Increased maternofetal calcium flux in parathyroid hormone-related protein-null mice

Abstract

The role of parathyroid hormone-related protein (PTHrP) in fetal calcium homeostasis and placental calcium transport was examined in mice homozygous for the deletion of the PTHrP gene (PTHrP-/- null; NL) compared to PTHrP+/+ (wild-type; WT) and PTHrP+/- (heterozygous; HZ) littermates. Fetal blood ionized calcium was significantly reduced in NL fetuses compared to WT and HZ groups at 18 days of pregnancy (dp) with abolition of the fetomaternal calcium gradient. In situ placental perfusion of the umbilical circulation at 18 dp was used to measure unidirectional clearance of (45)Ca across the placenta in maternofetal ((Ca)K(mf)) and fetoplacental ((Ca)K(fp)) directions; (Ca)K(fp) was < 5% of (Ca)K(mf) for all genotypes. At 18 dp, (Ca)K(mf) across perfused placenta and intact placenta ((Ca)K(mf(intact))) were similar and concordant with net calcium accretion rates in vivo. (Ca)K(mf) was significantly raised in NL fetuses compared to WT and HZ littermates. Calcium accretion was significantly elevated in NL fetuses by 19 dp. Placental calbindin-D(9K) expression in NL fetuses was marginally enhanced (P < 0.07) but expression of TRPV6/ECaC2 and plasma membrane Ca2+-ATPase (PMCA) isoforms 1 and 4 were unaltered. We conclude that PTHrP is an important regulator of fetal calcium homeostasis with its predominant effect being on unidirectional maternofetal transfer, probably mediated by modifying placental calbindin-D(9K) expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

Figure 1. PCR of paired placental cDNA (A) and fetal genomic DNA (B) from WT (PTHrP^+/+), HZ (PTHrP^+/−) and NL (PTHrP^−/−) fetuses of 3 individual litters using exon IV-specific primers, the major exon encoding murine PTHrP

Lane B, H₂O replacement of DNA. 100 base pair (bp) ladder is shown on left. Amplification product of the predicted size (138 bp) was visible in placental cDNA from all three genotypes, whilst genomic DNA demonstrated the predicted pattern of amplification with no product observable in NL fetuses.

Figure 2. Immunohistochemistry of PTHrP in the placenta of mouse fetuses which were WT (PTHrP^+/+) (A), HZ (PTHrP^+/−) (B) or NL (PTHrP^−/−) (C) for PTHrP allele

Sections were incubated with goat anti-human PTHrP antibody (2 μg ml⁻¹; A–C) or goat IgG (2 μg ml⁻¹, D) as negative control. Intense staining was observed within the IPYS (arrows) and labyrinthine zone (L) of placentas of WT and HZ fetuses. In contrast in the NL fetus, placental staining was negligible and comparable to negative control, confirming a lack of placental PTHrP protein expression in the null mutant. All scale bars in A–D are 50 μm. E, Western blot of WT mouse placental lysate (50 μg protein lane⁻¹) probed with either: +, affinity purified goat anti-human PTHrP antibody (1: 200; 1 μg ml⁻¹) alone giving a single immunoreactive signal of predicted size; or −, in the presence of 5× excess (1: 40) blocking peptide which abolished signal.

Figure 3. Western blots showing calcium transport protein expression in the placentas of WT (PTHrP^+/+), HZ (PTHrP^+/−) or NL (PTHrP^−/−) fetuses, matched from two litters

A, TRPV6 (ECaC2; 20 μg membrane protein lane⁻¹, 30 s exposure); B, calbindin-D_9K (20 μg PNS lane⁻¹, 15 s exposure); C, PMCA1 (30 μg membrane protein lane⁻¹, 5 min exposure); and D, PMCA4 (60 μg membrane protein lane⁻¹, 5 min exposure). For all blots the immunoreactive species detected accord with the predicted molecular mass of the target protein, with signal abolished by omission of primary antibody (not shown). E, densitometric analysis (mean +s.e.m.) of immunoreactive signal intensity for TRPV6 (ECaC2; n = 6), calbindin-D_9K (n = 9), PMCA1 (n = 6) and PMCA4 (n = 6) in placentas of WT, HZ and NL fetuses. Mean calbindin-D_9K expression in NL fetuses was ∼37% higher than in WT and HZ littermates (P < 0.07, Kuskal–Wallis). All signals fell within the linear range of detection. For PMCA1 and PMCA4 signals comprising the triplet and doublet species, respectively, were analysed collectively. Equal protein loading was confirmed by densitometric analysis of replicate blots probed for β-actin (data not shown).