Stress-induced expression of the γ subunit (FXYD2) modulates Na,K-ATPase activity and cell growth

Abstract

In kidney, the Na,K-ATPase is associated with a single span protein, the γ subunit (FXYD2). Two splice variants are differentially expressed along the nephron and have a differential influence on Na,K-ATPase when stably expressed in mammalian cells in culture. Here we used a combination of gene induction and gene silencing techniques to test the functional impact of γ by means other than transfection. NRK-52E cells (of proximal tubule origin) do not express γ as a protein under regular tissue culture conditions. However, when they were exposed to hyperosmotic medium, induction of only the γa splice variant was observed, which was accompanied by a reduction in the rate of cell division. Kinetic analysis of stable enzyme properties from control (α1β1) and hypertonicity-treated cultures (α1β1γa) revealed a significant reduction (up to 60%) of Na,K-ATPase activity measured under Vmax conditions with little or no change in the amounts of α1β1. This effect as well as the reduction in cell growth rate was practically abolished when γ expression was knocked down using specific small interfering RNA duplexes. Surprisingly, a similar induction of endogenous γa because of hypertonicity was seen in rat cell lines of other than renal origin: C6 (glioma), PC12 (pheochromocytoma), and L6 (myoblasts). Furthermore, exposure of NRK-52E cells to other stress inducers such as heat shock, exogenous oxidation, and chemical stress also resulted in a selective induction of γa. Taken together, the data imply that induction of γa may have adaptive value by being a part of a general cellular response to genotoxic stress.