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Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs.

ABSTRACT: We describe a protocol for a live-cell luciferase assay system for continuously monitoring fibroblast growth factor (FGF) signal disruption in human-induced pluripotent stem cells (iPSCs). Signal disrupting effects of chemicals are used as an indicator to evaluate toxicity. The assay is reliably predictive of the effects of limb malformation chemicals (AUC = 0.93). The current approach is limited to FGF signal disruption, and combinations with other types of signaling will be required to detect the effects of different toxicants. For complete details on the use and execution of this protocol, please refer to Kanno et al. (2022a).

SUBMITTER: Kanno S

PROVIDER: S-EPMC9168156 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs.

Kanno Seiya S Mizota Kashu K Okubo Yusuke Y Kageyama Tatsuto T Yan Lei L Fukuda Junji J

STAR protocols 20220604 2

We describe a protocol for a live-cell luciferase assay system for continuously monitoring fibroblast growth factor (FGF) signal disruption in human-induced pluripotent stem cells (iPSCs). Signal disrupting effects of chemicals are used as an indicator to evaluate toxicity. The assay is reliably predictive of the effects of limb malformation chemicals (AUC = 0.93). The current approach is limited to FGF signal disruption, and combinations with other types of signaling will be required to detect ...[more]

PMID: 35677614

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Project description:BackgroundThe Fibroblast Growth Factor (FGF) pathway is driving various aspects of cellular responses in both normal and malignant cells. One interesting characteristic of this pathway is the biphasic nature of the cellular response to some FGF ligands like FGF2. Specifically, it has been shown that phenotypic behaviors controlled by FGF signaling, like migration and growth, reach maximal levels in response to intermediate concentrations, while high levels of FGF2 elicit weak responses. The mechanisms leading to the observed biphasic response remains unexplained.ResultsA combination of experiments and computational modeling was used to understand the mechanism behind the observed biphasic signaling responses. FGF signaling involves a tertiary surface interaction that we captured with a computational model based on Ordinary Differential Equations (ODEs). It accounts for FGF2 binding to FGF receptors (FGFRs) and heparan sulfate glycosaminoglycans (HSGAGs), followed by receptor-phosphorylation, activation of the FRS2 adapter protein and the Ras-Raf signaling cascade. Quantitative protein assays were used to measure the dynamics of phosphorylated ERK (pERK) in response to a wide range of FGF2 ligand concentrations on a fine-grained time scale for the squamous cell lung cancer cell line H1703. We developed a novel approach combining Particle Swarm Optimization (PSO) and feature-based constraints in the objective function to calibrate the computational model to the experimental data. The model is validated using a series of extracellular and intracellular perturbation experiments. We demonstrate that in silico model predictions are in accordance with the observed in vitro results.ConclusionsUsing a combined approach of computational modeling and experiments we found that competition between binding of the ligand FGF2 to HSGAG and FGF receptor leads to the biphasic response. At low to intermediate concentrations of FGF2 there are sufficient free FGF receptors available for the FGF2-HSGAG complex to enable the formation of the trimeric signaling unit. At high ligand concentrations the ligand binding sites of the receptor become saturated and the trimeric signaling unit cannot be formed. This insight into the pathway is an important consideration for the pharmacological inhibition of this pathway.

Dataset Information

Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs.

Publications

Luciferase assay system to monitor fibroblast growth factor signal disruption in human iPSCs.

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