Equivalent anticancer activities of dietary vitamin D and calcitriol in an animal model of breast cancer: importance of mammary CYP27B1 for treatment and prevention.

Calcitriol [1,25(OH)2D3], the hormonally active form of vitamin D exerts anti-proliferative, pro-apoptotic, anti-inflammatory effects and other anticancer actions in breast cancer (BCa) cell cultures and animal models of BCa. Our research is focused on investigating the potential beneficial effects of dietary vitamin D3 compared to calcitriol and the underlying mechanisms in BCa treatment and chemoprevention. We recently found that dietary vitamin D3 exhibits significant tumor inhibitory effects in xenograft models of BCa that are equivalent to those elicited by the administration of the active hormone calcitriol. At the easily achievable dose tested in our studies, dietary vitamin D3 exhibited substantial tumor inhibitory activity and, unlike calcitriol, did not cause hypercalcemia demonstrating its relative safety. We found elevations in circulating calcitriol as well as increased CYP27B1 expression in the tumor and the intestine in tumor-bearing mice ingesting a vitamin D3-supplemented diet. We hypothesize that the elevation in circulating 25(OH)D induced by dietary vitamin D3 supplements stimulates local synthesis of calcitriol in the mammary tumor microenvironment and the ensuing paracrine/autocrine actions play a major role in the anticancer activity of dietary vitamin D3. Our findings suggest that the endocrine activity of calcitriol derived from tumor and other extra-renal sources such as the intestine, probably also plays a role in mediating the anticancer effects of dietary vitamin D3. Thus it appears that multiple sites of 1α-hydroxylation contribute to the anticancer effects of dietary vitamin D3. Our data strongly suggest that dietary vitamin D will be useful in the chemoprevention and treatment of BCa since it is a safe, economical and easily available nutritional agent that is equivalent to calcitriol in exerting anticancer effects, at least in mouse models. Furthermore, adequate vitamin D nutrition and avoidance of vitamin D deficiency appear to be important in reducing BCa risk. These findings warrant clinical trials in BCa patients and in women at high risk for BCa to evaluate the benefits of dietary vitamin D3 supplementation. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Bone marrow ablation demonstrates that excess endogenous parathyroid hormone plays distinct roles in trabecular and cortical bone.

Mice null for Cyp27b1, which encodes the 25-hydroxyvitamin D-1α-hydroxylase [1α(OH)ase(-/-) mice], lack 1,25-dihydroxyvitamin D [1,25(OH)(2)D] and have hypocalcemia and high parathyroid hormone (PTH) secretion. Intermittent, exogenous PTH is anabolic for bone. To determine the effect of the chronic excess endogenous PTH on osteogenesis and bone turnover, bone marrow ablations (BMX) were performed in tibiae and femurs of 6-week-old 1α(OH)ase(-/-) mice and in wild-type (WT) controls. Newly formed bone tissue was analyzed at 1, 2, and 3 weeks after BMX. BMX did not alter the higher levels of PTH in 1α(OH)ase(-/-) mice. In the marrow cavity, trabecular volume, osteoblast number, alkaline phosphatase-positive areas, type I collagen-positive areas, bone formation-related genes, and protein expression levels all increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. Osteoclast numbers and surface and ratio of RANKL/OPG-relative mRNA levels decreased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. In the cortex, alkaline phosphatase-positive osteoblasts and osteoclast numbers increased significantly after BMX in 1α(OH)ase(-/-) mice, compared with WT. These results demonstrate that chronic excess endogenous PTH exerts an anabolic role in trabecular bone by stimulating osteogenic cells and reducing bone resorption, but plays a catabolic role in cortical bone by enhancing bone turnover with an increase in resorption.

Expanding role for vitamin D in chronic kidney disease: importance of blood 25-OH-D levels and extra-renal 1alpha-hydroxylase in the classical and nonclassical actions of 1alpha,25-dihydroxyvitamin D(3).

Recent advances in the understanding of vitamin D have revolutionized our view of this old nutritional factor and suggested that it has much wider effects on the body than ever believed before. In addition to its well-known effects on calcium/phosphate homeostasis, vitamin D, through its hormonal form, 1alpha,25-dihydroxyvitamin D(3) or calcitriol, is a cell differentiating factor and anti-proliferative agent with actions on a variety of tissues around the body (e.g., skin, muscle, immune system). By influencing gene expression in multiple tissues, calcitriol influences many physiological processes besides calcium/phosphate homeostasis including muscle and keratinocyte differentiation, insulin secretion, blood pressure regulation, and the immune response. The incidence of various diseases including epithelial cancers, multiple sclerosis, muscle weakness as well as bone-related disorders has been correlated with vitamin D deficiency/insufficiency and has led to a re-evaluation of recommended daily intakes both in the normal subject and CKD patient. Critical developments have been the emergence of the value of blood 25-OH-D measurement as a tool in predicting vitamin D-related problems and this has in turn emphasized the importance of the extra-renal version of the 1alpha-hydroxylase, the enzyme responsible for the final step in vitamin D activation. The widespread expression of this extra-renal enzyme supports the view that it exists to boost intracellular concentrations of calcitriol within some target tissues in order to modulate a unique set of genes specifically in those tissues, a process which is therefore dependent upon circulating 25-OH-D. For CKD patients with their tendency to vitamin D substrate insufficiency coupled with their documented loss of the renal 1alpha-hydroxylase in late stages, this new information has profound implications. Physicians must start to manage the vitamin D insufficiency by vitamin D supplements throughout stages 1-5 whilst continuing to provide calcitriol replacement therapy, in the form of calcitriol or its analogs, in stages 3-5.

Exogenous 1,25-dihydroxyvitamin D3 exerts a skeletal anabolic effect and improves mineral ion homeostasis in mice that are homozygous for both the 1alpha-hydroxylase and parathyroid hormone null alleles.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and PTH each modulate calcium and skeletal homeostasis. To identify 1,25(OH)(2)D(3)-mediated skeletal and mineral ion actions independent of PTH, double-knockout mice, which are homozygous for both the 1alpha-hydroxylase and PTH null alleles, were treated with 1,25(OH)(2)D(3), sc, from d 4 to 14 and compared with vehicle-treated animals. Serum calcium rose in 1,25(OH)(2)D(3)-treated double-knockout mice, and messenger RNA and protein levels of the renal calcium transporters TRPV5, calbindin-D(28K), calbindin-D(9K), and Na(+)/Ca(2+) exchanger 1 were up-regulated. Parameters of endochondral bone formation, including long bone length, epiphyseal volume, chondrocyte proliferation and differentiation, and cartilage matrix mineralization, were all increased by 1,25(OH)(2)D(3), Exogenous 1,25(OH)(2)D(3) also increased both trabecular and cortical bone; augmented both osteoblast number and type I collagen deposition in bone matrix; and up-regulated expression levels of the osteoblastic genes alkaline phosphatase, type I collagen, and osteocalcin. Furthermore, in 1,25(OH)(2)D(3)-treated double mutants, osteoclastic bone resorption appeared to decline. The results indicate that administered 1,25(OH)(2)D(3) used intestinal and renal but not skeletal mechanisms to elevate serum calcium and that this sterol can promote endochondral and appositional bone increases independent of endogenous PTH.

Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vitamin D-dependent rickets type 1.

The secosteroid hormone, 1,25-dihydroxyvitamin D [1,25(OH)2D], plays a crucial role in normal bone growth, calcium metabolism, and tissue differentiation. The key step in the biosynthesis of 1,25(OH)2D is its 1 alpha-hydroxylation from 25-hydroxyvitamin D (25-OHD) in the kidney. Because its expression in the kidney is very low, we cloned and sequenced cDNA for 25-OHD-1 alpha-hydroxylase (P450c1 alpha) from human keratinocytes, in which 1 alpha-hydroxylase activity and mRNA expression can be induced to be much greater. P450c1 alpha mRNA was expressed at much lower levels in human kidney, brain, and testis. Mammalian cells transfected with the cloned P450c1 alpha cDNA exhibit robust 1 alpha-hydroxylase activity. The identity of the 1,25(OH)2D3 product synthesized in transfected cells was confirmed by HPLC and gas chromatography-mass spectrometry. The gene encoding P450c1 alpha was localized to chromosome 12, where the 1 alpha-hydroxylase deficiency syndrome, vitamin D-dependent rickets type 1 (VDDR-1), has been localized. Primary cultures of human adult and neonatal keratinocytes exhibit abundant 1 alpha-hydroxylase activity, whereas those from a patient with VDDR-1 lacked detectable activity. Keratinocyte P450c1 alpha cDNA from the patient with VDDR-1 contained deletion/frameshift mutations either at codon 211 or at codon 231, indicating that the patient was a compound heterozygote for two null mutations. These findings establish the molecular genetic basis of VDDR-1, establish a novel means for its study in keratinocytes, and provide the sequence of the key enzyme in the biological activation of vitamin D.

Vitamin D hydroxylases and their regulation in a naturally vitamin D-deficient subterranean mammal, the naked mole rat (Heterocephalus glaber).

The vitamin D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is generated by a series of hydroxylation steps in the liver and kidneys. We investigated whether naturally vitamin D-deficient subterranean mammals (naked mole rats, Heterocephalus glaber) employ the same enzymatic pathways, and whether these are regulated in a similar manner to that established for other mammals. Vitamin D3-25-hydroxylase in the liver and both 25-hydroxyvitamin D3-1-hydroxylase and 25-hydroxyvitamin D3-24 hydroxylase (1-OHase and 24-OHase) in the kidney were detectable in mole rats. As expected for vitamin D-deficient mammals, the 1-OHase activity predominated over the 24-OHase. After mole rats received a supraphysiological supplement of vitamin D3, 1-OHase activity was suppressed and 24-OHase activity was enhanced. Irrespective of vitamin D status, forskolin (a protein kinase A activator) and dibutyryl cyclic AMP did not alter the activity of either 1-OHase or 24-OHase. These findings suggest that the response of renal hydroxylases to parathyroid hormone was blunted. Phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol (OAG) (protein kinase C activators), suppressed 1-OHase activity. 24-OHase activity was induced by TPA but not by OAG. These effects were similar to those illicited by vitamin D3 supplementation but were additive in that they increased the responses shown in vitamin D-replete mole rats. These data confirm that naturally vitamin D-deficient mole rats can convert vitamin D3 to the hormone, 1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)