Aromatase in bone: roles of Vitamin D3 and androgens

We have mainly focused on the regulatory mechanism of cytochrome P450 aromatize in bone cells. Our previous study demonstrated a strong positive correlation of serum dehydroepiandrosterone sulfate (DHEA-S) and estrone (E1) with BMD in postmenopausal women but no correlation between serum estradiol (E2) and BMD in the same group. In addition, administration of DHEA to ovariectomized rat significantly increased BMD. These in vivo findings strongly suggested that circulating adrenal androgen may be converted to estrogen in osteoblast and may contribute to BMD maintenance. Actually, in cultured human osteoblast cells, DHEA was found to convert to androstenedione by 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and then androstenedione to estrone through the apparent aromatase activity. The aromatase activity in cultured human osteoblast cells was significantly increased by dexamethasone (DEX). Interestingly, DEX and 1α,25-dihydroxyvitamin D₃ (VD₃) synergistically enhanced aromatase activity as well as P450arom mRNA expression. A little stronger induction of aromatase activity by DEX and VD₃ was observed in cultured human fibroblasts. The increase of the aromatase activity by DEX and VD₃ was accompanied with the increase of luciferase activity of fibroblast cells transfected with Exon 1b-promoter-luciferase construct, but not of osteoblasts transfected with the same construct, suggesting a different regulatory mechanism of aromatase by DEX and 1α,25-dihydroxyvitamin D₃ (VD₃) between these two cells despite the same promotor usuage. In human bone cells, intracrine mechanism through aromatase activity, together with a positive regulation of aromatase activity by glucocorticoid and VD₃, may contribute to the local production of estrogens, thus leading to protective effect against osteoporosis especially after menopause. The effect of sex steroids (estrogen versus testosterone) in bone remodeling was also briefly reviewed based on several recent findings in this field.     

Vitamin D and bone

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.     

Vitamin D analogs and bone

Vitamin D analogs increase intestinal calcium absorption, and have been shown to possess antiresorptive and also bone anabolic properties in vivo. Therefore, the pharmacological profile of vitamin D analogs would be well suited for the treatment of osteoporosis. However, the calcemic side effects of this compound class, especially at higher doses, have hampered their wide use in osteoporotic patients. Nevertheless, the clear potential for bone anabolic properties together with oral availability have stimulated the interest in this substance class, and there is an active search for bone selective vitamin D compounds. After an overview of the physiological functions of vitamin D in bone, this article focuses on the effects of acute and chronic administration of pharmacological doses of vitamin D analogs on bone in animal models and humans. Furthermore, the endocrinological, cellular, and microanatomical mechanisms involved in the skeletal actions of vitamin D analogs are discussed. The final section briefly reviews the available data on possible bone selective vitamin D analogs.     

Aromatase inhibitors and bone

The decrease in estrogen levels with the use of aromatase inhibitors results in an increase in the rate of bone remodelling. This result in an acceleration of bone loss, and probably to an increase in the risk of fractures. The risk of fracture is particularly high in the older woman and in the woman with a low bone mineral density. We have a number of proven treatments for the treatment of postmenopausal osteoporosis and it is likely that some of these, particularly bisphosphonates, could be effective at preventing bone loss with aromatase inhibitors.     

维生素D_3与雄性生殖

维生素D3的经典作用是调节体内钙、磷的代谢平衡和维持骨的健康,有证据显示维生素D3也可调节动物生殖。维生素D代谢酶可合成和降解活性维生素D3及其代谢中间产物,活性维生素D3通过结合至目标组织的维生素D受体(vitamin D receptor,VDR)以发挥其生物学作用。VDR和维生素D代谢酶在雄性生殖系统中表达,表明维生素D3在雄性生殖生物学中起关键作用。本文对维生素D3与雄性生殖的研究进展作一综述,以期为推进维生素D3影响雄性生殖的分子机制和临床治疗男性不育的研究提供理论依据。     

Effects of pharmacological doses of Vitamin D3 on mineral balance and profiles of plasma Vitamin D3 metabolites in horses

Metabolism and functions of Vitamin D in horses differ from those in humans, pigs and rats. In horses, calcidiol and calcitriol concentrations in blood plasma are remarkably low (<10 nmol L(-1); 20-40 pmol L(-1), respectively). When a toxic amount of Vitamin D(3) is administered, the responsiveness of calcium and calcitriol concentrations in blood plasma is much reduced compared to the other domestic animal species but inorganic phosphate (Pi) response is much more marked, leading to an increase of the Ca x Pi product. Also, soft tissue calcifications have been observed to develop in horses during Vitamin D(3) intoxication. It was suggested that the elevation of the Ca x Pi product may play a causative role in this calcification process. To test this assumption, two horses were treated with 40,000 IU kg(-1) of Vitamin D(3) whilst dietary uptake of Ca and Pi was restricted to prevent or to diminish the increase of the Ca x Pi product. Distribution, number and severity of calcification centres were considerably less in these horses than in the control animals of a previous experiment which had received the same amount of Vitamin D(3) but where Ca and Pi intake was not restricted. It appears from these findings that in horses, the increase of the Ca x Pi product in blood plasma during a Vitamin D intoxication contributes to the soft tissue calcifications. It is further concluded that in the event of a Vitamin D intoxication, it is recommended to restrict the Ca and Pi uptake immediately. The authors believe that this may help to prevent or at least diminish soft tissue calcifications which are often fatal to the horse due to nephrocalcinosis.     

Vitamin D3 and calcium absorption in the chick

1. An attempt has been made to locate the site of action of vitamin D(3) as it affects the translocation of calcium across the intestine. 2. Calcium appears to be pumped out of cells by a process dependent on energy from metabolism. 3. The effects of cold, inhibitors and vitamin D(3) on the translocation of calcium by everted sacs of intestine were studied and compared with results obtained in vivo. 4. A model was proposed to explain the results which suggests that vitamin D(3) inhibits a metabolically operated pump that returns calcium from the mucosal cell to the lumen. 5. Some observations on the effect of sodium lauryl sulphate on the translocation of calcium in vivo and in vitro are reported.     

Differential effects of Vitamin D analogs on bone formation and resorption

Deficiency in Vitamin D and its metabolites leads to a failure in bone formation primarily caused by dysfunctional mineralization, suggesting that Vitamin D analogs might stimulate osteoblastic bone formation and mineralization. In this study, we compare the effect of selected Vitamin D analogs and active metabolite, 1alpha,25-dihydroxyvitamin D(3), 19-nor-1alpha, 25-dihydroxyvitamin D(2), and 1alpha-hydroxyvitamin D(2) or 1alpha,25-dihydroxyvitamin D(2) on bone formation and resorption. In a mouse calvariae bone primary organ culture system, all Vitamin D analogs and metabolite tested-stimulated collagen synthesis in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was the most efficacious among three. 19-nor-1alpha, 25-dihydroxyvitamin D(2) and 1alpha,25-dihydroxyvitamin D(2) showed similar potencies and 1alpha,25-dihydroxyvitamin D(3) was less potent than others. Osteocalcin was also up-regulated in a dose-dependent manner, suggesting that the three Vitamin D analogs have the equal potencies on bone formation. 25-Hydroxyvitamin D-24-hydroxylase expression was induced in a dose-dependent manner and 19-nor-1alpha, 25-dihydroxyvitamin D(2) was less potent than other two compounds. In a mouse calvariae organ culture, all induced a net calcium release from calvariae in a dose-dependent manner, but the potency is in the order of 1alpha,25-dihydroxyvitamin D(2) congruent with1alpha,25-dihydroxyvitamin D(3)>19-nor-1alpha, 25-dihydroxyvitamin D(2). In a Vitamin D/calcium-restricted rat model, all caused an elevation in serum calcium in a dose-dependent manner. There is no significant difference between 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) in potencies, but 19-nor-1alpha, 25-dihydroxyvitamin D(2) is at least 10-fold less potent than the other two compounds. Our results suggest that Vitamin D analogs have direct effects on bone resorption and formation, and 19-nor-1alpha, 25-dihydroxyvitamin D(2) may be more effective than 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(2) on stimulating anabolic bone formation.     

Investigating Transdermal Delivery of Vitamin D3

Transdermal delivery of therapeutic amounts of vitamin D₃ is proposed to overcome its variable oral bioavailability, especially for people who suffer from fat malabsorption. The main challenge for this delivery route is to overcome the barrier properties of skin, especially for very lipophilic compounds such as vitamin D₃. In this study, the effect of different penetration enhancers, such as oleic acid, dodecylamine, ethanol, oleic acid in propylene glycol, isopropyl myristate, octyldodecanol, and oleyl alcohol in propylene glycol were evaluated in vitro for their effectiveness in delivering vitamin D₃ through polyamide filter, polydimethylsiloxane membrane, and porcine skin. A diffusion cell was used to study the transdermal permeability of vitamin D₃. Ointment formulations of vitamin D₃ were prepared containing the most widely used penetration enhancers, oleic acid, and dodecylamine. The ointment containing oleic acid as chemical penetration enhancer did not improve delivery compared to control. On the other hand, the formulation containing dodecylamine as a penetration enhancer did improve the transdermal delivery of vitamin D₃. However, statistical significance and an amount high enough for nutritional supplementation purposes were reached only when the skin was pretreated with 50% ethanol. In these conditions, the ointment delivered an amount of 760-ng vitamin D₃ per cm² of skin. The research shows promise that transdermal delivery could be an effective administration route for vitamin D₃ when ethanol and dodecylamine are used as penetration enhancers.KEY WORDS: dodecylamine, ethanol, penetration enhancer, transdermal delivery, vitamin D₃     

Dental alveolar bone defects related to Vitamin D and calcium status

Vitamin D is important for skeletal development, growth, and homeostasis but has been sparsely studied in the oro-facial bone. Dental alveolar bone anchors teeth to mandible and maxilla bones via a periodontal ligament. Its formation and maintenance are strictly dependent on the presence of tooth organs and it is characterized by a high turnover rate. In order to study the role of Vitamin D and the calcium status on dental alveolar bone formation, microradiographic and histologic comparison of wild-type, Vitamin D receptor null mutant (VDR (-/-) hypo- and normo-calcemic mice and tissues were performed at 2 months. In hypo-calcemic VDR (-/-) mice, alveolar bone was hypomineralized and demonstrated a cellular and matrix organization, similar to the immature woven bone. In normo-calcemic VDR (-/-) mice, mineralization of dental alveolar bone appeared normal, but bone was morphologically abnormal in some specific anatomical locations. These data show that Vitamin D and calcium status may control the formation of dental alveolar bone. The differences of phenotype between hypo- and normo-calcemic VDR null mutant mice suggested a specific Vitamin D control of alveolar bone formation by the Vitamin D nuclear receptor pathway.     

Vitamin D and 1,25-dihydroxyvitamin D3 as modulators in the immune system

Treatment from weaning until old age with 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)) prevents diabetes in NOD mice. It is mainly through its actions on dendritic cells (DCs), that 1,25(OH)(2)D(3) changes the function of potentially autoreactive T lymphocytes. In contrast, early life treatment (from 3 to 70 days of age) of NOD mice with vitamin D or 1,25(OH)(2)D(3) did not influence final diabetes incidence at 200 days of age. Also in spontaneous diabetic BB rats, diabetes could not be prevented by early life treatment (from 3 to 50 days of age) with vitamin D (1000 IU per day) or 1,25(OH)(2)D(3) (0.2 microg/kg per day or 1 microg/kg per 2 days). However, when NOD mice were made vitamin D deficient in early life (until 100 days of age), diabetes onset occurred earlier and final incidence was increased. These data further support a role for vitamin D and its metabolites in the pathogenesis of type 1 diabetes in NOD mice.     

Expression of Cholinergic,Insulin, Vitamin D Receptors and GLUT 3 in the Brainstem of Streptozotocin Induced Diabetic Rats: Effect of Treatment with Vitamin D3

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B_max of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.     

Vitamin D3-implications for brain development

There is growing evidence that 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is active in the brain but until recently there was a lack of evidence about its role during brain development. Guided by certain features of the epidemiology of schizophrenia, our group has explored the role of 1,25(OH)(2)D(3) in brain development using whole animal models and in vitro culture studies. The expression of the vitamin D receptor (VDR) in the embryonic rat brain rises steadily between embryonic day 15-23, and 1,25(OH)(2)D(3) induces the expression of nerve growth factor and stimulates neurite outgrowth in embryonic hippocampal explant cultures. In the neonatal rat, low prenatal vitamin D(3) in utero leads to increased brain size, altered brain shape, enlarged ventricles, reduced expression of nerve growth factors, reduced expression of the low affinity p75 receptor and increased cellular proliferation. In summary, there is growing evidence that low prenatal levels of 1,25(OH)(2)D(3) can influence critical components of orderly brain development. It remains to be seen if these processes are of clinical relevance in humans, but in light of the high rates of hypovitaminosis D in pregnant women and neonates, this area warrants further scrutiny.     

Mechanisms of Neuroprotective Action of Vitamin D3

This review considers modern data on the mechanisms underlying the neuroprotective effect of the neurosteroid vitamin D(3) and its receptors in the nervous system. Special attention is paid to Ca2+ regulation, stimulation of neurotrophin release, interaction with reactive oxygen and nitrogen species, and neuroimmunomodulatory effects of calcitriol, the main biologically active form of vitamin D(3), in the nervous system.