Limits...
Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A - PLoS ONE (2009)

Bottom Line: Mathematical modeling suggests that NF-kappaB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IkappaBalpha.Perturbation of long-term NF-kappaB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model.We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-kappaB in the nucleus, and these oscillations are functionally advantageous.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. sungm@mail.nih.gov

ABSTRACT
NF-kappaB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-kappaB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-kappaB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-kappaB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-alpha stimulation. Mathematical modeling suggests that NF-kappaB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IkappaBalpha. The ability of NF-kappaB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-kappaB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-kappaB in the nucleus, and these oscillations are functionally advantageous.

Show MeSH

Related in: MedlinePlus

Two contrasting perturbations of NF-κB oscillations decouple nucleocytoplasmic shuttling from feedback-driven long-term dynamics.(A) Perturbed dynamics were simulated using the model in Fig. 3 and the parameter values from clusters 5, 7, and 8, with the additional modifications to nuclear export terms or the induced IκBα synthesis term for LMB or CHX effects, respectively (see text). The graphs show the time courses of free, IκBα-complexed, and total p65 in the nucleus, as predicted by the model. The red curves indicate the free and complexed components of a typical profile of nuclear p65. Inhibition of nuclear export allows one cycle of free p65 whereas inhibition of negative feedback induces constant activity. (B) Experimentally observed time lapse profiles of nuclear p65 level for single cells co-treated with LMB or CHX. Cells were treated with 10 ng/ml TNF-α and either 5 nM LMB or 2.5 µg/ml CHX, and followed by live cell imaging. (C) Nuclear export of free p65 was simulated using the model in Fig. 3 with the parameter values from clusters 5, 7, and 8, and s was reduced by 100-fold at t = 0 for the CHX effect. In addition, nuclear import parameters (iNF, iI) were reduced to 10−5 at t = 90 min after TNF-α and CHX co-treatment. The total nuclear p65 amount (which is close to the free p65 amount under this condition) is shown in the upper panel. The first 10 minute interval after inhibition of import is plotted on the expanded time axis on the right, to facilitate comparison to the experimentally measured time course in (D). (D) Fluorescence loss in photobleaching (FLIP) was performed to determine any ongoing exchange of p65 between the nucleus and the cytoplasm after its apparently complete nuclear translocation induced by TNF-α alone or in co-treatment with CHX or LMB. A circular spot in the cytoplasm was repeatedly bleached for 10 minutes while the nuclear mean intensity was monitored (inset). The relatively short timeframe for the FLIP protocol did not allow measurements up to complete loss of GFP signal. Error bars are S.E.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g004: Two contrasting perturbations of NF-κB oscillations decouple nucleocytoplasmic shuttling from feedback-driven long-term dynamics.(A) Perturbed dynamics were simulated using the model in Fig. 3 and the parameter values from clusters 5, 7, and 8, with the additional modifications to nuclear export terms or the induced IκBα synthesis term for LMB or CHX effects, respectively (see text). The graphs show the time courses of free, IκBα-complexed, and total p65 in the nucleus, as predicted by the model. The red curves indicate the free and complexed components of a typical profile of nuclear p65. Inhibition of nuclear export allows one cycle of free p65 whereas inhibition of negative feedback induces constant activity. (B) Experimentally observed time lapse profiles of nuclear p65 level for single cells co-treated with LMB or CHX. Cells were treated with 10 ng/ml TNF-α and either 5 nM LMB or 2.5 µg/ml CHX, and followed by live cell imaging. (C) Nuclear export of free p65 was simulated using the model in Fig. 3 with the parameter values from clusters 5, 7, and 8, and s was reduced by 100-fold at t = 0 for the CHX effect. In addition, nuclear import parameters (iNF, iI) were reduced to 10−5 at t = 90 min after TNF-α and CHX co-treatment. The total nuclear p65 amount (which is close to the free p65 amount under this condition) is shown in the upper panel. The first 10 minute interval after inhibition of import is plotted on the expanded time axis on the right, to facilitate comparison to the experimentally measured time course in (D). (D) Fluorescence loss in photobleaching (FLIP) was performed to determine any ongoing exchange of p65 between the nucleus and the cytoplasm after its apparently complete nuclear translocation induced by TNF-α alone or in co-treatment with CHX or LMB. A circular spot in the cytoplasm was repeatedly bleached for 10 minutes while the nuclear mean intensity was monitored (inset). The relatively short timeframe for the FLIP protocol did not allow measurements up to complete loss of GFP signal. Error bars are S.E.

Mentions: LMB blocks nuclear export of p65 into the cytoplasm, an enabling mechanism behind NF-κB oscillation. When cells are simultaneously treated with TNF-α and LMB, a single pulse of free nuclear NF-κB is expected to be followed by the formation of the NF-κB∶IκBα complex in the nucleus. This is also predicted by model simulations (Fig. 4A, upper panel; Parameter values were from cluster 5, 7, 8 in Fig. 3C, and all export rates were reduced by 105-fold at t = 0; An example decomposition plot for TNF-α alone is shown in Fig. S2C for comparison). Time lapse imaging confirmed that total nuclear p65 accumulates in the nucleus (Fig. 4B, upper panel). The nuclear retention of IκBα-bound inactive NF-κB by LMB matches previous reports well [16].


Sustained oscillations of NF-kappaB produce distinct genome scanning and gene expression profiles.

Sung MH, Salvatore L, De Lorenzi R, Indrawan A, Pasparakis M, Hager GL, Bianchi ME, Agresti A - PLoS ONE (2009)

Two contrasting perturbations of NF-κB oscillations decouple nucleocytoplasmic shuttling from feedback-driven long-term dynamics.(A) Perturbed dynamics were simulated using the model in Fig. 3 and the parameter values from clusters 5, 7, and 8, with the additional modifications to nuclear export terms or the induced IκBα synthesis term for LMB or CHX effects, respectively (see text). The graphs show the time courses of free, IκBα-complexed, and total p65 in the nucleus, as predicted by the model. The red curves indicate the free and complexed components of a typical profile of nuclear p65. Inhibition of nuclear export allows one cycle of free p65 whereas inhibition of negative feedback induces constant activity. (B) Experimentally observed time lapse profiles of nuclear p65 level for single cells co-treated with LMB or CHX. Cells were treated with 10 ng/ml TNF-α and either 5 nM LMB or 2.5 µg/ml CHX, and followed by live cell imaging. (C) Nuclear export of free p65 was simulated using the model in Fig. 3 with the parameter values from clusters 5, 7, and 8, and s was reduced by 100-fold at t = 0 for the CHX effect. In addition, nuclear import parameters (iNF, iI) were reduced to 10−5 at t = 90 min after TNF-α and CHX co-treatment. The total nuclear p65 amount (which is close to the free p65 amount under this condition) is shown in the upper panel. The first 10 minute interval after inhibition of import is plotted on the expanded time axis on the right, to facilitate comparison to the experimentally measured time course in (D). (D) Fluorescence loss in photobleaching (FLIP) was performed to determine any ongoing exchange of p65 between the nucleus and the cytoplasm after its apparently complete nuclear translocation induced by TNF-α alone or in co-treatment with CHX or LMB. A circular spot in the cytoplasm was repeatedly bleached for 10 minutes while the nuclear mean intensity was monitored (inset). The relatively short timeframe for the FLIP protocol did not allow measurements up to complete loss of GFP signal. Error bars are S.E.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747007&req=5

pone-0007163-g004: Two contrasting perturbations of NF-κB oscillations decouple nucleocytoplasmic shuttling from feedback-driven long-term dynamics.(A) Perturbed dynamics were simulated using the model in Fig. 3 and the parameter values from clusters 5, 7, and 8, with the additional modifications to nuclear export terms or the induced IκBα synthesis term for LMB or CHX effects, respectively (see text). The graphs show the time courses of free, IκBα-complexed, and total p65 in the nucleus, as predicted by the model. The red curves indicate the free and complexed components of a typical profile of nuclear p65. Inhibition of nuclear export allows one cycle of free p65 whereas inhibition of negative feedback induces constant activity. (B) Experimentally observed time lapse profiles of nuclear p65 level for single cells co-treated with LMB or CHX. Cells were treated with 10 ng/ml TNF-α and either 5 nM LMB or 2.5 µg/ml CHX, and followed by live cell imaging. (C) Nuclear export of free p65 was simulated using the model in Fig. 3 with the parameter values from clusters 5, 7, and 8, and s was reduced by 100-fold at t = 0 for the CHX effect. In addition, nuclear import parameters (iNF, iI) were reduced to 10−5 at t = 90 min after TNF-α and CHX co-treatment. The total nuclear p65 amount (which is close to the free p65 amount under this condition) is shown in the upper panel. The first 10 minute interval after inhibition of import is plotted on the expanded time axis on the right, to facilitate comparison to the experimentally measured time course in (D). (D) Fluorescence loss in photobleaching (FLIP) was performed to determine any ongoing exchange of p65 between the nucleus and the cytoplasm after its apparently complete nuclear translocation induced by TNF-α alone or in co-treatment with CHX or LMB. A circular spot in the cytoplasm was repeatedly bleached for 10 minutes while the nuclear mean intensity was monitored (inset). The relatively short timeframe for the FLIP protocol did not allow measurements up to complete loss of GFP signal. Error bars are S.E.
Mentions: LMB blocks nuclear export of p65 into the cytoplasm, an enabling mechanism behind NF-κB oscillation. When cells are simultaneously treated with TNF-α and LMB, a single pulse of free nuclear NF-κB is expected to be followed by the formation of the NF-κB∶IκBα complex in the nucleus. This is also predicted by model simulations (Fig. 4A, upper panel; Parameter values were from cluster 5, 7, 8 in Fig. 3C, and all export rates were reduced by 105-fold at t = 0; An example decomposition plot for TNF-α alone is shown in Fig. S2C for comparison). Time lapse imaging confirmed that total nuclear p65 accumulates in the nucleus (Fig. 4B, upper panel). The nuclear retention of IκBα-bound inactive NF-κB by LMB matches previous reports well [16].

Bottom Line: Mathematical modeling suggests that NF-kappaB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IkappaBalpha.Perturbation of long-term NF-kappaB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model.We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-kappaB in the nucleus, and these oscillations are functionally advantageous.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America. sungm@mail.nih.gov

ABSTRACT
NF-kappaB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-kappaB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-kappaB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-kappaB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-alpha stimulation. Mathematical modeling suggests that NF-kappaB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IkappaBalpha. The ability of NF-kappaB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-kappaB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-kappaB in the nucleus, and these oscillations are functionally advantageous.

Show MeSH
Related in: MedlinePlus