Journal
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
Volume 289, Issue 5, Pages H2103-H2111Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpheart.00393.2005
Keywords
signal transuction; gene expression; heart; cytokines
Funding
- NHLBI NIH HHS [P01 HL-70709, T32-HL-072751, HL-69057] Funding Source: Medline
- NIAMS NIH HHS [T32-AR-07592] Funding Source: Medline
- NIA NIH HHS [AG-14637] Funding Source: Medline
- NIGMS NIH HHS [R25-GM-55036, T32-GM-008181, GM-069431, GM-066855] Funding Source: Medline
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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-kappa production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser(536), 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 Ser(536)-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.
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