222 Tankyrase inhibition suppresses expression of Hippo signaling pathway components in bovine embryonic stem cells

The chemical compound IWR-1 plays a vital role in the derivation of pluripotent stem cells in multiple species, especially in livestock. It is recognised as a canonical WNT inhibitor through tankyrase-inhibition-mediated β-catenin deconstruction. Recent studies in both human and bovine embryonic stem cells (bESC) revealed that tankyrase inhibition may also act on other pathways that are essential for maintaining cell potency. Neither knocking out β-catenin nor supplementing other WNT inhibitors can replace the use of tankyrase inhibitors in the embryonic stem cell (ESC) culture medium. In this study, we aimed at understanding the action mechanism of IWR-1 in bESC. Bovine ESC lines were derived from individual blastocysts and cultured in complete mTeSR1 medium (STEMCELL Technologies) supplemented with IWR-1 and antibiotics and maintained on growth-arrested mouse embryonic fibroblasts. Cell lines were considered as replicates for all experiments. To investigate the suppressive effects of IWR-1 on lineage differentiation, we compared expression of lineage markers before and after induction of differentiation, which was performed by withdrawing IWR-1 from the culture medium. Cells were routinely propagated in complete mTeSR1 plus IWR-1 for over 20 times before starting the treatment. During the treatment period, cells were cultured in IWR-1 minus medium for 1 month when their “dome”-like colony morphology diminished. Cells were sampled before and after treatment, and RNA extraction and cDNA synthesis were performed simultaneously. The quantitative PCR (qPCR) results showed that IWR-1 withdrawal caused elevated (P < 0.05) expression of lineage markers including CXCR4 (endoderm), MEOX1 (mesoderm) and NESTIN (ectoderm), although other lineage markers FOXA1, MIXL1, PAX6, SNAI1, SOX1, EOMES and SOX17 remained unaffected (n = 4). These data suggested that IWR-1 can prevent bESC from differentiating into certain lineages of the three germ layers. Next, we investigated inhibitory effects of IWR-1 on tankyrase activity. Abundance of TNKS1 and TNKS2 protein was assessed in bESC treated with or without IWR-1 for 1 day by western blotting. Results showed that TNKS2 but not TNKS1 level was reduced (P < 0.05) if IWR-1 was withdrawn from the medium. We confirmed that the decreased TNKS2 was not due to transcriptional regulation by qPCR. These data indicated that IWR-1 mitigated the poly-ADP-ribose polymerase activity of TNKS2, and therefore likely disrupted self-poly(ADP-ribosyl)ation and degradation of TNKS2. Subsequently, bulk RNA-seq analysis was performed on bESC treated plus/minus IWR-1 for 1 day (n = 5). We noticed that a WNT/Hippo targeted gene CYR61 and a transcription factor of WNT pathway TCF7 were both up-regulated (P < 0.05) in IWR-1 minus cells. Transcription of four genes coding the Hippo pathway components TEAD4, AMOTL2, WWC2, and AJUBA were stimulated (P < 0.05) by IWR-1 removal as well. Treatment effects on the transcription of these genes except AJUBA were validated by qPCR in another cell culture experiment (n = 6–8). Changes in TEAD4 protein expression were further confirmed by western blotting (n = 3). In all, our study suggests that IWR-1 negatively regulates Hippo signaling pathway besides disrupting the canonical WNT pathway to sustain cell potency in bovine, possibly through inhibition of TNKS2 activity. This highlights an important role of Hippo pathway activity in regulation of pluripotency, and perhaps also provides targets for improving the current culture system of ESC in livestock.