The effects of YAP activity on retinoic acid signaling in the embryonic epicardium

Abstract

Retinoic acid (RA), a metabolite of vitamin A, is primarily produced by epicardial cells and is required for the proper differentiation of epicardial cells into cardiac fibroblasts and VSMCs during heart development. Recent literature has suggested that both mechanotransduction, which is mechanical stimuli eliciting chemical responses within the cell, and the Hippo signaling pathway could potentially regulate RA-signaling. The transcriptional co-activator YAP is regulated by the Hippo-pathway and mechanotransduction and elicits changes in gene transcription through the TEAD transcription factor. Recently it was found that there were TEAD motifs in the Dhrs3 gene and YAP showed specific binding to the Dhrs3 enhancer via chromatin immunoprecipitation combined with qPCR (ChIP-PCR) in the mouse epicardial cell line MEC1. However, beyond its effects on the expression of Dhrs3 it is not clear if YAP actually affects RA-signaling. We hypothesized that YAP regulates retinoic acid signaling in embryonic epicardial cells. The first aim was to assess if YAP regulates the transcription of enzymes and receptors involved in RA metabolism or signaling in MEC1 cells. Using RT-qPCR we investigated the effect of constitutively active YAP, or conversely suppressed YAP activity on Dhrs3, Raldh2, RAR𝛽, Cyp26A1, and Cyp26B1 expression. To suppress YAP activity, a YAP small interfering RNA (siRNA), or the YAP-TEAD chemical inhibitor, Verteporfin, were utilized but these manipulations did not show significant effects on YAP targets, or retinoid receptors and enzymes in MEC1 cells. To overexpress YAP, a constitutively active form of YAP, YAP25SA, was transfected into MEC1 cells. We were able to confirm a direct relationship between Cyp26a1 expression and YAP activity as YAP25SA increased Cyp26a1 mRNA expression by 37.8-fold within 6 hours of transfection. As well, we were able to determine that YAP25SA affected Cyp26a1 expression through a TEAD mechanism; as co-transfection of YAP25SA with YTIP-GFP, a plasmid expressing a peptide that interferes with YAP-TEAD interaction significantly diminished the effects of YAP25SA on Cyp26a1 expression. The second aim investigated if YAP regulates the metabolism of retinol into RA by utilizing the two-hybrid Gal4-RAR;UAS-tk-Gaussia luciferase reporter system that estimate the amount of RA produced by monitoring the activation of the Gal4RAR reporter. Transfection of MEC1 cells with YAP25SA allowed more retinol to be converted to RA and for a longer period of time when compared to the control. Since the activity of YAP influences the expression of Cyp26a1 and metabolism of retinol, it was concluded that YAP activity plays a role in RA-signaling.