VGLL4-TEAD1 promotes vascular smooth muscle cell differentiation from human pluripotent stem cells via TET2

https://doi.org/10.1016/j.yjmcc.2023.01.005Get rights and content

Highlights

  • The expression of VGLL4 and TEAD1 increased when hESCs were differentiated into vascular smooth muscle cells (VSMCs).

  • VGLL4 and TEAD1 promotes the VSMC differentiation from hESCs.

  • VGLL4 might interact with TEAD1 to promote the expression of TET2 and facilitate the VSMC differentiation.

Abstract

The Hippo signaling pathway plays a critical role in cardiovascular development and stem cell differentiation. Using microarray profiling, we found that the Hippo pathway components vestigial-like family member 4 (VGLL4) and TEA domain transcription factor 1 (TEAD1) were upregulated during vascular smooth muscle cell (VSMC) differentiation from H1 ESCs (H1 embryonic stem cells). To further explore the role and molecular mechanisms of VGLL4 in regulating VSMC differentiation, we generated a VGLL4-knockdown H1 ESC line (heterozygous knockout) using the CRISPR/Cas9 system and found that VGLL4 knockdown inhibited VSMC specification. In contrast, overexpression of VGLL4 using the PiggyBac transposon system facilitated VSMC differentiation. We confirmed that this effect was mediated via TEAD1 and VGLL4 interaction. In addition, bioinformatics analysis revealed that Ten-eleven-translocation 2 (TET2), a DNA dioxygenase, is a target of TEAD1, and a luciferase assay further verified that TET2 is the target of the VGLL4-TEAD1 complex. Indeed, TET2 overexpression promoted VSMC marker gene expression and countered the VGLL4 knockdown-mediated inhibitory effects on VSMC differentiation. In summary, we revealed a novel role of VGLL4 in promoting VSMC differentiation from hESCs and identified TET2 as a new target of the VGLL4-TEAD1 complex, which may demethylate VSMC marker genes and facilitate VSMC differentiation. This study provides new insights into the VGLL4-TEAD1-TET2 axis in VSMC differentiation and vascular development.

Introduction

Vascular smooth muscle cells (VSMCs) are the major cell type in the medial layer of the blood vessels and play an essential role in vascular homeostasis and disease. VSMCs are not terminally differentiated cells and retain significant plasticity to switch their phenotype between contractile and synthetic. Under pathological conditions such as vascular injury, VSMCs dedifferentiate from a quiescent contractile state into a proliferative synthetic state [1]. Therefore, clarifying the molecular mechanisms of VSMC phenotype differentiation and dedifferentiation is important for better understanding of vascular development and development of new therapeutic strategies for vascular diseases. Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have unlimited proliferative capacity and the potential to differentiate into various somatic cell types. Thus, they serve as powerful tools for studying disease modeling, tissue engineering, drug screening, and cell lineage differentiation including that of VSMCs [2,3]. Human PSC-derived VSMCs are an unlimited, immune-compatible cell source without ethical implications, which are becoming a popular model system for basic research and therapeutic applications in vascular disease [[4], [5], [6]].

Although the complex mechanisms of VSMC differentiation have been studied, detailed molecular mechanisms still need to be fully elucidated. Recent studies have shown that the Hippo pathway plays an important regulatory role in cardiovascular development and is involved in the regulation of VSMC differentiation [7]. For example, Pagiatakis reported that transcriptional co-activator with PDZ-binding motif (TAZ) is induced by transforming growth factor-β (TGF-β) signaling and cooperates with serum response factor (SRF) to express VSMC genes and promote VSMC differentiation [8]. Knockout of Yes-associated protein (YAP) and TAZ has been reported to inhibit VSMC differentiation from neural crest cells [9]. However, Wang  et al. found that overexpression of YAP inhibited myocardin transcription and attenuated VSMC differentiation from PSCs [10]. Therefore, the role of YAP/TAZ in VSMC differentiation is controversial, and whether other components of the Hippo pathway regulate VSMC differentiation needs to be explored. Vestigial-like family member 4 (VGLL4) of the Hippo pathway has been shown to compete with YAP to interact with TEA domain transcription factor (TEAD) and inhibit YAP function in cancer [11]. In addition, VGLL4 is also involved in regulating cell differentiation such as muscle myoblast and osteoblast differentiation [12,13]. However, whether VGLL4 modulates VSMC differentiation remains unclear. Interestingly, recent evidence has revealed that TEAD1, a VGLL4 binding partner, targets PITX2c and myocardin to promote VSMC differentiation and vascular development in vivo [14]. Previous studies have shown that VGLL4 is a transcriptional cofactor that physically interacts with different transcription factors including TEADs; therefore, VGLL4 acts as a tumor suppressor or gene expression regulator in various biological processes, such as cell differentiation. For example, Feng et al. reported that VGLL4 acts as a co-activator of TEAD4 to promote MyoG transactivation and facilitates myoblast differentiation in a YAP-independent manner [15]. Therefore, it is possible that VGLL4 coordinates with TEAD to regulate VSMC differentiation.

Recent evidence indicated that epigenetic regulation plays a vital role in VSMC differentiation and phenotype switching, including histone modification and DNA methylation [1,16]. Interestingly, Martin et al. discovered that Ten-eleven-translocation 2 (TET2), a dioxygenase family protein with DNA demethylation activity, regulates VSMC differentiation [17]. The TET family of enzymes includes TET1, TET2, and TET3 [[18], [19], [20]]. These proteins function as 5-methylcytosine (5-mC) dioxygenases, which can iteratively oxidize 5-mC to form 5-hydroxymethylcytosine (5-hmC) and then transform the 5-hmC demethylene group into an unmodified cytosine via the DNA glycosidase TDG-mediated base excision repair (BER) pathway [18]. TET enzymes play important roles in embryonic development, cell lineage specification, and cancer [21]. Recently, Wu et al. discovered that the Hippo pathway effector YAP induced TET1 expression and then interacted with TEAD to cause regional DNA demethylation and promote liver growth and tumorigenesis [22]. They revealed that YAP signaling in health and diseases is controlled by epigenetic remodeling. However, whether VGLL4, a competitor of YAP, regulates TET-mediated epigenetic modifications or is involved in VSMC differentiation remains unknown.

In this study, we revealed that VGLL4 expression increases during mesoderm-mediated VSMC differentiation from H1 ESCs. Overexpression of VGLL4 promotes, whereas knockdown of VGLL4 inhibits VSMC differentiation. Importantly, our data indicate that VGLL4 forms a complex with TEAD1 and upregulates TET2 expression. Overexpression of TET2 also promotes VSMC differentiation and counters the inhibitory effect of VGLL4 knockdown during VSMC differentiation from H1 ESCs.

Section snippets

Human pluripotent stem cell culture and mesoderm-mediated VSMC differentiation

The H1 ESC line was obtained from Wicell Research Institute (Madison, WI). To maintain the long-term undifferentiated state, the H1 ESCs (generations 60–80) were grown on Matrigel (Corning, #356234) in Nuwacell hESCs medium (Nuwacell, #RP01001) at 37 °C and 5% CO2. Culture medium was changed daily and cells were passaged every 4–6 days with Versene solution (Gibco, #5040066) with ROCK inhibitor Y-27632 (5 μM, Selleck, #S1049) at a dilution of 1:10. For the induction of mesoderm and VSMC, we

VSMC differentiation from H1 ESCs

We used a chemically induced differentiation protocol to generate VSMCs from H1 ESCs using a mesodermal progenitor intermediator (Fig. 1A) [23]. From day 2–4, mesodermal progenitors were induced by treating H1 ESCs with CP21R7 and BMP4 (Fig. S1A), and both mRNA and protein expression levels of marker genes, such as T and EOMES, were transiently upregulated (Fig. 1B, D-E). The expression of pluripotency gene OCT4 gradually decreased during this process (Fig. 1C). After treatment with PDGF-BB and

Discussion

Our study revealed that VGLL4 expression increases during VSMC differentiation from H1 ESCs. Overexpression of VGLL4 promoted, whereas knockdown of VGLL4 inhibited VSMC differentiation. Moreover, our data indicate that VGLL4 facilitated VSMC marker gene expression by interacting with TEAD1 to form a transcriptional complex, which targeted the TET2 promoter region to induce its expression and mediate VSMC differentiation.

During vascular development, VSMCs are derived from multiple embryonic

Author contributions

YW contributed to the conception, design and financial support of the study. ZW, YQ, MH, YX, YC performed the experiments, data analysis, and interpretation. PJ, JM, XC, JF, XF, and YG assisted with the experiments. YW and ZW wrote the paper. YW and ML edit the paper. All authors read and approved the final manuscript.

Declaration of Competing Interest

The authors have no conflicts of interest to declare.

Acknowledgements

We would like to thank all the members in Institute of Hypoxia Medicine who contribute to this work. We appreciate the great help from Dr. Lei Zhang (Institute of Biochemistry and Cell Biology at Chinese Academy of Sciences), Dr. Jiaxi Zhou (State Key Laboratory of Experimental Hematology in China), Dr. Fa-xing Yu (Institute of Pediatrics, Children's Hospital of Fudan University) for this project. This work was supported by Natural Science Foundation of Zhejiang Province (LY21C120003), National

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