INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PROINFLAMMATORY CYTOKINES are a class of secretory polypeptides that are synthesized and released locally by macrophages, leukocytes and endothelial cells in response to injury (1). Nitric oxide (NO) has been reported to play an important role as an effector molecule in cytokine signal transduction in a variety of cell types (7). NO is formed from the amino acid L-arginine by a distinct family of NO synthases (NOS) (17). Two different constitutive isoforms of NOS have been cloned and sequenced from brain and endothelium (3, 8). Proinflammatory cytokines have been shown to induce a third isoform of this enzyme [inducible NOS (iNOS)] in a variety of other cell types, including cardiac myocytes (CM) (2).

Tumor necrosis factor- (TNF-) is a proinflammatory cytokine that is primarily secreted by activated macrophages in response to stress (1). The biological effects of TNF- are caused by activation of specific cell-surface receptors (25). The multiple second messenger systems of TNF--receptor activation include protein kinase C (PKC), phospholipase A₂, phosphatidylcholine-specific phosphlipase C, sphingomyelinase, a ceramide-activated protein kinase, and mitogen-activated protein (MAP) kinases (25, 27).

Both myocardial macrophages and CM have been shown to synthesize TNF- (16). TNF- has been implicated in myocardial dysfunction and cardiac myocyte death in a variety of experimental and clinical conditions, including congestive heart failure (CHF) (15, 21). TNF- has been reported to regulate CM by NO-dependent and NO-independent mechanisms (5, 12, 16, 30).

We and others (9, 18, 22) have shown that interleukin-1 (IL-1) stimulates iNOS mRNA expression, iNOS protein synthesis, and NO production by neonatal rat CM in culture. The effects of TNF- on IL-1-stimulated NO production by CM have not been characterized. We now report that TNF- enhances IL-1-induced NO production by CM through a novel mechanism involving MAP kinase-mediated activation of NF-B.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise indicated. Cytokines were purchased from Genzyme (Boston, MA). The concentrations used were described in units per milliliter with the specific activity for IL-1 as 10⁸ U/mg protein and TNF- as 10⁷ U/mg protein. These values correspond to 5 ng/ml of IL-1 for 500 U/ml and 100 ng/ml of TNF- for 1,000 U/ml, as reported by others (9, 22) in the literature.

Animal experiments were performed in compliance with the guidelines of the National Institutes of Health and the Animal Care and Use Committee of the Robert C. Byrd Health Sciences Center of West Virginia University.

Isolation of CM. Myocytes were prepared from the ventricles of 1- to 2-day-old rat pups as we (18) have previously described. Briefly, the ventricles of 30-50 hearts obtained from three different litters were minced in Ca²⁺- and Mg²⁺-free Hanks' balanced salt solution (HBSS) and digested for 15-min periods in 10 ml of a solution containing 0.1% trypsin (GIBCO), 15 U/ml collagenase, and 0.1 mg/ml DNase (Worthington Biochemical, Freehold, NJ) in HBSS. Digestion was stopped by adding 10 ml of DMEM-Ham's F-12 (DMEM-F-12; GIBCO) containing 5% calf serum. Cycles were repeated until all of the tissue was digested. Myocytes were cultured in DMEM-F-12 culture medium supplemented with 5% calf serum, penicillin (50 U/ml), and streptomycin (50 mg/ml). Cells were seeded at a density of 1.25 × 10⁵ cells/cm² on various dishes (Falcon Plastics, Cockeysville, MD; Costar, Cambridge, MA) according to the experimental requirements. Culture medium was changed to fresh serum-free DMEM-F-12 containing insulin, transferrin, selenium, and BSA 48 h after plating was completed. Myocytes formed confluent monolayers of spontaneously beating cells 24 h later. These cells were washed, and fresh serum-free DMEM-F-12 was added. IL-1 (Genzyme), N^G-monomethyl-L-arginine (L-NMMA), and TNF- were added at this time and incubated as indicated in figures.

Assay for NO₂ production. NO₂ assays on neonatal rat cardiac myocyte cell culture supernatants were performed, as we (20) described previously. Briefly, the stable metabolic end product of NO synthesis, NO₂ was used as a measure of NO production. Cell culture supernatants from 48-well plates were mixed with an equal volume of Greiss reagent for 1 h. The absorbance at 550 nm was measured with a microplate reader (Molecular Devices). We previously demonstrated that the ratio between NO₂ and total NO₂ + NO₃ did not significantly change throughout the various experiments. Thus the NO₂ levels accurately reflected the total amount of NO produced.

MAP kinase in-gel assay. A myelin basic protein (MBP) in-gel kinase assay was performed as previously described (10). Twenty micrograms of protein per lane from cell lysates underwent electrophoresis on a SDS gel containing 0.4 mg/ml MBP (Sigma M-2016). MAP kinase activity was quantified by densitometry using the Optimas software program run on a Gateway 2000 personal computer (Optimas, Bothell, WA).

Electrophoretic mobility shift assay. Nuclear extracts were prepared as previously described by Ye and Samuels (29) and stored at 80°C before use. The double-stranded oligonucleotide containing a consensus NF-B binding site 5'-AGTTGA AGG C-3' (Santa Cruz Biotechnology, Santa Cruz, CA) was used to detect NF-B activity. Oligonucleotides were end-labeled with [-³²P]ATP (3,000 Ci/mmol; Amersham) and T4 polynucleotide kinase (Promega, Madison, WI). ³²P-labeled oligonucleotides (~30,000 counts/min) and 10 µg of nuclear protein were incubated for 20 min at room temperature in a total volume of 25 µl in the presence of 2 mM Tris · HCl (pH 7.5), 8 mM NaCl, 0.2 mM EDTA, 0.2 mM -mercaptoethanol, 0.8% glycerol, and 1 µg poly(dI-dC). Protein-DNA complexes were resolved by electrophoresis on nondenaturing 5% polyacrylamide gels and visualized by autoradiography.

Northern blot analysis. Northern blots were prepared as previously described (10). After exposure of cells (2.5 × 10⁶ cells/60-mm dish) to experimental conditions, total RNA was extracted using Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacture's instructions. A 10-µg sample of total RNA per lane was subjected to electrophoresis on 1.2% agarose gels containing 2.2 M Formalin. RNAs were transferred onto Zeta-probe blotting membranes (Bio-Rad, Hercules, CA) using Vacuum Blotter (model 785, Bio-Rad), and ultraviolet auto-cross-linked (GS Gene linker, Bio-Rad). Membranes were hybridized 16 h at 62°C with HS-114 hybridization solution (Molecular Research Center, Cincinnati, OH) containing murine iNOS (Alexis, San Diego, CA) and human GAPDH (Cayman Chemical, Ann Arbor, MI) cDNA probes labeled with deoxy-[-³²P]CTP (3,000 Ci/mM; Amersham) by random priming (Megaprime DNA labeling system; Amersham). The hybridized membranes were serially washed at 55°C using 1× sodium citrate- sodium chloride, and 1% SDS solution and exposed to Kodak XAR-5 film overnight at 70°C with an intensifying screen.

Western blot analysis. Western blots were performed as previously described (10). CM were lysed directly in each plate (1.25 × 10⁶ cells in a 30-mm plate) by application of a buffer containing 10 mM Tris · HCl (pH 7.4), 150 mM NaCl, 2 mM EGTA, 2 mM 1,4-dithiothreitol, 1 mM sodium orthovanadate, 100 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Protein concentrations were determined by the Bradford assay. The samples were treated with 2× Laemmli loading buffer and boiled for 5 min. Equal amounts (20 µg) of the denatured proteins per lane were subjected to 12% SDS-PAGE, transferred to a nitrocellulose membrane, and reversibly stained with Ponceau red to verify equal loading. The blots were probed with a 1:2,000 dilution of mouse monoclonal antibodies specific for iNOS (Alexis). The iNOS protein was detected using the Amersham ECL system.

Protein kinase C assay. PKC activity was quantified by using a commercially available protein kinase kit (Calbiochem, La Jolla, CA).

Statistical methods. Data represent means ± SE of 9-15 different determinations derived from 3 individual cells from each of 3-5 completely separate myocyte preparations of 30-50 individual neonatal rat pup hearts/preparation from 3 litters/preparation. A total of 15 different litters of 150-250 rat pup hearts were used for n = 5 preparations (Fig. 1). A total of 9 different litters of 90-150 rat pup hearts were used for n = 3 preparations (Figs. 2-4). ANOVA and the Student-Newman-Keuls test were used for multigroup comparisons. Values of P < 0.05 were considered statistically significant.

	RESULTS

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Exposure of CM to IL-1 alone (500 U/ml) resulted in a significant increase in NO₂ production at 48 h as we and others (9, 18, 22) have previously reported (P < 0.01, n = 5). Exposure of CM to TNF- alone (from 10 to 1,000 U/ml) had no effect on NO₂ production over vehicle alone, as was previously reported by others (22) [P = not significant (NS), n = 5]. The addition of TNF- to IL-1 resulted in a statistically significant increase in NO₂ production compared with IL-1 alone, as was previously reported by others (22) (P < 0.01, n = 5) (Fig. 1). Potential mechanisms involved in TNF- enhancement of IL-1-stimulated NO production have not been previously reported, however. The TNF--mediated increase in IL-1-stimulated NO₂ production was totally abolished by the addition of PD-98059 (20 µM), a selective MAP kinase kinase inhibitor (26) (P < 0.01, n = 5) (Fig. 1). The addition of the PKC inhibitor calphostin C (Cal C, 200 nM) had no effect on TNF- enhancement of IL-1-stimulated NO₂ production (P = NS, n = 5) (Fig. 1). The same concentration of Cal C (200 nM) completely inhibited TNF--stimulated PKC activity in CM in preliminary experiments (basal = 1.82 ± 0.12; TNF- = 3.43 ± 0.18, TNF- + Cal C = 1.92 ± 0.25 U/mg protein; P < 0.01, n = 6).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effects of interleukin-1 (IL-1; 500 U/ml) alone, or tumor necrosis factor- (TNF-; 1,000 U/ml) alone or IL-1 + TNF- on nitric oxide (NO2) production, and effects of mitogen-activated protein (MAP) kinase inhibitor PD-98059 or protein kinase C (PKC) inhibitor calphostin C (Cal C) on IL-1 + TNF--stimulated NO2 production by neonatal rat cardiac myocytes (CM). Values are means ± SE of 15 different determinations derived from 3 wells each from 5 separate CM preparations from 15 different litters (n = 5). * P < 0.01 vs. vehicle; ** P < 0.01 vs. IL-1.

The addition of TNF- alone to CM only induced negligible iNOS mRNA expression detectable by Northern analyses and no protein synthesis detectable by Western analyses. However, the addition of TNF- to IL-1 considerably enhanced both iNOS mRNA expression and iNOS protein synthesis compared with IL-1 alone (Fig. 2, A and B). PD-98059 did not reduce IL-1-stimulated iNOS mRNA levels. However, PD-98059 greatly reduced the enhancement by TNF- of IL-1-stimulated iNOS mRNA expression and protein synthesis. Cal C had no effect on TNF--stimulated iNOS mRNA expression or protein synthesis.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   A: representative Northern blot analysis of inducible nitric oxide synthase (iNOS) mRNA expression in CM exposed to IL-1, TNF-, or IL-1 + TNF-, and effects of PD-98059 and Cal C on IL-1 + TNF--stimulated iNOS expression. Amount of iNOS mRNA expression was determined by densitometry. Values are means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3) (from left to right: 3.2 ± 0.2, 22 ± 1.7, 9 ± 1.1, 60.6 ± 1.0, 31 ± 4.3, 61.3 ± 7.1, 19 ± 0.6). Equal RNA loading was confirmed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). B: representative Western blot analysis of iNOS protein in CM exposed to IL-1, TNF-, or IL-1 + TNF-, and effects of PD-98059 and Cal C on IL-1 + TNF--induced iNOS protein. Amount of iNOS protein was determined by densitometry. Values are means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3) (from left to right: 6.4 ± 0.2, 16.1 ± 0.9, 6.3 ± 0.2, 42.6 ± 2.3, 15.2 ± 1.8, 43.6 ± 3.2, 14 ± 1.5).

The role of MAP kinase activation in TNF- enhancement of IL-1-stimulated NO production was further confirmed by enzymatic assay. TNF- significantly increased MAP kinase activity that was reduced by the addition of the MAP kinase kinase inhibitor, PD-98059 (Fig. 3). However, the addition of Cal C did not reduce the increase in MAP kinase activity stimulated by TNF- (Fig. 3).

View larger version (60K): [in this window] [in a new window]   Fig. 3.   Representative MAP kinase in-gel assay illustrating MAP kinase activation by IL-1, TNF-, and IL-1 + TNF-, and inhibition of this effect by known MAP kinase kinase inhibitor PD-98059 but not known PKC inhibitor Cal C. MAP kinase activity was quantified by densitometry. Values are means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3) (from left to right: 2.7 ± 0.3, 4.0 ± 0.1, 6.3 ± 0.1, 10.0 ± 0.12, 5.2 ± 0.1, 10.6 ± 0.4, 4.9 ± 0.2).

We have previously shown by immunohistochemistry that nuclear translocation of NF-B is essential for IL-1-stimulated NO production by CM (19). We now studied the effect of TNF- and IL-1 on NF-B activation determined by electrophoretic mobility shift assay (Fig. 4). IL-1 and TNF- each increased NF-B activity at 2 h. The addition of TNF- and IL-1 together greatly potentiated NF-B activation to a greater extent than the addition of either alone (Fig. 4A). PD-98059, but not Cal C, reduced NF-B activity that followed exposure to both IL-1 and TNF- (Fig. 4A). Interestingly, PD-98059 did not inhibit NF-B activity stimulated by IL-1 alone. Gel supershift assay using anti-p65 antibody confirmed NF-B activation (Fig. 4B).

View larger version (73K): [in this window] [in a new window]   Fig. 4.   A: representative NF-B activity reflected in electrophoretic mobility shift assay illustrating NF-B activation by IL-1, TNF-, and IL-1 + TNF-, and inhibition of this effect by PD-98059 but not Cal C. NF-B activity was quantified by densitometry. Values are means ± SE in relative optical units derived from 3 separate experiments from 9 different litters (n = 3) (from left to right: 58 ± 5.5, 123.2 ± 17.2, 66.6 ± 6.0, 241.3 ± 13.9, 129.6 ± 10.7, 253.6 ± 16.5, 112.6 ± 5.8). B: identity of NF-B band was repeatedly confirmed by gel supershift assay using anti-p65 antibody. Mutant oligonucleotide in 1st column was substituted for oligonucleotide containing a consensus NF-B binding site.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Both IL-1 and TNF- have been shown to activate multiple second messenger signaling pathways including MAP kinase and PKC pathways by binding to specific cell surface receptors (1, 25). Therefore, we examined the potential involvement of MAP kinase and/or PKC in TNF--mediated NO production in CM. PD-98059, a specific inhibitor of MAP kinase kinase, completely abolished TNF- enhancement of IL-1-stimulated NO production. Cal C, a PKC inhibitor, did not reduce TNF--enhanced NO production (Fig. 1), although it did reduce TNF--stimulated PKC activity in CM (see RESULTS). Both Northern and Western analyses were consistent with an inhibitory effect of PD-98059 and the absence of an effect of Cal C on TNF- enhancement of IL-1-induced iNOS mRNA expression and protein synthesis (Fig. 2).

TNF-- and IL-1-activated MAP kinase activity was further studied by the "in-gel" MAP kinase assay. TNF- greatly increased MAP kinase activity which was inhibited by PD-98059 but not Cal C (Fig. 3). Together, our data indicate that TNF- enhances NO production stimulated by IL-1 through a PKC-independent MAP kinase pathway. Our findings are consistent with reports of both PKC-dependent and -independent MAP kinases in neonatal rat CM (24). More study is warranted before drawing definitive conclusions regarding the PKC independence of this particular MAP kinase that participates in iNOS regulation in CM. Activation of this MAP kinase alone by TNF- may be sufficient to induce transient expression of iNOS mRNA. However, it is clearly not sufficient to stimulate iNOS protein synthesis and NO production in CM.

IL-1 and TNF- receptor signaling have each been shown to lead to NF-B activation (14). We previously reported that nuclear translocation of NF-B is essential for IL-1-stimulated NO production in neonatal rat CM (19). More recently, an elegant series of experiments were published that provided compelling evidence that NF-B is required for TNF-- and IL-1-induced iNOS mRNA expression (23). In addition, four NF-B-enhancer elements were identified upstream in the human iNOS promoter that confer inducibility to TNF- and IL-1 (23). An effect of MAP kinase in the regulation of these NF-B-enhancer elements has not been reported. Therefore, we investigated the role of MAP kinase cascades in NF-B activation. IL-1 and TNF- each increased NF-B activity. The effect of IL-1 on NF-B activity was greater than TNF- as indicated by electrophoretic mobility shift assay (Fig. 4). The addition of TNF- to IL-1 greatly potentiated the effect of either cytokine alone on NF-B activity. PD-98059, but not Cal C, reduced TNF- plus IL-1-stimulated NF-B activity (Fig. 4). Our data indicate that both IL-1 and TNF- increase NF-B activity. However, TNF- alone only stimulated minimal iNOS mRNA expression, without iNOS protein synthesis (Fig. 2). Thus activation of NF-B is necessary for iNOS mRNA expression, but not sufficient for iNOS protein synthesis.

We recently also reported induction of iNOS mRNA expression without resulting in iNOS protein synthesis by neonatal rat CM after exposure to norepinephrine (10). The addition of norepinephrine to IL-1 also enhanced iNOS mRNA expression, protein synthesis, and nitrite production (10). These modulatory effects of TNF- and norepinephrine on IL-1-induced NO production may suggest potentially important mechanisms to control cardiac myocyte NO production under physiological and/or pathological conditions. It is interesting to note that elevated circulating levels of TNF- and norepinephrine have each independently been associated with a poorer prognosis in patients with CHF (4, 15). The pathophysiological relevance of these TNF- and norepinephrine levels and/or their effect on cardiac myocyte NO production remains to be determined (6).

PD-98059 did not inhibit NF-B activity stimulated by IL-1 alone (Fig. 4). This observation suggests that IL-1 stimulates NF-B activity through a different signaling pathway than TNF-. It has been reported in HepG2 cells that IL-1 can activate three MAP kinase cascades, namely, p46/54(JNK), p38 (MAPK) and Erk-1/2, with maximal activation of 25-fold with p38, and only threefold with Erk-1/2 (13). Inhibition of p38 MAP kinase has been shown to block NF-B nuclear translocation and activation in a rat heart model of ischemia (11). This is particularly interesting in view of a report (9) that chronic hypoxia inhibits both IL-1-stimulated NO production and NF-B stimulation by neonatal rat CM in culture. The identification of the specific MAP kinases involved in iNOS regulation in CM warrants further study.

Elevated circulating levels of TNF- have been described in a variety of clinical and experimental conditions associated with myocardial dysfunction, including CHF (15). The pathophysiological relevance of TNF- and NO in clinical conditions such as CHF is unclear. These newly identified endogenous mediators may contribute to myocardial dysfunction via direct depression of contractility and/or induction of myocyte apoptosis. These effects of TNF- on myocardial dysfunction could be caused by NO-dependent and/or NO-independent mechanisms. NO has been reported to have a negative inotropic effect on the heart and to be involved in the regulation of apoptosis (5, 28). On the other hand, cytokines and NO may serve a compensatory role in human disease that may not be as apparent in isolated, healthy cells. It is clear, however, that cytokine-induced NO production in CM is very tightly controlled through an intricate series of integrated pathways. Such complexity and integration alone suggests physiological significance. Therefore, exploring the mechanisms involved in cytokine-mediated NO production by CM may provide novel insights relevant to designing management strategies for patients with myocardial dysfunction. The MAP kinase described in this study may represent one such potentially novel therapeutic target.