Intestinal calcium transporter genes are upregulated by estrogens and the reproductive cycle through vitamin D receptor-independent mechanisms.

UNLABELLED: 1alpha,25(OH)2-vitamin D strongly regulates the expression of the epithelial calcium channel CaT1. CaT1 expression is reduced in ERKOalpha mice and induced by estrogen treatment, pregnancy, or lactation in VDR WT and KO mice. Estrogens and vitamin D are thus independent potent regulators of the expression of this calcium influx mechanism, which is involved in active intestinal calcium absorption. INTRODUCTION: Active duodenal calcium absorption consists of three major steps: calcium influx into, transfer through, and extrusion out of the enterocyte. These steps are carried out by the calcium transport protein 1 (CaT1), calbindin-D9K, and the plasma membrane calcium ATPase (PMCA1b), respectively. We investigated whether estrogens or hormonal changes during the female reproductive cycle influence the expression of these genes, and if so, whether these effects are vitamin D-vitamin D receptor (VDR) dependent. MATERIALS AND METHODS: We evaluated duodenal expression patterns in estrogen receptor (ER)alpha and -beta knockout (KO) mice, as well as in ovariectomized, estrogen-treated, pregnant, and lactating VDR wild-type (WT) and VDR KO mice. RESULTS: Expression of calcium transporter genes was not altered in ERKObeta mice. CaT1 mRNA expression was reduced by 55% in ERKOalpha mice, while the two other calcium transporter genes were not affected. Ovariectomy caused no change in duodenal expression pattern of VDR WT and KO mice, whereas treatment with a pharmacologic dose of estrogens induced CaT1 mRNA expression in VDR WT (4-fold) and KO (8-fold) mice. Pregnancy enhanced CaTI expression equally in VDR WT and KO mice (12-fold). Calbindin-D9K and PMCA1b expression increased to a lesser extent and solely in pregnant VDR WT animals. In lactating VDR WT and KO mice, CaT1 mRNA expression increased 13 times, which was associated with a smaller increase in calbindin-D9K protein content and PMCA1b mRNA expression. CONCLUSIONS: Estrogens or hormonal changes during pregnancy or lactation have distinct, vitamin D-independent effects at the genomic level on active duodenal calcium absorption mechanisms, mainly through a major upregulation of the calcium influx channel CaT1. The estrogen effects seem to be mediated solely by ERalpha.

Vitamin D deficiency in guinea pigs: exacerbation of bone phenotype during pregnancy and disturbed fetal mineralization, with recovery by 1,25(OH)2D3 infusion or dietary calcium-phosphate supplementation.

Vitamin D (D) deficiency during human pregnancy appears to disturb fetal growth and mineralization, but fetal development is normal in D-deficient rats and vitamin D receptor gene-ablated mice. We used the guinea pig model to investigate maternal and fetal effects of D deficiency. Pregnant (Pr) and nonpregnant (NPr) animals were fed a D-replete (+D) or D-deficient diet (-D) for 8 weeks. We further studied whether the effects of a -D diet are reversed by continuous 1,25(OH)2D3 infusion (-D+1,25) and/or by a lactose-, Ca- and P-enriched D-deficient diet (-D+Ca/P). Bone analyses included histomorphometry of the proximal tibiae, dual-energy X-ray absorptiometry (DXA), and quantitative computed tomography (QCT) of the femora. Depletion of 25(OH)D3 and 1,25(OH)2D3 levels and the D-deficiency syndrome were more severe in pregnant animals. Indeed, Pr/-D but not NPr/-D guinea pigs were hypophosphatemic, and showed robust increases in growth plate width and osteoid surface and thickness; in addition, bone mineral density on DXA was lower in Pr/-D animals only, which was exclusively in cortical bone on QCT. Bone phenotype was partly normalized in Pr/-D+1,25 and Pr/-D+Ca/P animals. Compared with +D fetuses, -D fetuses had very low or undetectable 25(OH)D3 and 1,25(OH)2D3, were hypercalcemic and hypophosphatemic, and had lower osteocalcin levels. In addition, body weight and total body bone mineral content were 10-15% lower; histomorphometry showed hypertrophic chondrocyte zone expansion and hyperosteoidosis. 1,25(OH)2D3 levels were restored in -D+1,25 fetuses, and the phenotype was partially corrected. Similarly, the fetal +D phenotype was rescued in large part in -D+Ca/P fetuses, despite undetectable circulating 25(OH)D3 and 1,25(OH)2D3. We conclude that pregnancy markedly exacerbates D deficiency, and that augmenting Ca and P intake overrides the deleterious effects of D deficiency on fetal development.

Dietary calcium and phosphate restriction in guinea-pigs during pregnancy: fetal mineralization induces maternal hypocalcaemia despite increased 1 alpha,25-dihydroxycholecalciferol concentrations.

Guinea-pig fetuses at term are mineralized to a degree comparable with human fetuses, which makes the guinea-pig an attractive animal model to study maternal-fetal interactions with regard to Ca and phosphate (P) homeostasis. We studied non-pregnant and pregnant (day 57) vitamin D-replete guinea-pigs, fed either a normal guinea-pig chow with 9.6 g Ca/kg and 4.9 g P/kg or a study diet with 2 g Ca/kg and 1 g P/kg (low-Ca-P diet) for 7-8 weeks. Both pregnancy and the low-Ca-P diet decreased plasma concentrations of 25-hydroxycholecalciferol (25(OH)D3), but increased total and free 1 alpha,25-dihydroxycholecalciferol (1,25(OH)2D3), strongly suggesting an additive stimulation of 1 alpha-hydroxylase activity. Maternal and fetal 25(OH)D3 and 1,25(OH)2D3 levels were highly correlated (r 0.82 and 0.92 respectively, P < 0.001). Dual-energy absorption X-ray absorptiometry (DXA) showed that both pregnancy and the low-Ca-P diet decreased bone mineral density (BMD) of the maternal femur, particularly at the distal metaphysis. Despite higher 1,25(OH)2D3 concentrations and lower BMD, pregnant animals on the low-Ca-P diet were hypocalcaemic; blood Ca2+ levels were inversely correlated with the number of fetuses in this group (r -0.93, P < 0.001). Fetal growth as well as mineralization (assessed by whole-body and femoral DXA, bone histomorphometry and plasma-bone osteocalcin measurements) were unaltered in the low-Ca-P group. In conclusion, fetal mineralization proceeds normally but induces maternal hypocalcaemia in guinea-pigs with dietary restriction of Ca and P.

Pathogenesis of fetal hypomineralization in diabetic rats: evidence for delayed bone maturation.

There is some evidence that fetuses of diabetic rats (FDR) are hypomineralized. To explore the pathogenic role of decreased maternal duodenal Ca absorption, fetal hypotrophy, and decreased placental calbindin-D9K, respectively, spontaneously diabetic rats fed a 1.0% Ca diet were compared with diabetic rats treated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (15 ng/ 100 g) during week 3 of pregnancy, which restored duodenal calbindin-D9K concentrations to normal; with nondiabetic rats semistarved during week 3, which resulted in similar fetal hypotrophy; and with nondiabetic rats fed high cation diets (1.5% Ca-1.5% Sr and 1.5% Ca-3.5% Sr) during week 3, the latter of which repressed duodenal and placental calbindin-D9K toward concentrations measured in diabetic rats. In addition, fetal tibiae were studied histologically. Ca content was lower in 21.5-d-old FDR than in control fetuses. FDR had lower plasma osteocalcin (OC) levels and, on histomorphometry, increased hypertrophic cartilage width, indicating retarded bone maturation. Maternal 1,25(OH)2D3 treatment did not change Ca content and hypertrophic cartilage width in FDR. Fetuses of semistarved rats had plasma OC levels and hypertrophic cartilage width comparable to those of control fetuses. Fetuses of rats fed the 1.5% Ca-3.5% Sr diet were more severely hypomineralized than FDR but had higher plasma OC than both FDR and control fetuses, compatible with fetal Ca deficiency. Whereas diabetic placentas showed weak but homogeneous staining of calbindin-D9K in the labyrinth on immunohistology, degenerative zones were present in placentas of rats fed the 1.5% Ca-3.5% Sr diet. Thus, there is no mineralization defect in FDR caused by disturbed maternal duodenal Ca absorption or transplacental Ca transport, but a delay in bone maturation that is unexplained by their lower body weight.