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Am J Physiol Heart Circ Physiol 289: H2103-H2111, 2005. First published June 24, 2005; doi:10.1152/ajpheart.00393.2005
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Inhibitor-{kappa}B kinase-{beta} regulates LPS-induced TNF-{alpha} production in cardiac myocytes through modulation of NF-{kappa}B p65 subunit phosphorylation

Gentzon Hall,1 Ishwar S. Singh,2 Lisa Hester,2 Jeffery D. Hasday,1,2 and Terry B. Rogers1

Departments of 1Biochemistry and Molecular Biology and 2Medicine, University of Maryland School of Medicine, Baltimore, Maryland

Submitted 21 April 2005 ; accepted in final form 17 June 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-{kappa}B family of transcription factors are essential regulators of myocardial TNF-{alpha} gene expression, recent developments in our understanding of the modulation of NF-{kappa}B activity through posttranslational modification of NF-{kappa}B subunits suggest that the present view of NF-{kappa}B-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-{alpha} production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-{alpha} production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-{beta} reduced LPS-induced TNF-{alpha} production 38-fold, TNF-{alpha} mRNA levels 6-fold, and I{kappa}B-{alpha} phosphorylation 5-fold and degraded I{kappa}B-{alpha} 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-{alpha} production 3.5-fold, whereas overexpression of dominant-negative IKK-{beta} reduced LPS-induced TNF-{alpha} production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-{alpha} had no effect on p65 phosphorylation or TNF-{alpha} production, revealing that IKK-{beta}, not IKK-{alpha}, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-{alpha} production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-{alpha} gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-{kappa}B signaling in TNF-{alpha} gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.

signal transuction; gene expression; heart; cytokines

IN RECENT YEARS, PROINFLAMMATORY cytokines such as TNF-{alpha}, IL-1{beta}, and IL-6 have emerged as significant contributors to myocardial dysfunction after stress, injury, or infection (12, 22, 27–29, 33). Myocardial cytokine production has been linked to the pathogenesis of several cardiomyopathies, including ischemic heart disease, congestive heart failure, and sepsis-induced myocardial dysfunction (12, 22, 33, 49, 50). Additionally, compelling evidence from the study of transgenic mice genetically engineered for cardiac-restricted overexpression of TNF-{alpha} demonstrates that chronic exposure to elevated levels of locally produced TNF-{alpha} leads to ventricular chamber dilation and heart failure (4, 20). Given the severity of the effects of these potent proinflammatory molecules on myocardial performance and viability, it is important to characterize the signaling cascades that link proinflammatory agonists to cytokine production in heart.

Despite the importance of cytokines in cardiac pathology, relatively little is known about the molecular events underlying their stress-induced production in cardiac myocytes. Several studies, including our own, have identified the transcription factor NF-{kappa}B as a key regulator of TNF-{alpha} gene activation (14, 33, 51, 54). The NF-{kappa}B pathway can be activated by diverse proinflammatory stimuli, including reactive oxygen intermediates, cytokines, hypoxia, ischemia, ultraviolet radiation, and viral or bacterial products (19, 51). In previous studies, we demonstrated that LPS, a component of the gram-negative bacterial cell wall, is a potent stimulator of NF-{kappa}B signaling and cytokine gene expression (54). In nonmyocytes, the LPS moiety is recognized by the Toll-like receptor 4 (TLR4) and signals via a CD14- and MD-2-dependent mechanism to trigger the assembly of a multiprotein signaling apparatus at the cytoplasmic face of TLR4. This assembly of adaptor molecules, such as myeloid differentiation factor 88 and TNF receptor-associated factor 6, facilitates activation of the IL receptor-associated kinase, which signals through the TAK1-TAB kinase complex to phosphorylate and activate IKK (2). Several of the components of this signaling cascade have been identified in heart and are linked to LPS-induced cytokine production and other pathophysiological responses in cardiac myocytes (34, 45).

The NF-{kappa}B family of transcription factors exists in the cytoplasm of unstimulated cells as homo- or heterodimers complexed to the I{kappa}B proteins. On stimulation, phosphorylation of I{kappa}B by IKK triggers its degradation. This step unmasks a nuclear localization sequence on NF-{kappa}B that promotes its translocation to the nucleus followed by binding and activation of various genes including TNF-{alpha} (13, 14). This classic model of NF-{kappa}B activation, although it is documented in heart (14, 24, 26, 54, 55), now appears to be a simplified view of the process. For instance, the p65/RelA subunit of NF-{kappa}B is modified extensively by phosphorylation in a highly cell type- and stimulus-specific manner. p65 is known to be phosphorylated at seven separate sites by diverse kinases, with each modification exerting distinct effects on its transcriptional activity (1, 5–7, 11, 16, 25, 30, 37, 41–44, 53, 58). Despite the roles of phosphorylation of p65, little is known about such reactions in cardiac myocytes.

In an effort to identify key signaling and regulatory mechanisms that underlie myocardial cytokine expression at the molecular level, we examined the regulation of TNF-{alpha} production using an established model of cultured mouse neonatal cardiac myocytes (54). The focus was to examine the signaling linked to phosphorylation of p65 at a key regulatory site, Ser536, in its COOH-terminal transactivation domain (TAD). The results reported here show that IKK-{beta} activity is involved in the phosphorylation of p65 at Ser536. Furthermore, we demonstrate that phosphorylation of p65 is important in the NF-{kappa}B-dependent activation of myocardial TNF-{alpha} gene expression. Taken together, these findings reveal a central role for IKK-{beta} in the modulation of NF-{kappa}B activation and TNF-{alpha} gene expression in heart and provide further support for the potential therapeutic benefit of targeted IKK-{beta} inhibition in the management of various cardiomyopathies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Culture of cardiac myocytes. Mouse neonatal cardiac myocytes were cultured following the methods described by Wright et al. (54). Acute dissociation of adult mouse ventricular cardiac myocytes was performed according to the methods described by DuBell et al. (10). All cell culture reagents were certified free of endotoxin.

Adenoviruses and infections. Dominant-negative kinase-dead [by site-directed mutagenesis of the ATP-binding domain (K44M)] mutants of IKK-{alpha} (dnIKK-{alpha}) and IKK-{beta} (dnIKK-{beta}) were the generous contributions of Dr. Yibin Wang (57). The dominant-negative mutant of p65 (dnp65), made transcriptionally inactive by deletion of COOH-terminal TAD 1 and 2, was kindly provided by Dr. Matthew P. Soares (45). The {beta}-galactosidase ({beta}-Gal)-expressing adenovirus (Ad-{beta}-Gal) was generously provided by Dr. William Randall. The optimal infective concentration for each virus was determined by measuring the number of viral particles per cell that resulted in the maximal expression of the desired construct by Western blot. The effective viral doses were 300–500 particles/cell. Day 1 myocyte cultures were infected for 2 h in serum-free medium before the addition of serum-supplemented medium. After 24 h, cells were washed and allowed to incubate for an additional 48 h in serum-supplemented medium before experiments were performed.

Immunoblotting and reagents. Cultures were infected with Ad-dnIKK-{beta}, Ad-dnIKK-{alpha}, Ad-dnp65, or Ad-{beta}-Gal 48 h before stimulation. After stimulation, cardiac myocytes were placed on ice and washed twice in ice-cold 1x PBS. Cells were harvested using a rubber policeman and transferred to a 1.5-ml Eppendorf tube. Cells were then centrifuged for 30 s at 7,000 rpm and resuspended in 2x Laemelli solution (100 mM Tris·HCl, pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, and 0.01% bromphenol blue) supplemented with phosphatase inhibitors (50 mM NaF, 1 µM calyculin A, and 200 µM orthovanadate) and protease inhibitor cocktail (Sigma Chemical, St. Louis, MO). After SDS-PAGE using NuPAGE 10% Bis-Tris precast gels (Invitrogen, Carlsbad, CA), the cells were transferred to 0.45-µm-pore polyvinylidene difluoride membrane (Immobilon, Billerica, MA). The antibodies for Western blotting were as follows: a 1:2,000 dilution of mouse monoclonal anti-phospho-p65 (Ser536), a 1:500 dilution of anti-phospho-IKK-{alpha}/{beta} (Ser180/181), a 1:1,000 dilution of anti-I{kappa}B-{alpha}, a 1:1,000 dilution of anti-phospho-I{kappa}B-{alpha} (Cell Signaling Technologies, Beverly, MA), and a 1:2,000 dilution of mouse monoclonal anti-p65 (BD Biosciences, San Jose, CA). The pharmacological reagents IKK-{beta} inhibitor IV, SC-514, and IKK-{beta} inhibitor V (IMD-0354) were obtained from Calbiochem (San Diego, CA).

Measurement of TNF-{alpha} release. TNF-{alpha} in culture supernatants was quantified in the University of Maryland Cytokine Core Laboratory using standard two-antibody ELISA with commercial antibody pairs and recombinant standards (Pierce-Endogen, Boston, MA). Polystyrene plates (Maxisorb, Nunc) were coated with capture antibody in PBS overnight at 25°C. The plates were washed four times with 50 mM Tris-0.2% Tween 20, pH 7.2, and then blocked for 90 min at 25°C with assay buffer (PBS containing 4% bovine serum albumin and 0.01% thimerosal, pH 7.2). The plates were washed, and 50 µl of assay buffer were added to each along with 50 µl of sample or standard prepared in assay buffer and incubated at 37°C for 2 h. After the plates were washed, streptavidin-peroxidase polymer in casein buffer (Research Diagnostics, Mount Pleasant, NJ) was added, and the plates were incubated at 25°C for 30 min. The plates were washed, 100 µl of substrate (tetramethyl benzidine; Dako, Carpentaria, CA) were added, and the plates were incubated for 20–30 min. The reaction was stopped with 100 µl of 2 N HCl, and the optical density (OD) at 450 nm (OD450) – OD at 650 nm (OD650) was read on a microplate reader (Molecular Devices, Sunnyvale, CA). The data were analyzed using a computer program (SoftPro, Molecular Devices). The assay had a lower detection limit of 8 pg/ml.

Real-time PCR methods. After treatment, cell culture medium was removed, and 1 ml of Trizol reagent (Invitrogen) was added to each dish. Cell suspension was then transferred to 15-ml Eppendorf tubes, and total mRNA was isolated by chloroform extraction and ethanol precipitation. Total mRNA pellets were resuspended in diethylpyrocarbonate-treated water and spectrophotometrically assayed for concentration. One microgram of total mRNA was then reverse-transcribed into cDNA using the Superscript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. TNF-{alpha} and hypoxanthine guanine phosphoribosyltransferase (HPRT, used as a "housekeeping gene" to normalize sample variations) mRNA levels were quantified by real-time PCR using an ABI Prism 7700 sequence detector according to manufacturer’s instructions. Real-time PCR was performed in 25-µl reaction volumes containing the SYBR green PCR Master Mix buffer (Applied Biosystems, Foster City, CA), 15 µg of cDNA, and TNF-{alpha} or HPRT primers at a final concentration of 3 nM to detect the expression of TNF-{alpha} and HPRT mRNAs.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assay was performed using a kit from Upstate Biotechnology (Lake Placid, NY) according to the manufacturer’s instructions. Unless otherwise stated, all reagents were provided in the kit. Briefly, cells were treated with LPS (1 µg/ml) and fixed by addition of 37% formaldehyde solution (Sigma Chemical) to the medium to a final concentration of 1%. After 15 min, the cells were washed with PBS containing protease inhibitors and collected by centrifugation. Cell pellets were resuspended in SDS lysis buffer and sonicated by nine 20-s bursts using duty cycle and output settings of 20 and 3, respectively. Sonicated cell lysates were diluted 10-fold using ChIP dilution buffer and precleared for 1 h at 4°C using 75 µl of 50% slurry of protein A-agarose beads saturated with salmon sperm DNA. Immunoprecipitation was carried out at 4°C overnight, and immune complexes were collected with salmon sperm DNA-saturated protein A-agarose beads preincubated with mouse monoclonal phospho-p65 (Ser536) antibody (Cell Signaling) at a 1:50 dilution. The beads were washed three times with immune complex wash buffers and twice with Tris-EDTA buffer, and the complexes were eluted with 0.1 M NaHCO3 and 1% SDS. Protein-DNA cross-links were reverted by incubation at 65°C overnight. After proteinase K digestion, DNA was isolated via phenol-chloroform extraction and purified via ethanol precipitation. PCR was performed (40 cycles, denaturing at 95°C for 30 s, annealing at 63°C for 30 s, and extension at 68°C for 30 s) using primers complementary to two sites flanking a ~300-bp fragment of the murine TNF-{alpha} promoter between nucleotides –434 and –722 relative to the transcription start site and encoding {kappa}B sites 2 and 2a: 5'-AGTCATACGGATTGGGAGAAATCCTG-3' (forward) and 5'-AGTTCTTGGAGGAAGTGGCTGAAGGCA-3' (reverse).

Statistical analysis. Values are means ± SD. The statistical significance of differences between control and experimental groups was calculated by one-way analysis of variance followed by Newman-Keuls test by the use of the GB-STAT statistical software program (Dynamic Microsystems). P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Bacterial LPS induces phosphorylation of NF-{kappa}B p65 at Ser536 in cultured murine neonatal cardiac myocytes. Previous studies from several groups, including our own, have shown that proinflammatory agonists, such as LPS, activate the nuclear translocation of NF-{kappa}B in cardiac myocytes (14, 24, 54). More recent studies in other cell types have demonstrated additional posttranslational events that participate in the activation of NF-{kappa}B-responsive genes, including phosphorylation of specific serine residues in the NF-{kappa}B p65 subunit. One such phosphorylation event at NF-{kappa}B p65 Ser536, which has been shown to markedly enhance the transactivation potential of p65 in other cell types (25, 26), was analyzed in cardiac myocytes exposed to LPS. Western blot analysis using an antibody specific for Ser536-phosphorylated p65 revealed that p65 is rapidly phosphorylated at Ser536 after exposure to LPS. In cultured neonatal cardiac myocytes, phosphorylation of p65 Ser536 was evident as early as 15 min after LPS exposure, peaked at 30–45 min, and subsequently waned without any apparent change in total p65 levels (Fig. 1, A and B). Similar results were obtained in freshly isolated adult ventricular myocytes (Fig. 1C).



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  		Fig. 1. LPS induces phosphorylation of p65 at Ser536 in cultured murine neonatal cardiac myocytes. A: cultured myocytes were stimulated with LPS (1 µg/ml) in the presence of 10 nM calyculin A for 0–90 min. Reactions were terminated, and whole cell protein extracts were prepared. Proteins were resolved on SDS gels and subjected to Western blot analysis with antiphospho-Ser536 p65 or anti-p65. B: summary data (means ± SD of 3 separate experiments) of the ratio of phospho-p65 to total p65 from densitometric analysis of Western blot images. OD, optical density. C: acutely dissociated adult mouse ventricular myocytes were stimulated with LPS for 0–60 min. Protein extracts were prepared and analyzed as described in A for phospho-Ser536 p65 by Western blot.

 
Pharmacological and molecular genetic inhibition of IKK-{beta} attenuates p65 phosphorylation and I{kappa}B-{alpha} phosphorylation and degradation in LPS-stimulated neonatal cardiac myocytes. To understand the molecular features of the NF-{kappa}B cascade in heart, it was important to identify the kinase responsible for this phosphorylation. In HeLa cells, studies have shown that IKK-{beta} and IKK-{alpha} are capable of phosphorylating Ser536 of p65 (42). Western blot analysis of neonatal cardiac myocytes using anti-phospho-IKK was used to assess IKK activation (9, 35). These results demonstrated that IKK-{beta} and IKK-{alpha} are phosphorylated within 10 min of LPS treatment and that phosphorylation is sustained for ≥60 min (Fig. 2A). To determine the contribution of IKK-{beta} to the phosphorylation of p65 in cardiac myocytes, the capacity of pharmacological inhibitors of IKK-{beta} to block LPS-induced p65 phosphorylation was assessed by phosphospecific Western blotting (Fig. 2, B and C). The phosphorylation signals were stabilized by blocking endogenous serine/threonine phosphatases with 10 nM calyculin A. Control experiments revealed that calyculin A, in the absence of LPS, had only a minimal effect on Ser536 phosphorylation (Fig. 2B). All three IKK inhibitors reduced LPS-induced p65 phosphorylation (Fig. 2B, lanes 4–6). Whereas IKK-{beta} inhibitor IV almost completely inhibited Ser536 phosphorylation, reduction of p65 phosphorylation by SC-514 or IMD-0354 was more modest (Fig. 2B). Specifically, the competitive ATP analog SC-514 blocked p65 phosphorylation by 21% and IMD-0354 and IKK-{beta} inhibitor IV by 22% and 87%, respectively (Fig. 2C). Time course studies revealed that the partial effect of SC-514 was not due to a shift in the time to maximum phosphorylation (data not shown).



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  		Fig. 2. LPS activates IKK-{beta} and p65 phosphorylation is attenuated by pharmacological inhibition of IKK-{beta} in neonatal cardiac myocytes. A: cultured myocytes were stimulated with LPS for 0–60 min. Reactions were terminated, and whole cell protein extracts were prepared. Proteins were resolved on SDS gels and subjected to Western blot analysis and developed with antiphospho-IKK. B: myocyte cultures were pretreated with IKK-{beta} inhibitors for 1 h before stimulation with LPS for 30 min. Whole cell extracts were collected and subjected to Western blot analysis as described in Fig. 1. C: summary data (means ± SD of 3 experiments) from inhibitor studies calculated as the ratio of intensities of phospho-p65 to total p65 bands in densitometric scans. Significantly different from LPS- and calyculin A-treated controls: *P < 0.05; {dagger}P < 0.01.

 
From diverse previous studies, it is predicted that IKK-{beta} phosphorylates I{kappa}B-{alpha} and initiates its degradation as well. Thus a series of experiments parallel to those in Fig. 2 were performed to examine this possibility. As shown in Fig. 3, A and B, pharmacological inhibition of IKK-{beta} reduced LPS-evoked I{kappa}B-{alpha} phosphorylation and degradation. Specifically, SC-514 reduced I{kappa}B-{alpha} phosphorylation and degradation by 11% and 28%, respectively, IMD-0354 reduced phosphorylation and degradation of I{kappa}B-{alpha} by 31% and 51%, respectively, and IKK-{beta} inhibitor IV reduced I{kappa}B-{alpha} phosphorylation and degradation by 83% and 60%, respectively. Taken together, these data support the view that IKK-{beta} activity is associated with the phosphorylation of I{kappa}B-{alpha} at Ser32 in addition to p65 phosphorylation at Ser536.



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  		Fig. 3. LPS-induced phosphorylation and degradation of I{kappa}B-{alpha} is attenuated by pharmacological inhibition of IKK-{beta} in neonatal cardiac myocytes. Myocyte cultures were pretreated with IKK-{beta} inhibitors for 1 h before stimulation with LPS for 30 min. Whole cell extracts were collected and subjected to Western blot analysis. A: summary data (means ± SD of 3 separate experiments) from inhibitor studies calculated as the relative ratio of intensities of I{kappa}B-{alpha} bands in densitometric scans. *Significantly different (P < 0.01) from LPS- and calyculin A-treated controls. B: summary data (means ± SD of 3 separate experiments) from inhibitor studies calculated as the relative ratio of intensities of Ser32-phosphorylated I{kappa}B-{alpha} bands in densitometric scans. *Significantly different (P < 0.01) from LPS- and calyculin A-treated controls. NS designates a nonspecific band that migrates at a higher molecular weight than phospho-I{kappa}B-{alpha} (A) and total I{kappa}B-{alpha}, which migrate identically to a ~37-kDa band.

 
Because pharmacological agents often exhibit off-target activities, the effects of the pharmacological IKK inhibitors were confirmed by genetic interruption of IKK signaling through expression of dnIKK-{beta} and dnIKK-{alpha} compared with {beta}-Gal (control) adenoviral expression constructs (57). As shown by phosphospecific Western blot analysis, LPS-induced p65 phosphorylation was almost completely attenuated by hemagglutinin (HA)-tagged dnIKK-{beta}, whereas HA-tagged dnIKK-{alpha} and the control adenoviral construct each had no detectable effect on LPS-induced p65 phosphorylation (Fig. 4). Western blotting with anti-HA antibody demonstrated similar expression of dnIKK-{alpha} and dnIKK-{beta} (see Fig. 6). Taken together, the pharmacological and molecular genetic data suggest that IKK-{beta}, but not IKK-{alpha}, is predominantly responsible for the phosphorylation of p65 at Ser536 after LPS stimulation of cardiac myocytes.



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  		Fig. 4. Expression of dominant negative IKK-{beta} (dnIKK-{beta}) attenuates LPS-evoked phosphorylation of p65 in cardiac myocytes. A: cultures infected with Ad-dnIKK-{alpha}, Ad-dnIKK-{beta}, or Ad-{beta}-Gal for 48 h before treatment with LPS for 0–50 min. Whole cell extracts were collected and subjected to Western blot analysis for phospho-Ser536 p65 and total p65 as described in Fig. 1 legend. B: summary data (means ± SD of 3 experiments) presented as the ratio of phospho-p65 to p65 band intensities normalized to the value obtained at 0 min for dnIKK-{beta}-infected ({blacktriangleup}) and {beta}-Gal-infected ({blacksquare}) cells. *Significant difference (P < 0.05) between dnIKK-{beta}- and {beta}-Gal-infected cultures.

 


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  		Fig. 6. Expression of dominant negative-IKK-{beta} attenuates TNF-{alpha} production in cardiac myocytes. A: cardiac myocyte cultures were infected with Ad-dnIKK-{beta} or Ad-{beta}-Gal (300 particles/cell) 48 h before treatment with LPS for 5 h. At the end of the incubation period, 200 µl of cell culture medium were collected, frozen, and then analyzed for TNF-{alpha} protein. Values are means ± SD of 3 separate experiments. To verify expression of hemagglutinin (HA)-tagged dnIKK-{beta} cells, extracts were prepared and analyzed by Western blot analyses and developed with anti-HA. dnIKK-{beta} was detected in infected cultures. *Significantly different from {beta}-Gal (P < 0.05). B: parallel experiments identical to those in A, except cells were infected with Ad-dnIKK-{alpha}.

 
Inhibition of IKK-{beta} attenuates TNF-{alpha} release in LPS-stimulated neonatal cardiac myocytes. We previously showed that NF-{kappa}B signaling is required for maximal LPS-induced TNF-{alpha} generation in mouse cardiac myocytes (54). The contribution of IKK-{alpha} and IKK-{beta} to LPS-induced TNF-{alpha} generation in cardiac myocytes was analyzed using the same complementary pharmacological and molecular genetic strategies used for analysis of p65 phosphorylation. To maintain consistency with earlier experiments, all experiments were performed in the presence of 10 nM calyculin A. Control studies demonstrated that calyculin A alone did not promote TNF-{alpha} release (Fig. 5). As we previously reported (54), incubating cardiac myocytes with 1 µg/ml LPS for 4–5 h stimulates release of >300 pg/ml TNF-{alpha} (Fig. 5). The IKK-{beta} inhibitors IMD-0354 and IKK-{beta} inhibitor IV blocked TNF-{alpha} generation by 95% and 97%, respectively, whereas SC-514 was less effective (43%) in attenuating TNF-{alpha} release. Thus SC-514 produced the least inhibition of TNF-{alpha} release, whereas IMD-0354 and IKK-{beta} inhibitor IV produced progressively greater inhibition.



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  		Fig. 5. LPS-induced TNF-{alpha} production is attenuated by pharmacological inhibition of IKK-{beta} in cardiac myocytes. Neonatal myocyte cultures were pretreated with IKK-{beta} inhibitors for 1 h before treatment with LPS in the presence of 10 nM calyculin A for an additional 5 h. At the end of the incubation period, 200 µl of cell culture medium were collected and frozen and subsequently assayed for released TNF-{alpha} protein. Values are means ± SD of 3 separate experiments. Significantly different from LPS- and calyculin A-treated controls: *P < 0.05; {dagger}P < 0.01.

 
The pharmacological inhibitor studies were complemented by the same molecular genetic approach utilized in the analysis of p65 phosphorylation (Fig. 6). As was seen for p65 phosphorylation, expression of dnIKK-{beta} reduced TNF-{alpha} expression by 51% compared with LPS-stimulated control ({beta}-Gal construct) cardiac myocytes (Fig. 6A), whereas expression of the dnIKK-{alpha} construct failed to alter TNF-{alpha} expression (Fig. 6B). Taken together, these data are consistent with the pattern of kinase inhibition on p65 phosphorylation and confirm that IKK-{beta}, but not IKK-{alpha}, is important in the LPS-induced signaling that leads to proinflammatory cytokine expression in cardiac myocytes.

The importance of the p65 TAD was further examined through the use of a dnp65 construct, a COOH-terminal deletion mutant of p65 lacking TADs 1 and 2 but retaining the ability to dimerize and bind DNA (45). As shown in Fig. 7, expression of dnp65 reduced TNF-{alpha} expression by 71%.



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  		Fig. 7. Expression of dominant-negative-p65 (dnp65) attenuates TNF-{alpha} production in cardiac myocytes. A: cardiac myocyte cultures were infected with Ad-p65 or Ad-{beta}-Gal (300 particles/cell) 48 h before treatment with LPS for 5 h. At the end of the incubation period, 200 µl of cell culture medium were collected and then analyzed for TNF-{alpha} protein. Values are means ± SD of 3 separate experiments.

 
Given the known activities of NF-{kappa}B as a transcriptional activator (14, 24, 26, 54), the results suggested that abrogation of TNF-{alpha} release resulted from reduced transcription. Accordingly, using real-time PCR analysis, we analyzed the effects of the IKK-{beta} inhibitors on steady-state TNF-{alpha} mRNA (Fig. 8). TNF-{alpha} mRNA was undetectable in untreated cardiac myocytes but accumulated after 1.5 h of stimulation with 1 µg/ml LPS. Although IMD-0354 and IKK-{beta} inhibitor IV each markedly inhibited LPS-induced TNF-{alpha} mRNA accumulation, SC-514, which exerted the least inhibition of TNF-{alpha} release, unexpectedly enhanced TNF-{alpha} mRNA expression (see DISCUSSION).



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  		Fig. 8. Pharmacological inhibition of IKK-{beta} inhibits LPS-induced TNF-{alpha} mRNA production in neonatal cardiac myocytes. Cultures were pretreated for 1 h with inhibitors before stimulation with LPS for 1.5 h. Total cellular mRNA was collected and reversed transcribed into cDNA for assay of TNF-{alpha} message using real-time PCR. Values are means ± SD from 3 separate experiments. *Significantly different from LPS- and calyculin A-treated controls (P < 0.01).

 
The effects of IKK-{beta} inhibition on TNF-{alpha} mRNA accumulation (Fig. 8) suggest that IKK-{beta} activates TNF-{alpha} expression, at least in part, by stimulating the recruitment of phospho-p65-containing NF-{kappa}B complexes to their cognate binding site(s) on the TNF-{alpha} promoter. Accordingly, we developed a ChIP assay that utilized anti-phospho-p65 antibody to immunoprecipitate phospho-p65-DNA complexes and PCR primers that amplified the TNF-{alpha} promoter region containing {kappa}B sites 2 and 2a (21) (see MATERIALS AND METHODS). As shown in Fig. 9, LPS stimulates the recruitment of phospho-p65 to the TNF-{alpha} promoter within 20 min, and this response is blocked in cells expressing dnIKK-{beta}. These results underscore the importance of phospho-Ser536 p65 in cytokine expression in cardiac cells.



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  		Fig. 9. Phospho-p65 recruitment to the TNF-{alpha} promoter in intact cardiac myocytes. Cardiac cells were infected with Ad-dnIKK-{beta} or Ad-{beta}-Gal (300 particles/cell) for 48 h before treatment with LPS for 0–20 min. Cell lysates were analyzed by chromatin immunoprecipitation, which included anti-phospho-Ser536. Ethidium bromide-stained agarose gel of final PCR products is shown. Lanes M and C, molecular weight marker and PCR product from TNF-{alpha} promoter template control, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
TNF-{alpha} is a pleiotropic cytokine that exerts multiple potentially harmful effects on cardiac myocytes. Myocardial production of cytokines, including TNF-{alpha}, is now recognized as a pathological component of the cardiac tissue response to stress, injury, or infection (12, 22, 32, 50). Several laboratories, including our own, have identified the NF-{kappa}B family of transcription factors as central regulators of proinflammatory cytokine production in heart (14, 24, 26, 54). We previously showed that exposure to LPS, a proinflammatory component of gram-negative bacterial outer membrane, stimulates the canonical NF-{kappa}B activation pathway in cultured cardiac myocytes through targeted degradation of I{kappa}B-{alpha} and the nuclear translocation of p65-containing NF-{kappa}B dimers, a prerequisite to transcriptional activation of NF-{kappa}B-responsive genes (54). However, although these central features of the NF-{kappa}B activation cascade have been described in heart, emerging evidence reveals that the NF-{kappa}B signaling is more complex than this simple model implies. For example, the NF-{kappa}B subunit p65/RelA is an established substrate of multiple kinases involved in stress-activated signaling, including PKA, mitogen- and stress-activated kinase-1, casein kinase II, IKK, ribosomal S6 kinase-1, PKC-{zeta}, and glycogen synthase kinase-3{beta} (for review see Ref. 44). Although these covalent modifications significantly influence p65 activity in several cell types, little is known of these processes in heart. Therefore, the goal of the present study was to examine the role of p65 phosphorylation by focusing on Ser536, a site located in the COOH-terminal TAD 1, known to enhance the ability of p65 to activate gene expression in multiple cell types (16, 25, 56). In the present study, we extended our previous observations by using an established model of NF-{kappa}B-activated TNF-{alpha} expression, cultured neonatal mouse myocytes (54). The main conclusions are that LPS activates the phosphorylation of Ser536 and that LPS stimulates IKK-{alpha} and IKK-{beta}, but only the latter appears to contribute to p65 Ser536 phosphorylation and TNF-{alpha} gene expression.

Although the results reported here focus on Ser536, it is recognized that there are several additional phosphorylation sites on p65. p65 is known to be phosphorylated at seven sites, including Ser529, Ser535, and Ser536, which are known to be targets of casein kinase II, calmodulin-dependent kinase IV, and IKK, respectively. Phosphorylation of each of these sites within the COOH-terminal TAD 1 of p65 has been shown to enhance the transactivation potential of the complex (1, 7, 25, 44). Additionally, p65 can be phosphorylated by PKA and mitogen- and stress-activated kinase-1 at Ser276, an essential modification for the subsequent binding of the histone acetylase p300/cAMP response element binding protein (CREB) binding protein (CBP) (37, 44, 52). The atypical PKC isoform PKC-{zeta} has also been shown to phosphorylate p65 at Ser311, which leads to an enhancement of p65 transactivation and p300/CBP binding (11, 44). Phosphorylation of p65 at Thr254 creates a binding site for Pin-1, which, through its interaction with p65, disrupts the binding of I{kappa}B and promotes p65 nuclear translocation (41, 44). Finally, glycogen synthase kinase-3{beta} phosphorylates Ser468 within the COOH-terminal TAD 2 of p65, which represses the transactivation potential of p65 (5, 44). The growing list of kinases capable of modulating the activity of NF-{kappa}B highlights the role of this protein complex as an integrator of stress-activated signaling cascades.

It is important to note that calyculin A was added to stabilize the p65 phosphorylation signal in LPS-stimulated cells. Interestingly, the addition of this phosphatase inhibitor enhanced TNF-{alpha} release and mRNA production in the presence of SC-514. We observed this effect in previous studies (54) and attribute this apparent synergism to a general upregulation of stimulus-induced NF-{kappa}B signaling. Given that the pathway is regulated by phosphorylation events at multiple levels, it is likely that the addition of a phosphatase inhibitor potentiates the activity of the pathway. However, control experiments revealed that, under the conditions used, the addition of calyculin A alone does not activate NF-{kappa}B signaling in cardiac myocytes.

Given the variety of kinases capable of phosphorylating p65, an important goal was to identify the kinase(s) that underlies the LPS-induced phosphorylation of Ser536. In an elegant series of studies, Sakurai et al. (42) demonstrated that two kinases of the IKK complex, IKK-{alpha} and IKK-{beta}, will each phosphorylate p65 Ser536 in HeLa cells stimulated with TNF-{alpha}. Other studies underscore the view that p65 phosphorylation at Ser536 can be cell type and stimulus dependent. For instance, O’Mahony et al. (38) identified IKK-{alpha}, and not IKK-{beta}, as the kinase that phosphorylates p65 in mouse embryonic fibroblasts expressing the human T cell leukemia virus type 1 Tax oncoprotein. Conversely, studies by Yang et al. (56) showed that IKK-{beta}, rather than IKK-{alpha}, directly phosphorylates Ser536 of p65 in TNF-{alpha}-stimulated uninfected mouse fibroblasts. Our initial finding that LPS rapidly activates IKK-{alpha} and IKK-{beta} (Fig. 3A) motivated a series of interrelated experiments to determine whether one or both of the IKK subunits phosphorylates p65 at Ser536 in cardiac myocytes. The results with a range of pharmacological inhibitors of IKK-{beta} supported the view that this kinase is involved in the response. These studies were complemented by dominant-negative experiments, which also supported a role for IKK-{beta}, findings that are in direct agreement with a recent report that shows that IKK-{beta} directly phosphorylates p65 at Ser536 in vitro and that this event is attenuated in an IKK-{beta}-knockout mouse embryonic fibroblast cell line (15). In fact, a recent in vitro study showed that p65 is at least as good a substrate for IKK-{beta} as the canonical substrate I{kappa}B-{alpha} (18). Taken together, these studies define a key role for IKK-{beta} in LPS-induced p65 phosphorylation in heart and, combined with the results of other investigators, are consistent with the conclusion that IKK-{beta} is the kinase that phosphorylates Ser536 in response to LPS.

There is growing interest in developing compounds that are specific inhibitors of IKK to limit progression of diverse ailments including heart disease. This study capitalized on a few of the family of inhibitors reported to be selective for IKK-{beta} over IKK-{alpha} (for review see Ref. 17). The salycilate compound IMD-0354 has been developed as an active site-directed IKK-{beta} inhibitor. In the present study, IMD-0354 markedly reduced TNF-{alpha} production while displaying more modest inhibitory effects on p65 phosphorylation at Ser536 and on I{kappa}B-{alpha} phosphorylation at Ser32. In this regard, it is interesting to note that Onai et al. (39) showed that although IMD-0354 significantly reduces NF-{kappa}B-dependent expression of IL-1{beta} and macrophage inflammatory protein-1 in cardiac myocytes, it has only modest effects on inhibiting I{kappa}B-{alpha} phosphorylation. Taken together, these inconsistencies suggest that the mechanism of IMD-0354 action includes other targets beyond IKK-{beta}. For example, given its potent effects on NF-{kappa}B-dependent gene expression, it is possible that this compound affects the translocation or DNA binding of NF-{kappa}B dimers. IKK-{beta} inhibitor IV, a ureidothiophenecarboxamide derivative, inhibits IKK-{beta} with an IC50 of 18 nM and is selective for IKK-{beta} over several kinases including IKK-{alpha}, JNK, and p38 MAPK (17, 40). As reported here, IKK-{beta} inhibitor IV markedly reduced LPS-activated p65 phosphorylation and TNF-{alpha} mRNA and protein production in cardiac myocytes. SC-514 is a selective, reversible, and competitive ATP analog inhibitor of IKK-{beta}, showing marked specificity for IKK-{beta} over IKK-{alpha} and IKK-i, two additional components of the IKK complex, and 30 other kinases, with IC50 of ~3–12 µM (15, 17, 18). In the present study, SC-514 partially reduced LPS-induced p65 phosphorylation and TNF-{alpha} production. These findings are consistent with a previous study of Kishore et al. (18) in which neither I{kappa}B-{alpha} phosphorylation and degradation nor nuclear transport of p65 was completely inhibited by this agent. In fact, autoinactivation of the IKK complex was slowed in SC-514-treated fibroblasts, a finding that might be related to the increases in TNF-{alpha} mRNA in cardiac cells in response to SC-514 (18). Taken together, the results obtained from the use of these compounds support a role for IKK-{beta} but also underscore potential side reactions that might limit their utility.

Thus to further strengthen the conclusions of this study, we employed a complementary molecular genetic strategy of IKK inhibition. Specifically, overexpression of dnIKK-{beta}, but not dnIKK-{alpha}, reduced p65 phosphorylation and TNF-{alpha} production in LPS-stimulated cardiac myocytes. Importantly, control experiments demonstrated that the selective effects were not due to the trivial possibility of differences in the expression levels of the two kinase-dead mutants. Although not likely, we cannot completely exclude the possibility that the differences might be due to greater affinity of dnIKK-{beta} for p65 compared with dnIKK-{alpha}, making dnIKK-{beta} a better inhibitor of p65 phosphorylation and TNF-{alpha} gene expression. The known lethality of IKK knockouts would make genetic manipulations unsuitable for further study of these issues in heart (23, 46, 47). However, the use of conditional knockouts or small interfering RNA strategies to selectively decrease levels of IKK-{alpha} or IKK-{beta} offers promising approaches.

Whereas the importance of phosphorylation of p65 Ser536 to NF-{kappa}B-responsive gene expression has been demonstrated in other cells (31), we established, for the first time, a link between this phosphorylation event and transcription of the TNF-{alpha} gene in cardiac myocytes. Real-time PCR showed that cytokine generation was inhibited at the level of mRNA accumulation. Although steady-state mRNA levels are determined by transcription rate and transcript stability and TNF-{alpha} is known to be regulated by both processes (3, 8, 36, 48, 59), several lines of evidence suggest that the predominant effect of IKK-{beta} is to increase TNF-{alpha} transcription. 1) IKK-{beta} phosphorylation of Ser536 is known to increase the transactivation potential of p65 but, to the best of our knowledge, has not been shown to modify transcript stability (31). 2) We have demonstrated the importance of p65 as a regulator of cardiomyocyte TNF-{alpha} production through the overexpression of dnp65, which significantly reduced LPS-induced TNF-{alpha} release. 3) Using ChIP assays, we have confirmed that Ser536-phosphorylated p65 was indeed recruited to the TNF-{alpha} promoter after LPS stimulation of cardiac myocytes. The data presented strongly implicate the role of IKK-{beta} in transcriptional activation of TNF-{alpha} in response to LPS stimulation of cardiac myocytes. Taken together, these results underscore the importance of posttranslational modification of p65 in the NF-{kappa}B-dependent activation of proinflammatory cytokine expression in heart.

In conclusion, we have confirmed our previous report that LPS can activate NF-{kappa}B and TNF-{alpha} expression in cardiac myocytes by stimulating multiple posttranslational modifications in NF-{kappa}B signaling molecules. Given the emerging role of TNF-{alpha} and other pleiotropic cytokines as significant contributors to myocardial dysfunction, a more complete understanding of the molecular mechanisms governing its production and release is warranted. The results of these studies define specific signaling intermediates and key molecular events within the NF-{kappa}B signaling pathway as potentially valuable therapeutic targets.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Student support was provided by National Institute of General Medical Sciences Initiative for Minority Student Development Grant R25-GM-55036. Additionally, this work was supported in part by National Institutes of Health Grants AG-14637 and P01 HL-70709 (T. B. Rogers), GM-069431 (I. S. Singh), GM-066855 and HL-69057 (J. D. Hasday), Department of Veterans Affairs Merit Review Awards (I. S. Singh and J. D. Hasday), National Institutes of Health Training Grants T32-GM-008181, T32-AR-07592, and T32-HL-072751, and the University of Maryland MD/PhD Program (G. Hall).


   ACKNOWLEDGMENTS
 
We thank Dr. Matthew Fenton and Dr. Subhendu Basu for assistance with real-time PCR studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. B. Rogers, Dept. of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201 (E-mail: trogers{at}som.umaryland.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Bae JS, Jang MK, Hong SH, An WG, Choi YH, Do Kim H, and Cheong JH. Phosphorylation of NF-{kappa} B by calmodulin-dependent kinase IV activates anti-apoptotic gene expression. Biochem Biophys Res Commun 305: 1094–1098, 2003.[CrossRef][Web of Science][Medline]
  2. Basu S and Fenton MJ. Toll-like receptors: function and roles in lung disease. Am J Physiol Lung Cell Mol Physiol 286: L887–L892, 2004.[Abstract/Free Full Text]
  3. Brook M, Sully G, Clark AR, and Saklatvala J. Regulation of tumour necrosis factor-{alpha} mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett 483: 57–61, 2000.[CrossRef][Web of Science][Medline]
  4. Bryant D, Becker L, Richardson J, Shelton J, Franco F, Peshock R, Thompson M, and Giroir B. Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-{alpha}. Circulation 97: 1375–1381, 1998.[Abstract/Free Full Text]
  5. Buss H, Dorrie A, Schmitz ML, Frank R, Livingstone M, Resch K, and Kracht M. Phosphorylation of serine 468 by GSK-3{beta} negatively regulates basal p65 NF-{kappa}B activity. J Biol Chem 279: 49571–49574, 2004.[Abstract/Free Full Text]
  6. Buss H, Dorrie A, Schmitz ML, Hoffmann E, Resch K, and Kracht M. Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK-{beta}, IKK-{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription. J Biol Chem 279: 55633–55643, 2004.[Abstract/Free Full Text]
  7. Chantome A, Pance A, Gauthier N, Vandroux D, Chenu J, Solary E, Jeannin JF, and Reveneau S. Casein kinase II-mediated phosphorylation of NF-{kappa}B p65 subunit enhances inducible nitric-oxide synthase gene transcription in vivo. J Biol Chem 279: 23953–23960, 2004.[Abstract/Free Full Text]
  8. Dean JL, Wait R, Mahtani KR, Sully G, Clark AR, and Saklatvala J. The 3'-untranslated region of tumor necrosis factor-{alpha} mRNA is a target of the mRNA-stabilizing factor HuR. Mol Cell Biol 21: 721–730, 2001.[Abstract/Free Full Text]
  9. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, and Karin M. A cytokine-responsive I{kappa}B kinase that activates the transcription factor NF-{kappa}B. Nature 388: 548–554, 1997.[CrossRef][Medline]
  10. DuBell WH, Gigena MS, Guatimosim S, Long XL, Lederer WJ, and Rogers TB. Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes. Am J Physiol Heart Circ Physiol 282: H38–H48, 2002.[Abstract/Free Full Text]
  11. Duran A, Diaz-Meco MT, and Moscat J. Essential role of RelA Ser311 phosphorylation by {zeta}-PKC in NF-{kappa}B transcriptional activation. EMBO J 22: 3910–3918, 2003.[CrossRef][Web of Science][Medline]
  12. Feldman AM, Combes A, Wagner D, Kadakomi T, Kubota T, Li YY, and McTiernan C. The role of tumor necrosis factor in the pathophysiology of heart failure. J Am Coll Cardiol 35: 537–544, 2000.[Abstract/Free Full Text]
  13. Ghosh S. NF-{kappa}B: a mediator of inflammatory responses. J Vasc Surg 33: 1288, 2001.
  14. Haudek SB, Bryant DD, Horton JW, and Giroir BP. NF-{kappa}B activation in mouse heart after LPS and/or TNF-{alpha} stimulation (Abstract). Shock 11: 11, 1999.[Medline]
  15. Jeong SJ, Pise-Masison CA, Radonovich MF, Park HU, and Brady JN. A novel NF-{kappa}B pathway involving IKK{beta} and p65/RelA Ser536 phosphorylation results in p53 inhibition in the absence of NF-{kappa}B transcriptional activity. J Biol Chem 280: 10326–10332, 2005.[Abstract/Free Full Text]
  16. Jiang X, Takahashi N, Matsui N, Tetsuka T, and Okamoto T. The NF-{kappa}B activation in lymphotoxin {beta}-receptor signaling depends on the phosphorylation of p65 at serine 536. J Biol Chem 278: 919–926, 2003.[Abstract/Free Full Text]
  17. Karin M, Yamamoto Y, and Wang QM. The IKK NF-{kappa}B system: a treasure trove for drug development. Nat Rev Drug Discov 3: 17–26, 2004.[CrossRef][Web of Science][Medline]
  18. Kishore N, Sommers C, Mathialagan S, Guzova J, Yao M, Hauser S, Huynh K, Bonar S, Mielke C, Albee L, Weier R, Graneto M, Hanau C, Perry T, and Tripp CS. A selective IKK-2 inhibitor blocks NF-{kappa}B-dependent gene expression in interleukin-1{beta}-stimulated synovial fibroblasts. J Biol Chem 278: 32861–32871, 2003.[Abstract/Free Full Text]
  19. Kopp E and Ghosh S. Inhibition of NF-{kappa}B by sodium salicylate and aspirin. Science 265: 956–959, 1994.[Abstract/Free Full Text]
  20. Kubota T, McTiernan CF, Frye CS, Slawson SE, Lemster BH, Koretsky AP, Demetris AJ, and Feldman AM. Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-{alpha}. Circ Res 81: 627–635, 1997.[Abstract/Free Full Text]
  21. Kuprash DV, Udalova IA, Turetskaya RL, Kwiatkowski D, Rice NR, and Nedospasov SA. Similarities and differences between human and murine TNF promoters in their response to lipopolysaccharide. J Immunol 162: 4045–4052, 1999.[Abstract/Free Full Text]
  22. Levine B, Kalman J, Mayer L, Fillit HM, and Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart-failure. N Engl J Med 323: 236–241, 1990.[Abstract]
  23. Li Q, Van Antwerp D, Mercurio F, Lee KF, and Verma IM. Severe liver degeneration in mice lacking the I{kappa}B kinase 2 gene. Science 284: 321–325, 1999.[Abstract/Free Full Text]
  24. Liu SF, Ye XB, and Malik AB. Inhibition of NF-{kappa}B activation by pyrrolidine dithiocarbamate prevents in vivo expression of proinflammatory genes. Circulation 100: 1330–1337, 1999.[Abstract/Free Full Text]
  25. Madrid LV, Mayo MW, Reuther JY, and Baldwin AS. Akt stimulates the transactivation potential of the RelA/p65 subunit of NF-{kappa}B through utilization of the I{kappa}B kinase and activation of the mitogen-activated protein kinase p38. J Biol Chem 276: 18934–18940, 2001.[Abstract/Free Full Text]
  26. Madrid LV, Wang CY, Guttridge DC, Schottelius AJG, Baldwin AS, and Mayo MW. Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-{kappa}B. Mol Cell Biol 20: 1626–1638, 2000.[Abstract/Free Full Text]
  27. Malave HA, Lee-Jackson D, Samuels PD, Smith DK, Blaustein AS, and Mann DL. Elevated circulating levels of tumor necrosis factor correlate with cardiac remodeling in patients with heart failure (Abstract). J Am Coll Cardiol 35: 195A, 2000.
  28. Malave HA, Taylor AA, Lee-Jackson D, and Mann DL. Elevated circulating levels of tumor necrosis factor correlate with depressed heart rate variability in patients with heart failure. Circulation 100: 206, 1999.
  29. Malave HA, Taylor AA, Nattama J, Deswal A, and Mann DL. Circulating levels of tumor necrosis factor correlate with indexes of depressed heart rate variability. A study in patients with mild-to-moderate heart failure. Chest 123: 716–724, 2003.[CrossRef][Web of Science][Medline]
  30. Mattioli I, Sebald A, Bucher C, Charles RP, Nakano H, Doi T, Kracht M, and Schmitz ML. Transient and selective NF-{kappa}B p65 serine 536 phosphorylation induced by T cell costimulation is mediated by I{kappa}B kinase-{beta} and controls the kinetics of p65 nuclear import. J Immunol 172: 6336–6344, 2004.[Abstract/Free Full Text]
  31. Mayo MW, Madrid LV, Westerheide SD, Jones DR, Yuan XJ, Baldwin AS, and Whang YE. PTEN blocks tumor necrosis factor-induced NF-{kappa}B-dependent transcription by inhibiting the transactivation potential of the p65 subunit. J Biol Chem 277: 11116–11125, 2002.[Abstract/Free Full Text]
  32. Meldrum DR. Tumor necrosis factor in the heart. Am J Physiol Regul Integr Comp Physiol 274: R577–R595, 1998.[Abstract/Free Full Text]
  33. Meldrum DR, Shenkar R, Sheridan BC, Cain BS, Abraham E, and Harken AH. Hemorrhage activates myocardial NF-{kappa}B and increases TNF-{alpha} in the heart. J Mol Cell Cardiol 29: 2849–2854, 1997.[CrossRef][Web of Science][Medline]
  34. Meng XZ, Ao LH, Song Y, Raeburn CD, Fullerton DA, and Harken AH. Signaling for myocardial depression in hemorrhagic shock: roles of Toll-like receptor 4 and p55 TNF-{alpha} receptor. Am J Physiol Regul Integr Comp Physiol 288: R600–R606, 2005.[Abstract/Free Full Text]
  35. Mercurio F, Zhu HY, Murray BW, Shevchenko A, Bennett BL, Li JW, Young DB, Barbosa M, and Mann M. IKK-1 and IKK-2: cytokine-activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278: 860–866, 1997.[Abstract/Free Full Text]
  36. Misquitta CM, Iyer VR, Werstiuk ES, and Grover AK. The role of 3'-untranslated region (3'-UTR)-mediated mRNA stability in cardiovascular pathophysiology. Mol Cell Biochem 224: 53–67, 2001.[CrossRef][Web of Science][Medline]
  37. Okazaki T, Sakon S, Sasazuki T, Sakurai H, Doi T, Yagita H, Okumura K, and Nakano H. Phosphorylation of serine 276 is essential for p65 NF-{kappa}B subunit-dependent cellular responses. Biochem Biophys Res Commun 300: 807–812, 2003.[CrossRef][Web of Science][Medline]
  38. O’Mahony AM, Montano M, Van Beneden K, Chen LF, and Greene WC. Human T-cell lymphotropic virus type 1 tax induction of biologically active NF-{kappa}B requires I{kappa}B kinase-1-mediated phosphorylation of RelA/p65. J Biol Chem 279: 18137–18145, 2004.[Abstract/Free Full Text]
  39. Onai Y, Suzuki J, Kakuta T, Maejima Y, Haraguchi G, Fukasawa H, Muto S, Itai A, and Isobe M. Inhibition of I{kappa}B phosphorylation in cardiomyocytes attenuates myocardial ischemia/reperfusion injury. Cardiovasc Res 63: 51–59, 2004.[Abstract/Free Full Text]
  40. Podolin PL, Callahan JF, Bolognese BJ, Li YH, Carlson K, Davis TG, Mellor GW, Evans C, and Roshak AK. Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of I{kappa}B kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell proliferation. J Pharmacol Exp Ther 312: 373–381, 2005.[Abstract/Free Full Text]
  41. Ryo A, Suizu F, Yoshida Y, Perrem K, Liou YC, Wulf G, Rottapel R, Yamaoka S, and Lu KP. Regulation of NF-{kappa}B signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 12: 1413–1426, 2003.[CrossRef][Web of Science][Medline]
  42. Sakurai H, Chiba H, Miyoshi H, Sugita T, and Toriumi W. I{kappa}B kinases phosphorylate NF-{kappa}B p65 subunit on serine 536 in the transactivation domain. J Biol Chem 274: 30353–30356, 1999.[Abstract/Free Full Text]
  43. Sakurai H, Suzuki S, Kawasaki N, Nakano H, Okazaki T, Chino A, Doi T, and Saiki I. Tumor necrosis factor-{alpha}-induced IKK phosphorylation of NF-{kappa}B p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway. J Biol Chem 278: 36916–36923, 2003.[Abstract/Free Full Text]
  44. Schmitz ML, Mattioli I, Buss H, and Kracht M. NF-{kappa}B: a multifaceted transcription factor regulated at several levels. Chembiochem 5: 1348–1358, 2004.[CrossRef][Web of Science][Medline]
  45. Soares MP, Muniappan A, Kaczmarek E, Koziak K, Wrighton CJ, Steinhauslin F, Ferran C, Winkler H, Bach FH, and Anrather J. Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis. J Immunol 161: 4572–4582, 1998.[Abstract/Free Full Text]
  46. Takeda K, Takeuchi O, Tsujimura T, Itami S, Adachi O, Kawai T, Sanjo H, Yoshikawa K, Terada N, and Akira S. Limb and skin abnormalities in mice lacking IKK-{alpha}. Science 284: 313–316, 1999.[Abstract/Free Full Text]
  47. Tanaka M, Fuentes ME, Yamaguchi K, Durnin MH, Dalrymple SA, Hardy KL, and Goeddel DV. Embryonic lethality, liver degeneration, and impaired NF-{kappa}B activation in IKK-{beta}-deficient mice. Immunity 10: 421–429, 1999.[CrossRef][Web of Science][Medline]
  48. Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, and Ghosh S. I{kappa}B-{beta} regulates the persistent response in a biphasic activation of NF-{kappa}B. Cell 80: 573–582, 1995.[CrossRef][Web of Science][Medline]
  49. Torre-Amione G, Bozkurt B, Deswal A, and Mann DL. An overview of tumor necrosis factor-{alpha} and the failing human heart. Curr Opin Cardiol 14: 206–210, 1999.[CrossRef][Web of Science][Medline]
  50. Torre-Amione G, Kapadia S, Lee J, Durand JB, Bies RD, Young JB, and Mann DL. Tumor necrosis factor-{alpha} and tumor necrosis factor receptors in the failing human heart. Circulation 93: 704–711, 1996.[Abstract/Free Full Text]
  51. Valen G, Yan ZQ, and Hansson GK. Nuclear factor-{kappa}B and the heart. J Am Coll Cardiol 38: 307–314, 2001.[Abstract/Free Full Text]
  52. Vermeulen L, De Wilde G, Van Damme P, Vanden Berghe W, and Haegeman G. Transcriptional activation of the NF-{kappa}B p65 subunit by mitogen- and stress-activated protein kinase-1 (MSK1). EMBO J 22: 1313–1324, 2003.[CrossRef][Web of Science][Medline]
  53. Wang D, Westerheide SD, Hanson JL, and Baldwin AS. Tumor necrosis factor-{alpha}-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem 275: 32592–32597, 2000.[Abstract/Free Full Text]
  54. Wright G, Singh IS, Hasday JD, Farrance IK, Hall G, Cross AS, and Rogers TB. Endotoxin stress-response in cardiomyocytes: NF-{kappa}B activation and tumor necrosis factor-{alpha} expression. Am J Physiol Heart Circ Physiol 282: H872–H879, 2002.[Abstract/Free Full Text]
  55. Xuan YT, Tang XL, Banerjee S, Takano H, Li RCX, Han H, Qiu YM, Li JJ, and Bolli R. Nuclear factor-{kappa}B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits. Circ Res 84: 1095–1109, 1999.[Abstract/Free Full Text]
  56. Yang F, Tang E, Guan KL, and Wang CY. IKK-{beta} plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide. J Immunol 170: 5630–5635, 2003.[Abstract/Free Full Text]
  57. Zandi E, Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The I{kappa}B kinase complex (IKK) contains two kinase subunits, IKK-{alpha} and IKK-{beta}, necessary for I{kappa}B phosphorylation and NF-{kappa}B activation. Cell 91: 243–252, 1997.[CrossRef][Web of Science][Medline]
  58. Zhong HH, Voll RE, and Ghosh S. Phosphorylation of NF-{kappa}B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1: 661–671, 1998.[CrossRef][Web of Science][Medline]
  59. Zou W, Amcheslavsky A, Takeshita S, Drissi H, and Bar-Shavit Z. TNF-{alpha} expression is transcriptionally regulated by RANK ligand. J Cell Physiol 202: 371–378, 2005.[CrossRef][Web of Science][Medline]



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00393.2005v1
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