Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Regulation of NF-kappaB responses by epigenetic suppression of IkappaBalpha expression in HCT116 intestinal epithelial cells.

Intestinal epithelial cells play critical roles in regulating mucosal immunity. Since epigenetic factors such as DNA methylation and histone modifications are implicated in aging, carcinogenesis, and immunity, we set out to assess any role for epigenetic factors in the regulation of intestinal epithelial cell immune responses. Experiments were conducted using the HCT116 cell line, and a subclone was genetically engineered to lack DNA methyltransferases (DNMT). The induction of the chemokine interleukin-8 and the antiapoptotic protein cFLIP by tumor necrosis factor-alpha were markedly less in HCT116 cells lacking DNMT than in parental cells. These effects were accompanied by lower monocyte chemotaxis and higher caspase signaling in HCT116 cells lacking DNMT than parental cells. Tumor necrosis factor-alpha-induced NF-kappaB activation was blocked and IkappaBalpha expression was higher in HCT116 cells lacking DNMT than in parental cells. A CpG island in the IkappaBalpha gene promoter region was found to contain variable levels of methylation in parental HCT116 cells. Chromatin immunoprecipitation analysis of histone proteins bound to the IkappaBalpha gene promoter revealed that higher levels of IkappaBalpha expression in HCT116 cells lacking DNMT compared with parental cells were accompanied by more chromatin marks permissive to gene transcription. These findings show that epigenetic factors influence the NF-kappaB system in intestinal epithelial cells, resulting in a previously unrecognized mechanism of innate immune regulation.