The roles of PLD and DGK isoforms in PIP2 homeostasis during PLC signaling

项目总结

PROJECT SUMMARY Increases in phospholipase C (PLC) activity have been associated with diseases such as hypertension, and the current first-line of antihypertensive drugs all target Ca2+ influx that results from PLC activity. However, about 10% of patients with hypertension show resistant hypertension, where blood pressure remains uncontrolled despite the use of 3+ different antihypertensive drug classes. There is a significant need to find alternative modulators of PLC activity that are less susceptible to resistance. The downstream lipid produced by PLC, phosphatidic acid (PA), can regulate PLC signaling by replenishing PLC’s substrate phosphatidylinositol 4,5- bisphosphate (PIP2). PA is produced downstream of PLC activity by the diacylglycerol kinases (DGK through DGK) or phospholipase D enzymes (PLD1 and PLD2). The PA then helps to resynthesize PIP2 through metabolic pathways and through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K). Based on this, the objective of this proposal is to determine how the various DGK and PLD isozymes contribute to PIP2 levels and PLC regulation. The rationale of the proposed research is to identify new, druggable, isoform-specific targets to regulate PLC activity in resistant hypertension. The central hypothesis is that PLC signaling activates a particular subset of DGK and PLD isozymes that are necessary for PI recycling and PIP5K activity, respectively. We will test this hypothesis using two specific aims: 1) define the DGK isozymes that function in phosphatidylinositol recycling and 2) determine the role of the DGK and PLD isozymes in PIP5K activation, PIP2 replenishment and downstream Ca2+ signaling. Our research design includes endogenously-tagging the DGK and PLD isozymes using a CRISPR/Cas9 approach to see how the different isozymes are activated by PLC signaling. We will then knockdown the PLC-activated isoforms to see how they affect lipid levels, PIP5K activity, and Ca2+ influx. The proposed research is significant because it will demonstrate how the specific isoforms of DGK and PLD regulate PLC activity. This is a critical first step in identifying isoform-specific targets for antihypertensive drug therapies.