Identification and Characterization of the TGFβ/Hippo Crosstalk through Mathematical Modeling

Transforming Growth Factor-beta (TGFβ) superfamily members control a myriad of cellular activities and are key regulators of most developmental and homeostatic processes. The Hippo pathway is a major regulator of tissue growth and organ size. Hippo and TGFβ pathways are intimately interconnected. Hippo pathway mediators Taz/Yap interact with the TGFβ regulated Smads and when Hippo is activated, the cytoplasmically localized Taz/Yap reduces or prevents Smad nuclear accumulation thereby dampening TGFβ induced transcriptional activity. In the nucleus, Taz/Yap can transcriptionally cooperate with Smads to alter target genes regulation. With the aim of identifying the TGFβ/Hippo crosstalk, I used experimental data and computational modeling of Hippo and TGFβ crosstalk. Quantitative high-content immunofluorescence (IF) microscopy showed that Taz/Yap abrogation reduces nuclear accumulation of Smads and the reduction is not due to degradation of Smad/Taz/Yap proteins or receptors. Immunoblotting experiments revealed that Taz/Yap knock down alters phosphorylation/dephosphorylation of Smads. IF microscopy showed that there is no difference in the export and dephosphorylation rates in cells transfected with siCTL versus siTaz/Yap while TGFβ receptor activity is inhibited. IF microscopy, immunoblotting and statistical analysis showed that Taz/Yap levels in the cytoplasm or nucleus is constant regardless of dose of TGFβ or time of treatment when the Hippo pathway activity is constant. Mathematical modeling predicted that the differential accumulation of Smads at cells transfected with siCTL and siTaz/Yap might be due to both receptor activity alteration and existence of a Taz/Yap regulated retention factor. As a candidate retention factor, Med18 the subunit 18 of the mediator complex was identified. IF experiment results confirmed that knocking down Med18 reduces nuclear accumulation of Smads. Analysis of RNAseq data for cells transfected with siCTL or siTaz/Yap showed that Hippo pathway perturbation yielded differential effects. Mathematical modeling revealed that TGFβ target genes might have constant or time varying mRNA degradation rate. In addition, Taz/Yap could modulate the temporal expression patterns of TGFβ target genes by selectively changing the kinetic parameters of the gene expression model. Analysis of the gene expression patterns of selected genes showed that TGFβ/Hippo pathway crosstalk might be a mechanism to control the sequence of gene activation.

Department dc:contributor.department

Biochemistry

Year dc:date.issued

2020

Author dc:creator

• Labibi, Bita

Advisors dc:contributor.advisor

• Attisano, Liliana
• Wrana, Jeffrey L

• Mathematical modelling
• Smad nuclear accumulation
• TGFβ/Hippo crosstalk
• Transforming Growth Factor-beta

Handle dc:identifier.uri

http://hdl.handle.net/1807/103440

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/103440