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, αKlotho, 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-β1C. 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.

Keywords: G protein-coupled receptor (GPCR); NHERF1; NPT2A; PDZ Protein; alternative splicing; fibroblast growth factor receptor (FGFR); klotho; parathyroid hormone; transport.

FIGURE 1.

NPT2A, NHERF1, PTHR, and FGFR1 expression in RPTECs. A, distribution of NPT2A (green), NHERF1 (red), and nuclei (blue) in filter-grown RPTECs. Single optical sections from the apical region of the cell are shown. Arrows, NPT2A association with cilia. B, magnified views of a single cell. C, x-z plane depicting PTHR (green) and nuclear (blue) staining of RPTECs grown on coverslips. D, FGFR1 (green) and F-actin (red) staining of RPTECs grown on filters. The x-z plane is shown.

FIGURE 2.

FGFR mRNA and protein expression in RPTECs. A, representative 3% agarose gel of FGFR expression in RPTECs determined by RT-PCR (Table 2). 200- and 100-bp markers are shown in the left lane. B, schematic representations of the FGFR1 gene and protein structure. The FGFR-β1c splice variant harbors two extracellular immunoglobulin-like domains (II and III) with alternatively spliced exon 9 in loop III, a transmembrane domain (TM), and two intracellular tyrosine kinase domains (TK). C, FGFR1-β splice variant in a characteristic 2% agarose gel with ladder in the left lane. The 500-bp PCR product is consistent with the β-splice variant lacking exon 3. D, FGFR1b/c splice variants. Lane 1, FGFR1b; lane 2, FGFR1c; lane 3, mRNA containing both exons 8 and 9. Lane 3 PCR was performed using the forward primer for FGFR1b and the reverse primer for FGFR1c (Table 1). An illustrative 2% agarose gel is shown with a 100-bp ladder in the left lane. The PCR product in lane 2 is consistent with the exon 9 FGFR1c splice variant. Lane 3 shows an mRNA species containing both exons 8 and 9. E, FGFR1c is the primary species of FGFR1 detected by immunoblotting. FGFR1 and FGFR1c in RPTECs were detected by non-selective and isotype-selective antisera, respectively, as described under “Experimental Procedures.” The results are illustrative of n = 3 independent experiments. F, MS/MS spectrum for the identified specific peptide ³⁵⁵SDFHSQMAVHKLAK³⁶⁸ from FGFR1c. The peak heights show the relative abundances of the corresponding fragmentation ions, with the annotation of the identified matched N terminus-containing b ions in blue and the C terminus-containing y ions in red. Charge state: +3, observed m/z: 538.93969, theoretical m/z: 538.94008, precursor mass error: −0.72, Xcorr: 0.719.

FIGURE 3.

PTH and FGF23 inhibit phosphate transport. A, RPTECs were treated for 2 h with 100 nm PTH(1–34) or FGF23. Phosphate uptake was measured for 10 min, as detailed under “Experimental Procedures.” Data represent the mean ± S.E. (error bars) of n = 6 independent experiments performed in triplicate. Data were normalized for each experiment, where phosphate uptake under control, untreated conditions, was defined as 0% inhibition. Data were fit to a sigmoidal relation, and K_d values were calculated with Prism. B, RPTECs on 12-well plates were treated for 2 h with the indicated concentrations of PTH or FGF23. Phosphate uptake was measured for 10 min, as outlined under “Experimental Procedures.” Data represent the mean ± S.E. of n = 6 independent experiments performed in triplicate. *, p < 0.05 versus control; **, p < 0.01 versus control. C, C-terminal FGF23(180–251) fragment blocks FGF23 but not PTH inhibitable phosphate uptake. Data are the mean ± S.E. of n = 4 experiments. **, p < 0.01 versus FGF23.

FIGURE 4.

Signaling pathways mediating PTH and FGF23 effects on phosphate transport. RPTECs were treated for 2 h with 100 nm PTH(1–34) (A) or FGF23 (B) in the presence or absence of the specified inhibitors: BisI (PKC), GSK-650394 (SGK-1), H89 (PKA), and PD (PD-98059, ERK1/2). Inhibitors were used at 1 μm except for GSK, where 10 μm was employed. Phosphate uptake was measured as before. Data represent the mean ± S.E. (error bars) of n = 6 experiments. *, p < 0.05; **, p < 0.01 versus PTH or FGF23.

FIGURE 5.

Effect of FGFR1 exon 9 and exon 8 knockdown on FGF23-regulated phosphate transport. A, FGFR1 knockdown by siRNA for exon 8 (si8), exon 9 (si9), or a scrambled control (scr) was assessed by immunoblotting using antibody that specifically recognizes FGFR1c. A representative result is presented. B, quantification of si8 and si9 knockdown of FGFR1c. Data represent the mean ± S.E. (error bars) of n = 4 experiments. C, siRNA effects on FGF23-sensitive phosphate transport. Data represent the mean ± S.E. of n = 4 experiments. *, p < 0.05; **, p < 0.01.

FIGURE 6.

αKlotho expression and function. A, αKlotho is expressed by RPTECs. Shown is an immunoblot of 20 μg of protein lysate duplicate wells probed with rabbit polyclonal anti-Klotho antibody. Molecular mass markers representing 50 and 75 kDa are shown. B, chlorate treatment of RPTECs interfered with FGF23 but not PTH inhibition of phosphate transport. RPTECs were treated overnight with 50 mm NaClO₄, followed by a 2-h treatment with 100 nm PTH(1–34) or FGF23, as indicated. Phosphate uptake was measured as described under “Experimental Procedures.” Data represent the mean ± S.E. (error bars) of n = 4 independent experiments. Data were normalized for each experiment, where phosphate uptake under control, untreated conditions was defined as 0% inhibition. **, p < 0.01 versus FGF23 alone.

FIGURE 7.

siRNA αKlotho knockdown blocks FGF23- but not PTH-inhibitable phosphate transport. αKlotho expression was knocked down in RPTECs using siRNA as described under “Experimental Procedures.” A, knockdown of αKlotho was assessed by immunoblotting. A representative experiment is depicted. The data for three siRNA duplexes plus a scrambled control (scr) are shown. B, the effects of αKlotho knockdown on PTH- and FGF23-dependent phosphate transport were measured. Data represent the mean ± S.E. (error bars) of n = 5 experiments. Data were normalized for each experiment, where phosphate uptake under control, untreated conditions was defined as 0% inhibition. **, p < 0.01; ***, p < 0.001 versus scrambled.

FIGURE 8.

shRNA NHERF1 knockdown inhibits FGF23- and PTH-regulated phosphate transport. NHERF1 expression was knocked down in RPTECs by shRNA transfection as described under “Experimental Procedures.” A, knockdown of NHERF1 was assessed by immunoblotting. A representative experiment is depicted. The data for NHERF1 shRNA and a scrambled control (scr) are shown. B, effects of NHERF1 knockdown on PTH- and FGF23-dependent phosphate transport were assessed. Data represent the mean ± S.E. (error bars) of n = 4 experiments. Data were normalized for each experiment, where phosphate uptake under control, untreated conditions was defined as 0% inhibition. **, p < 0.01 versus FGF23 or PTH.

FIGURE 9.

PTH and FGF23 down-regulation of NPT2A. A, NPT2A protein levels in RPTECs after a 2-h treatment with PTH or FGF23. A representative experiment is presented. B, immunofluorescence experiments depicting the change in localization of NPT2A in response to PTH or FGF23 treatment. RPTECs were left untreated (control) or treated for 2 h with 100 nm PTH or FGF23. Green, NPT2A; red, NHERF1; blue, nuclei. The x-z plane is depicted.