Induced stabilization of IκBα can facilitate its re-synthesis and prevent sequential degradation

The transcription factor NF-kappaB is responsible for regulating genes that can profoundly impact cell proliferation, apoptosis, inflammation, and immune responses. The NF-kappaB inhibitor IkappaBalpha is rapidly degraded and then re-synthesized after an NF-kappaB stimulus. We have found that the re-synthesis Of IkappaBalpha in a human colon-derived cell line (HT-29) includes the post-translational stabilization of newly synthesized IkappaBalpha. The TNF-alpha-induced stabilization of newly synthesized IkappaBalpha involves the C-terminal PEST region of the protein: N-terminal deletion mutants (lacking the IkappaB kinase phosphorylation sites) were readily stabilized by TNF-alpha, whereas deletion of the C-terminus resulted in a constitutively stable protein. The role of the C-terminus in stabilization was further supported by the finding that fusion of the IkappaBalpha C-terminus to GFP generated a protein that could also be stabilized by TNF-alpha. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 prevented stabilization Of IkappaBalpha and delayed the re-emergence Of IkappaBalpha following TNF-alpha-induced degradation. The IkappaBalpha stabilization pathway could prevent sequential rounds Of IkappaBalpha degradation without preventing IkappaBalpha phosphorylation. Analysis of two other cell lines (SW480 and THP-1) revealed similarities and cell-specific differences in the regulation Of IkappaBalpha stabilization. We propose that cytokine stabilization of newly synthesized IkappaBalpha in some cell types is a critical homeostatic mechanism that limits inflammatory gene expression. (C) 2003 Wiley-Liss, Inc.