1,25-二羟维生素D3的生物学效应

1,25-二羟维生素D3是维生素D3的活性形式,其生物学效应是由基因组与非基因组两种机制介导的。维生素D3除了具有钙磷代谢调节作用外,还具有其他更为广泛的生物学效应。1,25-二羟维生素D3能够抑制多种类型细胞的增殖,诱导细胞的凋亡和分化,调节机体免疫系统功能,保护中枢神经系统,以及保护基因等。     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in normal rats. Serum (S) levels and urinary excretion of Ca2+ (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. The animals were housed in metabolic cages and 24-hr urine specimens were collected. After 24 hr SCa2+ increased similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3, while 24,25(OH)2D3 alone did not change SCa2+. UCaV after 24 hr increased significantly less (P less than 0.025) with 1,25(OH)2D3 + 24,25(OH)2D3 than with 1,25(OH)2D3 alone. After 5 days of 1,25(OH)2D3, SCa2+ rose from 5.1 +/- 0.15 to 6.29 +/- 0.08 whereas 1,25(OH)2D3 + 24,25(OH)2D3 effected a greater increase in SCa2+ up to 6.63 +/- 0.09 (P less than 0.01). 24,25(OH)2D3 alone did not change SCa2+. UCaV after 5 days of treatment rose similarly with 1,25(OH)2D3 and with 1,25(OH)2D3 + 24,25(OH)2D3. After 10 days of 1,25(OH)2D3 SCa2+ was 6.17 +/- 0.15 meq/liter while with the combination SCa2+ rose to 6.74 +/- 0.2 (P less than 0.025). 24,25(OH)2D3 alone did not change SCa2+. These results show that (a) 24,25(OH)2D3 alone does not alter SCa2+ in normal rats, (b) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, and (c) it is suggested that the effect of 24,25(OH)2D3 on serum Ca2+ level, at least partly, may result from its hypocalciuric effect.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

24,25(OH)2D3 enhances the calcemic effect of 1,25(OH)2D3 in PTX rats

The effect of 24,25(OH)2D3 on 1,25(OH)2D3-induced hypercalcemia was studied in parathyroidectomized (PTX) rats for 10 days. Serum (S) and urinary Ca excretion (UCaV) were measured in (a) control rats, (b) rats receiving a daily sc injection of 54 ng 1,25(OH)2D3, (c) rats receiving 24,25(OH)2D3 in the same dose and same manner, and (d) rats receiving 1,25(OH)2D3 + 24,25(OH)2D3. Our results show that (i) 24,25(OH)2D3 alone does not increase SCa2+ in PTX rats, (ii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 enhances the hypercalcemic response to 1,25(OH)2D3 without a parallel increase in UCaV, (iii) combined administration of 1,25(OH)2D3 + 24,25(OH)2D3 reduces the rise in urinary excretion of Ca2+ compared with that of rats receiving 1,25(OH)2D3 alone for 10 days, and (iv) these alterations are independent of parathyroid hormone.     

The assessment of circulating 25(OH)D and 1,25(OH)2D: where we are and where we are going

The field of Vitamin D assay technology has progressed significantly over the past 4 decades. Further, the clinical utility of these measurements has moved from esoteric into mainstream clinical diagnosis. This movement has been fueled by the realization that Vitamin D is involved in bodily systems beyond skeletal integrity. The clinical assay techniques for circulating 25(OH)D and 1,25(OH)₂D have progressed away from competitive protein binding assay (CPBAs) that utilize tritium reporters to radioimmunoassay (RIAs) that utilize both I¹²⁵ and chemiluminescent reporters. These advances have allowed direct serum analysis of 25(OH)D in an automated format that provides a huge sample throughput. Detection of circulating 25(OH)D can also be achieved utilizing direct high-performance liquid chromatographic (HPLC) or liquid chromatography coupled with mass spectrometry (LC–MS) techniques. These methods are accurate, however, they require expensive equipment and restrict sample throughput in the large clinical laboratory. Direct serum detection of 1,25(OH)₂D is unlikely to occur for many reasons as a sample pre-purification will always be required. However, a semi-automated chemiluminescent detection system with automated sample preparation is in final development for the determination of circulating 1,25(OH)₂D. These advances will allow both 25(OH)D and 1,25(OH)₂D to be detected in an accurate, rapid fashion to meet the clinical demands we see emerging.     

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.     

Effects of 24,25(OH)2D3, 1,25(OH)2D3 and 25(OH)D3 on alkaline and tartrate-resistant acid phosphatase activities in fetal rat calvaria

The aim of this work was to evaluate the effects of 24,25-dihydroxyvitamin D3, 24,25(OH)2D3, on alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) activities in fetal rat calvaria cultures. These actions were compared with those of 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, and 25-hydroxyvitamin D3, 25(OH)D3, in similar experimental conditions. At 10 min, 30 min and at 24 h incubation time, 1,25(OH)2D3 (10(-10)M) and 25(OH)D3 (10(-7) M) produced a significant increase in AP and TRAP activities compared to control group (without vitamin D metabolites). However, 24,25(OH)2D3 (10(-7) M) only produced effects on phosphatase activities similar to those produced by 1,25(OH)2D3 and 25(OH)D3, after 24 h incubation time. These findings suggest that 1,25(OH)2D3 and 25(OH)2D3 could carry out actions in minutes (nongenomic mechanism), while 24,25(OH)2D3 needs longer periods of time to perform its biological actions (genomic mechanism).     

Biologic effect of 1,24(R)(OH)2D3 versus 1,25(OH)2D3 administration in chronic renal failure

1,24(R)(OH)2D3 is a synthetic analogue of 1,25(OH)2D3 which binds to the same receptors as the physiologic metabolite with a lower affinity. The aim of the present study was to compare the activity of 1,24(R)(OH)2D3 and 1,25(OH)2D3 on several target organs in patients with chronic renal failure. Treatment with 1,24(R)(OH)2D3 at doses of either 1 or 2 μg daily was carried out in two groups of 9 patients, with serum creatinine of 4.61 ± 1.59 and 4.66 ± 1.46 mg/dl, respectively. Doses of 1,25(OH)2D3 were 0.5 and 1 μg daily and were administered to 9 and 13 patients, serum creatinine of 4.52 ± 1.67 and 4.3 ± 1.16 mg/dl, respectively. Treatment periods were of 2 weeks. Administration of 1,25(OH)2D3, 1 μg, induced significant increments of intestinal calcium absorption (ICA), ionized calcium, osteocalcin, serum creatinine, urine Ca/GFR, and a decrease in iPTH. 1,25(OH)2D3, 0.5 μg, induced a significant increase in ICA and osteocalcin and a decrease in iPTH. Similarly 1,24(OH)2D3, 2 μg daily, significantly stimulated ICA and raised serum levels of osteocalcin and creatinine while lowering serum iPTH. In addition, 1,24(R)(OH)2D3 administration induced a significant fall of serum 1,25(OH)2D3. Following 1 μg, only osteocalcin increased. Therefore, the dose of 2 μg of 1,24(R)(OH)2D3 has biologic activity similar to 0.5 μg 1,25(OH)2D3 (4:1). However the activity ratio on osteocalcin production appears to be 2:1. In addition, 1,24(R)(OH)2D3 is able to inhibit renal tubular 1-hydroxylase. In conclusion 1,24(R)(OH)2D3 may prove to be useful in the treatment of metabolic bone disease.     

24,25(OH)2D3 attenuates the calcemic effect of 1,25(OH)2D3 in rats with reduced renal mass

The present study was undertaken to evaluate the effect of 24,25(OH)2D3 on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)2D3, (iii) rats treated with 24,25(OH)2D3, and (iv) rats treated with 1,25(OH)2D3 and 24,25(OH)2D3. After 4 days, serum calcium in the 1,25(OH)2D3-treated group was 7.13 +/- 0.32 meq/liter (P less than 0.001 vs control). With the combination of 1,25(OH)2D3 and 24,25(OH)2D3 serum calcium was higher than that in control, 6.25 +/- 0.5 meq/liter (P less than 0.001 vs control), but lower than that in rats receiving 1,25(OH)2D3 alone (P less than 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)2D3 alone. On the eighth day serum calcium in the 1,25(OH)2D3-treated group, 6.52 +/- 0.25, was higher than in the 1,25(OH)2D3 + 24,25(OH)2D3 group, 5.87 +/- 0.17 meq/liter, P less than 0.05, P less than 0.001 vs control. In both 1,25(OH)2D3- and 1,25(OH)2D3 + 24,25(OH)2D3-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)2D3-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)2D3 and 24,25(OH)2D3 attenuates the calcemic response to 1,25(OH)2D3 without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)2D3 on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)2D3 on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)2D3 on the bone.     

Platelet-rich plasma and 1,25(OH)2 vitamin D3 synergistically stimulate osteogenic differentiation of adult human mesenchymal stem cells

Mesenchymal stem cell proliferation and differentiation are regulated by growth factors and systemic hormones in a concerted manner. We have examined the effects of platelet-rich plasma (PRP) and its interaction with 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on the differentiation and proliferation of human mesenchymal stem cells (hMSCs). PRP stimulated cell proliferation in a manner inversely proportional to the dose. Exposure to PRP inhibited hMSC osteogenic differentiation. Co-treatment with PRP and 1,25(OH)₂D₃, however, synergistically stimulated osteogenic differentiation. Serial co-treatment with PRP and 1,25(OH)₂D₃ showed that this synergistic effect was due to the modulation of a 1,25(OH)₂D₃-induced differentiation by PRP rather than vice versa.     

1,25(OH)2 vitamin D3 sites of action in the brain

Summary After injection of ³H 1,25(OH)₂ vitamin D₃ to adult rats and mice, under normal or vitamin D deficient diet, the hormone was found to be accumulated in nuclei of neurons in certain brain regions. Nuclear concentration was prevented or diminished, when excess unlabeled 1,25 (OH)₂ vitamin D₃ was injected before ³H 1,25(OH)₂ vitamin D₃, while excess 25 (OH) vitamin D₃ did not prevent nuclear labeling.Highest nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is observed in certain neurons in the nucleus interstitialis striae terminalis, involving its septo-preoptic pars dorsolateralis and its anterior hypothalamic-thalamic portion, and in the nucleus centralis of the amygdala, all constituting a system of target neurons linked by a component of the stria terminalis. Nuclear concentration of ³H 1,25 (OH)₂ vitamin D₃ is also found in neurons in the periventricular nucleus of the preoptic-hypothalamic region, including its extensions, the parvocellular paraventricular and arcuate nucleus, in the ventromedial nucleus, supramammillary nucleus, reticular nucleus of the thalamus, ventral hippocampus, caudate nucleus, pallium, in the midbrain-pontine central gray, dorsal raphe nucleus, parabrachial nuclei, cranial motor nuclei, substantia gelatinosa of the sensory nucleus of the trigeminus, Golgi type II cells of the cerebellum, and others.The extensive distribution of target neurons suggests that 1,25(OH)₂ vitamin D₃ regulates the production of several aminergic and peptidergic messengers, and influences the activity of certain endocrine-autonomic, sensory and motor systems.     

The cellular and molecular regulation of 1,25(OH)2D3 production.

The synthesis of 1,25(OH)₂D₃ is a critical control point in the regulation of calcium metabolism, and possibly in the growth and differentiation of a number of cell types. This paper reviews our current understanding of the regulation of this process at the cellular and molecular levels, with the emphasis on the mechanisms of feedback control 1,25(OH)₂D₃ itself, control of parathyroid hormone, the roles of cyclic AMP dependent protein kinase and protein kinase C, and the interaction between the various intracellular regulators of 1,25(OH)₂D₃ production.     

Regulation of 1,25(OH)2D3 (calcitriol) receptor by vitamin B6

In vitamin B6-deficient rats the concentration of in vivo occupied nuclear and total cellular receptors of 1.25(OH)2D3 increases 1.3-1.7 times, whereas the binding of in vitro occupied receptors to DNA-cellulose increases by 40%. Pyridoxal-5'-phosphate (PLP) added in vitro to solubilized receptors of 1.25(OH)2D3 lowers the ligand binding by 15-25% but causes no dissociation of hormone-receptor complexes formed in vivo. The association of in vitro occupied receptors of 1.25(OH)2D3 with DNA-cellulose is suppressed by PLP (3.5-4.5-fold). It has been shown for the first time that vitamin B6 is a physiological regulator of 1.25(OH)2D3 receptor binding by chromatin and DNA which diminish the concentration of occupied receptors and thus suppress the hormonal response.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Homologous up-regulation of the 1,25 (OH)2 vitamin D3 receptor in rats

This study investigates the ability of vitamin D-metabolites to regulate 1,25(OH)2D3 receptors in vivo. Rats made vitamin D-deficient were treated with 1,25(OH)2D3 or vehicle for 1-5 days. In treated animals, receptors for 1,25(OH)2D3 in kidney increased dramatically compared with control levels. An increase in specific binding to 220% of control was seen after 2 doses of hormone, which reached to 336% after 5 days of treatment. Intestinal receptors increased to only 130% of control levels after 5 days of treatment. In vitamin D-replete animals, the difference between control and treated groups was slightly greater when endogenously occupied sites were measured by exchange (TPCK). However, significant changes were observed only after 4 days of hormone treatment. The data indicate that homologous up-regulation of the 1,25(OH)2D3 receptor occurs in vivo. The difference in response in kidney and in intestine suggests differential importance of up-regulation in various organs.     

Bone specific binding sites for 1,25(OH)2D3 in magnesium deficiency

It has been reported that some hypoparathyroid patients with magnesium deficiency showed altered responses to vitamin D treatment. In the same way, in vitro bone studies have demonstrated the existence of a decrease in the 1,25-dihydroxyvitamin D3-induced resorption in bone as a result of magnesium deficiency. These findings suggest some kind of alteration in the 1,25(OH)2D3 in bone in magnesium deficiency. In the present work, using a binding assay based on the 1,25(OH)2D3 and 3H-1,25(OH)2D3 competition for the hormone binding sites in rat calvaria homogenates, a significant decrease in the number of 1,25(OH)2D3 specific binding sites has been found in calvaria incubated in magnesium-deficient medium compared to magnesium-replete ones. Alterations in the hormone-receptor affinity were not found. These results suggest that an alteration in the 1,25(OH)2D3 action on magnesium-deficient bone could be due, at least in part, to a decrease in the number of available vitamin D receptors in bone cells.