The sodium hydrogen exchanger isoforms 1 (NHE1) is an important transporter involved in both intracellular pH homeostasis and anchoring actin filaments. The regulation of NHE1 is has been widely studied and is known to involve several signaling intermediates. Phospholipase D (PLD) is a key regulatory step in a wide array of signaling pathways. Two PLD isoforms have been identified based upon their specificity for activators. PLD1 is activated by RhoA, protein kinase C (PKC), and ARF, while PLD2 has a substantial basal level of activity but may be regulated by Arf and PKC in vivo. Preliminary research in our laboratory has identified a PLD activity in Chinese hamster lung fibroblasts (CCL39) that is stimulated by the a-adrenergic agonist, phenylephrine (PE). Stimulation of CCL39 cells by PE leads to the activation of NHE1, RhoA and extracellular signal regulated kinase (ERK). Furthermore, PE induces stress fiber formation, an indication of adrenergic involvement in cell growth. Additionally, stress fiber formation is blocked when the activity of NHE1, RhoA or ERK is inhibited. RhoA is a known activator of PLD1 and is the likely isoforms of PLD responding to PE in CCL39cells. The resulting PLD products can then lead to the activation of the ERK pathway by stimulating Raf. ERK activation will lead to an increase in NHE1 exchange activity. The increase in intracellular pH and the concomitant increase in local extracellular proton proposal focuses on an important novel role for PLD that will help explain some of the concentration and the enhanced ability of NHE1 to anchor actin fibers will increase both stress fiber formation and migration. To delineate the role of PLD in NHE1 regulation, this project aims to investigate: 1) which isoforms of PLD are expressed and activated by PE and to demonstrate the link between PLD and NHE1 activity. 2) The mechanism of PLD activation of NHEI. 3) Determine a novel role for PLD in the activation of NHE1 in early and late cell migration and invasion events. In short, these phenomena associated with PLD activity in cancer development. Finally, this research will be done at a predominantly undergraduate institution, Minnesota State University Moorhead, where students would be involved in all facets of this research proposal. In fact all of the preliminary data was collected by undergraduates, and the funding of this project would involve 10 to 16 undergraduate students each year over the three-year term of the proposal.
