Date on Master's Thesis/Doctoral Dissertation
Physiology and Biophysics
Lederer, Eleanor D.
Committee Co-Chair (if applicable)
Khundmiri, Syed J.
Clark, Barbara J.
Miller, Cynthia J.
Falcone, Jeff C.
Parathyroid hormone; Sodium phosphates
The type IIa sodium-phosphate cotransporter (Npt2a) is expressed in the apical membrane of the renal proximal tubule and is responsible for the reabsorption of the majority of the filtered load of phosphate. Parathyroid hormone (PTH) is secreted by the parathyroid gland in response to a decrease in serum calcium or an increase in serum phosphorus, and acutely induces phosphaturia through the rapid stimulation of endocytosis of Npt2a and its subsequent lysosomal degradation. Chronic PTH stimulation leads to sustained phosphaturia, but the mechanisms for the chronic regulation of Npt2a by PTH remain unclear. We hypothesize that PTH decreases Npt2a mRNA levels as a mechanism for inducing chronic phosphaturia. We address this hypothesis within three specific aims. The first specific aim addresses the kinetics and mechanisms of the PTH-stimulated decrease in Npt2a mRNA expression. The second aim details the signaling pathways involved in PTH-mediated downregulation of Npt2a mRNA. Lastly, the third aim characterizes the PTH-responsive phosphoproteome of the proximal tubule, and how changes in the expression and phosphorylation of RNA-binding proteins may affect Npt2a mRNA stability. In Aim 1, we found that PTH decreases Npt2a steady-state mRNA levels with a 50% loss in 2.2h compared to 8.6h in the absence of PTH. This effect is post-transcriptional, and is dependent on both transcription and translation. In Aim 2, we determined that PTH destabilizes Npt2a mRNA through both PKA- and PKC-dependent mechanisms. The rapid initial decline in Npt2a mRNA levels corresponds to and appears to be dependent on early PTH-stimulated PKA activation. In contrast, PTH-stimulated PKC activation occurs more gradually over several hours and likely contributes to the latter phase of Npt2a mRNA destabilization. Finally, in Aim 3, we found that PTH alters the phosphorylation status of almost 1200 proteins in the proximal tubule, including 68 RNA-binding proteins (RBPs). Two of those RBPs, KHSRP and Roquin-2, bind to Npt2a mRNA, and their expression varies inversely with Npt2a mRNA levels. From these studies, we conclude that PTH-mediated Npt2a mRNA destabilization likely occurs through PKA- and PKC-dependent modulation of RBP expression and activity, and that KHSRP and Roquin-2 are potential mediators of the PTH response.
Murray, Rebecca Dale, "Post-transcriptional regulation of the Type IIA sodium-phosphate cotransporter by parathyroid hormone." (2014). Electronic Theses and Dissertations. Paper 1749.