Elsevier

Biosystems

Volume 124, October 2014, Pages 75-85
Biosystems

Comparison of the molecular topologies of stress-activated transcription factors HSF1, AP-1, NRF2, and NF-κB in their induction kinetics of HMOX1

https://doi.org/10.1016/j.biosystems.2014.09.005Get rights and content

Highlights

  • We model the efficiency of HSF1, AP-1, NRF2, and NF-κB to remove inducers of stress.

  • The TF circuits were modelled at comparable levels of abstraction.

  • Two circuits (HSF1 and AP-1) seem especially suited for long-term removal of stress.

  • NRF2 seems most appropriate for cellular homeostasis by efficient ROS regulation.

Abstract

For cells, reacting aptly to changes in their environment is of critical importance. The protein Heme oxygenase-1 (HMOX1) plays a critical role as a guard of cellular homeostasis and is considered as a reliable indicator of cellular oxidative stress. A better insight in the regulation of HMOX1 would assist in understanding the physiological role of HMOX1 as well as improving functional interpretation of the gene as a biomarker in toxicogenomics. Remarkably, as many as four transcription factors are known to regulate the HMOX1 gene: HSF1, AP-1, NRF2, and NF-κB. To investigate induction kinetics of these transcription factors, we constructed mathematical simulation models for each of them. We included the topology of the known interactions of molecules involved in the activation of the transcription factors, and the feedback loops resulting in their down-regulation. We evaluate how the molecular circuitries associated with the different transcription factors differ in their kinetics regarding HMOX1 induction, under different scenarios of acute and less acute stress. We also evaluate the combined effect of the four transcription factors on HMOX1 expression and the resulting alleviation of stress. Overall, the results support the assumption of different biological roles for the four transcription factors, with AP-1 being a fast acting general stress response protein at the expense of efficiency, and NRF2 being important for cellular homeostasis in maintaining low levels of oxidative stress.

Introduction

Oxidative stress is believed to underlie the etiology of numerous human conditions. Under a wide range of stressful conditions, the protein Heme oxygenase-1 (HMOX1) plays a critical role in the maintenance of cellular homeostasis. HMOX1 is able to alleviate stress caused by reactive oxygen species (ROS) as its products have potent antioxidant and anti-inflammatory properties as well as signalling capabilities. As many diseases are accompanied by conditions of oxidative stress, and HMOX1 is one of the most sensitive and reliable indicators for several pathological conditions related to cellular oxidative stress, the modulation of HMOX1 activity is of potential therapeutic value (Ryter et al., 2006). In toxicogenomics, the up-regulation of Hmox1 is an important indicator for the skin sensitizing potential of a chemical (Natsch, 2009, van der Veen et al., 2013, Vandebriel et al., 2010). Possibly, the beneficial effects of HMOX1 in some systems may be limited to narrow windows of concentrations (Suttner and Dennery, 1999 Walther et al., 2012), increasing the need for a thorough understanding of its induction. A better insight in the regulation of HMOX1 may assist in understanding the biological role of HMOX1, as well as improving the functional interpretation of this gene as a biomarker. The variability in response could be caused by the multitude of stress-activated recognition sites contained within the Hmox1 promoter. No less than four known transcription factors are able to directly induce the expression of Hmox1 (Alam and Cook, 2007): HSF1, AP-1, NRF2, and NF-κB, which we will discuss in the following paragraphs.

HSF1 is a regulator of heat shock proteins (HSPs). The heat shock response is a molecular response to non-native and damaged or misfolded proteins (Szymanska and Zylicz, 2009). HSPs act either as chaperones to assist in proper folding, or as an escort for the proteins to the proteasome for degradation. Under normal conditions, HSF1 exists as an inactive monomer in a complex with the HSP Hsp70. Upon the formation of denatured proteins (DP), HSP70 is released from the complex to carry out its chaperone function with the DP. The now released HSF1 trimerizes and is transported into the nucleus where it is hyperphosphorylated. This enables HSF1 to bind to heat shock elements in the DNA, initiating the expression of HSPs. Its decline as an active transcription factor is initiated through a negative feedback loop with its gene product HSP70, which binds to the HSF1 transactivation domain. Although humans do not appear to have a consensus binding element for HSF1 in the promoter site for HMOX1 (Alam and Cook, 2007), there is evidence that a distal promoter site takes over this task under some conditions (Alam and Cook, 2007). To support this claim, a common expression pattern of heat shock induced proteins and HMOX1 in chemical induction of sensitization has been suggested (Pronk et al., 2013).

AP-1 is a transcription factor composed of proteins belonging to c-FOS, c-JUN and/or ATF family. It regulates gene expression in response to a variety of stimuli. Its regulation occurs in two ways: firstly by phosphorylative activation of the constituents of AP-1 that are already present, secondly by rapid and transient transcription (and translation) of the constituents based on cellular stimuli transferred by signalling pathways (Miller et al., 2010, Karin, 1995).

The NRF2 antioxidant response pathway is the primary cellular defence mechanism against the cytotoxic effects of oxidative stress. NRF2 increases the expression of several detoxifying and antioxidant enzymes. Under normal conditions, NRF2 is sequestered in the cytoplasm by KEAP1 leading to fast degradation. In the nucleus, the binding of NRF2 to the antioxidant response element (ARE) is inhibited by BACH1. Under oxidative stress conditions, ROS binds to KEAP1 and BACH1, releasing NRF2 to travel to the nucleus where it initiates transcription of anti-oxidative genes by binding to ARE (Reichard et al., 2007). When ROS is cleaned up, the system automatically returns to steady state as BACH1 and KEAP1 are no longer bound by ROS and resume the inhibition of NRF2 binding.

NF-κB is a group of related transcription factors found in almost all vertebrate animal cell types. It is involved in cellular responses to various stimuli. Under normal conditions, IκBA sequesters the NF-κB in an inactive state in the cytoplasm by masking nuclear localization signals. Its activation is initiated by the induction of activated IKK-dependent degradation of IκBA proteins. Activated NF-κB translocates to the nucleus, where it induces the expression of its own repressor IκBA, re-inhibiting NF-κB (for a detailed description, see Ruland, 2011).

Over the past decades, a reasonable understanding of the workings of the molecular circuitry of these four transcription factors in the induction by oxidative stress has been achieved. The details of their functioning in terms of kinetics and thresholds is hard to predict by their topology but can effectively be determined by modelling.

Models describing the activity of the individual transcription factors have been proposed. The response to unfolded protein by HSF1 has been modelled on several occasions (Szymanska and Zylicz, 2009, Petre et al., 2009, Peper et al., 1998, Rieger et al., 2005). The same applies to the NF-κB response (Ihekwaba et al., 2004, Lipniacki et al., 2004, Hoffmann et al., 2002). The AP-1 response was modelled by Miller et al. (2010). Although the model of NRF2 is well described in literature, we were not able to find an explicit mathematical model. Despite the usefulness of these models as they are, their difference in detail and difference in values of (fitted) parameters makes it difficult to compare them directly with regard to the induction kinetics of HMOX1. We therefore use the HSF1 model of Szymanska and Zylicz (2009) as a template, to model the molecular circuitries of the other transcription factors. This template is essentially as follows: a stressor (ROS or ROS-derived DP) leads to activation (by homo- or heteromeric complex formation or dissociation) of signalling protein or complex, which leads to transcriptional regulation of Hmox1 and (for some of the models) a signalling protein as well. This leads to reduced levels of the stressor, thus closing the circuitry. The resulting models have a low level of complexity, while they still capture the basic characteristics of the system. The novelty of this approach lies in the fact that the level of abstraction for each model now is comparable, as are the transcription-, translation- and biochemical reaction rates and protein abundances. This allows us to compare the transcription factors exclusively on the topology of the interacting molecules in their circuitry. This comparison also enables us to assess the value of the different transcription factors when combined in their effect on HMOX1 induction and ROS and DP removal under various stress situations, shedding light on their individual physiological role.

Section snippets

Parameters and molecular circuits

We take the model of Szymanska and Zylicz (2009) on the denatured protein response by the HSF1 molecular circuit as a template for our models. We adjust the model only slightly by introducing HMOX1 into the model, as a HSF-inducible molecule, in addition to HSP70. Moreover, parameter l4 (transcript degradation rate) is introduced. In the original model, this was integrated in parameter k4 (translation rate). The separation between transcript degradation rate and translation rate enables us to

Steady-state values

As a first step to study the robustness of the model, we determined how a model would approach a steady-state situation of a system under natural conditions. For NF-κB, literature states that 85% of IκBα is bound to NF-κB and 15% is free under normal conditions (Rice and Ernst, 1993). The model shows a similar result, as at steady state 84.4% IκBα is bound to NF-κB, while 15.6% is free. For HSP70, qualitative results are reported in literature stating that most HSP70 (HSP90) is bound to HSF1 at

Discussion

Although HMOX1 is an important molecule to prevent irreversible damage to cells caused by reactive oxygen species (ROS), much is unknown about its induction. To evaluate the kinetics of HMOX1 induction by its four transcription factors (Alam and Cook, 2007), we have made mathematical simulation models of the molecular circuits of HSF1, AP-1, NRF2, and NF-κB. All four transcription factors are capable of removing ROS by their capability of inducing HMOX1 (Alam and Cook, 2007). How these circuits

Acknowledgements

This work was supported by a grant of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (050-060-510) to the Netherlands Toxicogenomics Centre. We thank Wils A. L. Pronk and Sebastiaan Wesseling for their help concerning biochemical reaction rates.

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