Yes-Associated Protein and PDZ Binding Motif: A Critical Signaling Pathway in the Control of Human Pluripotent Stem Cells Self-Renewal and Differentiation

Introduction

Over the past two decades, human pluripotent stem cells (hPSCs) have gained attention due to their proliferative capacity and pluripotent characteristics that enables the generation of almost all cells (Dakhore et al., 2018; Farzaneh et al., 2017, 2019; Romito and Cobellis, 2016). hPSCs, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are an alternative approach toward stem cell biology, drug discovery, disease modeling, tissue engineering, and regenerative medicine (Cader et al., 2019; Dutta et al., 2017; Fatehullah et al., 2016; Grandy et al., 2019; Gupta et al., 2017; Kolagar et al., 2019; Nirmalanandhan and Sittampalam, 2009; Singh et al., 2015; Takahashi and Yamanaka, 2013). hESCs are commonly derived from the inner cell mass (ICM) of preimplantation embryos and can maintain their pluripotency in appropriate culture media (Dakhore et al., 2018).

Mouse epiblast stem cells (the primed pluripotent state) have similar properties with hESCs (the naive pluripotent state) (Davidson et al., 2015; Nichols and Smith, 2009; Vallier et al., 2009; Zhou et al., 2017). Both cell types can self-renew by dividing asymmetrically with the activation of transforming growth factor b (TGFb) and basic fibroblast growth factor pathways and can differentiate into other types of cells when stimulated with a proper set of internal and external signals (Nichols and Smith, 2009; Vallier et al., 2009). hPSCs interact with multiple soluble and insoluble factors including growth factors, small molecules, neighboring cells, and the extracellular matrix (Klim et al., 2010; Murphy et al., 2014; Zaltsman et al., 2019). The generation of functional differentiated cells from hPSCs holds great promise for regenerative medicine approaches and the understanding of human diseases.

However, there are still several repetitive challenges in practice (Shcherbina et al., 2019). Hence, defining the signaling pathways governing pluripotent stem cells (PSCs) proliferation and differentiation represents a major task (Jiao et al., 2018; Perestrelo et al., 2018; Shcherbina et al., 2019; Vallier et al., 2009). The Hippo pathway is a major integrator of cell surface-mediated signals and play an essential role in biological processes across many different species (Harvey and Hariharan, 2012; Kwon et al., 2013; Yu and Guan, 2013) and in regulating function of hPSCs (Beyer et al., 2013; Chen et al., 2018; Mo et al., 2014; Ramos and Camargo, 2012; Sun et al., 2014; Yan et al., 2019). Yes-associated protein (YAP) and WW domain-containing transcription regulator 1 (WWTR1) or TAZ (PDZ binding motif) are critical downstream transcriptional coactivators in the Hippo pathway (Du et al., 2018; Plouffe et al., 2018).

Most previous studies have revealed the role of YAP/TAZ pathway in angiogenesis (Azad et al., 2019; Boopathy and Hong, 2019), tumorigenesis (Zhao et al., 2010), homeostasis (Varelas, 2014), regeneration (Lu et al., 2018), growth, cancer (Van Haele et al., 2019; Yang et al., 2019), and chromatin remodeling (Hillmer and Link, 2019). In this review article, we summarize the function of YAP/TAZ and focus on the regulation of YAP/TAZ in self-renewal and differentiation of hESCs.

The Hippo signaling pathway

Two decades ago, the Hippo pathway was identified in Drosophila melanogaster (Meng et al., 2016). The core components of the canonical signaling Hippo pathway include two serine–threonine kinase, mammalian Ste20-like kinases 1/2 (MST1/2; homologs of Drosophila Hippo [Hpo]), and the MAP4K family of kinases that stimulate large tumor suppressor 1/2 (LATS1/2; homologs of Drosophila Warts [Wts]) (LATS on) (Han, 2019; Meng et al., 2016). The Hippo signaling pathway acts by binding of MST1/2 to the Salvador homolog 1 (SAV1) as a regulatory protein and MOB kinase activator 1A (MOB1) (Du et al., 2018). The Hippo kinase by promoting the cytoplasmic accumulation of YAP and TAZ (two homologs of Drosophila Yorkie [Yki]) negatively controls their activity (Seo and Kim, 2018).

Neurofibromatosis type 2 (NF2) with the MST/SAV complex enhances the LATS1/2 phosphorylation (Yin et al., 2013). Subsequently, the activated LATS1/2 promotes the formation of phosphorylated YAP and TAZ in the cytoplasm by interaction with α-catenin and 14-3-3 protein. The phosphorylated YAP and TAZ are going to degrade or interact with 14-3-3 protein (Kanai et al., 2000; Zhao et al., 2007). There are several domains and motifs including the WW domain(s), the TEA domain family member 1 (TEAD) transcription factor-binding domain, the SH3-binding domain, the coiled-coil (CC) domain, the TAD domain, and the PDZ-binding motif within YAP and TAZ (Dong et al., 2019; Holden and Cunningham, 2018; Pobbati et al., 2012; Varelas, 2014).

When Hippo signaling is inactive (LATS off), the activated YAP and TAZ translocate into the nucleus to bind the TEAD1-4 transcription factor (as DNA binding partners of YAP/TAZ; homologs of Drosophila Scalloped [Sd]). Then, TEAD stimulates cell proliferation, survival, migration, and antiapoptosis-related genes such as CTGF, CYR61, and ITGB2 (Wu et al., 2008; Zhao et al., 2008). In the nucleus, several transcription factors such as SMAD, RUNX2, TBX5, and ERBB4 can interact with YAP/TAZ (Du et al., 2018; Kim et al., 2018).

Many recent studies have revealed that the culture conditions have effects on the cytoplasmic or nuclear YAP/TAZ localization. In addition, the activity of Hippo pathway is influenced by cell density and shape, mechanical tension, and biochemical signals (Das et al., 2016; Seo and Kim, 2018). For instance, the accumulation of YAP/TAZ in the nucleus under stiff surfaces such as Matrigel and Collagen I is promoted; whereas under soft surfaces, YAP–TAZ are maintained in the cytoplasm (Aragona et al., 2013; Dupont et al., 2011; Sun et al., 2014).

Besides, the accumulation of YAP/TAZ in the cytoplasm under high cell density is more than low cell density (Varelas et al., 2010). Recent studies have demonstrated that F-actin polymerization or G-actin depolymerization regulator of the Hippo pathway (Seo and Kim, 2018). The accumulation of YAP/TAZ in the nucleus is inhibited by Rho-associated kinase (Rho GTPases) inhibitor Y27632 and myosin light chain kinase inhibitor (ML-7) (Das et al., 2016; Wada et al., 2011). Therefore, both culture conditions and the cytoskeletal matrix can regulate YAP/TAZ localization (Fig. 1).

OPEN IN VIEWER

FIG. 1.

The core of the canonical signaling Hippo pathway. The core components of the canonical signaling Hippo pathway include two serine–threonine kinase, mammalian Ste20-like kinases 1/2 (MST1/2; homologs of Drosophila Hippo [Hpo]), and the MAP4K family of kinases that stimulates large tumor suppressor 1/2 (LATS1/2; homologs of Drosophila Warts [Wts]) (LATS on). The Hippo signaling pathway acts by binding of MST1/2 to the SAV1 as a regulatory protein and MOB1. The Hippo kinase by promoting the cytoplasmic accumulation of YAP and TAZ (two homologs of Drosophila Yorkie [Yki]) negatively controls their activity. NF2 with the MST/SAV complex enhances LATS1/2 phosphorylation. Activated LATS1/2 promotes the formation of phosphorylated YAP and TAZ in the cytoplasm by interaction with α-catenin and 14-3-3 protein. Phosphorylated YAP and TAZ are going to degrade or interact with 14-3-3 protein. There are several domains and motifs including the WW domain(s), the TEAD transcription factor-binding domain, the SH3-binding domain, the CC domain, the TAD domain, and the PDZ-binding motif within YAP and TAZ. When Hippo signaling is inactive (LATS off), the active YAP and TAZ translocate into the nucleus to bind the TEAD1-4 transcription factor. Then, TEAD stimulates cell proliferation, survival, migration, and antiapoptosis-related genes such as CTGF, CYR61, and ITGB2. In the nucleus, several transcription factors such as SMAD, RUNX2, TBX5, and ERBB4 can interact with YAP/TAZ. CC, coiled coil; TAZ, PDZ binding motif; YAP, Yes-associated protein.

YAP/TAZ signals can regulate hESC self-renewal and differentiation

hESCs as naive PSCs can proliferate and self-renew indefinitely to generate cells like themselves with a normal karyotype and give rise to all somatic cell types derived from ectoderm, endoderm, and mesoderm in proper conditions, even after prolonged culture (Dakhore et al., 2018; Ohgushi et al., 2015). An unprecedented amount of information is directed toward investigating the role of signaling pathways such as TGFb, FGF/ERK, Lin28/Let7, Wnt/β-catenin, and BMPs in hESC self-renewal and differentiation (Farzaneh et al., 2019, 2019; Kolagar et al., 2019; Singh et al., 2012).

Previous studies have shown that YAP as a potent transcription coactivator by binding to the TEAD plays an essential role in regulating hESC fate decisions (Lian et al., 2010). Lian et al. (2010) have found that during hESC differentiation, Lats kinase as a key component of the Hippo tumor suppressor pathway inhibits YAP protein and leads to a loss of embryonic stem cell pluripotency (Ramos and Camargo, 2012). A recent study has shown that the survival of hESCs depends on the A-kinase anchoring protein (AKAP)-Lbc/GTPase Rho signaling. The AKAP-Lbc/Rho signaling by regulating actin microfilament organization maintains the nuclear activity of YAP and TAZ (Ohgushi et al., 2015).

It has been shown that the WNT and Hippo pathways can regulate the expression of pluripotency factor OCT4 (Sato et al., 2004; Ying et al., 2008). YAP is necessary for β-catenin action (a mediator of WNT signaling) to control cardiomyocytes differentiation (Heallen et al., 2011; Sato et al., 2004). Also, Bejoy et al. (2016) have found that Hippo/YAP signaling through the interactions with Wnt signaling can control neural tissue patterning of hPSCs. YAP/TAZ pathway can prevent differentiation of hPSCs and promote reprogramming efficiency (Ramos and Camargo, 2012). It seems that YAP/TAZ as a stemness-promoting factor controls hepatic, intestine, and epidermal cell proliferation (Camargo et al., 2007; Schlegelmilch et al., 2011).

Hindley et al. revealed that the Hippo/YAP pathway that is associated with phenotypes of tumorigenesis and cellular overgrowth with retinoic acid signaling enhanced hESC-derived neural crest cell fate decisions and migration. They observed a significant increase in the expression of markers consistent with a neural crest phenotype or neural epithelial–mesenchymal transition such as SLUG, TWIST, AP2, and FOXD3 (Hindley et al., 2016). Low-density culture conditions as a modulator of Hippo pathway activity increased nuclear YAP expression and changed neural cell fate. Therefore, under this condition, hESCs could be differentiated into adipocytes, chondrocytes, osteoblasts, and peripheral neurons as neural crest progenitors (Hindley et al., 2016).

It has been demonstrated that YAP–TEAD complexes can activate Oct4 expression and improve the proliferation of hPSCs (Lian et al., 2010). The effect of the Hippo pathway on hPSC was also studied by Papaspyropoulos et al. (2018). They found that RASSF1A as a key player of cell fate and a natural barrier to stem cell self-renewal, by switching YAP from the β-catenin/TCF pluripotency complex, enhanced hESCs differentiation (Papaspyropoulos et al., 2018). Therefore, RASSF1A mediates the transcription factor selection of YAP in stem cells and acts as a functional “switch” between pluripotency and differentiation (Papaspyropoulos et al., 2018). These data support the functional importance of the YAP–TAZ in hESC cell fate (Fig. 2).

OPEN IN VIEWER

FIG. 2.

The functional importance of the YAP-TAZ in hESC cell fate. hESCs are commonly derived from the ICM of preimplantation embryos and can maintain their self-renewal in appropriate culture media. These cells can differentiate into adipocytes, chondrocytes, cardiomyocytes, and peripheral neurons. YAP–TAZ as potent transcription coactivators have an essential role in regulating hESC fate decisions. When the Hippo pathway is on (LATS on), MST1/2 phosphorylates SAV1 and activates MOB1A/B and LATS1/2. Therefore, the phosphorylated YAP and TAZ are maintained in the cytoplasm for proteasomal degradation by the 14-3-3 phosphopeptide binding proteins. As a result, the TEAD with VGL4 inhibits target gene expression. When the Hippo pathway is off (LATS off), unphosphorylated YAP and TAZ accumulate in the nucleus and in complex with TEADs enhance the expression of target genes.

Conclusion

Extensive studies have established an important role of intrinsic and extrinsic signaling pathways in controlling human embryonic development and hESCs fate decisions. Recent findings regarding the functions of the Hippo/YAP pathway in hESCs self-renewal and differentiation enhanced the significance of this pathway. The YAP/TAZ can promote propagation, self-renewal, and also regulate lineage commitment of hESCs into multiple mature somatic cell types. According to the importance of the Hippo/YAP pathway as a vital modulator of pluripotency in hESCs, the exact mechanism of YAP/TAZ in the regulation of hESCs fate should be considered. Also, the crosstalk of Hippo pathway-dependent transcription factors with other molecular signaling pathways that control hESC proliferation and maturation should be studied.