Fetal Calcium Homeostasis Research Articles

Bone Is a Major Target of PTH/PTHrP Receptor Signaling in Regulation of Fetal Blood Calcium Homeostasis.

The blood calcium concentration during fetal life is tightly regulated within a narrow range by highly interactive homeostatic mechanisms that include transport of calcium across the placenta and fluxes in and out of bone; the mechanisms of this regulation are poorly understood. Our findings that endochondral bone-specific PTH/PTHrP receptor (PPR) knockout (KO) mice showed significant reduction of fetal blood calcium concentration compared with that of control littermates at embryonic day 18.5 led us to focus on bone as a possibly major determinant of fetal calcium homeostasis. We found that the fetal calcium concentration of Runx2 KO mice was significantly higher than that of control littermates, suggesting that calcium flux into bone had a considerable influence on the circulating calcium concentration. Moreover, Runx2:PTH double mutant fetuses showed calcium levels similar to those of Runx2 KO mice, suggesting that part of the fetal hypocalcemia in PTH KO mice was caused by the increment of the mineralized bone mass allowed by the formation of osteoblasts. Finally, Rank:PTH double mutant mice had a blood calcium concentration even lower than that of the either Rank KO or PTH KO mice alone at embryonic day 18.5. These observations in our genetic models suggest that PTH/PTHrP receptor signaling in bones has a significant role of the regulation of fetal blood calcium concentration and that both placental transport and osteoclast activation contribute to PTH's hypercalcemic action. They also show that PTH-independent deposition of calcium in bone is the major controller of fetal blood calcium level.

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

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 45Ca across the placenta in maternofetal (CaKmf) and fetoplacental (CaKfp) directions; CaKfp was < 5% of CaKmf for all genotypes. At 18 dp, CaKmf across perfused placenta and intact placenta (CaKmf(intact)) were similar and concordant with net calcium accretion rates in vivo. CaKmf 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-D9K 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-D9K expression. In situ perfusion of mouse placenta is a robust methodology for allowing detailed dissection of placental transfer mechanisms in genetically modified mice.

PTH/PTHrP activity and the programming of skeletal development in utero.

There is increasing evidence that nutritional deficiency in utero adversely affects bone development and the risk of developing osteoporosis in later life. Although the mechanisms involved are unknown, circumstantial evidence points to an important role of PTH/PTHrP activity. It is recognized that PTH and PTHrP are critically involved in regulating fetal calcium homeostasis, actions that are mediated at least in part by PPR. As well as playing a central role in the maintenance of calcium homeostasis in the fetus, studies in transgenic mice show that PTH, PTHrP, and PPR exert similar effects on skeletal development in utero, acting to increase the size of the trabecular envelope and decrease that of the cortical envelopes. Taken together, these observations raise the possibility that stimulation of PTH/PTHrP activity in the fetus in response to calcium deficiency acts to increase the size of the trabecular envelope but to reduce that of the cortical envelope. Although any increase in trabecular bone at birth is likely to be relatively transient, a decrease in size of the cortical envelope may have a persistent effect on the trajectory of bone growth in subsequent childhood. Consistent with this proposal, preliminary findings from birth cohort studies suggest that maternal calcium intake and cord blood calcium levels are positively related to bone mass of the offspring as assessed later in childhood. Further studies are justified to determine whether alterations in fetal PTH/ PTHrP activity caused by calcium stress lead to a reduction in size of the cortical envelope at birth that persists into childhood and later adult life and to identify modifiable maternal factors that are responsible for these changes.

Fetal parathyroids are not required to maintain placental calcium transport

We used Hoxa3 knockout mice and other mouse models to study the role of the fetal parathyroids in fetal calcium homeostasis. Hoxa3-null fetuses lack parathyroid glands, and absence of parathyroid hormone (PTH) was confirmed with a rodent PTH immunoradiometric assay. The ionized calcium level of Hoxa3-null fetuses was significantly lower than that of wild-type or heterozygous littermates or of the mother. Both the rate of placental calcium transfer and the plasma PTHrP level were normal in Hoxa3 mutants and their heterozygous siblings. Because we had previously observed an increase in placental calcium transfer in PTH/PTHrP receptor 1-null (Pthr1-null) fetuses, we assayed plasma PTHrP in those mice. Pthr1-null fetuses had plasma PTHrP levels 11-fold higher than those of their littermates. Northern analysis, immunohistochemical, and in situ hybridization studies of Pthr1-null fetuses indicated that liver and placenta had increased expression of PTHRP: In summary, loss of fetal parathyroids in Hoxa3-null fetuses caused marked hypocalcemia but did not alter placental calcium transfer or the circulating PTHrP level. The findings in the Pthr1-null fetuses indicate that several tissues may contribute to the circulating PTHrP level in fetal mice.

Exploring the normal regulation of fetal-placental calcium metabolism through the use of targeted gene ablation in fetal mice

Fetal calcium and bone metabolism differs from that of the adult, having been uniquely adapted to meet the specific needs of this developmental period. If these regulatory processes in the fetus were more fully elucidated and understood, the insight gained might lead to the development of novel therapies for osteoporosis and other metabolic bone diseases. We have explored the regulation of normal fetal calcium physiology through the use of gene targeting models. These techniques permit the study of fetal models that cannot be created by surgical or pharmacological techniques, such as fetal mice whose parathyroids produce parathyroid hormone (PTH) but not PTH-related protein (PTHrP), and vice versa. This article briefly reviews what we have learned from these studies, in the context of what is already known about fetal calcium homeostasis. Drug Dev. Res. 49:167–173, 2000. © 2000 Wiley-Liss, Inc.

Fetal calcium homeostasis

The mammalian fetus is maintained hypercalcaemic relative to its mother primarily by the action of a placental calcium pump located in the basal plasma membrane of the trophoblast. It is suggested that the activity of this pump is stimulated by a mid-molecular fragment of parathyroid hormone-related protein [PTHrP(38-94NH2)], produced in the placenta (and also in the parathyroid glands of fetal lambs and calves) as a result of post translational processing. In the sheep, calcitriol is an important determinant of fetal calcium homeostasis and it, too, stimulates the transport of calcium across the placenta. Fetal bone resorption is under the control of calcitriol, parathyroid hormone (PTH) and PTHrP. This resorption is modulated by the inhibitory effect of calcitonin. PTHrP also plays an important role in the regulation of endochondral ossification and chondrocyte maturation.

Tissue distribution of human gp330/megalin, a putative Ca(2+)-sensing protein.

We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution.

Midmolecular parathyroid hormone-related peptide in serum during pregnancy, lactation and in umbilical cord blood

Indications of an important physiological role of parathyroid hormone-related peptide (PTHrP) for fetal calcium homeostasis, maternal-fetal calcium transport and reproduction have accumulated over recent years. The PTHrP concentrations were measured by an earlier developed midregion radio-immunoassay in serum from lactating healthy females and umbilical cord blood and compared with levels in age-matched non-pregnant or lactating females. The PTHrP concentrations could be measured in all samples after silica cartridge C18 extraction of 10-12 ml of serum. The concentrations were significantly higher during lactation (mean +/- SD: 0.72 +/- 0.14 pmol/l, N = 22) and in umbilical cord blood (0.85 +/- 0.18 pmol/l, N = 12) compared with healthy age-matched women (0.48 +/- 0.09 pmol/l, N = 10, p < 0.001). The molecular forms of PTHrP were also studied in an age-matched control group, in pregnant women and in umbilical cord blood by gel chromatography in a fast protein liquid chromatography system of Sep-Pak-extracted pooled serum. In all three groups we found heterogeneity of the molecular forms with two predominant peaks. The smallest fragment had a molecular weight of 4-6 kD while the largest form appeared as a high-molecular-weight molecule. In conclusion, the concentrations of midmolecule PTHrP fragments in serum are elevated during lactation and in umbilical cord blood. Because the midregion of PTHrP has unique actions, our results indicate that PTHrP may play an important physiological role for the mother and for the maternal-fetal calcium transport.

Humoral hypercalcemia of malignancy

Studies on humoral hypercalcemia of malignancy have shown that tumors produce a protein that acts through the parathyroid hormone (PTH) receptor but is immunologically distinct from PTH. We have recently purified and cloned a parathyroid hormone-related protein (PTHrP) from a human lung cancer cell line. Full length cDNA clones were isolated and found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino terminal residues are identical with human PTH, although antisera directed at the amino terminus of PTHrP do not recognize PTH. A 34-amino acid synthetic peptide, PTHrP(1-34), was several times more potent than bovine or human PTH(1-34) in bioassays promoting the formation of cAMP and plasminogen activity in osteogenic sarcoma cells and activation of adenylate cyclase in chick kidney membranes. PTHrP(1-34) was also more potent than PTH(1-34) in stimulating cAMP and phosphate excretion and reducing calcium excretion in the isolated perfused rat kidney. PTHrP has been consistently demonstrated by immunohistochemistry in squamous cell carcinomas and in keratinocytes present in normal skin, but not in normal or hyperplastic parathyroid tissues or other tumors. PTHrP-like activity has been extracted from ovine placenta and fetal parathyroid tissue, suggesting that PTHrP may play a role in fetal calcium homeostasis.

Transplacental 45Ca and 32P flux in the guinea pig: effect of maternal hypercalcaemia and hypocalcaemia.

Transplacental 45Ca and 32P flux was measured across the in situ perfused guinea-pig placenta under conditions of acute maternal hypocalcaemia and hypercalcaemia. Maternal hypercalcaemia induced acutely by calcium gluconate infusion caused an increase in maternal-to-fetal 45Ca flux which was proportional to the increase in maternal plasma ionized calcium concentration. Acute maternal hypocalcaemia was induced by EGTA infusion and resulted in a decrease in maternal plasma ionized calcium concentration proportional to a corresponding decrease in transplacental 45Ca transfer. A bolus of calcium gluconate caused a transient decrease in 32P flux, whereas EGTA administration was without significant effect on transplacental 32P transfer. Calcium transport across the placenta is not saturated under conditions of maternal normocalcaemia and may be altered according to acute changes in maternal plasma calcium concentration. Thus, control of maternal-to-fetal calcium transfer does not appear to be at the placental level. This suggests that fetal calcium homeostasis may be regulated by the fetus itself.

The transfer of calcium during perfusion of the placenta in intact and thyroparathyroidectomized sheep

Placental perfusions were carried out in six ewes during the last two weeks of gestation. Perfusions were carried out using autologous fetal blood and the flow rates adjusted to give a perfusion pressure of 50--70 mmHg. Perfusion plasma calcium concentrations rose steadily throughout the perfusions achieving a mean increase of 1.65 mmol/1 above initial concentration within 100 minutes. A further three ewes in the last two weeks of gestation were thyroparathyroidectomized and normal plasma calcium concentration maintained by an intravenous infusion of calcium borogluconate. After three days, placental perfusions were carried out as before. The mean perfusion plasma calcium concentration achieved by those three ewes in a period of 100 minutes showed an increase of 1.25 mmol/1. It is concluded that the presence of the fetus is not necessary for the continued active transfer of calcium across the placenta from mother to fetus. The reduced rate of accumulation of calcium in the perfusate in TXPTX ewes is attributed to a decline in 1,25-DHCC concentrations in both maternal and fetal circulations. The implications of these results in relation to fetal calcium homeostasis and the placental transfer of calcium are discussed.

Placental Impermeability to Parathormone 125I in the Ewe

Placental permeability and metabolism of parathyroid hormone (PTH) 125I was studied in 5 gravid ewes during the last trimester. The mean plasma half-life was 38 +/- 0.8 and 22.6 +/- 2.7 min in the fetus and ewe, respectively. There was minimal placental transfer of PTH 125I in either the fetal to maternal and maternal to fetal directions. Fetal calcium homeostasis is probably not affected directly by changes in maternal serum PTH levels, and maternal calcium homeostasis probably is not affected directly by changes in fetal serum PTH concentrations.

FETAL‐MATERNAL PLASMA CALCIUM RELATIONSHIPS IN THE RABBIT

Plasma total calcium levels have been studied in pregnant rabbits and their fetuses from 16 days gestation to term. Between 16 and 21 days gestation the observed fetal plasma calcium level falls from the maternal level or above to 1·6 mg/100 ml. below the maternal level. Thereafter, the fetal plasma calcium rises and at 30 days gestation exceeds the maternal level by 1·9 mg/100 ml. There is a high correlation between fetal and maternal levels at 21 days which is lost by 30 days. Fetal skeletal calcification was studied using an alizarin stain technique. The earliest centres of calcification were present in the clavicle and mandible at 16 days. It is concluded that the observed fall in fetal plasma calcium near mid‐gestation may be due to the onset of skeletal calcification and the increase after 21 days to one or more of a variety of factors. Five 21 days pregnant animals were infused with 1 per cent calcium chloride solution (1 g anhydrous calcium chloride in 100 g aqueous solution) for up to 2½ hr. The maternal plasma calcium concentration was elevated by 7·7 mg/100 ml. during the infusion but the fetal level was only raised by 2·0 mg/100 ml. The correlation between fetal and maternal levels was lost during the infusion. These findings are compatible with either slow calcium transfer across the placenta or fetal calcium homeostasis.