Requirement of thyrotropin-dependent complex formation of protein kinase A catalytic subunit with inhibitor of {kappa}B proteins for activation of p65 nuclear factor-{kappa}B by tumor necrosis factor-{alpha}.
We previously demonstrated that TNF-alpha-dependent activation of p65 nuclear factor kappaB in rat thyroid FRTL-5 cells requires TSH. In the present study, we investigated the mechanism of this TSH action. Western blot analysis revealed that, in both the presence and absence of TSH, degradation of a cytosolic kappaB inhibitor (IkappaBalpha) occurred in response to TNF-alpha, resulting in nuclear translocation of p65 in both conditions. However, no DNA binding of p65 was detected in the absence of TSH, suggesting that posttranslational modification of p65 by TSH is required for its binding. Treatment of the cells cultured in the presence of TSH with a protein kinase A (PKA) inhibitor, H89, markedly reduced p65 binding and its transcriptional activity. However, transient block of TSH/cAMP-dependent activation of PKA catalytic subunit (PKAc) by adenylate cyclase inhibitor, SQ22536, had no effects on the p65 activation. Interestingly, it was found that PKAc formed a complex with IkappaBalpha and beta only in the presence of TSH, and this PKAc could be activated by TNF-alpha. TNF-alpha-dependent p65 activation was temporally associated with PKAc/IkappaBalpha complex formation. More than 3 h exposure of TSH was required for the complex formation and p65 activation. These results demonstrate that TSH induces the formation of PKAc/IkappaB complex in FRTL-5 cells and that this PKAc bound with IkappaB plays a critical role in TNF-alpha-dependent activation of p65.
Cao
Xia
X
Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya University, Nagoya 464-8601, Japan.
Kambe
Fukushi
F
Seo
Hisao
H
D021581:Active Transport, Cell Nucleus; D000262:Adenylyl Cyclases / Q000502:physiology; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D013972:Thyrotropin / Q000494:pharmacology*; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D013972:Thyrotropin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases