1A-C). These data indicate that interaction between YAP and CREB is important for human liver cancer cell growth and survival. An interesting question arising from our data was how YAP and CREB regulate with each other. We observed that YAP messenger RNA (mRNA) was reduced by the protein kinase A (PKA)/CREB inhibitor, H89 (Fig. 2A), whereas it was induced by the PKA/CREB activator, forskolin (Fig. 2B). YAP protein was also compromised, as measured by

immunoblotting analysis (Fig. 2C). Then, we investigated the mechanism underlying how YAP is regulated by CREB. A recent study[11] suggests that CREB binds to nucleotide (nt) −232/+115 containing a CRE of YAP promoter in HCC cells. However; our luciferase reporter gene assays revealed that YAP promoter activity was greatly enhanced by nt −608/−439 (Fig. 2D), which is also sensitive to both H89 and forskolin (Fig. 2E), because promoter activities from −608-Luc, but not −439-Luc,

Staurosporine research buy could be reduced by H89, whereas it was induced by forskolin (Fig 2E), suggesting the potential role of a novel cAMP responsive element (CRE) at this region. Furthermore, −608-Luc was inhibited when CREB was knocked down, whereas it was activated when CREB was ectopically expressed (Supporting Fig. 2A). Similarly, YAP protein can be up-regulated by CREB (Supporting Fig. 2B). Then, ChIP analysis was performed and it was demonstrated that CREB was able to bind to −608/−439 (R2); however, no enrichment was detected at an unrelated region (R1) (Fig. 2F). Taken together, YAP transcription is controlled by CREB through a novel Selleck Ensartinib promoter region. In both Bel-7402 and SMMC-7721 cells with YAP knocked down, we found that

transcription of known CREB target genes, such as Rab25,[12] HULC,[8] as well as the YAP target gene, CTGF,[13] was significantly inhibited (Fig. 3A), suggesting that YAP regulates CREB transcriptional activity. In addition, we observed that CREB correlated with YAP expression in almost all the cell lines detected, with highest YAP and CREB protein expression in HepG2 cells (Fig. 3B). Then, we tested whether YAP also regulates CREB protein expression. We found that cells with YAP knocked down had a much lower level of CREB, as compared to control, in both Bel-7402 and HepG2 cells (Fig. 3C). Furthermore, CREB was dose dependently up-regulated by an increasing Palmatine amount of YAP (Fig. 3D). Surprisingly, YAP knockdown did not significantly affect CREB mRNA levels (Fig. 3E), thus ruling out the possibility that YAP regulates CREB transcription. As previously described, that CREB is critical for HULC promoter activity,[8] we used this luciferase reporter system to confirm our hypothesis that YAP regulates CREB activity. We found that promoter activities from WT (with CRE) was greatly inhibited, whereas no obvious changes were detected from the Mut (without CRE) one in HepG2 cells with YAP knocked down, compared to the control (Fig. 3F).