Inhibition with antisense oligonucleotide suggests that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells.
The IkappaB proteins are important in the regulation of the NF-kappaB/Rel group of transcription factors which are pivotal in the inflammatory response. IkappaB-alpha is itself upregulated by activation of NF-kappaB and is postulated to be part of a negative feedback loop. This role of IkappaB-alpha has been challenged, however, by recent evidence that demonstrates (1) continued activation of NF-kappaB in mesangial and endothelial cells despite the resynthesis of IkappaB-alpha protein and (2) that inhibition of the transactivating activity of NF-kappaB by corticosteroids can be dissociated from a rise in IkappaB-alpha protein. We investigated the role of IkappaB-alpha in mesangial cells using a phosphorothioate antisense oligonucleotide directed against the translational start point of IkappaB-alpha. If IkappaB-alpha does function as a negative feedback inhibitor in these cells, then reducing IkappaB-alpha levels should lead to an increase in NF-kappaB activity. We first demonstrated that IkappaB-alpha protein resynthesis following stimulation could be specifically reduced. We then showed that NF-kappaB DNA binding was not increased with antisense treatment following stimulation. Finally, NF- kappaB-dependent gene signalling after stimulation (determined through an NF-kappaB luciferase reporter and upregulation of the mRNA of known NF-kappaB-responsive genes MCP-1 and IkappaB-alpha) was reduced rather than increased. These data suggest that IkappaB-alpha does not form a negative autoregulatory loop for NF-kappaB in mesangial cells and may actually reduce NF-kappaB activity. This may have relevance to therapies directed at inhibition of NF-kappaB activity in mesangial cell diseases.
Auwardt
R B
RB
Immunology Research Centre, St. Vincent's Hospital, Melbourne, Vic., Australia. auwardrb@svhm.org.au
Mudge
S J
SJ
Chen
C
C
Power
D A
DA
D000818:Animals; D002478:Cells, Cultured; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism; D006706:Homeostasis / Q000502:physiology*; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D020672:I-kappa B Proteins; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense