Convergent Signaling Pathways Regulate Parathyroid Hormone and Fibroblast Growth Factor-23 Action on NPT2A-mediated Phosphate Transport

Abstract

Parathyroid hormone (PTH) and FGF23 are the primary hormones regulating acute phosphate homeostasis. Human renal proximal tubule cells (RPTECs) were used to characterize the mechanism and signaling pathways of PTH and FGF23 on phosphate transport and the role of the PDZ protein NHERF1 in mediating PTH and FGF23 effects. RPTECs express the NPT2A phosphate transporter, ␣Klotho, FGFR1, FGFR3, FGFR4, and the PTH receptor. FGFR1 isoforms are formed from alternate splicing of exon 3 and of exon 8 or 9 in Ir-like loop 3. Exon 3 was absent, but mRNA containing both exons 8 and 9 is present in cytoplasm. Using an FGFR1c-specific antibody together with mass spectrometry analysis, we show that RPTECs express FGFR-␤1C. The data are consistent with regulated FGFR1 splicing involving a novel cytoplasmic mechanism. PTH and FGF23 inhibited phosphate transport in a concentration-dependent manner. At maximally effective concentrations, PTH and FGF23 equivalently decreased phosphate uptake and were not additive, suggesting a shared mechanism of action. Protein kinase A or C blockade prevented PTH but not FGF23 actions. Conversely, inhibiting SGK1, blocking FGFR dimerization, or knocking down Klotho expression disrupted FGF23 actions but did not interfere with PTH effects. C-terminal FGF23(180 -251) competitively and selectively blocked FGF23 action without disrupting PTH effects. However, both PTH and FGF23-sensitive phosphate transport were abolished by NHERF1 shRNA knockdown. Extended treatment with PTH or FGF23 down-regulated NPT2A without affecting NHERF1. We conclude that FGFR1c and PTHR signaling pathways converge on NHERF1 to inhibit PTH-and FGF23-sensitive phosphate transport and down-regulate NPT2A.Parathyroid hormone (PTH) 2 and FGF23 display two remarkable features: 1) PTH and FGF23 exhibit parallel inhibition of renal phosphate transport mediated by NPT2A (sodium-dependent phosphate transporter-2a) but opposing actions on 1,25(OH) 2 -vitamin D; 2) despite being structurally and functionally distinct classes of membrane-delimited receptors, PTH and FGF receptors activate kinases that obligatorily phosphorylate NHERF1 at conserved sites required for their phosphaturic action. Phosphorus is essential for growth and maintenance of the skeleton and for generating high energy phosphate compounds. Evolutionary adaptation in humans and other terrestrial vertebrates to phosphorus-rich diets involves cell and molecular mechanisms ensuring the efficient urinary elimination of excess inorganic phosphate. The renal proximal tubule is the primary site of phosphate homeostasis and hormone-dependent phosphate transport. The NPT2A sodium-dependent phosphate cotransporter (SLC34A1) in proximal tubules is regulated by PTH and FGF23 (1, 2). PTH and FGF23 reduce phosphate uptake by sequestering and down-regulating NPT2A, thereby enhancing urinary phosphate excretion (3, 4). PTH actions are mediated by its cognate G protein-coupled PTH receptor (PTHR) (5, 6). Both PKA and PKC have been implicated in PTH-dependent inhibition of NPT2A (7-12)....

Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport

“…Mutating the PDZ ligand disrupts binding to NHERF1 (9). PTH and FGF23 down-regulate NPT2A expression and function by distinct signaling pathways that converge at NHERF1 (10). Mice lacking NHERF1 display characteristic mineral-ion wasting and osteopenia (11,12).…”

Genetic variants disrupt human RGS14 binding to NHERF1 and regulation of NPT2A-mediated phosphate transport

“…We thus sought to determine the effect of RGS14 on basal and PTH-sensitive phosphate transport and the consequence of the various RGS14 mutations on this process. For these studies we employed opossum kidney (OK) cells, an accepted model for PTH-regulated phosphate transport (31) or human RPTEC cells, which also exhibit PTH-sensitive phosphate transport (32).…”

“…As shown in Fig To extend these findings, we sought to determine and characterize the action of RGS14 on PTH-sensitive phosphate transport in RPTEC, a non-transformed line of human proximal tubule cells (34). PTH inhibits phosphate transport in RPTEC (32). Our initial experiments failed to disclose an effect on PTH action.…”

“…48hr later, cells were serum-starved overnight and then treated for 2 h with 10 nM (RPTEC) or 100 nM (OK cells) PTH . Phosphate uptake was measured as described (32). The hormone-supplemented medium was aspirated, and the wells were washed three times with 1 ml of Nareplete wash buffer (140 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO 4 , 0.1 mM KH 2 PO 4 , 10 mM HEPES, pH 7.4).…”

Personalized Intervention in Monogenic Stone Formers

“…30 Heterozygous mutations of this gene may cause phosphate wasting, stones and osteomalacia. 6,7 Thus, this gene should be included with SLC34A3 and SLC34 A1 as part of sequencing panels.…”