The Putative Role of 1,25(OH)2D3 in the Association of Milk Consumption and Parkinson's Disease.

The consumption of dairy products, particularly of low fat milk, has been shown to be associated with the occurrence of Parkinson's disease. This association does not necessarily reflect a pathophysiological role of milk intake in the development of Parkinson's disease. Nevertheless, the present review discusses a potential mechanism possibly mediating an effect of milk consumption on Parkinson's disease. The case is made that milk is tailored in part to support bone mineralization of the suckling offspring and is thus rich in calcium and phosphate. Milk intake is thus expected to enhance intestinal calcium phosphate uptake. As binding to fatty acids impedes Ca2+ absorption, low fat milk is particularly effective. Calcium and phosphate uptake inhibit the formation of 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), the active form of vitamin D. Calcium inhibits 1,25(OH)2D3 production in part by suppressing the release of parathyroid hormone, a powerful stimulator of 1,25(OH)2D3 formation. Phosphate excess stimulates the release of fibroblast growth factor FGF23, which suppresses 1,25(OH)2D3 formation, an effect requiring Klotho. 1,25(OH)2D3 is a main regulator of mineral metabolism, but has powerful effects apparently unrelated to mineral metabolism, including suppression of inflammation and influence of multiple brain functions. In mice, lack of 1,25(OH)2D3 and excessive 1,25(OH)2D3 formation have profound effects on several types of behavior, such as explorative behavior, anxiety, grooming and social behavior. 1,25(OH)2D3 is produced in human brain and influences the function of various structures including substantia nigra. In neurons 1,25(OH)2D3 suppresses oxidative stress, inhibits inflammation and stimulates neurotrophin formation thus providing neuroprotection. As a result, 1,25(OH)2D3 is considered to favorably influence the clinical course of Parkinson's disease. In conclusion, consumption of milk could in theory accelerate the downhill course of neuronal function in Parkinson's disease. However, substantial additional experimentation is required to define the putative causal role of 1,25(OH)2D3 in the pathophysiology of Parkinson's disease and its sensitivity to milk consumption.

Glucocorticoid exposure induces preeclampsia via dampening 1,25-dihydroxyvitamin D3.

The pathogenesis of preeclampsia (PE) involves a number of biological processes that may be directly or indirectly affected by glucocorticoid (GC) and vitamin D. GC exposure increases the risk of PE, and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) deficiency may result in PE. The purpose of the present study was to confirm the involvement of GC/1,25-(OH)2D3 axis in the pathogenesis of PE. In the study, cortisol levels of PE patients were found to be higher than that of non-complicated pregnancies, while 1,25-(OH)2D3 were decreased in both PE women and GC-induced PE rats. Mechanically, GC reduced 1,25-(OH)2D3 levels via disturbing its biosynthetic and catabolic enzymes, including Cyp3a1,Cyp24a1 and Cyp27b1, especially enhancing the expressions of Cyp3a1, the dominant enzyme for vitamin D degeneration. Moreover, replenishing 1,25-(OH)2D3 ameliorated the symptoms and placental oxidative stress of GC-induced rat PE. The protective actions of 1,25-(OH)2D3 might be explained by its roles in antagonizing the effects of GC on trophoblast proliferation and apoptosis. Together, these findings suggest that GC exposure could lead to PE via dampening 1,25-(OH)2D3 biosynthesis, and GC/1,25-(OH)2D3 axis might represent a common pathway through which PE occurs.

Lutein Enhances Bone Mass by Stimulating Bone Formation and Suppressing Bone Resorption in Growing Mice.

Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxia-induced cell damage in the eye by removing free radicals. However, its role in other tissues is controversial, and the effects of lutein on bone tissues are unknown. To identify a possible role of lutein in bone tissues, we examined the effects of lutein on bone formation and bone resorption and on femoral bone mass in mice. Lutein enhanced the formation of mineralized bone nodules in cultures of osteoblasts. On the other hand, lutein clearly suppressed 1α, 25-dihydroxyvitamin D3-induced bone resorption as measured by pit formation in organ culture of mouse calvaria. In co-cultures of bone marrow cells and osteoblasts, lutein suppressed 1α, 25-dihydroxyvitamin D3-induced osteoclast formation. In cultures of bone marrow macrophages, lutein suppressed soluble RANKL, the receptor activator of nuclear factor-kappaB (NF-κB) ligand, induced osteoclast formation. When five-week-old male mice were orally administered lutein for 4 weeks, the femoral bone mass was clearly enhanced in cortical bone, as measured by bone mineral density in dual X-ray absorptiometry and micro computed tomography (µCT) analyses. The present study indicates that lutein enhances bone mass in growing mice by suppressing bone resorption and stimulating bone formation. Lutein may be a natural agent that promotes bone turnover and may be beneficial for bone health in humans.

Effect of 1,25(OH)2 D3 and 20(OH)D3 on interleukin-1ß-stimulated interleukin-6 and -8 production by human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Vitamin D-1,25(OH)2 D3 or 1,25D3-maintains healthy osseous tissue, stimulates the production of the antimicrobial peptide cathelicidin and has anti-inflammatory effects, but it can cause hypercalcemia. Evidence links diminished serum levels of 1,25D3 with increased gingival inflammation. Periodontitis progression is associated with increased local production of inflammatory mediators by immune cells and gingival fibroblasts. These include interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8, both regulated by signaling pathways, including NF-κB and MAPK/AP-1. The objectives were to determine the effects of 1,25D3 or a non-calcemic analog, 20-hydroxyvitamin D3 -20(OH)D3 or 20D3-on IL-1ß-stimulated IL-6 and IL-8 production, and NF-κB and MAPK/AP-1 activation, by human gingival fibroblasts. MATERIAL AND METHODS: Human gingival fibroblasts were incubated ± IL-1ß, with or without exposure to 1,25D3 or 20D3. IL-6 and IL-8 in culture supernatants were measured by enzyme-linked immunosorbent assay. NF-κB (p65) and AP-1 (phospho-cJun) and were measured in nuclear extracts via binding to specific oligonucleotides. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß-stimulated IL-6 and IL-8 levels were both significantly inhibited (40%-60%) (P<.045) by 1,25D3, but not 20D3 (0%-15% inhibition, not statistically significant). Both 1,25D3 and 20D3 significantly and similarly inhibited IL-1ß-stimulated nuclear levels of p65 and phospho-cJun (P<.02). CONCLUSION: Reduction of the activation of NF-κB and AP-1 alone is not able to inhibit strongly the IL-1ß stimulated IL-6 and IL-8 gene expression. 1,25D3 but not 20D3 may affect some of the many other factors/processes/pathways that in turn regulate the expression of these genes. However, the results suggest that topical application of ligands of the vitamin D receptor may be useful in the local treatment of periodontitis while reducing adverse systemic effects.

Drug-Mediated Gene Regulation of Vitamin D3 Metabolism in Primary Human Dermal Fibroblasts.

Vitamin D metabolism was studied in primary human dermal fibroblasts with focus on drug-mediated gene regulation related to adverse side effects of antiretroviral drugs used in HIV therapy. The fibroblasts expressed mRNA for cytochrome P450 (CYP) enzymes catalysing bioactivating (CYP2R1, CYP27A1 and CYP27B1) and catabolic reactions (CYP24A1). The cells produced both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 . The results demonstrate that primary dermal fibroblasts have an active vitamin D3 -metabolizing system. High incidence of low bone mineral density is a concern for HIV-infected patients treated with antiretroviral drugs. Osteomalacia and severe vitamin D deficiency have been reported. We investigated whether drug-mediated gene regulation could be a possible mechanism behind these adverse drug effects. Fibroblasts were treated with different drugs used in HIV therapy, and the 1α,25-dihydroxyvitamin D3 levels and relative mRNA levels for crucial enzymes were determined. Efavirenz, stavudine and ritonavir significantly down-regulated the bioactivating CYP2R1 and up-regulated the catabolic CYP24A1. The drugs reduced bioactivating enzyme activities and cellular levels of 1α,25-dihydroxyvitamin D3 . The current results indicate that effects on gene expression may lead to disturbed vitamin D metabolism and decreased cellular levels of active vitamin D3 . The data are consistent with the impaired bone health in patients treated with certain antiretroviral drugs.

1,25(OH)2D3 and VDR Signaling Pathways Regulate the Inhibition of Dectin-1 Caused by Cyclosporine A in Response to Aspergillus Fumigatus in Human Corneal Epithelial Cells.

BACKGROUND: The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition. METHODS: Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1ß and TNF-α) were detected by RT-PCR, western blot and ELISA. RESULTS: Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone. CONCLUSIONS: These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition.

Klotho-Dependent Cellular Transport Regulation.

Klotho is a transmembrane protein that in humans is encoded by the hKL gene. This protein is known to have aging suppressor effects and is predominantly expressed in the distal convoluted tubule of the kidney, parathyroid glands, and choroid plexus of the brain. The Klotho protein exists in both full-length membrane form and a soluble secreted form, which exerts numerous distinct functions. The extracellular domain of Klotho can be enzymatically cleaved off and released into the systemic circulation where it functions as ß-glucuronidase and a hormone. Soluble Klotho is a multifunction protein present in the biological fluids including blood, urine, and cerebrospinal fluid of mammals. Klotho deficiency leads to multiple organ failure accompanied by early appearance of multiple age-related disorders and early death, whereas overexpression of Klotho results in the opposite effects. Klotho, an enzyme and hormone, has been reported to participate in the regulation of cellular transport processes across the plasma membrane either indirectly through inhibiting calcitriol (1,25(OH)2D3) formation or other mechanism, or by directly affecting transporter proteins, including ion channels, cellular carriers, and Na(+)/K(+)-ATPase. Accordingly, Klotho protein serves as a powerful regulator of cellular transport across the plasma membrane. Importantly, Klotho-dependent cellular transport regulation implies stimulatory or inhibitory effects. Klotho has been shown to play a key role in the regulation of multiple calcium and potassium ion channels, and various cellular carriers including the Na(+)-coupled cotransporters such as NaPi-IIa, NaPi-IIb, EAAT3, and EAAT4, CreaT1 as well as Na(+)/K(+)-ATPase. These regulations are parts of the antiaging function of Klotho, which will be discussing throughout this chapter. Clearly, further experimental efforts are required to investigate the effect of Klotho on other transport proteins and underlying molecular mechanisms by which Klotho exerts its effect.

Structural Studies of Vitamin D Nuclear Receptor Ligand-Binding Properties.

The vitamin D nuclear receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 hormone (1,25(OH)2D3, or calcitriol), classically regulate mineral homeostasis and metabolism but also much broader range of biological functions, such as cell growth, differentiation, antiproliferation, apoptosis, adaptive/innate immune responses. Being widely expressed in various tissues, VDR represents an important therapeutic target in the treatment of diverse disorders. Since ligand binding is a key step in VDR-mediated signaling, numerous 1,25(OH)2D3 analogs have been synthesized in order to selectively modulate the receptor activity. Most of the synthetic analogs have been developed by modification of a parental compound and some of them mimic 1,25(OH)2D3 scaffold without being structurally related to it. The ability of ligands that have different size and conformation to bind to VDR and to demonstrate biological effects is intriguing, and therefore, ligand-binding properties of the receptor have been extensively investigated using a variety of biochemical, biophysical, and computational methods. In this chapter, we describe different aspects of the structure-function relationship of VDR in complex with natural and synthetic ligands coming from structural analysis. With the emphasis on the binding modes of the most promising compounds, such as secosteroidal agonists and 1,25(OH)2D3 mimics, we also highlight the action of VDR antagonists and the evidence for the existence of an alternative ligand-binding site within the receptor. Additionally, we describe the crystal structures of VDR mutants associated with hereditary vitamin D-resistant rickets that display impaired ligand-binding function.

New signalling pathway involved in the anti-proliferative action of vitamin D3 and its analogues in human neuroblastoma cells. A role for ceramide kinase.

1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3), a crucial regulator of calcium/phosphorus homeostasis, has important physiological effects on growth and differentiation in a variety of malignant and non-malignant cells. Synthetic structural hormone analogues, with lower hypercalcemic side effects, are currently under clinical investigation. Sphingolipids appear to be crucial bioactive factors in the control of the cell fate: the phosphorylated forms, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), are mitogenic factors, whereas sphingosine and ceramide (Cer) usually act as pro-apoptotic agents. Although many studies correlate S1P function to impaired cell growth, the relevance of C1P/Cer system and its involvement in neuroblastoma cells remain to be clarified. Here, we demonstrated the anti-proliferative effect of 1,25(OH)2D3 as well as of its structural analogues, ZK156979 and ZK191784, in human SH-SY5Y cells, as judged by [³H]thymidine incorporation, cell growth and evaluation of active ERK1/2 levels. The inhibition of ceramide kinase (CerK), the enzyme responsible for C1P synthesis, by specific gene silencing or pharmacological inhibition, drastically reduced cell proliferation. 1,25(OH)2D3 and ZK191784 treatment induced a significant decrease in CerK expression and C1P content, and an increase of Cer. Notably, the treatment of SH-SY5Y cells with ZK159222, antagonist of 1,25(OH)2D3 receptor, trichostatin A, inhibitor of histone deacetylases, and COUP-TFI-siRNA prevented the decrease of CerK expression elicited by 1,25(OH)2D3 supporting the involvement of VDR/COUP-TFI/histone deacetylase complex in CerK regulation. Altogether, these findings provide the first evidence that CerK/C1P axis acts as molecular effector of the anti-proliferative action of 1,25(OH)2D3 and its analogues, thereby representing a new possible target for anti-cancer therapy of human neuroblastoma.

Calcium supplementation prevents obesity, hyperleptinaemia and hyperglycaemia in adult rats programmed by early weaning.

It is known that Ca therapy may have anti-obesity effects. Since early weaning leads to obesity, hyperleptinaemia and insulin resistance, we studied the effect of dietary Ca supplementation in a rat model. Lactating rats were separated into two groups: early weaning (EW) - dams were wrapped with a bandage to interrupt lactation in the last 3 d of lactation and control (C) - dams whose pups had free access to milk during the entire lactation period (21 d). At 120 d, EW and C offspring were subdivided into four groups: (1) C, received standard diet; (2) CCa, received Ca supplementation (10 g of calcium carbonate/kg of rat chow); (3) EW, received standard diet; (4) EWCa, received Ca supplementation similar to CCa. The rats were killed at 180 d. The significance level was at P < 0·05. Adult EW offspring displayed hyperphagia (28 %), higher body weight (9 %) and adiposity (77 %), hyperleptinaemia (twofold increase), hypertriacylglycerolaemia (64 %), hyperglycaemia (16 %), higher insulin resistance index (38 %) and higher serum 25-hydroxyvitamin D3 (fourfold increase), but lower adiponectinaemia:adipose tissue ratio (44 %). In addition, they showed Janus tyrosine kinase 2 and phosphorylated signal transducer and activator of transcription 3 underexpression in hypothalamus (36 and 34 %, respectively), suggesting leptin resistance. Supplementation of Ca for 2 months normalised these disorders. The EW group had no change in serum insulin, thyroxine or triiodothyronine, and Ca treatment did not alter these hormones. In conclusion, we reinforced that early weaning leads to late development of some components of the metabolic syndrome and leptin resistance. Dietary Ca supplementation seems to protect against the development of endocrine and metabolic disorders in EW offspring, maybe through vitamin D inhibition.

On the mechanism underlying (23S)-25-dehydro-1alpha(OH)-vitamin D3-26,23-lactone antagonism of hVDRwt gene activation and its switch to a superagonist.

(23S)-25-Dehydro-1alpha(OH)-vitamin D(3)-26,23-lactone (MK) is an antagonist of the 1alpha,25(OH)(2)-vitamin D(3) (1,25D)/human nuclear vitamin D receptor (hVDR) transcription initiation complex, where the activation helix (i.e. helix-12) is closed. To study the mode of antagonism of MK an hVDR mutant library was designed to alter the free molecular volume in the region of the hVDR ligand binding pocket occupied by the ligand side-chain atoms (i.e. proximal to helix-12). The 1,25D-hVDR structure-function studies demonstrate that 1) van der Waals contacts between helix-12 residues Leu-414 and Val-418 and 1,25D enhance the stability of the closed helix-12 conformer and 2) removal of the side-chain H-bonds to His-305(F) and/or His-397(F) have no effect on 1,25D transactivation, even though they reduce the binding affinity of 1,25D. The MK structure-function results demonstrate that the His-305, Leu-404, Leu-414, and Val-418 mutations, which increase the free volume of the hVDR ligand binding pocket, significantly enhance MK antagonist potency. Surprisingly, the H305F and H305F/H397F mutations turn MK into a VDR superagonist (EC(50) approximately 0.05 nm) but do not concomitantly alter MK binding affinity. Molecular modeling studies demonstrate that MK antagonism stems from its side chain energetically preferring a pose in the VDR ligand binding pocket where its terminal C26-methylene atom is far removed from helix-12. MK superagonism results from an energetically favored increase in interaction between Leu-404/Val-418 and C26, resulting in an increase in the stability and population of the closed, helix-12 conformer. Finally, the results/model generated, coupled with application of a VDR ensemble allosterics model, provide an understanding for the species specificity of MK.

CYP1A1 and CYP3A4 modulation by dietary flavonoids in human intestinal Caco-2 cells.

Flavonoids have been proposed to exert beneficial effects in a multitude of disease states. However, evidence of potential toxic actions has also emerged. Since large doses of flavonoids can be encountered in the intestine simultaneously with ingested drugs and pollutants, this study aimed at investigating nine individual flavonoid compounds and their interactions with the major intestinal isoforms of cytochrome P450, i.e. CYPs 1A1 and 3A4, using human intestinal Caco-2 cells cultivated in a serum-free medium. Genistein, quercetin and chrysin provoked a dose-dependent inducing effect on the CYP1A1 activity, measured with the EROD assay. However, they did not affect the CYP1A1 mRNA expression, suggesting they are not aryl hydrocarbon receptor-ligands in intestinal cells and act at a post-transcriptional level. Chrysin, at 50 microM, was detected as a potent inhibitor of the TCDD-induced CYP1A1 activity, leading the activity to ca. 10% of the TCDD-control value (n=3), this effect involving, at least partly, direct interactions at the enzyme level. Quercetin was also shown to significantly inhibit the constitutive CYP3A4 activity, measured by the 6beta-(OH)-testosterone assay, and to impair its induction by 1,25-vitamin D(3). Chrysin, quercetin and genistein, were detected as significant inhibitors of the 1,25-vitamin D(3)-induced CYP3A4 activity. In vivo, these effects could result in reduced activation of procarcinogens and/or in drug bioavailability limitation. They underline the importance of intestinal studies to assess food safety and health risks linked to the ingestion of flavonoid-enriched supplements or functional foods.

Human CYP24 catalyzing the inactivation of calcitriol is post-transcriptionally regulated by miR-125b.

Human vitamin D3 hydroxylase (CYP24) catalyzes the inactivation of 1alpha,25-dihydroxyvitamin D3 (calcitriol), which exerts antiproliferative effects. CYP24 has been reported to be overexpressed in various cancers in which microRNA levels are dysregulated. In silico analysis identified a potential miR-125b recognition element (MRE125b) in the 3'-untranslated region of human CYP24 mRNA. We investigated whether CYP24 is regulated by miR-125b. In luciferase assays using a reporter plasmid containing MRE125b, transfection of the antisense oligonucleotide (AsO) for miR-125b increased the reporter activity in KGN cells, and transfection of precursor miR-125b decreased the reporter activity in MCF-7 cells. The endogenous CYP24 protein level was also increased by AsO for miR-125b in KGN cells and was decreased by precursor miR-125b in MCF-7 cells. These results suggested that human CYP24 is regulated by miR-125b. Immunohistochemical analysis revealed that the CYP24 protein levels in human breast cancer were higher than in adjacent normal tissues, without an accompanying CYP24 mRNA increase. On the other hand, the expression levels of miR-125b in cancer tissues were significantly (P < 0.0005) lower than those in normal tissues. It is noteworthy that the CYP24 protein levels in cancer tissues were inversely associated with the cancer/normal ratios of the miR-125b levels, indicating that the decreased miR-125b levels in breast cancer tissues would be one of the causes of the high CYP24 protein expression. In conclusion, this study clearly demonstrates that miR-125b post-transcriptionally regulates the CYP24, which serves as a possible mechanism for the high CYP24 expression in cancer tissues.

PTH(7-84) inhibits PTH(1-34)-induced 1,25-(OH)2D3 production in murine renal tubules.

To elucidate whether PTH(7-84), a degradation product of PTH(1-84), which inhibits PTH(1-84)-induced bone resorption, also exerts an antagonistic effect on the kidney, we studied the effect of PTH(7-84) on PTH(1-34)-induced production of 1,25-(OH)2D3 in primary cultured murine renal tubules. Neonatal mouse renal tubules cultured in serum-free MEM for 7 days were treated with PTH(1-34) and/or PTH(7-84). Three hours after addition of 25-OHD(3) (10(-6) M), 1,25-(OH)2D3 was determined. PTH(1-34) stimulated the conversion of 25-OHD3 to 1,25-(OH)2D3, and PTH(7-84) dose-dependently inhibited this process. Real-time PCR revealed that PTH(1-34) increased the expression level of 1alpha-hydroxylase mRNA, whereas PTH(7-84) did not affect the expression level 1alpha or 24-hydroxylase mRNA. These in vitro data suggest that PTH(7-84) elicits an antagonistic effect in renal tubules through receptors different from the type I PTH/PTHrP receptor. This may at least partly account for the decreased serum level of 1,25-(OH)2D in patients with severe primary hyperparathyroidism with renal failure.

9-Cis retinoic acid reduces 1alpha,25-dihydroxycholecalciferol-induced renal calcification by altering vitamin K-dependent gamma-carboxylation of matrix gamma-carboxyglutamic acid protein in A/J male mice.

Matrix gamma-carboxyglutamic acid protein (MGP), a vitamin K-dependent protein, is involved in regulation of tissue calcification. We previously reported that 9-cis retinoic acid (RA) mitigates 1alpha,25-dihydroxycholecalciferol [1,25(OH)(2)D3]-induced renal calcification in a 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung cancer A/J male mouse model. This raised the question if the mechanism(s) underlying this calcification involves vitamin K. We assessed expression and vitamin K dependent gamma-carboxylation of MGP and vitamin K concentrations [phylloquinone (PK), as well as its conversion product, menaquinone-4 (MK-4)] in tissues obtained from NNK-injected A/J male mice fed 1,25(OH)(2)D3 (2.5 microg/kg diet; D group) +/- RA (15 mg/kg diet) for 20 wk. Renal calcification was only observed in the D group (2/10; 20% of the group). Renal MGP mRNA and uncarboxylated MGP (ucMGP) increased in response to D (P < 0.05) but not in response to RA or RA + D. In contrast, gamma-carboxylated MGP increased to 2.2-fold of the control in response to D+RA (P < 0.05) but not in response to RA or D alone. Although all diets contained equal amounts of PK, the kidney MK-4 concentration was higher in the D group (P < 0.05) and lower in the RA group (P < 0.05) compared with the RA+D or control groups. Renal PK concentrations were lower in the RA and RA+D groups than in the control and D groups (P < 0.05). These data suggest that 9-cis RA mitigated 1,25(OH)(2)D3-induced renal calcification by modifying the 1,25(OH)(2)D3-induced increase in ucMGP. The mechanisms by which 9-cis RA and 1,25(OH)(2)D3 alter vitamin K concentrations warrant further investigation.

A 24-phenylsulfone analog of vitamin D inhibits 1alpha,25-dihydroxyvitamin D(3) degradation in vitamin D metabolism-competent cells.

The antimitotic, prodifferentiating, and proapoptotic steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], at supraphysiological levels has potential for tumor therapy. However, epithelial cells from tumor-prone organs such as colon, prostate, and breast express not only the vitamin D receptor, but also vitamin D hydroxylases. In contrast to normal cells, malignant cells have high basal levels of the hydroxylase 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) and, in addition, have the potential to induce CYP24 in response to 1alpha,25-(OH)(2)D(3). Because 24-hydroxylation by CYP24 would rapidly degrade the steroid hormone in the course of therapy, the enzyme activity in tumor cells should be inhibited. We demonstrate that a 24-phenylsulfone analog of 1alpha,25-(OH)(2)D(3), KRC-24SO(2)Ph-1 (S-4a), rapidly and potently inhibits 24-hydroxylase activity in human tumor cells derived from colon, prostate, and mammary gland. Although enzymatic inhibition is a consequence of direct interaction, S-4a as a vitamin D analog apparently binds to the vitamin D receptor and induces CYP24 mRNA, which, however, is not translated into increased enzymatic activity. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase expression is not affected at all by S-4a. When both 1alpha,25-(OH)(2)D(3) and S-4a are added to the cell culture, transcription of CYP24 is increased, possibly because of an increase in the half-life of the hormone. The colon cell line COGA-13 has very high levels of CYP24 and is, therefore, resistant to the action of vitamin D. Yet, S-4a imparts antimitotic activity to 1alpha,25-(OH)(2)D(3) and may therefore constitute a therapeutic to stimulate the antiproliferative potential of vitamin D-based antitumor activity.

Practical synthesis and evaluation of the biological activities of 1alpha,25-dihydroxyvitamin D3 antagonists, 1alpha,25-dihydroxyvitamin D3-26,23-lactams. Designed on the basis of the helix 12-folding inhibition hypothesis.

A practical synthetic route to novel vitamin D antagonists of DLAM (1alpha,25-dihydroxyvitamin D(3)-26,23-lactam) was developed from vitamin D(2) via the 1,3-dipolar cycloaddition reaction as a key step. Six DLAM derivatives (24 compounds) with a variety of nitrogen substituents and stereochemistries at C23 and C25 were synthesized. Among these new derivatives, (23S,25S)-DLAM isomers bound effectively to VDRs and showed antagonistic activity in the HL-60 cell differentiation inhibition assay. The importance of the substituent on the nitrogen of DLAMs for antagonistic activity was also suggested by computational docking studies.

Inhibition of apolipoprotein AI gene expression by 1, 25-dihydroxyvitamin D3.

Members of the steroid receptor superfamily are known to alter the transcription of apolipoprotein AI (apo AI), the major apoprotein of high-density lipoprotein (HDL). To assess the role of vitamin D receptor (VDR) in apo AI gene expression, we investigated the effect of 1alpha, 25-dihydroxycholecalciferol (1, 25-(OH)2 D3) as well as the vitamin D antagonist ZK-191784 (ZK), on apo AI gene expression and promoter activity in the human hepatoma cell line HepG2. Apo AI secretion and mRNA levels were both suppressed in a dose-dependent manner in HepG2 cells treated 1, 25-(OH)2 D3. This was accompanied by a similar decrease in apo AI promoter activity. Mapping of the vitamin D response element showed that suppression required a region of the apo AI gene promoter identified previously to contain site A. However, vitamin D treatment had no effect on nuclear factor binding to site A of the apo AI promoter. Treatment with vitamin D receptor antagonist ZK inhibited the ability of 1, 25-(OH)2 D3 to repress apo AI promoter activity, while higher doses of ZK increased apo AI promoter activity. ZK did not alter estradiol stimulated apo AI promoter activity. The VDR antisense ODN had no effect on apo AI promoter activity in control cells, however, it reversed the repression normally seen in cells treated with 1, 25-(OH)2D3. It is concluded that 1, 25-(OH)2 D3 suppresses apo A1 gene expression at the transcriptional level, possibly by altering coactivators or corepressors. This effect requires the VDR as well as a vitamin D response element in the apo AI promoter.

All-trans retinoic acid antagonizes the action of calciferol and its active metabolite, 1,25-dihydroxycholecalciferol, in rats.

An antagonistic interaction between retinol and calciferol has been established. However, the mechanism by which this antagonism occurs is unclear. One possibility is that retinol affects the metabolism of calciferol. To investigate this hypothesis, retinol- and calciferol-depleted rats were given various amounts of ergocalciferol, cholecalciferol, 1alpha,25-dihydroxycholecalciferol [1,25(OH)2D3], or 24,24-difluoro-1alpha,25-dihydroxycholecalciferol [24-F2-1,25(OH)2D3] in combination with various amounts of retinyl acetate or all-trans retinoic acid (ATRA) in a series of studies. Rats administered 1720 or 3440 microg retinyl acetate once every 3 d for 33 d in combination with 25.8 ng ergocalciferol or 25 ng cholecalciferol every 3 d had lower serum calcium and greater serum phosphorus concentrations than rats fed 0 or 11.4 mug retinyl acetate every 3 d. In addition, rats fed 400 microg ATRA/d in combination with 25.8 ng ergocalciferol every 3 d, 25 ng cholecalciferol every 3 d, 2-5 ng 1,25(OH)2D3/d, or 0.5-1 ng 24-F2-1,25(OH)2D3/d had significantly lower serum calcium and higher serum phosphorus concentrations than rats not given ATRA in the diet. Therefore, both retinyl acetate and ATRA are able to antagonize the action of ergocalciferol and cholecalciferol in vivo. Additionally, ATRA antagonizes the in vivo action of 1,25(OH)2D3 and an analog, 24-F2-1,25(OH)2D3, that cannot be 24-hydroxylated. Together, these results suggest that retinol does not antagonize the action of calciferol by altering the metabolism of calciferol or 1,25(OH)2D3, but does so by another mechanism.

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation.

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.