RESUMO
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.
Assuntos
Dioxigenases , Células-Tronco Pluripotentes , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição de Domínio TEA , Músculo Liso Vascular/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes/metabolismo , Miócitos de Músculo Liso/metabolismo , Proliferação de CélulasRESUMO
Transcription factors TEAD1 and TEAD4 play an important role in development, differentiation, cell growth and proliferation. To further understand the exact role of TEAD1 and TEAD4 in these processes. We generated TEAD1 and TEAD4 doxycycline-inducible expression human embryonic stem cell lines (WAe001-A-67 and WAe001-A-68) by PiggyBac transposon system. These cell lines retained normal morphology and karyotype, normal expression of pluripotent markers, and differentiation potential. These cell lines can be used to verify whether the TEAD1 and TEAD4 play a role in stem cell and cell lineage differentiation.
