The skeleton as an intracrine organ for vitamin D metabolism

The endocrine hormone, 1alpha,25-dihydroxyvitamin D(3) (1,25D) is an important regulator of calcium and phosphorus homeostasis. In this context, 1,25D is generally recognized as necessary for the maintenance of a healthy skeleton through its actions on the small intestine. In this review, we highlight the direct effects of 1,25D on the constituent cells of the bone, actions that are independent of effects on the intestine and kidney. We also consider the evidence that 25D levels, not 1,25D levels, correlate best with parameters of bone health, and that the bone itself is a site of metabolic conversion of 25D into 1,25D, by virtue of its expression of the 25-hydroxyvitamin D 1alpha-hydroxylase, CYP27B1. We review the evidence that at least osteoblasts and chondrocytes, and possibly also bone resorbing osteoclasts, are capable of such metabolic conversion, and therefore that these cells likely participate in autocrine and paracrine loops of vitamin D metabolism. We conclude that the skeleton is an intracrine organ for vitamin D metabolism, challenging the long-held notion that 1,25D is solely an endocrine hormone.     

Autocrine and paracrine actions of vitamin d

Vitamin D deficiency continues to attract considerable attention because of claims that an adequate status can reduce the risk of a wide range of diseases. The facts are that this hormone modulates the expression of a very large number of genes, possibly some 5 to 10% of the genome; that it has been subject to very strong evolutionary pressures; and that its biological activities are exerted across a wide range of tissues, and these all contribute to the plausibility that such claims may eventually be found to be valid. While the endocrine action of the active metabolite, 1,25-dihydroxyvitamin D, has been well-characterised to contribute to maintaining plasma calcium and phosphate homeostasis through regulation of intestinal absorption, recent research has focused on its autocrine and/or paracrine activities. Such activities of vitamin D have been best characterised in skin tissues and the immune system where it regulates cell differentiation and maturation as well as the innate immune system. Recent data are now available to implicate autocrine/paracrine activities in each of the major bone cell types where it also regulates cell proliferation and differentiation. In rodent models, adequate levels of serum 25-hydroxyvitamin D have been found to be critical to optimise bone health and to protect against osteoporosis. These findings are consistent with clinical data that such activity is present in humans. The introduction of an autocrine/paracrine paradigm for vitamin D has significant implications for critical levels of serum 25-hydroxyvitamin D for optimal health.     

Vitamin D: a hormone for all seasons--how much is enough?

Further understanding of its endocrine mechanisms and increased evidence for autocrine/paracrine actions has recently enhanced our knowledge of the biological activities of the vitamin D metabolite 1,25-dihydroxyvitamin D (1,25(OH)2D). The recognition of the contribution of vitamin D depletion to increased risk of osteoporosis, and most importantly the risk of hip fracture in the elderly, has increased the clinical significance of clinical laboratory testing for vitamin D status. Research has revealed that at least three genes contribute to vitamin D activity within tissues. These are the vitamin D receptor as well as two major vitamin D metabolising enzymes, CYP27B1, responsible for synthesis of 1,25(OH)2D and CYP24, responsible for catabolism of vitamin D metabolites. Current research focuses on the contribution of vitamin D metabolism to increasing vitamin D activity. This is of particular interest in bone forming cells where increased 1,25(OH)2D activity has been proposed to contribute to strengthening the skeleton. As well, solid tumours such as prostate, breast and colon cancers are another increasing area of vitamin D research. The major issues for the clinical laboratory in vitamin D testing include defining clinical decision limits for the interpretation of serum 25-hydroxyvitamin D (25OHD) levels and improving the precision and accuracy of this assay.     

1,25(OH)2-vitamin D3 receptors: gene regulation and genetic circuitry

Our understanding of how vitamin D mediates biological responses has entered a new era. It is now clear that the bulk of the biological responses supported by vitamin D occur as a consequence of its metabolism to its daughter metabolite 1 alpha,25-dihydroxyvitamin D3 (a steroid hormone). The fact that 1,25(OH)2D3 receptors are ubiquitous in tissue distribution opens the possibility for unforeseen biological functions of the vitamin D endocrine system. For example, 1,25(OH)2D3 serves as an immunoregulatory hormone and a differentiation hormone besides its classical role in mineral homeostasis. The avian 1,25)OH)2D3 receptor has recently been cloned and shown to be a member of the nuclear transacting receptor family that includes estrogen, progesterone, glucocorticoid, thyroxine (T3), aldosterone, and retinoic acid receptors. We have compiled an extensive number of RNA polymerase II-transcribed genes that are regulated by 1,25(OH)2D3. Classification of these genes on functional grounds identifies and formulates the several genetic circuits or biochemical systems in which 1,25(OH)2D3 plays an essential regulatory role. These systems include genes that govern oncogene and lymphokine expression as well as those involved in mineral homeostasis, vitamin D metabolism, and regulation of a set of replication-linked genes (c-myc, c-myb, and histone H4), which are critical for rapid cellular proliferation. An integrated analysis of the combinations of genetic circuits regulated by 1,25(OH)2D3 suggests that they may be collectively tied to a DNA replication-differentiation switch.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

Vitamin D and prostate cancer

Vitamin D and its metabolites are best known for their actions in calcium and bone metabolism. However, epidemiological studies have suggested that an increased prostate cancer risk is associated with decreased production of vitamin D. In vitro and in vivo studies have shown that the biologically active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25D), inhibits proliferation of cancer cells derived from multiple tissues, including the prostate. Although the mechanisms underlying the growth inhibitory effects of 1,25D have not been fully elucidated, in prostate cancer cells 1,25D reduces cell growth via a number of cellular pathways, including cell cycle arrest, induction of apoptosis, and altered activation of growth factor signaling. The hypercalcemia induced by 1,25D in vivo limits its use clinically as a therapeutic agent. However, several 1,25D analogs have been developed that reduce prostate tumor growth in rodent xenograft models without causing hypercalcemia. Additional studies are required in order to determine whether these 1,25D analogs will be useful therapeutic agents for the treatment of prostate cancer.     

The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents

The active form of vitamin D, 1,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)], is a secosteroid hormone that regulates calcium and bone metabolism, controls cell proliferation and differentiation, and exerts immunoregulatory activities. This range of functions has been exploited clinically to treat a variety of conditions, from secondary hyperparathyroidism to osteoporosis, to autoimmune diseases such as psoriasis. Recent advances in understanding 1,25(OH)(2)D(3) functions and novel insights into the mechanisms of its immunomodulatory properties suggest a wider applicability of this hormone in the treatment of autoimmune diseases and allograft rejection.     

The vitamin D story: a collaborative effort of basic science and clinical medicine

The discovery in 1919-1924 of vitamin D and its production in skin and foods by UV irradiation led to the elimination of rickets as a major medical problem. The identification and chemical preparation of vitamin D in the next decade provided large quantities of vitamin D to the physician for the treatment of a variety of metabolic bone diseases. Early in the 1960s, little was known about the function of vitamin D in causing mineralization of the skeleton, and hence in preventing the disease rickets in children and osteomalacia in adults. With the application of modern tools of biochemistry came the discovery that vitamin D must first be modified by 25-hydroxylation in the liver followed by 1 alpha-hydroxylation in the kidney to produce the vitamin D hormone 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. This process is strongly feedback-regulated and is one of the major endocrine systems regulating plasma calcium and phosphorus concentrations. Furthermore, it is a major endocrine system regulating bone mass and state. With the chemical synthesis of 1,25-(OH)2D3 and many of its analogs has come the possibility of treating a number of metabolic bone diseases not previously managed adequately, such as vitamin D-resistant rickets, hypoparathyroidism, renal osteodystrophy, and osteoporosis. By using 1,25-(OH)2D3, considerable work has been carried out to understand how this hormone facilitates calcium transport across the intestinal membrane. Modern work is described on the molecular mechanism of action of the vitamin D hormone in eliciting the cellular responses that result in mineral homeostasis. The possible use of the vitamin D analogs to bring about differentiation of myelocytic-type leukemias and in the treatment of psoriasis has been an important new development. This paper will thus be a blend of basic science of the vitamin D system and the application of that information to the treatment of disease.     

Vitamin D analogs: therapeutic applications and mechanisms for selectivity

The vitamin D endocrine system plays a central role in mineral ion homeostasis through the actions of the vitamin D hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], on the intestine, bone, parathyroid gland, and kidney. The main function of 1,25(OH)(2)D(3) is to promote the dietary absorption of calcium and phosphate, but effects on bone, kidney and the parathyroids fine-tune the mineral levels. In addition to these classical actions, 1,25(OH)(2)D(3) exerts pleiotropic effects in a wide variety of target tissues and cell types, often in an autocrine/paracrine fashion. These biological activities of 1,25(OH)(2)D(3) have suggested a multitude of potential therapeutic applications of the vitamin D hormone for the treatment of hyperproliferative disorders (e.g. cancer and psoriasis), immune dysfunction (autoimmune diseases), and endocrine disorders (e.g. hyperparathyroidism). Unfortunately, the effective therapeutic doses required to treat these disorders can produce substantial hypercalcemia. This limitation of 1,25(OH)(2)D(3) therapy has spurred the development of vitamin D analogs that retain the therapeutically important properties of 1,25(OH)(2)D(3), but with reduced calcemic activity. Analogs with improved therapeutic indices are now available for treatment of psoriasis and secondary hyperparathyroidism in chronic kidney disease, and research on newer analogs for these indications continues. Other analogs are under development and in clinical trials for treatment of various types of cancer, autoimmune disorders, and many other diseases. Although many new analogs show tremendous promise in cell-based models, this article will limit it focus on the development of analogs currently in use and those that have demonstrated efficacy in animal models or in clinical trials.     

Biological activities and binding properties of 23,23-difluoro-25-hydroxyvitamin D3 and its 1 alpha-hydroxy derivative

23,23-Difluoro-25-hydroxyvitamin D3 is 5-10 times less active than 25-hydroxyvitamin D3 in stimulating intestinal calcium transport, bone calcium mobilization, increasing serum phosphorus, mineralization of rachitic bone, and binding to the plasma transport protein in rats. It is converted to 23,23-difluoro-1 alpha, 25-dihydroxyvitamin D3 by chick renal 25-hydroxyvitamin D-1-hydroxylase. This compound is one-seventh as active as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Thus, fluoro substitution on carbon-23 of vitamin D has an unexpected and unexplained suppressive action on plasma binding and biological activity. However, since this substitution does not block the biological response of 25-hydroxyvitamin D3, these results provide additional evidence that 23-hydroxylation of vitamin D is not involved in biological function.     

24R,25-dihydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 are both indispensable for calcium and phosphorus homeostasis

The essential role of vitamin D throughout the life of most mammals and birds as a mediator of calcium homeostasis is well established. In view of the complex endocrine system existent for the regulated metabolism of vitamin D₃ to both 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 24R,25-dihydroxyvitamin D₃ [24R,25-(OH)₂D₃] (both produced by the kidney), an intriguing problem is to elucidate whether only one or both of these dihydroxyvitamin D₃ metabolites is required for the generation of all the biological responses mediated by the parent vitamin D₃. In contrast to the accumulated knowledge concerning the short term actions of 1,25(OH)₂-D₃ on stimulating intestinal calcium absorption and bone calcium reabsorption, relatively little is known of the biological function of 24,25(OH)₂D₃. We report now the results of a nine month study in which chicks were raised on a vitamin D-deficient diet from hatching to sexual maturity and received as their sole source of “vitamin D” either 24,25(OH)₂D₃ or 1,25(OH)₂D₃ singly or in combination. Specifically we are describing the integrated operation of the vitamin D endocrine system as quantitated by the individual measurement in all birds of 22 variables related to “vitamin D status” and as evaluated by the statistical procedure of multivariate discriminant analysis. Twelve of these variables involved detailed analysis of the bone including quantitative histology and the other 10 variables reflect various manifestations of vitamin D action, e.g. serum Ca²⁺ and Pi levels, vitamin D-dependent calcium binding protein (CaBP) in the intestine and kidney, egg productivity etc. As evaluated by the multivariate analysis, it is clear that 24,25(OH)₂D₃ and 1,25(OH)₂D₃ are simultaneously required for normalization of calcium homeostasis.     

The vitamin D deficiency pandemic and consequences for nonskeletal health: mechanisms of action

Vitamin D, the sunshine vitamin, is important for childhood bone health. Over the past two decades, it is now recognized that vitamin D not only is important for calcium metabolism and maintenance of bone health throughout life, but also plays an important role in reducing risk of many chronic diseases including type I diabetes, multiple sclerosis, rheumatoid arthritis, deadly cancers, heart disease and infectious diseases. How vitamin D is able to play such an important role in health is based on observation that all tissues and cells in the body have a vitamin D receptor, and, thus, respond to its active form 1,25-dihydroxyvitamin D. However, this did not explain how living at higher latitudes and being at risk of vitamin D deficiency increased risk of these deadly diseases since it was also known that the 1,25-dihydroxyvitamin D levels are normal or even elevated when a person is vitamin D insufficient. Moreover, increased intake of vitamin D or exposure to more sunlight will not induce the kidneys to produce more 1,25-dihydroxyvitamin D. The revelation that the colon, breast, prostate, macrophages and skin among other organs have the enzymatic machinery to produce 1,25-dihydroxyvitamin D provides further insight as to how vitamin D plays such an essential role for overall health and well being. This review will put into perspective many of the new biologic actions of vitamin D and on how 1,25-dihydroxyvitamin D is able to regulate directly or indirectly more than 200 different genes that are responsible for a wide variety of biologic processes.     

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.     

Involvement of 1,25-dihydroxyvitamin D3 in regulating myocardial calcium metabolism: physiological and pathological actions

The role of the prohormone vitamin D3 in regulating calcium and phosphate metabolism in the intestine, kidney, and bone has been known for several decades. Recent studies have provided evidence that vitamin D3, may also play an important role in regulating metabolism in other organs, including heart. This role has been suggested by the identification of a specific receptor for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, in these tissues, as well as the presence of a 1,25(OH)2D3-dependent calcium binding protein. Although administration of excessive quantities of vitamin D3 has been shown in many studies to produce myocardial calcinosis and heart failure, the importance of vitamin D3 in regulating myocardial metabolism under normal conditions has only recently been demonstrated. The purpose of the present review is to assess the current status of research regarding the pathological and physiological actions of vitamin D3 on the heart. The initial section of this report will focus on the pathological effects of excessive vitamin D3 on cardiovascular function, while the latter sections will describe recent studies related to the involvement of 1,25(OH)2D3 in regulating calcium homeostasis in ventricular cells and the relationship between vitamin D3 and myocardial contractility.     

Vitamin D analogues in the treatment of psoriasis.

Psoriasis is a chronic hyperproliferative skin disease in which inflammatory and immunologic processes may play important pathophysiologic roles. Recently the skin has been identified as a target tissue for vitamin D. Because 1,25-dihydroxy vitamin D3 inhibits epidermal proliferation and promotes epidermal differentiation, it has been introduced for the treatment of psoriasis vulgaris. In addition to 1,25-(OH)2-D3, synthetic vitamin D3 analogues have undergone clinical evaluation. Calcipotriol (INN) (calcipotriene [USAN]) has been studied most extensively. Compared with 1,25-(OH)2-D3, calcipotriol is about 200 times less potent in its effects on calcium metabolism, although similar in receptor affinity. Topical calcipotriol 50 micrograms/g applied twice daily is efficacious and safe for the treatment of psoriasis. Because topical calcipotriol is slightly more efficacious than betamethasone 17-valerate and dithranol, calcipotriol should be considered a first line drug in the management of psoriasis. These results illustrate that it is possible to separate the vitamin D effects on the cellular level from those on calcium metabolism not only in vitro, but also in a clinical setting.     

Vitamin D: its role in cancer prevention and treatment

Vitamin D, the sunshine vitamin, has been recognized for almost 100 years as being essential for bone health. Vitamin D provides an adequate amount of calcium and phosphorus for the normal development and mineralization of a healthy skeleton. Vitamin D made in the skin or ingested in the diet, however, is biologically inactive and requires obligate hydroxylations first in the liver to 25-hydroxyvitamin D, and then in the kidney to 1,25-dihydroxyvitamin D. 25-Hydroxyvitamin D is the major circulating form of vitamin D that is the best indicator of vitamin D status. 1,25-dihydroxyvitamin D is the biologically active form of vitamin D. This lipid-soluble hormone interacts with its specific nuclear receptor in the intestine and bone to regulate calcium metabolism. It is now recognized that the vitamin D receptor is also present in most tissues and cells in the body. 1,25-dihydroxyvitamin D, by interacting with its receptor in non-calcemic tissues, is able to elicit a wide variety of biologic responses. 1,25-dihydroxyvitamin D regulates cellular growth and influences the modulation of the immune system. There is compelling epidemiologic observations that suggest that living at higher latitudes is associated with increased risk of many common deadly cancers. Both prospective and retrospective studies help support the concept that it is vitamin D deficiency that is the driving force for increased risk of common cancers in people living at higher latitudes. Most tissues and cells not only have a vitamin D receptor, but also have the ability to make 1,25-dihydroxyvitamin D. It has been suggested that increasing vitamin D intake or sun exposure increases circulating concentrations of 25-hydroxyvitamin D, which in turn, is metabolized to 1,25-dihydroxyvitamin D(3) in prostate, colon, breast, etc. The local cellular production of 1,25-dihydroxyvitamin D acts in an autocrine fashion to regulate cell growth and decrease the risk of the cells becoming malignant. Therefore, measurement of 25-hydroxyvitamin D is important not only to monitor vitamin D status for bone health, but also for cancer prevention.