Vitamin D receptor (VDR) and metabolizing enzymes CYP27B1 and CYP24A1 in breast cancer.

Vitamin D Receptor (VDR), a nuclear steroid receptor, is a transcription factor with a primary physiologic role in calcium metabolism. It has also a physiologic role in breast tissues during development of the gland and postpartum. In addition, it is commonly expressed in breast cancer and has tumor suppressive effects. Cytochrome enzymes CYP27B1 and CYP24A1 that perform the final conversion of the circulating form of vitamin D, 25-hydroxyvitamin D (25-OHD) to the active VDR ligand, 1a,25-dihydroxyvitamin D and the catabolism of it to inactive 24,25-dihydroxyvitamin D, respectively, are also expressed in breast cancer tissues. Defective regulation of the receptor and the metabolic enzymes of VDR ligand is prevalent in breast cancer and leads to decreased VDR signaling. The expression and molecular defects of VDR, CYP27B1 and CYP24A1 that perturb physiologic function, the implications for breast cancer progression and therapeutic opportunities are discussed in this paper.

Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse.

Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

Absence of vitamin D receptor in mature osteoclasts results in altered osteoclastic activity and bone loss.

Mature osteoclasts express the vitamin D receptor (VDR) and are able to synthesise and respond to 1,25(OH)2D3 via CYP27B1 enzyme activity. Whether vitamin D signalling within osteoclasts is necessary for the regulation of osteoclastic bone resorption in an in vivo setting is unclear. To determine the requirement for the VDR- and CYP27B1-mediated activity in mature osteoclasts, conditional deletion mouse models were created whereby either Vdr or Cyp27b1 gene was inactivated by breeding either Vdrfl/fl or Cyp27b1fl/fl mice with Cathepsin K-Cre transgenic mice (CstkCre) to generate CtskCre/Vdr-/- and CtskCre/Cyp27b1-/- mice respectively. To account for potential CtskCre-meaited off-target deletion of Vdr, Dmp1Cre were also used determine the effect of Vdr deletion in osteocytes. Furthermore, CtskCre/Vdr-/- mice were ovariectomised (OVX) to assess the role of VDR in osteoclasts under bone-loss conditions and bone marrow precursor cells were cultured under osteoclastogenic conditions to assess osteoclast formation. Six-week-old CtskCre/Vdr-/- female mice demonstrated a 15% decrease in femoral BV/TV (p<0.05). In contrast, BV/TV remained unchanged in CtskCre/Cyp27b1-/- mice as well as in Dmp1Cre/VDR-/- mice. When CtskCre/Vdr-/- mice were subjected to OVX, the bone loss that occurred in CtskCre/Vdr-/- was predominantly due to a diminished volume of thinner trabeculae when compared to control levels. These changes in bone volume in CtskCre/Vdr-/- mice occurred without an observable histological change in osteoclast numbers or size. However, while cultured bone marrow-derived osteoclasts from CtskCre/Vdr-/- mice were marginally increased when compared to VDRfl/fl mice, elevated expression of genes such as Cathepsin K, Nfatc1 and VATPase was observed. Collectively, these data indicate that the absence of VDR in mature osteoclasts causes exacerbated bone loss in young mice and during OVX which is associated with enhanced osteoclastic activity and without increased osteoclastogenesis.

Endogenous Calcitriol Synthesis Controls the Humoral IgE Response in Mice.

The vitamin D receptor participates in the control of IgE class-switch recombination in B cells. The physiologic vitamin D receptor agonist, 1,25(OH)2D3 (calcitriol), is synthesized by the essential enzyme 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1), which can be expressed by activated immune cells. The role of endogenous calcitriol synthesis for the regulation of IgE has not been proven. In this study, we investigated IgE-responses in Cyp27b1-knockout (KO) mice following sensitization to OVA or intestinal infection with Heligmosomoides polygyrus Specific Igs and plasmablasts were determined by ELISA and ELISpot, Cyp27b1 expression was measured by quantitative PCR. The data show elevated specific IgE and IgG1 concentrations in the blood of OVA-sensitized Cyp27b1-KO mice compared with wild-type littermates (+898 and +219%). Accordingly, more OVA-specific IgG1-secreting cells are present in spleen and fewer in the bone marrow of Cyp27b1-KO mice. Ag-specific mechanisms are suggested as the leucopoiesis is in general unchanged and activated murine B and T lymphocytes express Cyp27b1 Accordingly, elevated specific IgE concentrations in the blood of sensitized T cell-specific Cyp27b1-KO mice support a lymphocyte-driven mechanism. In an independent IgE-inducing model, i.e., intestinal infection with H. polygyrus, we validated the increase of total and specific IgE concentrations of Cyp27b1-KO compared with wild-type mice, but not those of IgG1 or IgA. We conclude that endogenous calcitriol has an impact on the regulation of IgE in vivo. Our data provide genetic evidence supporting previous preclinical and clinical findings and suggest that vitamin D deficiency not only promotes bone diseases but also type I sensitization.

Effects of the Administration of 25(OH) Vitamin D3 in an Experimental Model of Chronic Kidney Disease in Animals Null for 1-Alpha-Hydroxylase.

The final step in vitamin D activation is catalyzed by 1-alpha-hydroxylase (CYP27B1). Chronic kidney disease (CKD) is characterized by low levels of both 25(OH)D3 and 1,25(OH)2D3 provoking secondary hyperparathyroidism (2HPT). Therefore, treatments with active or native vitamin D compounds are common in CKD to restore 25(OH)D3 levels and also to decrease PTH. This study evaluates the dose of 25(OH)D3 that restores parathyroid hormone (PTH) and calcium levels in a model of CKD in CYP27B1-/- mice. Furthermore, we compare the safety and efficacy of the same dose in CYP27B1+/+ animals. The dose needed to decrease PTH levels in CYP27B1-/- mice with CKD was 50 ng/g. That dose restored blood calcium levels without modifying phosphate levels, and increased the expression of genes responsible for calcium absorption (TRPV5 and calbindinD- 28K in the kidney, TRPV6 and calbindinD-9k in the intestine). The same dose of 25(OH)D3 did not modify PTH in CYP27B1+/+ animals with CKD. Blood calcium remained normal, while phosphate increased significantly. Blood levels of 25(OH)D3 in CYP27B1-/- mice were extremely high compared to those in CYP27B1+/+ animals. CYP27B1+/+ animals with CKD showed increases in TRPV5, TRPV6, calbindinD-28K and calbindinD-9K, which were not further elevated with the treatment. Furthermore, CYP27B1+/+ animals displayed an increase in vascular calcification. We conclude that the dose of 25(OH)D3 effective in decreasing PTH levels in CYP27B1-/- mice with CKD, has a potentially toxic effect in CYP27B1+/+ animals with CKD.

Inferences from genetically modified mouse models on the skeletal actions of vitamin D.

Vitamin D has pleiotropic extra-skeletal effects which have been noted in mouse models of deletion of either the 25-hydroxy vitamin D 1α-hydroxylase enzyme, cyp27b1 (1OHase(-/-) mice) or of the vitamin D receptor (Vdr(-/-) mice); these may be preventable or reversible by either restoring normal signaling of the 1,25(OH)2D/VDR system, or in some cases by restoring normal mineral homeostasis. However, effects on skeletal and mineral homeostasis are clearly the major phenotype observed in humans with loss-of-function mutations in either CYP27B1 or VDR. In mouse phenocopies of these human disorders, correction of hypocalcemia and hypophosphatemia reduce elevated circulating parathyroid hormone concentrations and normalize impaired bone mineralization, but restoration of normal 1,25(OH)2D/VDR signaling may be required for optimal bone formation. Induction of high endogenous 1,25(OH)2D concentrations in genetically modified mouse models may cause increased bone resorption and decreased mineralization. Transgenic Vdr overexpression and conditional Vdr deletion in cells of the osteoblastic lineage have also provided insights into the stages of osteoblast differentiation which may mediate these actions. These anabolic and catabolic effects of the 1,25(OH)2D system on bone may therefore be a function of both the ambient concentration of circulating 1,25(OH)2D and the stage of differentiation of the osteoblast. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

25-Hydroxyvitamin D3 1-α-hydroxylase-dependent stimulation of renal klotho expression by spironolactone.

BACKGROUND: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. METHODS: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). RESULTS: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. CONCLUSION: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor.

Vitamin D action: lessons from VDR and Cyp27b1 null mice.

Even one century after its discovery, there are still many gaps in the understanding of the vitamin D endocrine system. Inactivation of the vitamin D receptor (VDR) or the enzymes metabolizing its ligand (especially Cyp27bl) in mice has clearly demonstrated that the active form of vitamin D [1,25(OH)2D] is essential to stimulate calcium absorption in the gut during normal/low calcium intake, and as a consequence, that 1,25(OH)2D is required to maintain normal serum calcium, bone and growth plate homeostasis. These findings have resulted in clear clinical guidelines for the treatment of vitamin D-related bone diseases of infants, children and adults. Tissue-specific VDR or Cyp27b1 deletion in mice has also proven to be useful to define the precise role of 1,25(OH)2D action in cells belonging to the intestine, bone, growth plate and also to many non-classical target tissues. Indeed, experimental findings show that 1,25(OH)2D has numerous extraskeletal effects, and observational studies in man demonstrate that disturbances in the vitamin D pathway are associated with major human diseases such as cancer, infections, autoimmune diseases, cardiovascular and metabolic diseases, muscle function, reproduction and neurocognitive disorders. We will compare the findings in VDR- and Cyp27bi-null mice with findings in man to elucidate what is presently understood of the vitamin D endocrine system and to identify the still outstanding questions.

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.

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy.

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae-infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.

[Frontiers in vitamin D; basic research and clinical application. Vitamin D regulation of macrophage -dependent innate immunity].

Among various pleiotropic actions of vitamin D, its immunomodulatory role has been well recognized for more than 30 years. However, despite the presence of vitamin D receptors in immune cells including lymphocytes and monocytes/macrorphages, the impact of vitamin D system on immune regulation remained unclear. Recently, two key functions of vitamin D : induction of antimicrobial peptides and autophagy in cells of the monocyte/macrophage lineage, have been described, revealing a much clearer picture of the role of vitamin D as an immunomodulator, particularly in innate immunity against pathogens such as Mycobacterium tuberculosis. This review summarizes recent advances in research of vitamin D as a regulator of innate immunity, and clinical trials of vitamin D as a potential anti-infectious agent.

Vitamin D metabolism and action in the prostate: implications for health and disease.

Prostate cancer (PCa) is the second most common cancer in men worldwide. Epidemiological, molecular, and cellular studies have implicated vitamin D deficiency as a risk factor for the development and/or progression of PCa. Studies using cell culture systems and animal models suggest that vitamin D acts to reduce the growth of PCa through regulation of cellular proliferation and differentiation. However, although preclinical studies provide a strong indication for anti-cancer activity, proof of therapeutic benefits in men is still lacking. The anti-proliferative and pro-differentiating properties of vitamin D have been attributed to calcitriol [1,25(OH)(2)D(3)], the hormonally active form of vitamin D, acting through the vitamin D receptor (VDR). Metabolism of vitamin D in target tissues is mediated by two key enzymes: 1α-hydroxylase (CYP27B1), which catalyzes the synthesis of calcitriol from 25(OH)D and 24-hydroxylase (CYP24), which catalyzes the initial step in the conversion of calcitriol to less active metabolites. Many factors affect the balance of calcitriol synthesis and catabolism and several maneuvers, like combination therapy of calcitriol with other drugs, have been explored to treat PCa and reduce its risk. The current paper is an overview addressing some of the key factors that influence the biological actions of vitamin D and its metabolites in the treatment and/or prevention of PCa.

The role of vitamin D receptor mutations in the development of alopecia.

Hereditary Vitamin D Resistant Rickets (HVDRR) is a rare disease caused by mutations in the vitamin D receptor (VDR). The consequence of defective VDR is the inability to absorb calcium normally in the intestine. This leads to a constellation of metabolic abnormalities including hypocalcemia, secondary hyperparathyroidism and hypophosphatemia that cause the development of rickets at an early age in affected children. An interesting additional abnormality is the presence of alopecia in some children depending on the nature of the VDR mutation. The data indicate that VDR mutations that cause defects in DNA binding, RXR heterodimerization or absence of the VDR cause alopecia while mutations that alter VDR affinity for 1,25(OH)(2)D(3) or disrupt coactivator interactions do not cause alopecia. The cumulative findings indicate that hair follicle cycling is dependent on unliganded actions of the VDR. Further research is ongoing to elucidate the role of the VDR in hair growth and differentiation.

Mutation prediction by PolyPhen or functional assay, a detailed comparison of CYP27B1 missense mutations.

Vitamin D-dependent rickets type 1 (VDDR-I) is caused by mutation in CYP27B1. The glycine residue at codon 102 is not conserved between human (G(102)) and rodent (S(102)). G102E mutation results in 80% reduction in its enzymatic activity but PolyPhen predicts benign change. It is not known whether G102S has any damaging effect on 1α-hydroxylase activity. We investigated the effect of CYP27B1 (G102S) on its enzymatic activity and compared mutation prediction accuracy for all known CYP27B1 mutations among three free online protein prediction programs: PolyPhen, PolyPhen-2, and PSIPRED. G102S has no damaging effect on 1α-hydroxylase activity. G102D retained 30% enzymatic activity. All three programs correctly predicted damaging change for G102D. PolyPhen predicted benign change for G102S, whereas PolyPhen-2 and PSIPRED indicated possible damaging effect. Among 24 reported damaging mutations, PSIPRED, PolyPhen-2, and PolyPhen achieved 100%, 91.7% (22/24), and 75% (18/24) accuracy rate, respectively. The residues of incorrectly predicted mutations were not conserved. We conclude that G102D resulted in a significant reduction in 1α-hydroxylase activity, whereas G102S did not. PSIPRED and PolyPhen-2 are superior to PolyPhen in predicting damaging mutations.

Vitamin D and immune function: understanding common pathways.

Vitamin D, acting through its active metabolite 1,25(OH)(2)D(3), exerts its influence on many physiologic processes in addition to the regulation of calcium and phosphate homeostasis. These processes include the immune system. Both the adaptive and innate immune systems are affected by 1,25(OH)(2)D(3) and its receptor, and the cells involved express not only the vitamin D receptor but also, in most cases, the enzyme CYP27B1, which produces 1,25(OH)(2)D(3). Both the vitamin D receptor and CYP27B1 can be constitutive or induced by the ligands that activate the immune processes in these cells, providing feedback loops that help regulate the immune response. In general, 1,25(OH)(2)D(3) suppresses most elements of the adaptive immune system while inducing most elements of the innate immune system. Thus 1,25(OH)(2)D(3) may be protective against various autoimmune diseases and may limit graft rejection by suppressing adaptive immunity while enhancing the first line of defense against invading microorganisms via upregulation of innate immunity.

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.

Vitamin D in defense of the human immune response.

Defensin is a generic name reserved for an endogenously synthesized antimicrobial agent. The purpose of this review is to describe a series of discoveries that led to the proposal that 25-hydroxylated metabolites of vitamin D are key, intracellular regulators of the synthesis and action of naturally occurring defensin molecules against bacterial antigens. The discussion will (1) highlight the basic elements of human immune response that is responsive to vitamin D, (2) recount work relevant to the extrarenal expression of the vitamin D-1-hydroxlase (CYP27b1) in the macrophage as an initiator of the innate immune response, and (3) describe recent work on the relevance of the vitamin D intracrine-autocrine-paracrine system in a model of a common and devastating human disease, tuberculosis.

Megalin-mediated endocytosis of vitamin D binding protein correlates with 25-hydroxycholecalciferol actions in human mammary cells.

The major circulating form of vitamin D is 25-hydroxycholecalciferol [25(OH)D3], which is delivered to target tissues in complex with the serum vitamin D binding protein (DBP). We recently observed that mammary cells can metabolize 25(OH)D3 to 1,25-dihydroxycholecalciferol [1,25(OH)(2)D3], the vitamin D receptor (VDR) ligand, and the objective of our study was to elucidate the mechanisms by which the 25(OH)D3-DBP complex is internalized by mammary cells prior to metabolism. Using fluorescent microscopy and temperature-shift techniques, we found that T-47D breast cancer cells rapidly internalize DBP via endocytosis, which is blunted by receptor-associated protein, a specific inhibitor of megalin-mediated endocytosis. Endocytosis of DBP was associated with activation of VDR by 25(OH)D3 but not 1,25(OH)(2)D3 (as measured by induction of the VDR target gene, CYP24). We also found that megalin and its endocytic partner, cubilin, are coexpressed in normal murine mammary tissue, in nontransformed human mammary epithelial cell lines, and in some established human breast cancer cell lines. To our knowledge, our studies are the first to demonstrate that mammary-derived cells express megalin and cubilin, which contribute to the endocytic uptake of 25(OH)D3-DBP and activation of the VDR pathway.

Vitamin D-mediated hypercalcemia in slack skin disease: evidence for involvement of extrarenal 25-hydroxyvitamin D 1alpha-hydroxylase.

UNLABELLED: A case of granulomatous slack skin disease is presented in which we studied the possible involvement of extrarenal 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. Immunolocalization of 1alpha-OH in skin showed simultaneous dysregulation in epithelial and granulomatous cells. INTRODUCTION: Granuloma-forming diseases such as sarcoidosis are associated with extrarenal synthesis of active 1,25-dihydroxyvitamin D [1,25(OH)2D]. Here we describe a case of granulomatous slack skin disease in which we have studied the possible involvement of extrarenal synthesis of 1,25(OH)2D in the pathogenesis of the patient's hypercalcemia. The aim of the study was to clarify the etiology of hypercalcemia in this patient. MATERIALS AND METHODS: This was a case study of a 19-year-old man with a T-cell lymphoproliferative disorder diagnosed as granulomatous slack skin disease who presented with hypercalcemia and raised serum 1,25(OH)2D. Analysis of expression of the enzyme 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase), which catalyzes synthesis of 1,25(OH)2D, was carried out by immunohistochemical analysis of involved and uninvolved skin. Approval was granted by the Mayo Foundation Institutional Review Board and Biospecimens Subcommittee. RESULTS: In uninvolved skin, expression of 1alpha-hydroxylase was confined to the basal layer of the epidermis, whereas slack skin showed overexpression of the enzyme in dermal granulomata and basal cells of the epidermis. CONCLUSIONS: Hypercalcemia associated with granulomatous slack skin syndrome seems to be caused by dysregulation of 1alpha-hydroxylase expression in both epidermal and dermal granulomatous cells. This contrasts with psoriasis and sarcoidosis of the skin, in which overexpression of the enzyme is restricted to keratinocytes and granulomata, respectively.