4 | Article: Hoffman et al (2002) The IkappaB-NF-kappaB signaling module: temporal control and selective gene activation

Question 1

What is NFκB ?

Answer 1

transcription factor with key role in proliferation, cell death, development and innate / adaptive immune responses

Question 2

NFkB

what is it involved in?

Answer 2

involved in cellular responses to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens

plays a key role in regulating the immune response to infection.

Question 3

NFkB

What can incorrect regulation lead to?

Answer 3

linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development

Question 4

What is the main focus of this study on NF-κB signaling?

Answer 4

• temporal control of NF-κB activation
• by the coordinated degradation and synthesis of IκB proteins (α, β, and ε)
• how this affects gene expression specificity.

Question 5

How does IκBα contribute to NF-κB regulation?

Answer 5

• strong negative feedback
• allows for fast turn-off of NF-κB response
• mediates rapid NF-κB activation.

Question 6

What roles do IκBβ and IκBε play in NF-κB regulation?

Answer 6

• reduce system’s oscillatory potential, stabilize NF-κB responses during longer stimulations.

Question 7

What is the characteristic NF-κB activation profile in response to TNF-α stimulation?

Answer 7

• profile resembles strongly damped oscillations
• two phases of activation observed in various human and mouse cell lines.

Question 8

How does the NF-κB response differ in cells containing only IκBα compared to those with only IκBβ or IκBε?

Answer 8

• Only IκBα: highly oscillatory NF-κB response,
• Only IκBβ or IκBε: monotonic increase to plateau, no notable subsequent repression.

Question 9

What does the computational model reveal about the interplay between IκB isoforms?

Answer 9

• interplay between IκB isoforms can result in rapid responses to stimulation onset/cessation
• allows for a relatively stable NF-κB response during long-term stimulation.

Question 10

How do transient stimuli affect NF-κB activation duration?

Answer 10

• consistent NF-κB activation durations regardless of stimulus length within first hr
• equivalent to the first peak of persistently stimulated NF-κB activity.

Question 11

What are the bimodal signal processing characteristics of the IκB-NF-κB signaling module?

Answer 11

• long stimulations: NF-κB activation lasts as long as the stimulus.
• stimulations < 1 hr: duration of response is largely invariant → even short stimulations result in substantial NF-κB activation.

Question 12

What does the study suggest about IκB gene expression?

Answer 12

• The study indicates previously unrecognized crossregulation in expression of IκB genes
• IκBβ and IκBε mRNA synthesis parameters: ca sevenfold lower than in single-IκB models.

Question 13

How might cells or cell lineages alter the responsiveness of the NF-κB signal transduction pathway?

Answer 13

vary relative synthesis levels of IκBα, β, ε in response to environmental/developmental cues

→ alter NF-κB pathway responsiveness.

Question 14

How did the IκBα mutant (IκBβ-/-, IκBε-/-) respond to TNF-α stimulation?

Answer 14

The IκBα mutant exhibited a highly oscillatory NF-κB response with four equally spaced peaks over the course of the 6-hour experiment.

Question 15

How did the IκBβ mutant (IκBα-/-, IκBε-/-) respond to TNF-α stimulation?

Answer 15

The IκBβ mutant showed a monotonic increase in NF-κB activity to a plateau within 1 hour, with no notable subsequent repression.

Question 16

How did the IκBε mutant (IκBα-/-, IκBβ-/-) respond to TNF-α stimulation?

Answer 16

The IκBε mutant also showed a monotonic increase in NF-κB activity to a plateau within 1 hour, similar to the IκBβ mutant, with no notable subsequent repression.

Question 17

How do the different IκB isoforms work together in wild-type cells according to the model based on Figure 2b?

Answer 17

Interplay between IκB isoforms results in
- rapid responses to stimulation onset or cessation (IκBα)
- relatively stable NF-κB response during long-term stimulation (IκBβ and IκBε: dampening)

Question 18

Figure 2 shows model predictions made in the article “The IκB-NFκB Signaling Module: Temporal Control and Selective Gene Activation” by Hoffmann et al. (2002).

[shown are figures 2C and 2D from the paper
2C = model of mutants with one isoform
2D = model with all isoforms at varying expression levels]

Which mutants were simulated in Figure 2a? Explain how these mutants helped the authors to estimate the parameters of the model.

(2023_1, 2021_1, 2020_2)

Answer 18

[figure 2 overall shows experimental data and modelling after stimulation with TNF-alpha.
2C = model of mutants with one isoform
2D = model with all isoforms at varying expression levels, alpha is constant, beta and epsilon are increased from top to bottom ]

• knockouts → fibroblasts that contain only one IκB isoform.
• analysis → estimate specific roles and kinetic parameters of each IκB isoform in regulating NF-κB dynamics.
• allowed for fitting of model to experimental data
• provided insights into ODEs of IκBα, IκBβ, and IκBε.

Question 19

The figure shows model predictions made in the article “The IκB-NFκB Signaling Module: Temporal Control and Selective Gene Activation” by Hoffmann et al. (2002).

[shown are figures 2C and 2D from the paper
2C = model of mutants with one isoform
2D = model with all isoforms at varying expression levels]

According to the Figure 2b [=2D in paper] what are the roles of the different IκB isoforms?

(2023_1, 2021_1)

Answer 19

[figure 2 overall shows experimental data and modelling after stimulation with TNF-alpha.
2C = model of mutants with one isoform
2D = model with all isoforms at varying expression levels, alpha is constant, beta and epsilon are increased from top to bottom ]

IκBα:
- rapid NF-κB activation, strong negative feedback regulation

IκBβ and IκBε
- respond more slowly to IKK activation
- act to dampen long-term oscillations of the NF-κB response.
- only IκBβ or IκBε: NF-κB increases monotonically to plateau, no subsequent repression.

Question 20

Hoffman et al. - Fig 2E and F

How did the authors interpret the results shown in these figures?

(2022_1)

Answer 20

• The experimental data (Fig. 2E) shows complex NF-κB activation pattern
• The computational model (Fig. 2F) was able to reproduce this complex activation pattern with good qualitative and quantitative agreement.

→ justifies use as predictive tool in experimentation

(also: was found that mRNA synthesis parameters for IκBβ and IκBε in WT model about 7fold lower than single-IκB-isoform models, suggesting previously unrecognized cross-regulation in IκB gene expression.)

Question 21

Hoffman et al. - Fig 2E and F

How did the authors arrive at the model parametrization used in their simulation?

(2022_1, 2012_2)

Answer 21

multi-step approach:

• Used previously determined biochemical parameters from literature where available.
• Created genetically reduced systems (knockout cell lines) to constrain parameter values.
• Employed various parameter estimation methods, including:
  
  • Random search (most successful)
  • Steepest gradient descent
  • Genetic algorithm
• Manually adjusted params. within SD limits → improve qualit. fits, esposcillatory beh.
• For the wild-type model:
  
  • Combined models of simplified signaling modules
  • Initially used transcription rates from knockout studies
  • Adjusted IκBβ and IκBε mRNA synthesis parameters to fit wild-type experimental data
  • Found these parameters to be about 7-fold lower than in single-IκB models
• Focused on qualit. fitting rather than exact param. Est (concl. not dep. on precise values.)
• Primarily adjusted parameters for:
  
  • Rates of transcription and translation of IκB isoforms
  • Rate of IκB-NF-κB nuclear export
  • NF-κB nuclear import rate

Question 22

Hoffman et al. - Fig 2E and F

Name two aspects how the graphical presentation could be improved to allow for a better comparison of experimental data and simulation results.

(2022_1)

Answer 22

1. Use same time scale for experimental data and simulation results (currently, experimental data uses a non-linear time scale while simulations use a linear scale).
2. Present experimental data, simulation results on same graph for direct visual comparison.

Question 23

Hoffman et al. - Fig 2E and F

Name one aspect of the experimental data that the model simulations don’t reproduce.

(2022_1)

Answer 23

Maybe means the expression levels (synthesis parameters)?
[It was found that the mRNA synthesis parameters for IκBβ and IκBε in wild-type model were about sevenfold lower than in single-IκB models, suggesting previously unrecognized cross-regulation in IκB gene expression.]

or:
model simulations appear smoother and more idealized than the experimental data, which shows more variability not captured in the simulations.

Question 24

Explain the main question discussed in the article.

(2020_2)

Answer 24

Role of the three different IκB isoforms in regulating NFκB response

Question 25

State one main result of the article

(2020_2)

State two results of the article

(2012_2)

Answer 25

Roles of three isoforms:
- IκBα responsible for strong negative feedback → fast turn-off of NF-κB response
- IκBβ, IκBε function to reduce system’s oscillatory potential
→ together the three isoforms make make possible bimodal signal processing characteristics (both qualitative and quantitative)

Question 26

Use Figure 5 [ illustration of NFkB signalling module] to briefly summarize the biological system that was examined in the article “The IκB-NFκB Signaling Module: Temporal Control and Selective Gene Activation” by Hoffmann et al. (2002).

(2020_2)

Answer 26

The article examined the IκB-NFκB signaling module, which consists of:
- NFκB, TF that regulates numerous genes involved in cellular processes
- 3 IκB isoforms (IκBα, IκBβ, IκBε), inhibit NFκB by binding to it, keeping in cyt
- IKK (IκB kinase), which after being activated phosphorylates IκB proteins, leading to their degradation and allowing NFκB to enter the nucleus