Response to Comment on "Oscillations in NF-κB Signaling Control the Dynamics of Gene Expression"

Abstract

Using the Hoffmann model (4), we concluded by sensitivity analysis (3) that RelA overexpression would minimally perturb the system; therefore, we used RelA localization as our principal output. We estimated that average overexpression of RelA fusion proteins was 3 to 5 times that of endogenous RelA levels in transfected cells, with a distribution in the cell population. Using simulations, Barken et al. (5) suggest that RelA, IκBα, or RelA and IκBα expression (within the range suggested by our data) maintains oscillations but alters oscillation frequency and amplitude. They suggest [figure 1 in (5)], that simulated 4-fold overexpression of RelA delays the second peak of nuclear localization by 3.5 hours (normal 2-hour peak delayed to 5.5 hours). This does not fit our experimental data, because in both cell lines used, the second peak of nuclear localization with RelA overexpression alone was at around 3 hours [see figure 1 and supplemental material in (1)]. Reanalysis of our data from SK-N-AS cells (Fig. 1) and HeLa and Swiss 3T3 cells (data not shown) demonstrates no correlation between RelA-DsRed expression level and successive peak timing (also of amplitude, not shown) with cellular fluorescent levels that vary up to ∼20 fold. This clear inconsistency between their in silico results and our experimental data suggests that their computational model cannot faithfully reproduce all aspects of the system. Altering the computational model (1, 4) by replacing the second-order term for NF-κB–induced IκBα synthesis with a linear expression [reaction 28 in table S1 in (1)] results in a new model that reproduces all of the fundamental characteristics of our experimental data. This model shows much reduced sensitivity of period to RelA concentration (Fig. 2), which demonstrates that continued refinement of the Hoffmann model (6, 7) may well bring the simulations closer to observed biological phenomena.