The Dynamics of TGF-β Signaling Are Dictated by Receptor Trafficking via the ESCRT Machinery

Daniel S.J Miller,Robert D Bloxham,Ming Jiang,Ilaria Gori,Rebecca E Saunders,Debipriya Das,Probir Chakravarty,Michael Howell,Caroline S Hill

doi:10.1016/j.celrep.2018.10.056

Abstract

Signal transduction pathways stimulated by secreted growth factors are tightly regulated at multiple levels between the cell surface and the nucleus. The trafficking of cell surface receptors is emerging as a key step for regulating appropriate cellular responses, with perturbations in this process contributing to human diseases, including cancer. For receptors recognizing ligands of the transforming growth factor β (TGF-β) family, little is known about how trafficking is regulated or how this shapes signaling dynamics. Here, using whole genome small interfering RNA (siRNA) screens, we have identified the ESCRT (endosomal sorting complex required for transport) machinery as a crucial determinant of signal duration. Downregulation of ESCRT components increases the outputs of TGF-β signaling and sensitizes cells to low doses of ligand in their microenvironment. This sensitization drives an epithelial-to-mesenchymal transition (EMT) in response to low doses of ligand, and we demonstrate a link between downregulation of the ESCRT machinery and cancer survival.

Highlights

Cell communication, mediated via signal transduction pathways, underpins both embryonic development and adult tissue homeostasis, and deregulation of these pathways is the underlying cause of many human diseases, a prominent example being cancer (Weber et al, 2016)
The family has traditionally been divided into two branches, whereby the TGFbs, activins, and NODAL induce SMAD2 and SMAD3 phosphorylation, and the bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) induce SMAD1, SMAD5, and SMAD9 phosphorylation, some cross-talk between these branches occurs (Gronroos et al, 2012; Ramachandran et al, 2018)
Whole Genome Screening Identifies ESCRT Components as Key Regulators of transforming growth factor b (TGF-b) Signaling Dynamics Upon acute exposure of cells to TGF-b, levels of PSMAD2 increase for around 1 hr before attenuating to lower levels at later time points (Figure S1A). These signaling dynamics are seen with a range of TGF-b concentrations, and we have subsequently used 2 ng/mL for our experiments as an example of a saturating dose. These characteristic signaling dynamics are the result of the TGF-b receptors becoming depleted from the cell surface within 5–10 min of ligand exposure, rendering the cells refractory to further acute stimulation (Vizan et al, 2013)

Summary

Introduction

Cell communication, mediated via signal transduction pathways, underpins both embryonic development and adult tissue homeostasis, and deregulation of these pathways is the underlying cause of many human diseases, a prominent example being cancer (Weber et al, 2016). The ligand brings the two types of receptors together, which allows the constitutively active kinase domain of the type II receptor to phosphorylate an intracellular glycine- and serinerich (GS) domain of the type I receptor (Feng and Derynck, 2005). This activates the type I receptor and provides a binding site for the downstream substrates of the pathway, the receptor-regulated SMADs (R-SMADs), which are phosphorylated at their extreme C termini. The family has traditionally been divided into two branches, whereby the TGFbs, activins, and NODAL induce SMAD2 and SMAD3 phosphorylation, and the bone morphogenetic proteins (BMPs) and growth and differentiation factors (GDFs) induce SMAD1, SMAD5, and SMAD9 phosphorylation, some cross-talk between these branches occurs (Gronroos et al, 2012; Ramachandran et al, 2018)

Results

Discussion

Conclusion