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

Am J Physiol Heart Circ Physiol. 2005 Nov;289(5):H2103-11. doi: 10.1152/ajpheart.00393.2005. Epub 2005 Jun 24.


TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-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 IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-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-kappaB 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Animals, Newborn
  • Blotting, Western
  • Cells, Cultured
  • Chromatin / metabolism
  • Cytokines / biosynthesis
  • Immunoprecipitation
  • Lipopolysaccharides / pharmacology*
  • Mice
  • Mutagenesis, Site-Directed / drug effects
  • Myocardium / metabolism
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • NF-kappaB-Inducing Kinase
  • Phosphorylation
  • Protein Serine-Threonine Kinases / physiology*
  • Signal Transduction / drug effects
  • Tumor Necrosis Factor-alpha / biosynthesis*


  • Chromatin
  • Cytokines
  • Lipopolysaccharides
  • Tumor Necrosis Factor-alpha
  • Protein Serine-Threonine Kinases