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  • Thus while there is ample evidence to

    2021-09-18

    Thus, while there is ample evidence to rule out a generalized blockade of TGF-ß signaling by Hippo pathway activation upon establishment of cell–cell contacts, more recent studies have established that, in specific contexts, Hippo and TGF-ß signaling may either interfere or cooperate with each other to regulate cell fate. In a study comparing the response of various cell types to TGF-ß in relation to cell density, we provided evidence that mesenchymal as well as some epithelial cell types show similar TGF-ß responses, even sometimes enhanced, when cultured at either low or high cell density, irrespective of YAP/TAZ nucleo-cytoplasmic localization (Nallet-Staub et al., 2015). This indicates that activation of the Hippo pathway by cell density does not necessarily inhibit TGF-ß responses despite YAP/TAZ nuclear exclusion. Also, in several cell lines, we found that modulating YAP and TAZ levels by means of siRNAs did not affect the extent of TGF-ß signaling. In our studies, the only cell lines for which high cell density was associated with loss of TGF-ß responsiveness were those known to exhibit cell polarity with distinct apical and basolateral membrane domains when grown at high density. In these polarized epithelial cell lines, lack of a TGF-ß responsiveness was a direct reflection of basolateral TGF-ß receptors that render RI-1 mg unable to respond to apically delivered TGF-ß ligand. However, basolaterally delivered TGF-ß ligand promotes SMAD2/3 nuclear accumulation and subsequent transcriptional response, with no sign of cytoplasmic retention of activated SMAD complex by cytoplasmic YAP/TAZ (Fig. 3B). This phenomenon is restricted to polarized epithelial cells, as fibroblasts grown to confluency do not exhibit TßR relocalization (Nallet-Staub et al., 2015). We took advantage of the TAT-fusion cell-penetrating peptide technology to generate cell-permeable TAT-SMAD3 protein, subjected or not to in vitro phosphorylation by activated TßRI. Following transduction of polarized EpH4 cells, we observed that TAT-SMAD3 was almost exclusively localized in the cytoplasm, while a large portion of TAT-SMAD3P proteins rapidly accumulated in the nucleus of cells. These data, at odds with the model of cytoplasmic sequestration of active SMAD complexes by phosphorylated YAP/TAZ (Varelas et al., 2008), led us to propose a model whereby YAP/TAZ nuclear exclusion by cell contacts is ubiquitous, and that mechanisms driving the nucleo-cytoplasmic localization of TAZ and P-SMAD are independent. This model eventually translates into situations where both YAP/TAZ and R-SMADS are fortuitously localized in the same cellular sub-compartment, with no significant implications for the outcome of TGF-ß responses. Of note, in polarized epithelial cells, BMP signals are transmitted from BMP receptor complexes exclusively localized at the basolateral surface of the cell membrane (Saitoh et al., 2013). In line with the TGF-ß receptor relocalization model, a study using colonies human embryonic stem cells grown on a micropatterned substrate and differentiated with BMP4 found that in response to both BMP4 and TGF-ß/activin, nuclear accumulation of R-SMADs was restricted to the periphery of hESC colonies and directly linked to cell density (Etoc et al., 2016). This spatial restriction of the response to TGF-ß ligands was abolished in calcium-free culture conditions that promote disruption of tight junctions. The authors identified that only the cells on the colony edges had receptors localized to the portion of the membrane facing the extracellular medium, while cells in the center of the colonies, unresponsive to BMP4, exhibited exclusively lateral receptor localization, below the tight junctions. Thus, lateralization of TGF-ß receptors in hESCs leads to polarity- selective response to growth factors. Similar to what we described for polarized epithelial cell lines, high density cultures of hESCs were responsive to basolaterally delivered BMP4 and activin, while low-density cultures were equally responsive to both apical and basolateral presentation of ligands. In high density hESC, YAP/TAZ was mostly excluded from nuclei, ruling out the possibility of a contribution of these Hippo pathway substrates to the early TGF-ß responses. In addition to receptor relocalization, another negative feedback mechanism was identified, whereby Noggin, an early gene target of BMP4/SMAD1 in hESCs is upregulated and further restricts signaling to the edges of hESC micro-colonies. Both receptor lateralization as a function of cell density and Noggin induction were able to recapitulate the signaling dynamics of SMAD1 within gastruloid differentiation of hESC cell colonies, and were sufficient to predict fate patterning under various experimental conditions, independent of Hippo pathway activation (Deglincerti et al., 2016).