and is considered to play a major role in growth factor signals. The regulatory subunit contains two SH2 domains, and binding of these SH2 domains to tyrosine-phosphorylated proteins results in the activation of lipid kinase activity (4, 27). Our results from in vitro lipid kinase assay using anti-phosphotyrosine immunoprecipitates revealed that PI3-kinase activity was stimulated by TNF- in cardiac myocytes and also suggested that this kinase activity was due to activation of class 1a PI3-kinase, although it is possible that the other classes of PI3-kinase are also involved in TNF--induced signal transduction pathways. Although TNF- receptors do not contain protein tyrosine kinase activity (15, 26, 28), tyrosine kinases and proteins including phosphotyrosine might be necessary to induce the activation of PI3-kinase by TNF-. The induction of specific tyrosine phosphorylation is suggested to be associated with cellular responses to TNF-. In 3T3-L1 adipocytes, it has been reported that TNF- rapidly induces the activation of nonreceptor tyrosine kinases, Jak 1 and 2 and Tyk 2 (9), and stimulates the tyrosine phosphorylation of a group of cytoplasmic proteins, such as insulin receptor substrate-1 (8) and signal transducers and activators of transcription 1, 3, 5, and 6 (9). Therefore, it is possible that these kinases and substrates are involved in TNF--induced PI3-kinase activation, although the tyrosine kinases and target proteins responsible for the activation of PI3-kinase by TNF- in cardiac myocytes remain to be identified.

We also showed that TNF- caused activation of Akt/PKB in cardiac myocytes. It has been demonstrated that PI3-kinase is necessary and sufficient for growth factor-dependent activation of Akt/PKB (2, 5, 6), although other pathways independent of PI3-kinase for activation of Akt/PKB have been suggested (11, 23). In cardiac myocytes, Akt/PKB activation by TNF- was inhibited by LY-294002. LY-294002 inhibits not only class 1a PI3-kinases but also other classes of PI3-kinases to various extents, whereas class 2 PI3-kinases are relatively resistant to this inhibitor (27). Therefore, we used a more specific molecular tool, the adenovirus expressing a dominant negative mutant of PI3-kinase (AxCAp85), which is a mutant of a regulatory subunit p85 lacking a binding site for a catalytic subunit p110 of PI3-kinase. Our results with this mutant clearly indicated that Akt/PKB activation by TNF- depends on PI3-kinase activity and suggested that class 1a PI3-kinase plays an important role in the signal transduction pathways of TNF- in cardiac myocytes.

Although it has been demonstrated that TNF- increases protein synthesis in cardiac myocytes (19, 39), less is known about the signal transduction pathway responsible for the TNF--induced protein synthesis. Recent reports demonstrated an essential role of the signaling pathway from PI3-kinase to Akt/PKB not only in protection from apoptosis (2, 5, 6) but also in protein synthesis (10, 37). Therefore, we investigated the role of the PI3-kinase-Akt/PKB pathway in protein synthesis stimulated by TNF-. Using a PI3-kinase inhibitor, LY-294002, and the dominant negative mutant of PI3-kinase, we showed the requirement of PI3-kinase activity in TNF--stimulated protein synthesis in cardiac myocytes. We next examined whether Akt/PKB is a responsible effector for protein synthesis resulting from TNF--induced PI3-kinase activation. For this purpose, we used a dominant negative mutant of Akt/PKB (Akt-AA), because a pharmacological inhibitor specific for Akt/PKB has not yet been reported. Akt-AA, whose phosphorylation sites critical for enzymatic activation are substituted with alanine, lacks protein kinase activity and acts as a dominant interfering mutant against endogenous Akt/PKB (3, 7, 10, 32). The expression of this dominant negative mutant of Akt/PKB significantly inhibited bulk protein synthesis stimulated by TNF-, indicating that Akt/PKB is the target of PI3-kinase in the signal transduction pathway mediating TNF--induced protein synthesis. Because it has been reported that growth factors promote cell survival as a consequence of PI3-kinase-mediated activation of Akt/PKB (2, 5, 6), there is a possibility that the inhibition of the PI3-kinase-Akt/PKB pathway induces cell death and thereby inhibits cell reaction to TNF- in cardiac myocytes. However, the expression of the dominant negative mutant of PI3-kinase or Akt/PKB did not alter the viable cell number as examined by WST-1 assay or activation of extracellular signal-regulated kinase by TNF- (data not shown). Therefore, the inhibition of the PI3-kinase-Akt/PKB pathway by the procedures we used did not induce cell death of cardiac myocytes nor result in nonspecific suppression of responses to TNF-.

In summary, the results of the present study provide the first demonstration of an essential role of the PI3-kinase-Akt/PKB pathway in the protein synthesis stimulated by TNF- in cardiac myocytes. Identification of upstream and downstream elements of PI3-kinase-Akt/PKB activation will be an important issue to understand the roles of PI3-kinase and Akt/PKB in cellular functions induced by TNF- and to fully clarify the signaling pathways of TNF- in cardiac myocytes.