Actin networks modulate heterogenous NF-κB dynamics in response to TNFα
Abstract
The canonical NF-κB transcription factor RELA is a master regulator of immune and stress responses and is upregulated in PDAC tumours. In this study, we characterised previously unexplored endogenous RELA-GFP dynamics in PDAC cell lines through live single cell imaging. Our observations revealed that TNFα stimulation induces rapid, sustained, and non-oscillatory nuclear translocation of RELA. Through Bayesian analysis of single cell datasets with variation in nuclear RELA, we predicted that RELA heterogeneity in PDAC cell lines is dependent on F-actin dynamics. RNA-seq analysis identified distinct clusters of RELA-regulated gene expression in PDAC cells, including TNFα-induced RELA upregulation of the actin regulators NUAK2 and ARHGAP31. Further, siRNA-mediated depletion of ARHGAP31 and NUAK2 altered TNFα-stimulated nuclear RELA dynamics in PDAC cells, establishing a novel negative feedback loop that regulates RELA activation by TNFα. Additionally, we characterised the NF-κB pathway in PDAC cells, identifying how NF-κB/IκB proteins genetically and physically interact with RELA in the absence or presence of TNFα. Taken together, we provide computational and experimental support for interdependence between the F-actin network and the NF-κB pathway with RELA translocation dynamics in PDAC.
Data availability
All data generated for this study have been included as source data files.
Article and author information
Author details
Funding
Cancer Research UK (S_3567)
- Francesca Butera
Cancer Research UK supported by Stand Up to Cancer UK (C37275)
- Chris Bakal
Cancer Research UK supported by Stand Up to Cancer UK (A20146)
- Chris Bakal
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- John G Albeck, University of California, Davis, United States
Version history
- Received: January 8, 2023
- Accepted: August 5, 2024
- Accepted Manuscript published: August 7, 2024 (version 1)
Copyright
© 2024, Butera et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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