It was then found that TAZ

It was then found that TAZ, a YAP holomog, binds heteromeric SMAD2/3–4 complexes in a TGF-ß-dependent fashion, and is recruited to TGFβ response elements (Varelas et al., 2008). Knockdown experiments identified TAZ as critical for nuclear accumulation of SMAD2/3/4 complexes in response to TGF-ß, and subsequent gene target transactivation. It was proposed that TAZ nucleo-cytoplasmic shuttling defines a hierarchical system regulating both SMAD nuclear accumulation and coupling to the transcriptional machinery involved in the maintenance of self-renewal markers in human embryonic stem BVT 2733 mg (Varelas et al., 2008). The same group subsequently found that TAZ/YAP dictate the localization of active SMAD complexes in response to cell density-mediated formation of polarity complexes (Varelas et al., 2010). Specifically, cell density inhibits nuclear accumulation of TGF-ß-activated SMAD complexes. Mechanistically, in high-density epithelial cell cultures, activation of the Hippo pathway leads to nuclear exclusion of TAZ/YAP following phosphorylation by activated LATS, leading to cytoplasmic sequestration of active (phosphorylated) SMAD complexes by phospho-TAZ/YAP (Fig. 3A). Cell-density dependent interactions of YAP/TAZ with cell junctions, and in particular with several components of the Crumbs polarity complex, promote TAZ/YAP phosphorylation, cytoplasmic retention, and consequently suppress TGF-ß signaling. In vivo, a correlation between subcellular localization of TAZ/YAP and that of P-SMAD2/3 was observed during various stages of mouse embryogenesis. Interfering with the integrity of the Crumbs complex and destruction of cell–cell junctions in epithelial cells was found to TGF-ß-driven transcription as well as TGF-ß-dependent epithelial to mesenchymal transition. Indirect support for the activated SMAD sequestration model is provided in a study (Pefani et al., 2016) in which the authors demonstrated that the Hippo pathway scaffold protein RASSF1A is recruited to TGF-β receptor I in response to TGF-ß, where it is targeted for degradation by the co-recruited E3 ubiquitin ligase ITCH. This, in turn, inactivates the MST1/LATS kinase cascade and facilitates YAP/SMAD2 interaction and subsequent nuclear translocation. Several lines of evidence point to alternate models whereby cell density and/or cell polarity may or may not interfere with TGF-ß/SMAD signaling. For example, in polarized NMuMG mouse mammary gland epithelial cells, TGF-ß is capable of dissolving tight junctions and induce an EMT through a mechanism whereby PAR6, a regulator of cell polarity which associates with Crumbs, Pals and other proteins to regulate the assembly of tight junctions, interacts with TGF-ß receptors and is a substrate of TßRII (Ozdamar et al., 2005). TGF-ß-induced phosphorylation of PAR6 leads to Smurf1 recruitment and subsequent degradation of RhoA, leading to dissolution of tight junctions. Yet, abrogating PAR6 phosphorylation does not alter SMAD activation and downstream transcriptional responses despite maintenance of intact tight junctions. Likewise, the serine/threonine kinase salt-inducible kinase 1 (SIK) was recently identified as a TGF-ß target gene that induces tight junction dissolution through Par3 degradation (Vanlandewijck et al., 2018). It is worth noticing that a large body of the early literature on TGF-ß, at a time when both the SMAD and Hippo signaling cascades had yet to be uncovered, demonstrated active biological or transcriptional responses to TGF-ß in multiple cell types grown to confluency. These include for example the characterization of TGF-ß as a major player for extracellular matrix remodeling in various tissues such as skin, liver, bone and cartilage, that led to its characterization as a key factor for tissue repair and fibrosis. Outcomes that were measured include up- and down-regulation of various ECM components (Heino et al., 1989; Ignotz et al., 1989; Redini, et al., 1991; Wrana et al., 1991; Mauviel et al., 1994; Korang et al., 1995; Chung et al., 1996; Kon et al., 1999; Daniels et al., 2004), as well as the regulation of ECM degradation via modulation of the expression of matrix metalloproteinases and their inhibitors (Overall et al., 1989; Overall et al., 1991; Mauviel et al., 1996).