Modulating Temporal Control of NF-kappaB Activation: Implications for Therapeutic and Assay Selection

¹ West Virginia University

^* To whom correspondence should be addressed. E-mail: david.klinke{at}mail.wvu.edu.

Submitted on September 17, 2007
Revised on October 22, 2007
Accepted on 14 January 2008

	Abstract

The activation of transcription factor NF-B (nuclear factor-B) plays a central role in the induction of many inflammatory response genes. This process is characterized by either oscillations or stable induction of NF-B nuclear binding. Changes in dynamics of binding result in the expression of distinct subsets of genes leading to different physiological outcomes. We examined NF-B DNA binding activity in LPS-stimulated IC-21 cells by EMSA and NTFA assay and interpreted the results using a kinetic model of NF-B activation. Both assays detected damped oscillatory behavior of NF-B with differences in sensitivity and reproducibility. 3,4-Dichloropropionaniline (DCPA) was used to modulate the oscillatory behavior of NF-B following LPS stimulation. DCPA is known to inhibit production of two of NF-B inducible cytokines, IL-6 and TNF-, by reducing but not completely abrogating NF-B-induced transcription. DCPA treatment resulted in a potentiation of early LPS-induced NF-B activation. Results from the NTFA assay, with a higher signal-to-noise ratio than EMSA, combined with in silico modeling revealed changes in NF-B dynamics which have never been previously reported. These results highlight the importance of cell type and stimuli specificity in transcription factor activity assessment. In addition, assay selection has important implications for network inference and drug discovery.

Key Words: immunology, macrophages, reaction pathway modeling, signal transduction