Fas-associated death-domain protein inhibits TNF-alpha mediated NF-kappaB activation in cardiomyocytes.
Fas-associated death-domain protein (FADD) is an adaptor molecule that links death receptors to caspase-8 in many cell types including cardiomyocytes (CMs). Although FADD has previously been reported to play an important role in CM apoptosis, the effect of FADD on CM NF-kappaB signaling, which is a proinflammatory pathway, has not been delineated. To investigate the role of FADD in CM NF-kappaB activation, we utilized adenoviral gene transfer of wild-type FADD and a truncation mutant that lacks the death-effector domain (FADD-DED) in rat CMs in vitro TNF-alpha activated NF-kappaB in CMs as demonstrated by phosphorylation and degradation of inhibitory-kappaB (IkappaB)-alpha-enhanced nuclear p65 and NF-kappaB DNA-binding activity as well as increased mRNA for the NF-kappaB-dependent adhesion molecule VCAM-1 (19 +/- 4.1-fold) as measured by quantitative RT-PCR. Gene transfer of FADD inhibited TNF-alpha-induced IkappaB-alpha phosphorylation, decreased p65 nuclear translocation and NF-kappaB DNA-binding activity, and reduced VCAM-1 transcript levels by 53-65%. Interestingly, FADD-DED exhibited a similar but weaker inhibitory effect on NF-kappaB activation. The effects of FADD on NF-kappaB were cell-type specific. FADD expression also inhibited TNF-alpha-mediated NF-kappaB activation in human endothelial cells but not in rat pulmonary artery smooth muscle cells. In contrast, FADD expression actually activated NF-kappaB in human embryonic kidney (HEK)-293 cells. In CMs, FADD inhibited NF-kappaB activation as well as phosphorylation of IkappaB-alpha and IkappaB kinase (IKK)-beta in response to cytokine stimulation or expression of the upstream kinases NF-kappaB-inducing kinase and IKK-beta. These data demonstrate that FADD inhibits NF-kappaB activation in CMs, and this inhibition likely occurs at the level of phosphorylation and activation of IKK-beta.
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.
Wei
W
Chao
Yan
Y
Shen
Ling
L
Li
Huailong
H
Zhao
Steffen E
SE
Meiler
Stuart A
SA
Cook
Anthony
A
Rosenzweig
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D000831:Animals, Newborn; D001711:Biotransformation / Q000187:drug effects; D002352:Carrier Proteins / Q000494:pharmacology*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D056970:Co-Repressor Proteins; D003600:Cytosol / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D018832:Molecular Chaperones; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology*; D009687:Nuclear Proteins / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; D016328:NF-kappa B; D009687:Nuclear Proteins; D014409:Tumor Necrosis Factor-alpha