Inhibitor-kappaB kinase-beta regulates LPS-induced TNF-alpha production in cardiac myocytes through modulation of NF-kappaB p65 subunit phosphorylation.
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.
Hall
Gentzon
G
Dept. of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA.
Singh
Ishwar S
IS
Hester
Lisa
L
Hasday
Jeffery D
JD
Rogers
Terry B
TB
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D000097782:NF-kappaB-Inducing Kinase
D002843:Chromatin; D016207:Cytokines; D008070:Lipopolysaccharides; D017346:Protein Serine-Threonine Kinases; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase