Receptor stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for many physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular cardiac myocytes (NRVMs), where agonist stimulation triggers PLC dependent hydrolysis of the PIP2 precursor phosphatidylinositol 4-phosphate (PI4P) leading to DAG production, nuclear PKD activation and cardiac hypertrophy. We have seen that this newly identified pathway exists in other cell types as well, such as Mouse Embryo Fibroblasts (MEFs) and PANC-1 cells upon endothelin-1 (ET-1) and neurotensin (NT), respectively. To determine if PLC dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation we examined PI4P depletion and PKD activation in NRVMs, MEFs and PANC-1 cells. In our studies we used a PI 4-kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4-phosphatase, Sac1-K2A (Sac1) to deplete PI4P. PAO treatment in NRVMs and MEFs, showed a dramatic inhibition of the ET-1-dependent global PKD activation in NRVMs and MEFs but not NT-dependent PKD activation in PANC-1 cells. Sac1 expression showed reduced ET-1-dependent global PKD activation in NRVMs and MEFs but did not change the NT-dependent PKD activation in PANC-1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. This shows that PI4P hydrolysis occurs upon receptor stimulation and provides a novel signaling mechanism with numerous implications in physiology and disease. My project seeks to understand the role of PI4P hydrolysis as a source of DAG in cells for better understanding of PLC- and DAG- dependent processes such as ion channel regulation and kinase activation, while providing a new potential target for diseases, such as cardiac hypertrophy and cancer.
The main classes of cell surface receptors are the G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTK). A major mechanism that can integrate signals from both GPCRs and RTKs and is present in all mammalian cells is the Phospholipase C (PLC) pathway. Receptor stimulated PLC-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for many physiological processes, such as neurotransmission and proliferation. All PLC isoforms hydrolyze PIP2 at the plasma membrane into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Although DAG production and kinase activation on intracellular membranes has been studied since the late 1980's, it has still failed to reach a consensus on the actual mechanism of activation. In recent years, PI4P has been found to be widely distributed on intracellular membranes and its hydrolysis has been found to be a source of second messengers required for cardiac hypertrophy and ion channel regulation. PI4P hydrolysis is a novel pathway that needs to be explored and understood in detail. In our opinion, this pathway will lead to increased understanding of many different biological processes and diseases, and reevaluation of many known GPCR and RTK signaling events. Ultimately, PI4P hydrolysis could serve as a potential novel therapeutic target in diseases, including but not limited to cardiac hypertrophy and cancer.
