Regulation of renal vitamin D receptor is an important determinant of 1alpha,25-dihydroxyvitamin D(3) levels in vivo

The synthesis of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is most strongly regulated by dietary calcium and the action of parathyroid hormone to increase 1alpha-hydroxylase (1alpha-OHase) and decrease 24-hydroxylase (24-OHase) in kidney proximal tubules. This study examines the hypothesis that 1,25-(OH)(2)D(3) synthesis, induced by dietary calcium restriction, is also the result of negative feedback regulation blockade. Rats fed a low calcium (0.02%, -Ca) diet and given daily oral doses of vitamin D (0, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 microg) remained hypocalcemic despite increasing levels of serum calcium in relation to the vitamin D dose. Plasma levels of 1,25-(OH)(2)D(3) rose to high levels (1200 pg/ml) at the high vitamin D dose levels. As expected, thyroparathyroidectomy caused a rapid fall in serum 1,25-(OH)(2)D(3). In rats fed a 0.47% calcium diet (+Ca) supplemented with vitamin D (4 microg/day), exogenous 1,25-(OH)(2)D(3) suppressed renal 1alpha-OHase and stimulated the 24-OHase. In rats fed the -Ca diet, vitamin D was unable to suppress the renal 1alpha-OHase or stimulate the renal 24-OHase. In contrast, vitamin D was fully able to stimulate intestinal 24-OHase. Intestinal vitamin D receptor (VDR) was present under all circumstances, while kidney VDR was absent under hypocalcemic conditions and present under normocalcemic conditions. It appears that tissue-specific down-regulation of VDR by hypocalcemia blocks the 1,25-(OH)(2)D(3) suppression of the 1alpha-OHase and upregulation of the 24-OHase in the kidney, causing a marked accumulation of 1,25-(OH)(2)D(3) in the plasma.     

Conventional and tissue-specific inactivation of the 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1)

Mutations in the human 25-hydroxyvitamin-D(3)-1alpha-hydroxylase (CYP27B1) gene cause pseudo vitamin D deficiency rickets (PDDR). The kidney is the main site of expression of the CYP27B1 gene, but expression has been documented in other cell types, including chondrocytes. We engineered a tissue-specific and a conventional knockout of CYP27B1 in mice. The conventional knockout strain reproduced the PDDR phenotype. Homozygote mutant animals were treated with 1,25(OH)(2)D(3) or fed a high-calcium diet (2% calcium, 1.25% phosphate, 20% lactose) for 5 weeks post-weaning. Blood biochemistry revealed that both rescue treatments corrected the hypocalcemia and secondary hyperparathyroidism. Bone histomorphometry confirmed that rickets were cured. The rescue regimen restored the biomechanical properties of the bone tissue. Mice carrying the loxP-bearing allele were bred to transgenic animals expressing the Cre recombinase in chondrocytes under the control of the collagen type II promoter. Genotyping confirmed excision of exon 8 in chondrocytes. Serum biochemistry revealed that mineral ion homeostasis is normal in mutant animals. Preliminary observation of bone tissue from mutant mice did not reveal major changes to the growth plate. Precise histomorphometric analysis will be required to assess the impact of chondrocyte-specific inactivation of CYP27B1 on the maturation and function of growth plate cells in vivo.     

1,25-Dihydroxyvitamin D₃ contributes to regulating mammary calcium transport and modulates neonatal skeletal growth and turnover cooperatively with calcium

To assess the interaction of 1,25(OH)(2)D(3) and dietary calcium on mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate, female lactating 1α(OH)ase(+/-) or 1α(OH)ase(-/-) mice were fed either a high-calcium diet containing 1.5% calcium in the drinking water or a "rescue diet." Dietary effects on the expression of molecules mediating mammary calcium transport were determined in the dams, and the effects of milk calcium content were assessed on skeletal growth and turnover in 2-wk-old 1,25(OH)(2)D(3)-deficient pups. Results showed that the reduction of milk calcium levels in the 1α(OH)ase(-/-) dams and the elevation of milk calcium levels in dams fed the rescue diet were associated with the down- or upregulation of calbindin D(9k) and plasma membrane Ca(2+) ATPase isoform 2b expression, respectively, in mammary epithelial cells. The action of ambient calcium in stimulating skeletal growth in the neonates appeared to supercede the direct action of 1,25(OH)(2)D(3), and the response of chondrocytes in the neonates to elevated calcium was more sensitive in hypocalcemic animals. Osteopenia was more apparent in pups nursed by dams with lower milk calcium than in 1,25(OH)(2)D(3)-deficient pups nursed by dams with higher milk calcium. Bone formation parameters were increased significantly in all pups fed by dams on the rescue diet but were still lower in 1α(OH)ase(-/-) pups than in 1α(OH)ase(+/-) pups. Consequently, there is an important contributory role of calcium in conjunction with 1,25(OH)(2)D(3) to mammary calcium transport in lactating dams and skeletal growth and turnover in the neonate.     

Effects of calcium and of the Vitamin D system on skeletal and calcium homeostasis: lessons from genetic models

Targeted deletion of genes encoding the 1,25-dihydroxyVitamin D [1,25(OH)(2)D]-synthesizing enzyme, 25 hydroxyVitamin D-1alpha-hydroxylase [1alpha(OH)ase or CYP27B1], and of the nuclear receptor for 1,25(OH)(2)D, the Vitamin D receptor (VDR), have provided useful mouse models of the inherited human diseases, Vitamin D-dependent rickets types I and II. We employed these models and double null mutants to examine the effects of calcium and of the 1,25(OH)(2)D/VDR system on skeletal and calcium homeostasis. Optimal dietary calcium absorption required both 1,25(OH)(2)D and the VDR. Skeletal mineralization was dependent on adequate ambient calcium but did not directly require the 1,25(OH)(2)D/VDR system. Parathyroid hormone (PTH) secretion was also modulated primarily by ambient serum calcium but the enlarged parathyroid glands which the mutants exhibited and the widened cartilaginous growth plates could only be normalized by the combination of calcium and 1,25(OH)(2)D, apparently independently of the VDR. Optimal osteoclastic bone resorption and osteoblastic bone formation both required an intact 1,25(OH)(2)D/VDR apparatus. The results indicate that calcium cannot entirely substitute for Vitamin D in skeletal and mineral homeostasis but that the two agents have discrete and overlapping functions.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

Recent advances in our understanding of 1,25-dihydroxyvitamin D(3) regulation of intestinal calcium absorption

Calcium is required for many cellular processes including muscle contraction, nerve pulse transmission, stimulus secretion coupling and bone formation. The principal source of new calcium to meet these essential functions is from the diet. Intestinal absorption of calcium occurs by an active transcellular path and by a non-saturable paracellular path. The major factor influencing intestinal calcium absorption is vitamin D and more specifically the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). This article emphasizes studies that have provided new insight related to the mechanisms involved in the intestinal actions of 1,25(OH)(2)D(3). The following are discussed: recent studies, including those using knock out mice, that suggest that 1,25(OH)(2)D(3) mediated calcium absorption is more complex than the traditional transcellular model; evidence for 1,25(OH)(2)D(3) mediated active transport of calcium by distal as well as proximal segments of the intestine; 1,25(OH)(2)D(3) regulation of paracellular calcium transport and the role of 1,25(OH)(2)D(3) in protection against mucosal injury.     

Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis

We employed a genetic approach to determine whether deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D) and deficiency of the vitamin D receptor (VDR) produce the same alterations in skeletal and calcium homeostasis and whether calcium can subserve the skeletal functions of 1,25(OH)2D and the VDR. Mice with targeted deletion of the 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha(OH)ase-/-) gene, the VDR gene, and both genes were exposed to 1) a high calcium intake, which maintained fertility but left mice hypocalcemic; 2) this intake plus three times weekly injections of 1,25(OH)2D3, which normalized calcium in the 1alpha(OH)ase-/- mice only; or 3) a "rescue" diet, which normalized calcium in all mutants. These regimens induced different phenotypic changes, thereby disclosing selective modulation by calcium and the vitamin D system. Parathyroid gland size and the development of the cartilaginous growth plate were each regulated by calcium and by 1,25(OH)2D3 but independent of the VDR. Parathyroid hormone secretion and mineralization of bone reflected ambient calcium levels rather than the 1,25(OH)2D/VDR system. In contrast, increased calcium absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by the 1,25(OH)2D/VDR system. These studies indicate that the calcium ion and the 1,25(OH)2D/VDR system exert discrete effects on skeletal and calcium homeostasis, which may occur coordinately or independently.     

Pathogenic role of Fgf23 in Dmp1-null mice

Autosomal recessive hypophosphatemic rickets (ARHR), which is characterized by renal phosphate wasting, aberrant regulation of 1alpha-hydroxylase activity, and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). ARHR resembles autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemia (XLH), hereditary disorders respectively caused by cleavage-resistant mutations of the phosphaturic factor FGF23 and inactivating mutations of PHEX that lead to increased production of FGF23 by osteocytes in bone. Circulating levels of FGF23 are increased in ARHR and its Dmp1-null mouse homologue. To determine the causal role of FGF23 in ARHR, we transferred Fgf23 deficient/enhanced green fluorescent protein (eGFP) reporter mice onto Dmp1-null mice to create mice lacking both Fgf23 and Dmp1. Dmp1(-/-) mice displayed decreased serum phosphate concentrations, inappropriately normal 1,25(OH)(2)D levels, severe rickets, and a diffuse form of osteomalacia in association with elevated Fgf23 serum levels and expression in osteocytes. In contrast, Fgf23(-/-) mice had undetectable serum Fgf23 and elevated serum phosphate and 1,25(OH)(2)D levels along with severe growth retardation and focal form of osteomalacia. In combined Dmp1(-/-)/Fgf23(-/-), circulating Fgf23 levels were also undetectable, and the serum levels of phosphate and 1,25(OH)(2)D levels were identical to Fgf23(-/-) mice. Rickets and diffuse osteomalacia in Dmp1-null mice were transformed to severe growth retardation and focal osteomalacia characteristic of Fgf23-null mice. These data suggest that the regulation of extracellular matrix mineralization by DMP1 is coupled to renal phosphate handling and vitamin D metabolism through a DMP1-dependent regulation of FGF23 production by osteocytes.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection

The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the effect of 17beta-estradiol (17beta-E2), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and dietary Ca2+ on the expression of the duodenal Ca2+ transport proteins was investigated in vivo and analyzed using realtime quantitative PCR. Supplementation with 17beta-E2 increased duodenal gene expression of TRPV5 and TRPV6 but also calbindin-D9K and plasma membrane Ca2+-ATPase (PMCA1b) in ovariectomized rats. 25-Hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) knockout mice are characterized by hyperparathyroidism, rickets, hypocalcemia, and undetectable levels of 1,25(OH)2D3 and were used to study the 1,25(OH)2D3-dependency of the stimulatory effects of 17beta-E2. Treatment with 17beta-E2 upregulated mRNA levels of duodenal TRPV6 in these 1alpha-OHase knockout mice, which was accompanied by increased serum Ca2+ concentrations from 1.69 +/- 0.10 to 2.03 +/- 0.12 mM (P < 0.05). In addition, high dietary Ca2+ intake normalized serum Ca2+ in these mice and upregulated expression of genes encoding the duodenal Ca2+ transport proteins except for PMCA1b. Supplementation with 1,25(OH)2D3 resulted in increased expression of TRPV6, calbindin-D9K, and PMCA1b and normalization of serum Ca2+. Expression levels of duodenal TRPV5 mRNA are below detection limits in these 1alpha-OHase knockout mice, but supplementation with 1,25(OH)2D3 upregulated the expression to significant levels. In conclusion, TRPV5 and TRPV6 are regulated by 17beta-E2 and 1,25(OH)2D3, whereas dietary Ca2+ is positively involved in the regulation of TRPV6 only.     

Extra-renal 25-hydroxyvitamin D3-1alpha-hydroxylase in human health and disease

Although ectopic expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase) has been recognized for many years, the precise function of this enzyme outside the kidney remains open to debate. Three specific aspects of extra-renal 1alpha-OHase have attracted most attention: (i) expression and regulation in non-classical tissues during normal physiology; (ii) effects on the immune system and inflammatory disease; (iii) expression and function in tumors. The most well-recognized manifestation of extra-renal 1alpha-OHase activity remains that found in some patients with granulomatous diseases where locally synthesized 1alpha,25(OH)(2)D(3) has the potential to spill-over into the general circulation. However, immunohistochemistry and mRNA analyses suggest that 1alpha-OHase is also expressed by a variety of normal human tissues including the gastrointestinal tract, skin, vasculature and placenta. This has promoted the idea that autocrine/paracrine synthesis of 1,25(OH)(2)D(3) contributes to normal physiology, particularly in mediating the potent effects of vitamin D on innate (macrophage) and acquired (dendritic cell) immunity. We have assessed the capacity for synthesis of 1,25(OH)(2)D(3) in these cells and the functional significance of autocrine responses to 1alpha-hydroxylase. Data suggest that local synthesis of 1,25(OH)(2)D(3) may be a preferred mode of response to antigenic challenge in many tissues.     

Intestinal calcium absorption: Molecular vitamin D mediated mechanisms

Rickets and hyperparathyroidism caused by a defective Vitamin D receptor (VDR) can be prevented in humans and animals by high calcium intake, suggesting that intestinal calcium absorption is critical for 1,25(OH)(2) vitamin D [1,25-(OH)(2)D(3)] action on calcium homeostasis. We assessed the rate of serum (45)Ca accumulation within 10 min after oral gavage in two strains of VDR-knock out (KO) mice (Leuven and Tokyo KO) and observed a threefold lower area under the curve in both KO-strains. Moreover, we evaluated the expression of intestinal candidate genes, belonging to a new class of calcium channels (TRPV), involved in transcellular calcium transport. The calcium transport protein ECaC2 was more abundantly expressed at mRNA level than ECaC1 in duodenum, but both were considerably reduced (ECaC2 > 90%, ECaC1 > 60%) in the two VDR-KO strains on a normal calcium diet. Calbindin-D(9K) expression was only significantly decreased in the Tokyo KO, whereas PMCA(1b) expression was normal in both VDR-KOs. In Leuven wild type mice, a high calcium diet inhibited (> 90%), and 1,25(OH)(2)D(3) or low calcium diet induced (sixfold) duodenal ECaC2 expression and, to a lesser degree, ECaC1 and calbindin-D(9K) expression. In Leuven KO mice, however, high or low calcium intake decreased calbindin-D(9K) and PMCA(1b) expression, whereas both ECaC mRNA expressions remained consistently low on any diet. These results suggest that the expression of the novel duodenal epithelial calcium channels (in particular ECaC2 or TRPV6) is strongly vitamin D dependent and that calcium influx, probably interacting with calbindin-D(9K), should be considered as a rate-limiting step in the process of vitamin D dependent active calcium absorption.     

Vitamin D: A millenium perspective

Vitamin D is one of the oldest hormones that have been made in the earliest life forms for over 750 million years. Phytoplankton, zooplankton, and most plants and animals that are exposed to sunlight have the capacity to make vitamin D. Vitamin D is critically important for the development, growth, and maintenance of a healthy skeleton from birth until death. The major function of vitamin D is to maintain calcium homeostasis. It accomplishes this by increasing the efficiency of the intestine to absorb dietary calcium. When there is inadequate calcium in the diet to satisfy the body's calcium requirement, vitamin D communicates to the osteoblasts that signal osteoclast precursors to mature and dissolve the calcium stored in the bone. Vitamin D is metabolized in the liver and then in the kidney to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. 1,25(OH)(2)D receptors (VDR) are present not only in the intestine and bone, but in a wide variety of other tissues, including the brain, heart, stomach, pancreas, activated T and B lymphocytes, skin, gonads, etc. 1,25(OH)(2)D is one of the most potent substances to inhibit proliferation of both normal and hyperproliferative cells and induce them to mature. It is also recognized that a wide variety of tissues, including colon, prostate, breast, and skin have the enzymatic machinery to produce 1,25(OH)(2)D. 1,25(OH)(2)D and its analogs have been developed for treating the hyperproliferative disease psoriasis. Vitamin D deficiency is a major unrecognized health problem. Not only does it cause rickets in children, osteomalacia and osteoporosis in adults, but may have long lasting effects. Chronic vitamin D deficiency may have serious adverse consequences, including increased risk of hypertension, multiple sclerosis, cancers of the colon, prostate, breast, and ovary, and type 1 diabetes. There needs to be a better appreciation of the importance of vitamin D for overall health and well being.     

25-Hydroxyvitamin D-1alpha-hydroxylase activity is diminished in human prostate cancer cells and is enhanced by gene transfer

The hormone 1alpha,25-dihydroxyvitamin D (1alpha,25(OH)(2)D) inhibits growth and induces differentiation of prostate cells. The enzyme responsible for 1alpha,25(OH)(2)D synthesis, 25-hydroxyvitamin D (25(OH)D)-1alpha-hydroxylase (1alpha-OHase), has been demonstrated in human prostate cells. We compared the levels of 1alpha-OHase activity in prostate cancer cell lines, LNCaP, DU145 and PC-3 and in primary cultures of normal, cancerous and benign prostatic hyperplasia (BPH) prostate cells. We observed a marked decrease in 1alpha-OHase activity in prostate cancer cells, including an undetectable level of activity in LNCaP cells. Transient or stable transfection of 1alpha-OHase cDNA into LNCaP cells increased 1alpha-OHase activity from undetectable to 4.95pmole/mg+/-0.69pmole/mg and 5.8pmole/mg+/-0.7pmole/mg protein per hour, respectively. In response to 25(OH)D, the prohormone of 1alpha,25(OH)(2)D, the transfected LNCaP cells showed a significant inhibition of 3H-thymidine incorporation (37%+/-6% and 56%+/-4% at 10(-8)M for transiently and stably transfected cells, respectively). These findings support an important autocrine role for 1alpha,25(OH)(2)D in the prostate and suggest that the re-introduction of the 1alpha-OHase gene to prostate cancer cells, in conjunction with the systemic administration of 25(OH)D, constitutes an endocrine form of gene therapy that may be less toxic than the systemic administration of 1alpha,25(OH)(2)D.     

Effects of vitamin K2 administration on calcium balance and bone mass in young rats fed normal or low calcium diet

OBJECTIVE: The purpose of this study was to examine the effects of vitamin K2 administration on calcium balance and bone mass in young rats fed a normal or low calcium diet. METHODS: Forty female Sprague-Dawley rats, 6 weeks of age, were randomized by stratified weight method into four groups with 10 rats in each group: 0.5% (normal) calcium diet, 0.1% (low) calcium diet, 0.5% calcium diet + vitamin K2 (menatetrenone, 30 mg/100 g chow diet), and 0.1% calcium diet + vitamin K2. After 10 weeks of feeding, serum calcium and calciotropic hormone levels were measured, and intestinal calcium absorption and renal calcium reabsorption were evaluated. Bone histomorphometric analyses were performed on cortical bone of the tibial shaft and cancellous bone of the proximal tibia. RESULTS: Feeding a low calcium diet induced hypocalcemia, increased serum parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D] levels with decreased serum 25-hydrovyvitamin D [25(OH)D] level, stimulated intestinal calcium absorption and renal calcium reabsorption, and reduced cortical bone mass as a result of decreased periosteal bone gain and enlarged marrow cavity, but did not significantly influence cancellous bone mass. Vitamin K2 administration in rats fed a low calcium diet stimulated renal calcium reabsorption, retarded the abnormal elevation of serum PTH level, increased cancellous bone mass, and retarded cortical bone loss, while vitamin K2 administration in rats fed a normal calcium diet stimulated intestinal calcium absorption by increasing serum 1,25(OH)2D level, and increased cortical bone mass. CONCLUSION: This study clearly shows the differential response of calcium balance and bone mass to vitamin K2 administration in rats fed a normal or low calcium diet.     

Role of the vitamin D-endocrine system in the pathophysiology of postmenopausal osteoporosis

Impaired calcium absorption and impaired adaptation to a low calcium diet are common features of aging in women and these processes are even more severely impaired in patients with osteoporotic fractures. The calcium absorption defects are associated with several abnormalities of the vitamin D-endocrine system including secondary hyperparathyroidism, intestinal resistance to 1,25-dihydroxyvitamin D (1,25(OH)(2)D) action, decreased 1,25(OH)(2)D production due to impaired 25(OH)D 1alpha-hydroxylase activity, and, in some elderly persons, nutritional deficiency of vitamin D. However, in postmenopausal women, most of these abnormalities are normalized by administration of physiologic replacement dosages of estrogen and, thus, appear to be secondary consequences of estrogen deficiency. Nonetheless, a minority of them, especially nutritional vitamin D deficiency and impaired 25(OH)D 1alpha-hydroxylase activity late in life, appear to be primary and are independent of estrogen deficiency.     

Gene structure and regulation of the murine epithelial calcium channels ECaC1 and 2.

The recently discovered epithelial calcium channels ECaC1 and ECaC2 are thought to play an important role in active calcium absorption in the intestine and kidney. Vitamin D-responsive elements (VDRE) were detected in the promoter sequence of human ECaC1 and regulation of ECaC by the steroid hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) has been postulated. In this study we describe the structure of two murine ECaCs genes, each consisting of 15 exons localized on chromosome 6. Murine ECaC2 expression was found in many target tissues of 1,25-(OH)(2)D(3), including skin and osteoblastic cells, while ECaC1 expression is confined to the kidney. By screening the murine promoter sequences, we detected a putative VDRE in ECaC1 and an estrogen response element in ECaC2. However, experiments in mice with a mutant, nonfunctioning vitamin D receptor showed that expression of ECaC1 in the kidney and of ECaC2 in duodenum is regulated by calcium levels, but not by 1,25-(OH)(2)D(3). Also, estrogen-deficient ovariectomized (OVX) mice and OVX mice supplemented with estradiol showed unchanged duodenal ECaC2 expression compared with control mice. We conclude that ECaC expression in the kidney and the intestine is regulated by extracellular calcium but not by vitamin D or estrogen in vivo in mice.