IP3R Knockout Cell Models: Studies on Structure/Function and Cell Physiology
Abstract
Ca2+ signaling is a regulator of critical cellular processes such as proliferation, metabolism and transcription. In non-excitable cells, intracellular Ca2+ release is mediated by the PLC pathway and the ER resident inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). Upon agonist stimulation, phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate into membrane bound diacylglycerol and soluble IP3, which diffuses and binds IP3Rs causing ER Ca2+ stores to flood into the cytoplasm. Some of this Ca2+ is taken up by mitochondria via the mitochondrial Ca2+ uniporter (MCU) complex where it alters metabolism via regulation of TCA cycle dehydrogenases. More than simple ion channels, IP3Rs are large tetrameric proteins that serve as cellular signaling hubs regulated by a multitude of agonists, antagonists, and modulators. Recently, novel cell models were engineered to stably knockout all three isoforms of IP3Rs(TKO), and despite the biological importance of Ca2+ signaling, these cells display mild phenotypes. The use of TKO cell lines in both structure/function studies related to redox regulation and physiological studies about adaptations to chronic Ca2+ loss was the focus of the current work. Sensitization of IP3R mediated Ca2+ release associated with oxidative stress was thought the be regulated via redox state of IP3R thiols, thus we began by using TKO cells to investigate redox sensitive cysteine residues. Utilizing proteomics and mutagenesis, we identified approximately 20 relevant cysteines, induced mutation of those resides in IP3Rs, and reinserted the mutated receptors back into TKO cells. Ca2+ release assays on oxidant treated mutant and WT receptors revealed a cluster of redox sensitive residues located in the suppressor domain of IP3R1. Pivoting from structure/function studies, TKO cells were then investigated as a model of adaptation to chronic loss of Ca2+ signaling. Biochemical and metabolomic approaches were used to investigate bioenergetic parameters of TKO, as well as MCUKO, cell lines. Broadly, TKO models displayed higher levels of AMPK-independent autophagy, decreased oxygen consumption rate, and slower proliferation. Conversely, MCUKO cells showed increased OCR and growth, in addition to upregulation of key metabolic pathways including glycolysis and TCA cycle flux in both oxidative and reductive directions. Neither TKO nor MCUKO cells had significant changes in ATP levels, however, MCUKO cells were unable to survive when relying solely on the TCA cycle. The final area of research was to discover the genetic mechanisms underlying the phenotype of TKO cells. Luciferase reporter assay, immunoblotting, and siRNA approaches were used to characterize how known Ca2+ sensitive transcription factors, NFAT, AP-1, CREB, and NF-κB, adapted to loss of Ca2+ regulation. Though all four were elevated at baseline in TKO cells, differential effects were found as NFAT had been functionally inactivated, AP-1 was constitutively activated, and CREB showed enhanced reliance on Ca2+ insensitive PKC isoforms for phosphorylation. Despite loss of Ca2+ feedback activation on PLC, both DAG production and Ras signaling were maintained and showed no significant difference from WT. Finally, increased H2O2 production, decreased superoxide production, and enhanced expression of ROS scavenging enzymes were measured in TKO models. We suggest that TKO cells have adapted to loss of the IP3 arm of PLC signaling by upregulating the DAG/novel PKC arm.
Subject Area
Cellular biology|Biochemistry|Physiology
Recommended Citation
Young, Michael Philip, "IP3R Knockout Cell Models: Studies on Structure/Function and Cell Physiology" (2023). ProQuest ETD Collection - Thomas Jefferson University. AAI30319074.
https://jdc.jefferson.edu/dissertations/AAI30319074
