Transcriptional regulation in the absence of Inositol Trisphosphate Receptor Calcium Signaling

The activation of IP ₃ receptor (IP ₃ R) Ca ²⁺ channels generates agonist-mediated Ca ²⁺ signals that regulate a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP ₃ R 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 Ca ²⁺ 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 IP ₃ R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression (DEG) of 828 and 311 genes in IP ₃ R 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 Ca ²⁺ -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 Ca ²⁺ 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.