Acute Parathyroid Hormone Injection Increases C-Terminal but Not Intact Fibroblast Growth Factor 23 Levels

Abstract

The acute effects of parathyroid hormone (PTH) on fibroblast growth factor 23 (FGF23) in vivo are not well understood. After a single subcutaneous PTH (1-34) injection (50 nmol/kg) in mice, FGF23 levels were assessed in plasma using assays that measure either intact alone (iFGF23) or intact/C-terminal FGF23 (cFGF23). Furthermore, FGF23 messenger RNA (mRNA) and protein levels were assessed in bone. In addition, we examined the effects of PTH treatment on FGF23 production in vitro using differentiated calvarial osteocyte-like cells. cFGF23 levels increased by three- to fivefold within 2 hours following PTH injection, which returned to baseline by 4 hours. In contrast, iFGF23 levels remained unchanged for the first 2 hours, yet declined to ∼60% by 6 hours and remained suppressed before returning to baseline after 24 hours. Using homozygous mice for an autosomal dominant hypophosphatemic rickets-FGF23 mutation or animals treated with a furin inhibitor, we showed that cFGF23 and iFGF23 levels increased equivalently after PTH injection. These findings are consistent with increased FGF23 production in bone, yet rapid cleavage of the secreted intact protein. Using primary osteocyte-like cell cultures, we showed that PTH increased FGF23 mRNA expression through cyclic adenosine monophosphate/protein kinase A, but not inositol triphosphate/protein kinase C signaling; PTH also increased furin protein levels. In conclusion, PTH injection rapidly increases FGF23 production in bone in vivo and in vitro. However, iFGF23 is rapidly degraded. At later time points through an unidentified mechanism, a sustained decrease in FGF23 production occurs.

Figure 1.

Acute effects of a single PTH injection in WT mice. Baseline cFGF23 levels in different experiments ranged between 218 ± 19 and 345 ± 34 pg/mL, and peak cFGF23 levels ranged between 578 ± 114 and 1244 ± 110 pg/mL; therefore, changes in cFGF23 (and iFGF23) levels are reported as percentage of change from baseline. (a) Plasma phosphate levels 2, 6, and 24 hours after PTH or vehicle injection. (b) Plasma cFGF23 levels, expressed as percentage over preinjection baseline, at 2, 4, 6, and 24 hours after PTH injection. (c) Plasma iFGF23 levels, expressed as percentage over preinjection baseline, at 2, 4, 6, and 24 hours after PTH injection. n = 5 or more mice per group. (d) Ratio of cleaved/total FGF23 was calculated as follows: (value measured by cFGF23 ELISA − value measured by iFGF23 ELISA)/value measured by cFGF23 ELISA. n = 7–8 per group. *P < 0.05.

Figure 2.

Dose-response effects of cFGF23 and iFGF23 after PTH injection. PTH doses of 12.5 and 50 nmol/kg were injected, and (a) peak cFGF23 levels (at 2 hours) and (b) nadir iFGF23 levels (at 6 hours) are shown. Absolute values are shown from an individual experiment. n = 5 or more mice per group. Bars show significant response by analysis of variance.

Figure 3.

Effects of PTH injection in ADHR mice or in animals pretreated with the furin inhibitor 537076 (at concentration 7.5 μg/g) or PBS vehicle. (a) cFGF23 and (b) iFGF23 changes after PTH injection (50 nmol/kg), or vehicle (10 mm citric acid, 150 mm NaCl, 0.05% Tween 80, pH 5.0) in ADHR mutant mice. n = 6–8 per group. Baseline cFGF23 levels in ADHR mutant mice in different experiments ranged between 190 ± 13 and 281 ± 19 pg/mL, and peak cFGF23 levels ranged between 485 ± 78 and 1242 ± 239 pg/mL, whereas baseline iFGF23 levels in ADHR mutant mice in different experiments ranged between 262 ± 23 and 345±18 pg/mL, and peak iFGF23 levels ranged between 450 ± 25 and 975 ± 149 pg/mL. Changes in cFGF23 and iFGF23 levels are reported as percentage of change from baseline. (c) cFGF23 and (d) iFGF23 levels in WT mice all injected with PTH; furin refers to mice pretreated with furin inhibitor, and vehicle refers to mice pretreated with vehicle as described. n > 4 mice per group. *P < 0.05. Hashed line shows 100% of baseline.

Figure 4.

mRNA and protein levels in bone from mice WT after PTH or vehicle injection. (a) mRNA expression in femurs from WT adult mice treated with PTH or vehicle. n = 3–4. Sost = sclerostin. (b) Western blot probed with biotin-conjugated anti-FGF23 antibody (Immutopics), upper panel, or actin, lower panel, in bone lysates from mice injected with vehicle or PTH. *P < 0.05.

Figure 5.

mRNA expression in cultured calvaria osteocytes. (a) and (b) show FGF23 and Nurr1 mRNA expression in calvarial osteocytes treated with PTH, vehicle, PKA inhibitor (15 nM PKA inhibitor P6062), PKC inhibitor (5 µM PKC inhibitor SCP0214), or both. Expression normalized to housekeeping gene ornithine decarboxylase antizyme 1 (see Materials and Methods). Data shown are mean of triplicate. *P < 0.05.

Figure 6.

Protein levels in calvarial osteocyte-like cells. (a) Shows Western blot analysis of Fam20C, GALNT3, and furin protein levels in calvarial osteocyte-like cells 2 hours after PTH or vehicle treatment. The top part shows expression levels normalized to actin using Image J, and the lower part shows the Western blot data. (b) Shows immunohistochemical staining of differentiated calvarial osteocyte-like cells after 21 days in culture treated with PTH or vehicle. The staining is normalized to vehicle. GALNT3, Furin, or Fam20c protein levels are shown. Magnification is 40-fold. *P < 0.05.

Figure 7.

Proposed model of PTH action on FGF23 production at early and late time points.