Caroline Hill

Novel regulators of the TGF-β signalling pathway

Members of the transforming growth factor β (TGF-β) superfamily of extracellular ligands control many key biological processes in both embryonic development and adult organisms, through their ability to regulate cell proliferation, migration, adhesion, differentiation and survival. Moreover, aberrant TGF-β signalling is responsible for several serious human diseases, including cancer, where it can act both as a tumour suppressor and tumour promoter.

Ligand stimulation induces complex formation between specific type I and type II receptors, which are serine/threonine kinases. The constitutively active type II receptor phosphorylates and activates the type I receptor, which in turn phosphorylates and activates a subset of receptor-mediated Smads (R-Smads), examples of which are Smad2 and Smad3, which act downstream of the TGF-β, Activin and Nodal ligands. The activated R-Smads form homomeric complexes and heteromeric complexes with Smad4, which accumulate in the nucleus where they directly regulate the transcription of target genes in conjunction with other DNA-binding transcription factors.

In my laboratory we are studying these pathways in early vertebrate development where they function normally, focusing mainly on the Xenopus system, although more recently we have additionally begun to use zebrafish. We are also trying to understand how TGF-β signalling is perturbed in tumorigenesis and have mainly studied this to date in model tissue culture systems.

Although we know in outline how the TGF-β signalling pathway works, it is clear that it is subject to many levels of regulation, of which we have very little understanding. To identify novel components and regulators of the TGF-β signalling pathway, we have performed high-throughput siRNA screens, using libraries of siRNA oligonucleotide SMARTpools. We have performed three different types of screen. Two of them have utilised human cell lines stably expressing an enhanced GFP fusion of the R-Smad, Smad2 (EGFP-Smad2).

We used automated microscopy to screen for factors regulating Smad2 subcellular localisation, assaying for siRNAs that either prevented TGF-β-induced nuclear accumulation of EGFP-Smad2, or prevented EGFP-Smad2 accumulation in the cytoplasm following receptor inhibition. In this context we have screened libraries of phosphatases, nuclear import and export regulators, and nuclear membrane components.

We have also performed screens using a transcriptional readout for TGF-β signalling. To do this, we generated a HaCaT cell line that stably expresses CAGA12-Luc, a Smad3/Smad4-dependent reporter containing 12 copies of the CAGAC site from the PAI-1 promoter ². The cell line additionally contains a Renilla reporter driven by the thymidine kinase (TK) promoter to act as an internal control for siRNAs that affect cell viability and survival, as well as those that have general effects on transcription. Using this transcriptional readout we have screened libraries of phosphatases, kinases, E3 ubiquitin ligases and deubiquitinating enzymes ¹.

As a result of these screens, we have identified potential new regulators of TGF-β superfamily signalling, in particular a number of interesting phosphatases, E3 ubiquitin ligases and deubiquitinating enzymes that have hitherto not been thought to be involved in TGF-β signalling. Some of these appear to act positively in the pathway, while others are repressors. The project will involve characterising a selection of these potential regulators in detail and working out how they act in the TGF-β signalling pathway. This work will be done using both tissue culture systems and early vertebrate embryos.

The project will aim to determine where in the pathway they function and their mechanism of action. Once we understand how these factors regulate TGF-β signalling, we will investigate whether they represent good targets through which to modulate the TGF-β pathway for therapeutic ends, and also whether they are deregulated in human tumours.

References

1. Levy L, Howell M, Das D, Harkin S, Episkopou V and Hill CS. Arkadia activates Smad3/Smad4-dependent transcription by triggering signal-induced SnoN degradation. Mol Cell Biol 2007; 27: 6068-6083.
2. Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S and Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF β-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998; 17: 3091-3100.