Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Effects of the vitamin D3 analog 1α,25-dihydroxyvitamin D3-3β-bromoacetate on rat osteosarcoma cells: Comparison with 1α,25-dihydroxyvitamin D3

The actions of the hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃ [1α,25-(OH)₂D₃], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1α,25-(OH)₂D₃ in many cell types including osteoblastic cells. We have compared the effects of 1α,25-(OH)₂D₃ and a novel 1α,25-(OH)₂D₃ bromoester analog (1,25-(OH)₂-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D receptor were studied to investigate genomic and nongenomic actions. In ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 × 10⁻⁸ M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)₂-BE but not the natural ligand with a significant increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 × 10⁻⁸ M, both 1α,25-(OH)₂D₃ and 1,25-(OH)₂-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)₂-BE are similar to those of 1α,25-(OH)₂D₃, and since 1,25-(OH)₂-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors. © 1996 Wiley-Liss, Inc.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25-dihydroxyvitamin D₃ (VD), RAR, RXR, and VDR are ligand-inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor-α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2-type sequence, but not on a DR5-type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR-mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997. © 1997 Wiley-Liss, Inc.     

Mechanism of vitamin D3-induced transcription of phospholipase D1 in HaCat human keratinocytes

1α,25-Dihydroxyvitamin D₃ (VitD₃) increases protein and gene expression of phospholipase D1 (PLD1), but not PLD2, in HaCaT human keratinocytes. We show that VitD₃ increases PLD1 gene expression through a vitamin D responsive element (VDRE) on the 5′ PLD1 promoter (−260 bp to −246 bp from exon 1). Similar results were obtained by transfecting VitD₃ receptor (VDR) into HEK293 cells, which are originally VitD₃-unresponsive. Electrophoresis mobility shift assays (EMSA) and chromatin immunoprecipitation (CHIP) assays showed that the complex of VitD₃, VDR and retinoid X receptor α (RXRα) binds to the VDRE and increases PLD1 gene expression in HaCaT cells.     

Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25-dihydroxyvitamin D3

1α,25-Dihydroxyvitamin D₃ (10^?12 M to 10^?8 M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1α,25-dihydroxyvitamin D₃ at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1α,25(OH)₂D₃′s inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1β,25-dihydroxyvitamin D₃, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

Synthesis of 2α-substituted-14-epi-previtamin D3 and its genomic activity

We synthesized and isolated 2α-substituted analogs of 14-epi-previtamin D₃ after thermal isomerization at 80 °C for the first time. The VDR binding affinity and transactivation activity of osteocalcin promoter in HOS cells were evaluated, and the 2α-methyl-substituted analog was found to have greater genomic activity than 14-epi-previtamin D₃.     

Design and synthesis of tetraol derivatives of 1,12-dicarba-closo-dodecaborane as non-secosteroidal vitamin D analogs

Vitamin D receptor (VDR), a nuclear receptor for 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃, 1), is a promising target for multiple clinical applications. We recently developed non-secosteroidal VDR ligands based on a carbon-containing boron cluster, 1,12-dicarba-closo-dodecaborane (p-carborane), and examined the binding of one of them to VDR by means of crystallographic analysis. Here, we utilized that X-ray structure to design novel p-carborane-based tetraol-type vitamin D analogs, and we examined the biological activities of the synthesized compounds. Structure–activity relationship study revealed that introduction of an ω-hydroxyalkoxy functionality enhanced the biological activity, and the configuration of the substituent significantly influenced the potency. Among the synthesized compounds, 4-hydroxybutoxy derivative 9a exhibited the most potent activity, which was equal to that of the secosteroidal vitamin D analog, 19-nor-1α,25-dihydroxyvitamin D₃ (2).     

Clonal Differences in Expression of 25-Hydroxyvitamin D3-1α-hydroxylase, of 25-Hydroxyvitamin D3-24-hydroxylase, and of the Vitamin D Receptor in Human Colon Carcinoma Cells: Effects of Epidermal Growth Factor and 1α,25-Dihydroxyvitamin D3

Human colon carcinoma cells express 25-hydroxyvitamin D₃-1α-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D₃ (1,25-D3), which can be metabolized by 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.     

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)₂D₃ which have been modified on the A-ring and C,D-ring side chain (1α-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YA = 3a) and 1β-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D₃ (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)₂D₃. Chondrocyte proliferation ([³H]-thymidine incorporation), proteoglycan production ([³⁵S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)₂D₃, 24,25-(OH)₂D₃, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)₂D₃ and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)₂D₃ having a greater effect. 24,25-(OH)₂D₃ caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)₂D₃, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation–dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. J. Cell. Biochem. 66:457–470, 1997. © 1997 Wiley-Liss, Inc.