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W H Okamura

Publications and source records attributed to W H Okamura.

At least 19 recordsLinked to original sources
PubMed2001

1alpha,25-Dihydroxyvitamin D3 modulates human adipocyte metabolism via nongenomic action.

We reported recently that suppression of the renal 1alpha,25-dihyroxyvitamin D3 (1lpha,25-(OH)2-D3) production in aP2-agouti transgenic mice by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, inhibits lipogenesis, and reduces adiposity. However, it was not clear whether this modulation of adipocyte metabolism by dietary calcium is a direct effect of inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i. Accordingly, we have now evaluated the direct role of 1alpha,25-(OH)2-D3. Human adipocytes exhibited a 1alpha,25-(OH)2-D3 dose-responsive (1-50 nM) increase in [Ca2+]i (P<0.01). This action was mimicked by 1alpha,25-dihyroxylumisterol3 (1alpha,25-(OH)2-lumisterol3) (P<0.001), a specific agonist for a putative membrane vitamin D receptor (mVDR), and completely prevented by 1b,25-dihydroxyvitamin D3 (1beta,25-(OH)2-D3), a specific antagonist for the mVDR. Similarly, 1alpha,25-(OH)2-D3 (5 nM) caused 50%-100% increases in adipocyte fatty acid synthase (FAS) expression and activity (P<0.02), a 61% increase in glycerol-3-phosphate dehydrogenase (GPDH) activity (P<0.01), and an 80% inhibition of isoproterenol-stimulated lipolysis (P<0.001), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. Notably, 1alpha,25-(OH)2-lumisterol3 exerted more potent effects in modulating adipocyte lipid metabolism, with 2.5- to 3.0-fold increases in FAS expression and activity (P<0.001) and a threefold increase in GPDH activity (P<0.001). Also 1alpha,25-(OH)2-lumisterol3 was approximately twice as potent in inhibiting basal lipolysis (P<0.025), whereas 1beta,25-(OH)2-D3 completely blocked all these effects. These data suggest that 1alpha,25-(OH)2-D3 modulates adipocyte Ca2+ signaling and, consequently, exerts a coordinated control over lipogenesis and lipolysis. Thus, a direct inhibition of 1alpha,25-(OH)2-D3-induced [Ca2+]i may contribute to an anti-obesity effect of dietary calcium, and the mVDR may represent an important target for obesity.

Adipocytes↗
PubMed2000

Characterization of a novel analogue of 1alpha,25(OH)(2)-vitamin D(3) with two side chains: interaction with its nuclear receptor and cellular actions.

The hormone 1alpha,25(OH)(2)-vitamin D(3) (125D) binds to its nuclear receptor (VDR) to stimulate gene transcription activity. Inversion of configuration at C-20 of the side chain to generate 20-epi-1alpha,25(OH)(2)D(3) (20E-125D) increases transcription 200-5000-fold over 125D with its 20-normal (20N) side chain. This enhancement has been attributed to the VDR ligand-binding domain (LBD) having different contact sites for 20N and 20E side chains that generate different VDR conformations. We synthesized 1alpha, 25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3) (Gemini) with two six-carbon side chains (both 20N and 20E orientations). Energy minimization calculations indicate the Gemini side chain possesses significantly more energy minima than either 125D or 20E-125D (2346, 207, and 127 minima, respectively). We compared activities of 125D, 20E-125D, and Gemini, respectively, in several assays: binding to wild-type (100%, 147%, and 38%) and C-terminal-truncated mutant VDR; transcriptional activity (of the transfected osteopontin promoter in ROS 17/2.8 cells: ED(50) 10, 0.005, and 1.0 nM); mediation of conformational changes in VDR assessed by protease clipping (major trypsin-resistant fragment of 34, 34, and 28 kDa). For inhibition of cellular clonal growth of human leukemia (HL-60) and breast cancer (MCF7) cell lines, the ED(50)(125D)/ED(50)(Gem) was respectively 380 and 316. We conclude that while Gemini readily binds to the VDR and generates unique conformational changes, none of them is able to permit a superior gene transcription activity despite the presence of a 20E side chain.

Animals↗
PubMed1999

Three-dimensional model of the ligand binding domain of the nuclear receptor for 1alpha,25-dihydroxy-vitamin D(3).

A three-dimensional model for residues 142-427 of the ligand binding domain (LBD) of the human nuclear receptor for 1alpha, 25-dihydroxy-vitamin D(3) [VDR] has been generated based on the X-ray crystallographic atomic coordinates of the LBD of the rat alpha1 thyroid receptor (TR). The VDR LBD model is an elongated globular shape comprised of an antiparallel alpha-helical triple sandwich topology, made up of 12 alpha-helical elements linked by short loop structures; collectively these structural features are similar to the characteristic secondary and tertiary structures for six nuclear receptors with known X-ray structures. The model has been used to describe the interaction of the conformationally flexible natural hormone, 1alpha,25-dihydroxy-vitamin D(3) [1alpha, 25(OH)(2)D(3)], and a number of related analogs with the VDR LBD. The optimal orientation of the 1alpha,25(OH)(2)D(3) in the LBD is with its A-ring directed towards the interior and its flexible side chain pointing towards and interacting with helix-12, site of the activation function-2 domain (AF-2) of the VDR. Mapping of four natural and one experimental point mutations of the VDR LBD, which result in ligand-related receptor dysfunction, indicates the close proximity of these amino acids to the bound ligand.

Amino Acid Sequence↗
PubMed1999

1alpha,25-dihydroxy-3-epi-vitamin D3: in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats.

We recently identified 1alpha,25-dihydroxy-3-epi-vitamin D3 as a major in vitro metabolite of 1alpha,25-dihydroxyvitamin D3, produced in primary cultures of neonatal human keratinocytes. We now report the isolation of 1alpha,25-dihydroxy-3-epi-vitamin D3 from the serum of rats treated with pharmacological doses of 1alpha,25-dihydroxyvitamin D3. 1alpha,25-dihydroxy-3-epi-vitamin D3 was identified through its co-migration with synthetic 1alpha,25-dihydroxy-3-epi-vitamin D3 on both straight and reverse phase high performance liquid chromatography systems and by mass spectrometry. Along with 1alpha,25-dihydroxy-3-epi-vitamin D3, other previously known metabolites, namely, 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxo-vitamin D3 and 1alpha,25-dihydroxyvitamin D3-26,23-lactone, were also identified. Thus, our study for the first time provides direct evidence to indicate that 1alpha,25-dihydroxy-3-epi-vitamin D3 is an in vivo metabolite of 1alpha,25-dihydroxyvitamin D3 in rats.

Animals↗
PubMed1999

1Alpha,25-dihydroxy-3-epi-vitamin D3, a natural metabolite of 1alpha,25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion.

1Alpha,25(OH)2D3 is an important negative regulator of parathyroid hormone (PTH) gene transcription. In parathyroid cells, as in other target tissues, 1alpha,25(OH)2D3 is degraded by side chain oxidation by the inducible 24-hydroxylase. We have previously shown that one metabolite of this pathway, 1alpha,23(S),25-(OH)3-24-oxo-D3, potently suppresses PTH synthesis and secretion in cultured bovine parathyroid cells (bPTC). Further examination of the metabolites of 1alpha,25(OH)2D3 in bPTC has revealed another compound that is less polar than 1alpha,25(OH)2D3. By HPLC analysis and mass spectrometry, this metabolite was identified as 1alpha,25(OH)2-3-epi-D3. The activity of this metabol ite on PTH gene transcription was assessed by the steady-state PTH secretion by bPTC after 72-h treatment with concentrations from 10(-11) M to 10(-7) M. 1Alpha,25(OH)2-3-epi-D3 was found to be only slightly, but not significantly, less active than the native 1alpha,25(OH)2D3 in suppressing PTH secretion despite having 30 times lower affinity for the bPTC VDR. Both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-3-epi-D3 maximally suppressed PTH secretion by 50%. Along with 1alpha,25(OH)2-3-epi-D3, the activities of the other two A-ring diastereomers were assessed. 1beta,25(OH)2D3 suppressed PTH only at 10(-7) M with a decrease of only 30%, in good agreement with its low VDR affinity. Surprisingly, 1beta,25(OH)2-3-epi-D3 stimulated PTH secretion by 30-50% at concentrations from 10(-11) M to 10(-8)M and fell to control (untreated) rates at 10(-7) M. The mechanism for this increase in PTH secretion is under investigation. Metabolism studies performed in bPTC cells using high concentrations of 1alpha,25(OH)2D3 substrate showed that in some incubations, the concentration of 1alpha,25(OH)2-3-epi-D3 was even higher than that of the parent substrate 1alpha,25(OH)2D3. This finding indicates a slower rate of metabolism for this diastereomer. Thus, production and accumulation of 1alpha,25(OH)2-3-epi-D3, as a major stable metabolite of 1alpha,25(OH)2D3 in parathyroid glands, may contribute to the prolonged suppressive effect of 1alpha,25(OH)2D3 on PTH gene transcription.

Animals↗
PubMed1999

Production of 1alpha,25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into calcitroic acid through the carbon 24 (C-24) oxidation pathway. It is now well established that the C-24 oxidation pathway plays an important role in the target tissue inactivation of 1alpha,25(OH)2D3. Recently, we reported that 1alpha,25(OH)2D3 is also metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] through the carbon 3 (C-3) epimerization pathway in human keratinocytes, human colon carcinoma cells (Caco-2), and bovine parathyroid cells. In a previous study, it was demonstrated that 1alpha,25(OH)2-3-epi-D3 when compared to 1alpha,25(OH)2D3 was less active in stimulating intestinal calcium absorption, calcium mobilization from bone, and induction of calbindin D28k. These findings suggest that the C-3 epimerization pathway, like the C-24 oxidation pathway, may play a role in the target tissue inactivation of 1alpha,25(OH)2D3. In this study, we determined the relationship between the C-24 oxidation and the C-3 epimerization pathways by investigating the metabolism of 1alpha,25(OH)2D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8). These two cell lines differ from each other in their ability to metabolize 1alpha,25(OH)2D3 through the C-24 oxidation pathway. It has been previously reported that the C-24 oxidation pathway is expressed only in UMR 106 cells but not in ROS 17/2.8 cells. The results of our present study provide new evidence that both cell lines possess the ability to metabolize 1alpha,25(OH)2D3 into 1alpha,25(OH)2-3-epi-D3 through the C-3 epimerization pathway. Our results also reconfirm the findings of previous studies indicating that UMR 106 cells are the only ones which express the C-24 oxidation pathway out of the two cell lines studied. Furthermore, this study reveals for the first time that the C-3 epimerization pathway may become an alternate metabolic pathway for the target tissue inactivation of 1alpha,25(OH)2D3 in some cells, such as ROS 17/2.8, in which the C-24 oxidation pathway is not expressed.

Animals↗
PubMed1998

Stimulation of phosphorylation of mitogen-activated protein kinase by 1alpha,25-dihydroxyvitamin D3 in promyelocytic NB4 leukemia cells: a structure-function study.

Recent studies have shown that 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] actions in cell growth and differentiation are mediated by both its nuclear receptor (VDRnuc) and its rapid membrane-related effects. In the present study, we investigated the effect of 1alpha,25-(OH)2D3 on p42mapk phosphorylation using human acute promyelocytic leukemia cells (NB4). 1Alpha,25-(OH)2D3 (10[-8] M) significantly increased p42mapk phosphorylation in a time- and dose-dependent manner, with the earliest response detectable at 30 sec. Because 1alpha,25-(OH)2D3 is a conformationally flexible molecule, we have used a series of conformationally locked (6-s-cis vs. 6-s-trans) analogs to evaluate which shape is optimal for activation. Four 6-s-cis-locked analogs (HF, JM, JN, and JP) and two 6-s-trans-locked analog (JB and JD) were studied. HF, JM, JN, and JP all increased p42mapk phosphorylation at 1 and 5 min (10[-8] M), but JB and JD had little effect. Analog HL [1beta,25-(OH)2D3], a specific antagonist for only the rapid effects of 1alpha,25-(OH)2D3, attenuated 1alpha,25-(OH)2D3-induced p42mapk phosphorylation 65-90%. To assess the potential involvement of the VDRnuc in mediating the analog's action, the relative abilities of the analogs to compete with [3H]1alpha,25-(OH)2D3 for binding in vitro to the VDRnuc of NB4 cells was measured. All 6-s-cis analogs bound poorly to VDRnuc (relative competitive index, 0.5-2%) compared with 1alpha,25-(OH)2D3 (relative competitive index, 100%). The present studies demonstrate for the first time that in NB4 cells 1alpha,25-(OH)2D3 rapidly activates the p42mapk pathway, and that this effect can be selectively mediated by analogs that can assume a 6-s-cis conformation.

Calcitriol↗
PubMed1997

Inhibitors of 25-hydroxyvitamin D3-1alpha-hydroxylase: thiavitamin D analogs and biological evaluation.

Six A-ring analogs of 1alpha,25-dihydroxyvitamin D3 (1, 1alpha,25-(OH)2-D3) 3-deoxy-3-thia-1alpha,25-(OH)2-D3 (3), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3alpha-oxide (6), 3-deoxy-3-thia-1alpha,25-(OH)2-D3-3beta-oxide (7) and the 5,6-trans counterparts 5, 8, and 9, respectively--were tested for their ability to inhibit 25-hydroxy-D3-1alpha-hydroxylase (1-OH-ase) in vitro in mitochondria isolated from kidneys of vitamin D deficient chicks. The six analogs were also evaluated in terms of their ability to bind to the chicken intestinal nuclear receptor (VDR) in comparison to the natural hormone 1alpha,25-(OH)2-D3. Analog 7 is not only the best inhibitor of the 1-OH-ase but it also binds effectively to the chick intestinal receptor. It is established that vitamin D analogs must have a 1alpha oxygen group for effective inhibition of the 1-OH-ase. This functional group is also needed for effective binding to the chick intestinal VDR.

Animals↗
PubMed1997

Comparison of 6-s-cis- and 6-s-trans-locked analogs of 1alpha,25-dihydroxyvitamin D3 indicates that the 6-s-cis conformation is preferred for rapid nongenomic biological responses and that neither 6-s-cis- nor 6-s-trans-locked analogs are preferred for genomic biological responses.

The hormone 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] generates biological responses via both genomic and rapid, nongenomic mechanisms. The genomic responses utilize signal transduction pathways linked to a nuclear receptor (VDRnuc) for 1alpha,25(OH)2D3, while the rapid responses are believed to utilize other signal transduction pathways that may be linked to a putative membrane receptor for 1alpha,25(OH)2D3. The natural seco steroid is capable of facile rotation about its 6,7 single carbon bond, which permits generation of a continuum of potential ligand shapes extending from the 6-s-cis (steroid like) to the 6-s-trans (extended). To identify the shape of conformer(s) that can serve as agonists for the genomic and rapid biological responses, we measured multiple known agonist activities of two families of chemically synthesized analogs that were either locked in the 6-s-cis (6C) or 6-s-trans (6T) conformation. We found that 6T locked analogs were inactive or significantly less active than 1alpha,25(OH)2D3 in both rapid responses (transcaltachia in perfused chick intestine, 45Ca2+ influx in ROS 17/2.8 cells) and genomic (osteocalcin induction in MG-63 cells, differentiation of HL-60 cells, growth arrest of MCF-7 cells, promoter transfection in COS-7 cells) assays. In genomic assays, 6C locked analogs bound poorly to the VDRnuc and were significantly less effective than 1alpha,25(OH)2D3 in the same series of assays designed to measure genomic responses. In contrast, the 6C locked analogs were potent agonists of both rapid response pathways and had activities equivalent to the conformationally flexibile 1alpha,25(OH)2D3; this represents the first demonstration that 6-s-cis locked analogs can function as agonists for vitamin D responses.

Animals↗
PubMed1996

Differing shapes of 1 alpha,25-dihydroxyvitamin D3 function as ligands for the D-binding protein, nuclear receptor and membrane receptor: a status report.

1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] is the principal mediator of a wide array of biological responses through the far reaching network of the vitamin D endocine system (VDE). The steroid hormone 1 alpha,25(OH)2D3 is delivered to the various target organs of the VDE via a specific plasma transport protein, the vitamin D binding protein (DBP). Also 1 alpha,25(OH)2D3 is known to initiate biological responses through a nuclear receptor, the nVDR (50 kDa) which regulates selected gene transcription and, in addition in some target tissues, through a second receptor located in the cell membrane, the mVDR (approximately 60 kDa), which is linked to protein kinase C and/or voltage-gated Ca2+ channels so as to generate biological responses very rapidly. 1 alpha,25(OH)2D3 as a ligand is unusually conformationally flexible due to the eight carbon side chain, the seco B-ring which permits rotation about the 6-7 single carbon bond, and the A-ring which undergoes chair-chair conformational interconversion characteristic of cyclohexane rings. This paper reviews the evidence that different shapes of the 1 alpha,25(OH)2D3 satisfy the optimal requirements of the ligand binding domains of the DBP, nVDR and mVDR. The presence of a relatively rigid side chain (composed by the presence of an aromatic ring) enhances ligand interaction 2-3 fold with the DBP, but diminishes ligand affinity for the nVDR by 100 fold. The mVDR responds effectively to analogs of 1 alpha,25(OH)2D3 which are 6-s-cis locked [e.g. 1 alpha,25(OH)2-previtamin D3 or 1 alpha,25(OH)2-provitamin D3], but these same analogs have only 1-2% of the activity of 1 alpha,25(OH)2D3 in regulating gene transcription. Finally the 6-s-trans analog, 1 alpha,25(OH)2-tachysterol3, had <0.1% of the activity of 1 alpha,25(OH)2D3 in regulating gene transcription.

Animals↗
PubMed1995

Chemistry and conformation of vitamin D molecules.

1 alpha,25-Dihydroxyvitamin D3 (1,25) is a structurally unique steroid hormone because it not only possesses the complete 25-hydroxycholesterol side chain, but most notably, it possesses a seco-B triene structure (it lacks a B-ring and is usually depicted in a non-steroidal, extended conformation). In contrast, the classical steroid hormones possess a truncated side chain (progesterone, cortisol, and aldosterone) or no side chain (estradiol and testosterone) and they all possess the fully intact ABCD steroid rings. These structural differences render the seco-B-steroid 1,25 considerably more conformationally flexible. Since 1,25 is now known to target a myriad of tissues where specific interactions occur to produce an array of biological responses, it is of interest to determine whether different topologies of 1,25 (resulting from different conformational orientations of 1,25) are necessary to interact effectively at the different target sites. The array of biological responses include both non-genomic and genomic effects and there is considerable promise for the efficacy of 1,25 analogs as chemotherapeutic agents in a variety of human disease states. For the non-genomic calcium transport response of transcaltachia, the finding that two 6-s-cis locked analogs, 1 alpha,25-dihydroxyprevitamin D3 (pre-1,25) and 1 alpha,25-dihydroxylumisterol3 (1,25-Lumi), are equipotent to 1,25, points strongly to the involvement of the 6-s-cis conformer of 1,25 as the biologically active conformer. Since there is a continuum of easily interconvertible 6,7-single bond conformers of the seco-B ring available to 1,25, conformational minima (either local or global) may have little to do with the manner in which 1,25 is bound to receptor. For the genomic calcium transport response, and for other genomic (or non-genomic) effects, there is no clear evidence whether the steroidal (s-cis) or non-steroidal (s-trans) conformer of 1,25 is involved. In order to address this matter further, efforts are underway to evaluate other conformationally locked analogs of 1,25 which might mimic either the planar 6-s-trans-1,25 or some intermediate conformer between it and the planar-6-s-cis form.

Animals↗
PubMed1994

Identification of a specific binding protein for 1 alpha,25-dihydroxyvitamin D3 in basal-lateral membranes of chick intestinal epithelium and relationship to transcaltachia.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) elicits biological responses by both genomic and nongenomic pathways. This report describes purification of a receptor for 1 alpha,25-(OH)2D3 (VDR) located in the basal-lateral membrane (BLM) of vitamin D-replete chick intestinal epithelium, which is implicated in the nongenomic stimulation of calcium transport (transcaltachia). The BLM-VDR exhibited saturable binding for [3H]1,25-(OH)2D3 (KD = 0.72 x 10(-9)M, Bmax = 0.24 pmol/mg of protein). A 4500-fold purification of the BLM-VDR receptor was achieved. In addition, saturable binding was observed for [3H]24R,25-(OH)2D3 at physiologically relevant levels (KD = 19 x 10(-9) M, Bmax = 2.4 pmol/mg of protein) to a component apparently distinct from the 1 alpha,25-(OH)2D3 BLM-VDR. A functional correlation between the BLM-VDR and transcaltachia was observed in three experimental situations: (i) vitamin D deficiency, which suppresses transcaltachia, resulted in reduced specific [3H]1 alpha,25-(OH)2D3 binding in the BLM-VDR, relative to corresponding fractions from vitamin D-replete chicks; (ii) the BLM-VDR exhibited down-regulation of specific [3H]1 alpha,25-(OH)2D3 binding following exposure to nonradioactive 1 alpha,25-(OH)2D3; and (iii) the relative potencies of two "6-s-cis" analogs of 1 alpha,25-(OH)2D3 to initiate transcaltachia and their ability to compete with [3H]1 alpha,25-(OH)2D3 for binding to the BLM-VDR were parallel. The combined results support the existence of a plasmalemal 1 alpha,25-(OH)2D3 receptor which is a prime candidate for signal transduction in transcaltachia.

Animals↗
PubMed1994

1 alpha,25-Dihydroxy-20-epi-vitamin D3: an extraordinarily potent inhibitor of leukemic cell growth in vitro.

We have evaluated seven recently synthesized vitamin D3 analogs for their abilities to inhibit clonal growth of leukemic cells, to induce leukemic cell differentiation, to stimulate clonal growth of normal myeloid committed stem cells, and to transactivate a reporter gene having a 1,25(OH)2D3 response element (VDRE). The 1,25(OH)2-20-epi-D3 showed extraordinary activity; at 10(-11) mol/L it inhibited clonal growth of 87% of HL-60 myeloblast cells, 60% of S-LB1 cells (human T-cell lymphotropic virus type 1 [HTLV-1]-immortalized human T-lymphocyte cell line) and 50% of leukemic clonogenic cells (colony-forming unit-leukemia) obtained from patients with acute myelogenous leukemia. No effect of either 1,25(OH)2D3 or 1,25(OH)2-20-epi-D3 was observed on the clonal proliferation of an HTLV-1-immortalized human T-lymphocyte cell line (Ab-VDR) having nonfunctional 1,25 (OH)2D3 cellular receptors (VDR). The abilities of 1,25(OH)2-20-epi-D3 to induce differentiation of HL-60 cells, as measured by generation of superoxide and nonspecific esterase production, was less than its antiproliferative activities. This analog stimulated colony-forming unit-granulocyte-macrophage growth from normal human bone marrow. To gain insights into the remarkable antileukemic activities of 1,25(OH)2-20-epi-D3, we examined its ability to enter HL-60 cells, bind to the VDR, and interact with a transfected VDRE attached upstream of a TK promoter-driven reporter gene (chloramphenicol acetyl transferase [CAT]). The 1,25(OH)2-20-epi-D3 potently increased CAT activity (> 16-fold, as compared with cells transfected with control receptor having no VDRE); paradoxically, 1,25(OH)2-20-epi-D3 was of equal potency to 1,25(OH)2D3 in transactivating the VDRE-containing reporter gene, even though the analog had a 1,000-fold greater antileukemic effect as compared with 1,25(OH)2D3. In summary, we have identified an extremely potent 1,25(OH)2D3 analog with antiproliferative and differentiating effects on leukemic cells and that may be clinically useful. This analog appears to generate biologic responses via the classical VDR pathway, but further studies are required to elucidate the mechanism by which this analog produces its prominent activities.

Calcitriol↗
PubMed1994

14-epi stereoisomers of 25-hydroxy- and 1 alpha,25-dihydroxyvitamin D3: synthesis, isomerization to previtamins, and biological studies.

The C-14 epimers of vitamin D, 14-epi-25-hydroxyvitamin D3 (4) and 14-epi-1 alpha, 25-dihydroxyvitamin D3 (5), were synthesized, and their isomerization via [1,7]-sigmatropic hydrogen shifts to the corresponding previtamin forms (4' and 5', respectively) was studied. The activation parameters of the [1,7]-sigmatropic hydrogen shifts were found to be similar to those of other vitamin D analogues, although epimerization at C-14 shifts the equilibrium of the triene chromophore to the previtamin form. The in vivo biological activities of 4, 4', 5, and 5' in the chick in terms of their ability to elicit intestinal calcium absorption and bone calcium mobilization were determined. These vitamin D analogues, the first in the natural steroid series modified at the C-14 position, were essentially devoid of activity. The relative competitive indices (RCIs), derived in an in vitro assay reflecting the ability of these analogues to bind to the chick intestinal nuclear receptor, were determined. Analogues 4, 4', 5, and 5' had RCI values of 0.08, 0.01, 15, and 1.6, respectively, in comparison to the natural ligand, 1 alpha, 25-dihydroxyvitamin D3 (3), whose value is 100 by definition. Thus, the in vivo and in vitro data were somewhat at variance, particularly for 5, which bound significantly to the chick intestinal receptor. In vitro binding studies with the human serum vitamin D binding protein (DBP) were also conducted. The RCI values for human DBP reflects the ability of an analogue to bind to this protein in comparison to the hormone 3, whose value is 100. The measured RCI values for 4, 4', 5, and 5' were 3450, 90, 12, and 2.2, respectively. It is noteworthy that analogue 4 binds approximately 35 times more effectively than the parent hormone 3, but approximately 20 times less effectively than 25-hydroxyvitamin D3 (2).

Animals↗
PubMed1994

Nonnuclear effects of the steroid hormone 1 alpha,25(OH)2-vitamin D3: analogs are able to functionally differentiate between nuclear and membrane receptors.

The steroid hormone 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] stimulates biological responses via both genomic mechanisms and nongenomic mechanisms (opening of voltage-gated Ca2+ channels). We report here that the two closed B-ring steroid analogs of 1 alpha,25(OH)2D3, 1 alpha,25(OH)2-7-dehydrocholesterol and 1 alpha,25(OH)2-lumisterol3, are able to generate the nongenomic response, transcaltachia, without the ability to compete with the natural metabolite for binding to its nuclear receptor. We propose that the nongenomic membrane associated receptor can accept the ligand in its closed "6-s-cis" conformation whereas the nuclear receptor prefers the extended "6-s-trans" conformer.

Animals↗
PubMed1994

Profile of ligand specificity of the vitamin D binding protein for 1 alpha,25-dihydroxyvitamin D3 and its analogs.

The profile of structural preference for the ligand binding domain of the human vitamin D binding protein (DBP) was determined by steroid competition assay of 71 analogs of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. The following categories of structural modification were evaluated [values represent fold change; R = reduction, I = increase in binding to the DBP from the reference 1 alpha, 25(OH)2D3]: (1) deletion in the A ring of the 1 alpha-hydroxyl-(20-1600I); (2) conversion of the triene system to the previtamin form (6-40R); (3) addition of substituents to carbon 11 of the C ring (4-14R); (4) inversion of the C/D ring junction (8-20R); (5) unsaturation of the D ring (16-ene; 4-140R); (6) replacement of hydrogen with deuterium atoms (no effect); alteration of the side chain by (7) adding or deleting carbon atoms (5-12R); (8) addition of fluorines (0.2-10R); (9) presence of unsaturation (22-ene, 0-5R; 23-ene, 3R-10I; 23-yne, 5-20R); (10) addition of hydroxyls (2-100R); and (11) addition of an aromatic ring (0-20I). Thus the DBP ligand binding domain could tolerate only modest changes to the structure of 1 alpha,25(OH)2D3 without a reduction in binding of the analog. The increases in binding seen in the aromatic side chain and with a triple bond at carbon-23 may be indicative of a preferred conformation of the flexible 1 alpha, 25(OH)2D3 side chain. In addition, a comparison was made of the DBP ligand binding domain with that of the human HL-60 cell 1 alpha, 25(OH)2D3 nuclear receptor. Both ligand binding domains could equivalently accommodate to the presence of (1) a side-chain cyclopropyl group, (2) 22-ene or 23-yne, (3) lengthening the side chain by two carbons, (4) presence of four to six fluorine atoms, (5) substitution of an oxygen for carbon 22, and (6) presence of a 22-[m-(dimethylhydroxymethyl)phenyl] aromatic group in the side chain. The DPB could tolerate better than the HL-60 cell receptor the presence of a 22-(p-hydroxyphenyl) aromatic group in the side chain and the absence of the 1 alpha-hydroxyl. In contrast, the HL-60 cell receptor could tolerate better than the DBP the following structural modifications: presence of a 16-ene, or 16-ene plus 23-yne unsaturation, and presence of an 11 beta-hydroxyl.

Binding, Competitive↗