KSRP and Roquin‐2: Mediators of PTH‐Induced Npt2a mRNA Destabilization?

Abstract

Parathyroid hormone (PTH) potently induces phosphaturia through the downregulation of the type IIa sodium-phosphate cotransporter (Npt2a), at the protein and the mRNA level. We previously identified 68 RNA-binding proteins (RBPs) whose phosphorylation status significantly changed in response to 30m or 2h PTH. Preliminary in silico analysis predicts that two of those RBPs – Roquin-2 and KSRP – are capable of interacting with the 3'UTR of Npt2a mRNA. We hypothesize that PTH stimulates Npt2a mRNA degradation through alterations in Roquin-2 and KSRP activity and/or expression. To address this hypothesis, we examined Roquin-2 and KSRP expression in opossum kidney (OK) cells, a proximal tubule cell culture model, treated with 100nM PTH for 15m to 2h. Western blot demonstrated a time-dependent increase in expression of both proteins in response to PTH and a decrease in expression in response to low phosphate media. Nuclear expression of KHSRP increased 10-fold following 2h PTH. Nuclear expression of Roquin-2 increased 207% after 2h PTH, and cytosolic expression increased 342%. Immunoprecipitation of either Roquin-2 or KSRP from control and PTH-treated cells was followed by RNA isolation. RT-qPCR analysis identified Npt2a mRNA from both Roquin-2 and KSRP pulldowns. We conclude that KSRP and Roquin-2 bind Npt2a mRNA and their expression is modulated by known regulators of Npt2a expression. These findings suggest that KSRP and Roquin-2 potentially mediate Npt2a mRNA destabilization in response to PTH. Funding for this research provided by VA to EDL.