Persistent Tumor Necrosis Factor Signaling in Normal Human Fibroblasts Prevents the Complete Resynthesis of IκB-α

David M Poppers,Paul Schwenger,Jan Vilček

doi:10.1074/jbc.m002806200

Abstract

Transcription factor NF-kappa B is normally sequestered in the cytoplasm, complexed with I kappa B inhibitory proteins. Tumor necrosis factor (TNF) and interleukin-1 induce I kappa B-alpha phosphorylation, leading to I kappa B-alpha degradation and translocation of NF-kappa B to the nucleus where it activates genes important in inflammatory and immune responses. TNF and interleukin-1 actions are typically terminated by desensitization, and I kappa B-alpha reappearance normally occurs within 30-60 min. We found that in normal human FS-4 fibroblasts maintained in the presence of TNF, I kappa B-alpha protein failed to return to base-line levels for up to 15 h. Removal of TNF at any time during the 15-h period resulted in complete I kappa B-alpha resynthesis, suggesting that I kappa B-alpha reappearance was prevented by continued TNF signaling. Long term exposure of FS-4 fibroblasts to TNF led to a persistent presence of I kappa B-alpha mRNA, sustained I kappa B kinase activation, continuous proteasome-mediated degradation of I kappa B-alpha, and sustained nuclear localization of NF-kappa B. Continuous exposure of FS-4 cells to TNF did not lead to a sustained activation of p38 or ERK mitogen-activated protein kinases, suggesting that not all TNF-induced signaling pathways are persistently activated. These findings challenge the notion that all cytokine-mediated signals are rapidly terminated by desensitization and illustrate the need to elucidate the process of deactivation of TNF-induced signaling.

Highlights

The transcription factor NF-␬B1 is important in the regulation of genes involved in the immune and inflammatory responses, including genes encoding inflammatory cytokines (e.g. Tumor necrosis factor (TNF), IL-1, IL-6, and IL-8), cell adhesion molecules
We found that in normal human FS-4 fibroblasts maintained in the presence of TNF, I␬B-␣ protein failed to return to base-line levels for up to 15 h
Degradation and Reappearance of I␬B-␣ Protein in Human Fibroblasts Stimulated with TNF and IL-1—Normal human diploid FS-4 fibroblasts were treated with either TNF or IL-1 for periods ranging from 15 min to 15 h

Summary

Persistent TNF Signaling in Human Fibroblasts

Receptor-mediated endocytosis [17,18,19], by TNFR shedding (20 –22), or by mechanisms that have not been fully characterized [23]. TNF induces I␬B-␣ degradation within 15 min, which is followed by I␬B-␣ resynthesis and complete reappearance of I␬B-␣ protein within approximately 30 min to 2 h [12, 26]. Complete reappearance of I␬B-␣ protein commonly occurs even if cells are maintained in the continuous presence of TNF [27,28,29], and this has been ascribed to the previously mentioned autoregulatory NF-␬B loop and to cellular desensitization to TNF action. We show here that in normal human diploid FS-4 fibroblasts stimulated with TNF, I␬B-␣ is rapidly degraded but I␬B-␣ reappearance is incomplete because cells do not become desensitized to TNF signaling, and newly synthesized I␬B-␣ continues to be phosphorylated and degraded. Our results challenge the paradigm that TNF signaling (and cytokine signaling in general) is always rapidly terminated by desensitization

EXPERIMENTAL PROCEDURES

RESULTS

Continuous TNF Signaling Does Not Lead to Persistent ERK

DISCUSSION