Inhibitor-[kappa]B kinase-[beta] regulates LPS-induced TNF-[alpha] production in cardiac myocytes through modulation of NF-[kappa]B p65 subunit phosphorylation

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 [Ser.sup.536], a modification associated with enhancement of p65 trans-activation 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 [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-[alpha], not IKK-[beta], plays a central role in regulation of p65 phosphorylation at [Ser.sup.536] and TNF-[alpha] production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of [Ser.sup.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. signal transuction; gene expression; heart; cytokines