Transcriptional regulation in the absence of Inositol Trisphosphate Receptor Calcium Signaling

The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that regulate a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB, AP-1 and NFκb) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition the role of protein kinase C (PKC) was investigated with inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression (DEG) of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. In summary three main adaptations in TKO cells are identified in this study: 1) increased basal activity of NFAT, CREB, AP-1 and NFκb; 2) an increased reliance on Ca2+-insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g. PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone. ### Competing Interest Statement The authors have declared no competing interest. * [Ca2+]c : cytosolic [Ca2+] CaN : Calcineurin PKC : protein kinase C ETC : electron transport chain COX2 : cyclooxygenase-2 ETS1 : E26 transformation specific sequence-1 IRS-2 : insulin receptor suabstrate-2 NFAT : nuclear factor of activated T-cells CREB : cAMP response element-binding protein AP1 : activator protein-1 NFκb : Nuclear factor kappa-light-chain-enhancer of activated B cells