Crosstalk between Hippo and TGFβ: Subcellular Localization of YAP/TAZ/Smad Complexes

The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways. (C) 2015 Elsevier Ltd. All rights reserved.