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

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