Fas-associated death-domain protein inhibits TNF-? mediated NF-?B activation in cardiomyocytes

Abstract

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 TNF-? signaling, which is a proinflammatory pathway, has not been delineated. To investigate the role of FADD in CM NF-kB 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-? activated NF-kB in CMs as demonstrated by phosphorylation and degradation of inhibitory-kB (IkB)-?-enhanced nuclear p65 and NF-kB DNA-binding activity as well as increased mRNA for the NF-kB-dependent adhesion molecule VCAM-1 (19 � 4.1-fold) as measured by quantitative RT-PCR. Gene transfer of FADD inhibited TNF-?-induced IkB-? phosphorylation, decreased p65 nuclear translocation and NF-kB DNA-binding activity, and reduced VCAM-1 transcript levels by 53-65%. Interestingly, FADD-DED exhibited a similar but weaker inhibitory effect on NF-kB activation. The effects of FADD on NF-kB were cell-type specific. FADD expression also inhibited TNF-?-mediated NF-kB activation in human endothelial cells but not in rat pulmonary artery smooth muscle cells. In contrast, FADD expression actually activated NF-kB in human embryonic kidney (HEK)-293 cells. In CMs, FADD inhibited NF-kB activation as well as phosphorylation of IkB-? and IkB kinase (IKK)-? in response to cytokine stimulation or expression of the upstream kinases NF-kB-inducing kinase and IKK-?. These data demonstrate that FADD inhibits NF-kB activation in CMs, and this inhibition likely occurs at the level of phosphorylation and activation of IKK-?. Copyright � 2005 the American Physiological Society.