Parathyroid hormone decreases renal vitamin D receptor expression in vivo

Abstract

The vitamin D receptor (VDR) is a nuclear transcription factor responsible for mediating the biological activities of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. Renal and parathyroid gland VDR content is an important factor in calcium homeostasis, vitamin D metabolism, and the treatment of secondary hyperparathyroidism and renal osteodystrophy. In these tissues, VDR expression is highly regulated by the calcium and vitamin D status. Although 1,25(OH)(2)D(3) up-regulates VDR expression, hypocalcemia and vitamin D deficiency result in drastically reduced expression of the receptor. The generation of 25-hydroxyvitamin D(3)-1alpha-hydroxylase-null mice, which are incapable of endogenously producing 1,25(OH)(2)D(3), has allowed us to investigate the influence of parathyroid hormone (PTH) on VDR expression independent of PTH-mediated increases in 1,25(OH)(2)D(3). Administration of human PTH (1-34) (110 microg/kg per day) for 48 h reduced renal VDR levels from 515 to 435 fmol/mg protein (15%, P < 0.03) in wild-type mice. In the 25-hydroxyvitamin D(3)-1alpha-hydroxylase-null mice, PTH administration strongly reduced renal VDR levels, from 555 to 394 fmol/mg protein (29%, P < 0.001). These results demonstrate that PTH is a potent down-regulator of VDR expression in vivo.

Fig. 1.

Serum calcium and phosphorus. 1α-Hydroxylase^+/+ and 1α-hydroxylase^-/- mice were maintained on a 20% lactose diet containing 2.0% calcium, 1.25% phosphorus, and 1 ng of 1,25(OH)₂D₃ per mouse per day. Either PTH (110 μg/kg per day) or vehicle was administered by microosmotic pump for 48 h. At death, blood was collected and serum was isolated. Assays were performed as described in Materials and Methods. (A) Serum calcium. (B) Serum phosphorus. All values are reported as the mean ± SEM (n = 10). ^*, significance at P < 0.005 vs. vehicle-treated mice; †, significance at P < 0.03.

Fig. 2.

1α-Hydroxylase transcript levels. 1α-Hydroxylase^+/+ and 1α-hydroxylase^-/- mice were maintained on a 20% lactose diet containing 2.0% calcium, 1.25% phosphorus, and 1 ng of 1,25(OH)₂D₃ per mouse per day. Either PTH (110 μg/kg per day) or vehicle was administered by microosmotic pump for 48 h. One kidney per mouse was harvested into Tri Reagent, and total RNA was prepared. After reverse transcription, 1α-hydroxylase mRNA was quantified by using real-time PCR, and standardized to β-actin mRNA. (Top) Values are charted as relative mRNA with a maximal value of 1. Values are reported as the mean ± SEM (n = 10). ^*, significance at P < 0.001 vs. vehicle-treated mice. N.D., not detectable. (Middle and Bottom) A representative sample from each group was electrophoresed through an ethidium bromide-stained gel and was UV-illuminated. M.W., pGEM (Promega) molecular weight markers. Lane 1, +/+, vehicle; lane 2, +/+ PTH; lane 3, -/- vehicle; lane 4, -/- PTH.

Fig. 3.

Suppression of renal VDR by PTH. 1α-Hydroxylase^+/+ and 1α-hydroxylase^-/- mice were maintained on a 20% lactose diet containing 2.0% calcium, 1.25% phosphorus, and 1 ng of 1,25(OH)₂D₃ per mouse per day. PTH (110 μg/kg per day) or vehicle was administered by microosmotic pump for 48 h. One kidney per mouse was harvested into isotonic buffer, whole-cell extract was prepared, and VDR content was determined by ELISA and standardized to total protein, as described in Materials and Methods. Each group contained 10 mice. The values represent the mean ± SEM. ^*, significance at P < 0.03 vs. vehicle-treated mice; †, significance at P < 0.001.

Fig. 4.

VDR transcript levels. 1α-Hydroxylase^+/+ and 1α-hydroxylase^-/- mice were maintained on a 20% lactose diet containing 2.0% calcium, 1.25% phosphorus, and 1 ng of 1,25(OH)₂D₃ per mouse per day. Either PTH (110 μg/kg per day) or vehicle was administered by microosmotic pump for 48 h. One kidney per mouse was harvested into Tri Reagent, and total RNA was prepared. After reverse transcription, VDR mRNA was quantified by using real-time PCR and was standardized to β-actin mRNA. The mean ± SEM (n = 10) is expressed as relative mRNA with a maximal value of 1.