Quantitative analysis reveals crosstalk mechanisms of heat shock-induced attenuation of NF-κB signaling at the single cell level

<div><p>Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor κB (NF-κB) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-κB p65 response to Tumor Necrosis Factor α (TNFα) treatment. The degree of inhibition of the NF-κB response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-κB responses while being consistent with the observed population-level responses. In particular “all-or-nothing” encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and IκBα phosphorylation, while others involving transport were “analogue”. In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-κB p65 subunit. The single cell responses exhibited “all-or-nothing” encoding. While most cells did not respond to TNFα stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKKα and IKKβ subunits in whole cell lysates. However, a combination of “all-or-nothing” crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-κB responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.</p></div

Mathematical modeling discriminates different single cell HS encoding mechanisms.

<p>(<b>A</b>) HS effect is modeled via a time-dependent attenuation function y(t). Each model simulation consists of three steps: (I) randomization of the attenuation coefficient <i>R</i> from the gamma distribution, (II) calculation of the attenuation function y(t) for the given <i>R</i>, (III) NF-κB model simulation. (<b>B</b>) List of considered cross-talk mechanisms. Roman numerals refer to mechanisms depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006130#pcbi.1006130.g002" target="_blank">Fig 2</a>. Reverse effect indicates activation. (<b>C</b>) Schematic diagram of NF-κB model. Colored arrows indicate simulated cross-talk mechanism from B. (<b>D</b>) Simulation of hypothetical mechanisms involved in the NF-κB and HS pathway cross-talk. Simulations performed for 60 min HS exposure before TNFα stimulation. Shown are a sample of 50 time courses of simulated nuclear NF-κB levels (colored lines) and average nuclear NF-κB levels (black bold line), calculated from 1,000 single cell simulations for cells treated with TNFα. (<b>E</b>) Scatterplots of the maximum nuclear NF-κB against the attenuation coefficient <i>R</i> per simulated cell. Color scheme refers to “all-or-nothing” or “analogue” encoding, as well as mechanisms where a population-level fit was not obtained (no effect).</p

IKK depletion was not sufficient to recapitulate observed responses.

<p><b>(A)</b> Western blot analysis of soluble (S) and insoluble (IS) NF-κB p65, IKKα and IKKβ proteins level in WT U2OS cells. Cells were either cultured under normal conditions (37°C) or subjected to 15, 30 and 60 min of HS at 43°C. β-actin was used as a loading control. (<b>B</b>) Quantification of the blotting data from A. (<b>C</b>) Schematic representation of the IKK modulation in the model. (<b>D</b>) List of considered models (as indicated with red arrows in schematics C). (<b>E</b>) Simulation of the HS-dependent 60% IKK depletion (mechanism b*). Shown are 50 time courses of simulated nuclear NF-κB levels (green lines) and average nuclear NF-κB levels (black bold line), calculated from 1,000 single cell simulations for cells treated with TNFα after 60 min HS exposure. <b>(F)</b> Comparison of the cellular IKK levels after 60 min HS exposure. Shown are mean (±SD) levels for models assuming no inhibition (a, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006130#pcbi.1006130.g003" target="_blank">Fig 3</a>), as well as HS-mediated IKK depletion (mechanism b*) and IKK degradation (b, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006130#pcbi.1006130.g003" target="_blank">Fig 3</a>).</p

Temporal encoding of HS crosstalk.

<p>(<b>A</b>) Simulation of HS cross-talk assuming IKK depletion and inhibition of IKK activation (model b*+c from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006130#pcbi.1006130.g007" target="_blank">Fig 7</a>). Shown are a sample of 50 time courses of simulated nuclear NF-κB levels (colored lines) and average nuclear NF-κB levels (black bold line), calculated from 1,000 single cell simulations for cells treated with TNFα after different HS exposure times. <b>(B)</b> Percentage (%) of responding (yellow) and non-responding (blue) cells from A. <b>(C)</b> Characteristics of NF-κB trajectories in responding cells from B. Left panel: the distribution of the maximum nuclear NF-κB. Right panel: time to first response. <b>(D)</b> Scatterplots of the maximum nuclear NF-κB level per cell against (I) attenuation coefficient <i>R</i>, (II) IKKn level before HS and (III) IKKn at time t₀ (after 60 min HS, before TNFα treatment). <b>(E)</b> Fitted IKKn levels after different HS exposure times (calculated from data in A).</p

Shape (<i>k</i>) and scale (<i>θ</i>) parameters of gamma distribution for the attenuation coefficient.

<p>Note that for <i>k = 1</i>, θ corresponds to the average value of the attenuation coefficient <i>R</i>.</p

HS cross-talk involves multiple “all-or-nothing” mechanisms.

<p>(<b>A</b>) Schematic representation of the IKK modulation schemes in the model. (<b>B</b>) List of considered cross-talk mechanisms (as indicated with colored arrows in A). (<b>C</b>) Simulation of HS cross-talk mechanisms. All models assume HS-mediated 60% depletion of IKK (mechanism b*) in combination with other mechanisms as in B. Shown are a sample of 50 time courses of simulated nuclear NF-κB levels (colored lines) and average nuclear NF-κB levels (black bold line), calculated from 1,000 single cell simulations for cells treated with TNFα after 60 min HS exposure. <b>(D)</b> Percentage (%) of responding (yellow) and non-responding (blue) cells from data in C or <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1006130#pcbi.1006130.g006" target="_blank">Fig 6E</a> (model b*). <b>(E)</b> Characteristics of NF-κB responses in cells from C. Left panel: the distribution of the maximum nuclear NF-κB. Right panel: time to first response. Shown are characteristics of responding cells for (b*+c) and (b*+d), and of all simulated cells for other mechanisms. (<b>F</b>) Scatterplots of the maximum nuclear NF-κB level against the attenuation coefficient <i>R</i> per cell for different models considered as in C. <b>(G)</b> Analysis of the HS-mediated inhibition across different mechanisms. (Left) Shown are the average (±SD) values of the mechanism-specific attenuation coefficient <i>R</i> across models in C. (Right): Fold change of the mechanism specific attenuation coefficient <i>R</i> across different models (calculated with respect to the corresponding <i>R</i> for a model without IKK depletion).</p

Schematic diagram of the HS-induced NF-κB regulation.

<p>HS may lead to: (i) IKK inactivation due to conformational changes or denaturation, or actions of HSPs and upstream kinases; (ii) inhibition of IκB phosphorylation; (iii) modulation of importin-dependent nuclear transport; (iv) destabilization of protein complexes involved in transcription; (v) inhibition of translation via molecular crowding and stress granules formation.</p

HS inhibits single cell p65-EGFP translocation.

<p>Confocal microscopy images of representative U2OS cells stably expressing p65-EGFP fusion protein. Top panel: cells maintained at 37°C and stimulated with TNFα. Bottom panel: cells exposed to 60 min 43°C HS prior to TNFα stimulation. Time of stimulation is displayed in minutes. Nuclear p65-EGFP translocation is depicted with an asterisk (*). Scale bar, 10 μm.</p

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