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A W Norman

Publications and source records attributed to A W Norman.

At least 19 recordsLinked to original sources
PubMed1999

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1alpha,25-dihydroxyvitamin D3.

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.

Animals↗
PubMed1999

Mitogen-activated protein kinase is involved in the degradation of p53 protein in the bryostatin-1-induced differentiation of the acute promyelocytic leukemia NB4 cell line.

Overexpression of mutant p53 has been reported to promote tumorigenicity in several cancers. However, despite its potential importance, the signals regulating mutant p53 protein expression are not known. Here we show that a form of p53 that is incapable of binding DNA is overexpressed in the acute promyelocytic leukemia NB4 cell line. Our results demonstrate that treatment of NB4 cells with bryostatin-1, which induces differentiation in this cell line, leads to hyperphosphorylation of this DNA binding-impaired form of p53 via mitogen-activated protein kinase. After this phosphorylation, the p53 protein is degraded by the ubiquitin/proteasome pathway. Furthermore, we show that inhibition of p53 hyperphosphorylation blocks p53 protein degradation and cell differentiation. In addition, inhibition of the ubiquitin/proteasome pathway also blocks p53 protein degradation and cell differentiation. These findings suggest a role for mitogen-activated protein kinase in the degradation of the DNA binding-impaired form of p53 protein and in the bryostatin-induced differentiation observed in this cell line. The implications of these results with respect to the functional significance of p53 phosphorylation and degradation in cell differentiation are discussed.

Bryostatins↗
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↗
PubMed1999

Bryostatin-1 and 1alpha,25-dihydroxyvitamin D3 synergistically stimulate the differentiation of NB4 acute promyelocytic leukemia cells.

One of the objectives of treatment for patients with acute promyelocytic leukemia (APL) is to induce tumor cell differentiation and block cell proliferation. Acute promyelocytic leukemia cells (NB4) responded to the combination treatment of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] plus phorbol 12-myristate 13-acetate (PMA) and differentiated into monocyte/macrophage-like cells, as well as expressed strong alkaline phosphatase (ALP) activities. Since PMA has limited clinical application due to its tumor-promoting effect, another protein kinase C activator, bryostatin-1, was currently tested for its interaction with 1alpha,25(OH)2D3 to induce NB4 cell differentiation and block cell proliferation. Bryostatin-1 alone, but not 1alpha,25(OH)2D3 alone, significantly inhibited cell proliferation and induced NB4 cell differentiation into monocyte/macrophages; however neither bryostatin-1 nor 1alpha,25(OH)2D3 alone induced ALP expression. Like PMA, bryostatin-1 synergistically interacted with 1alpha,25(OH)2D3 to stimulate ALP expression 30-fold over the control (P < 0.001) and further promote appearance of monocyte/macrophage-like cells. The ALP stimulation was both time- and dose-dependent. Thus, we demonstrate for the first time that the combination of bryostatin-1 and 1alpha,25(OH)2D3 strongly affect NB4 cell differentiation and proliferation. Therefore, this proposed combination treatment may be an alternatively potential therapeutic regimen for APL patients and assay of ALP may be a more sensitive and facile way to monitor the possible remission of APL patients.

Alkaline Phosphatase↗
PubMed1998

1alpha,25(OH)2-vitamin D3 signaling in chick enterocytes: enhancement of tyrosine phosphorylation and rapid stimulation of mitogen-activated protein (MAP) kinase.

The steroid hormone 1alpha,25(OH)2-vitamin D3 (1alpha,25(OH)2D3) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1alpha,25(OH)2D3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1alpha,25(OH)2D3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42-44, 55-60, and 105-120 Kda. The 42-44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01-10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1alpha,25(OH)2D3 since its metabolic precursors 25(OH)D3 and vitamin D3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1alpha,25(OH)2D3-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1alpha,25(OH)2D3 analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans (6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1beta,25(OH)2D3, a known antagonist of 1alpha,25(OH)2D3-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1alpha,25(OH)2D3 and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes.

Animals↗
PubMed1998

Evidence for a 1 alpha,25-dihydroxyvitamin D3 receptor/binding protein in a membrane fraction isolated from a chick tibial fracture-healing callus.

Previous biological studies have implicated two vitamin D metabolites, 1 alpha,25(OH)2-vitamin D3[1 alpha,25(OH)2-D3] and 24R,25(OH)2-vitamin D3 [24R,25(OH)2D3] in the process of skeletal fracture-healing. While a nuclear receptor for 1 alpha,25(OH)2D3 is known to be present in osteoblast and absent in osteoclast cell lines, no systematic study has been carried out on the callus tissue which is formed during fracture-healing. The present report shows that a binding protein/receptor for 1 alpha,25(OH)2D3 resides both in a postnuclear membrane fraction and in a high speed cytosol fraction of the callus tissue obtained 10 days after imposition of a tibial fracture. The dissociation constant, KD, for 1 alpha,25(OH)2D3 was 0.83 +/- 0.34 M and 0.66 +/- 0.38 nM respectively, for the membrane and cytosol fractions. Results from a panel of steroid competition assays indicate that both receptor/binding proteins greatly prefer 1 alpha-hydroxylated ligands as compared to 1 alpha-deoxy or 24-hydroxylated ligands. The presence of 1 alpha,25(OH)2D3 receptors in the fracture-healing callus is consistent with the known biological effects of the metabolite on the fracture-healing process.

Animals↗
PubMed1998

1Alpha,25-dihydroxyvitamin D3 and phorbol ester mediate the expression of alkaline phosphatase in NB4 acute promyelocytic leukemia cells.

Both 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and phorbol 12-myristate 13-acetate [PMA] are cytodifferentiation agents regulating myeloid cell line differentiation along the monocyte/macrophage pathway. Alkaline phosphatase (ALP) activity has been reported to be absent or present at low levels in monocytes/macrophages. In the present study, the acute promyelocytic leukemia cell line (NB4) was found to undergo monocyte/macrophage differentiation and strongly express ALP activity after exposure to the combination of 1alpha,25(OH)2D3 and PMA treatment. The ALP stimulation was both dose- and time-dependent with the ED50 doses for 1alpha,25(OH)2D3 and PMA at 2 x 10(-10) M and 4 x 10(-11) M, respectively. Assessment of the cell morphology via cell adherence, phagocytosis and ALP staining confirmed that NB4 cells treated by 1alpha,25(OH)2D3 plus PMA showed macrophage characteristics, but were also strongly stained with ALP. The present study is the first report that NB4 cells express ALP activity as a consequence of combination treatment with 1alpha,25(OH)2D3 and PMA.

Alkaline Phosphatase↗
PubMed1998

Studies on 24R,25-dihydroxyvitamin D3: evidence for a nonnuclear membrane receptor in the chick tibial fracture-healing callus.

The effect(s) of 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] on fracture healing was studied in a vitamin D-depleted chick model. 24R,25(OH)2D3, together with another hormonally active vitamin D metabolite, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], improved bone mechanical strength parameters (torsional strength, angular deformation, and stiffness) and the ash content. The synthetic epimer 24S,25-dihydroxyvitamin D3 [24S,25(OH)2D3] was not as potent as the natural 24R,25(OH)2D3. In light of the ability of the fracture-healing callus to discriminate between 24R,25(OH)2D3 and 24S,25(OH)2D3, a search was initiated in fracture-healing callus tissue for the presence of a specific 24R,25(OH)2D3 receptor. No evidence was obtained for a classical nuclear/cytosol receptor for 24R,25(OH)2D3 in the fracture-healing callus. A specific receptor/binding protein for 24R,25(OH)2D3 was found in the callus membrane fraction, which showed different ligand binding affinities [KD = 18.3 +/- 1.9 nmol/L, Bmax = 43.9 +/- 6.0 fmol/mg; relative competitive index (RCI) for 24R,25(OH)2D3/24S,25(OH)2D3/25(OH)D3/1alpha,25(OH)2D3 = 100/37/401/2.0] compared with the ubiquitous serum vitamin D-binding protein (RCI = 100/99/219/5). Also, a callus membrane-binding protein/receptor for 1alpha,25(OH)2D3 was detected with a KD = 0.83 +/- 0.35 nmol/L and a Bmax = 35.5 +/- 5.2 fmol/mg. Thus, we have demonstrated a biological role for 24R,25(OH)2D3 in fracture healing and described the presence of its receptor/binding protein in a callus membrane fraction.

24,25-Dihydroxyvitamin D 3↗
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

Stimulation by 1alpha,25(OH)2-vitamin D3 of whole cell chloride currents in osteoblastic ROS 17/2.8 cells. A structure-function study.

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3) can generate biological responses via genomic and nongenomic mechanisms. This article reports for the first time the effects of 1alpha,25(OH)2D3 and structurally related analogs on whole cell chloride currents in osteoblastic cells. 1alpha,25(OH)2D3 promoted the rapid enhancement of outwardly rectifying Cl- currents in 93% of the osteoblasts in a concentration-dependent manner, with a maximal increase of about 4-fold between 0.5 and 5 nM. This effect of 1alpha,25(OH)2D3 was blocked by 1 nM stereoisomer 1beta,25(OH)2D3 when added to the bath before 1alpha,25(OH)2D3. On the other hand, 1 nM of the 6-s-cis locked analog 1alpha,25(OH)2-lumisterol3 significantly increased by about 2.2-fold outward Cl- currents in the ROS 17/2.8 cells, whereas the increase promoted by same concentration of the 6-s-trans locked analog 1alpha,25(OH)2-tachysterol (0.8-fold) was significantly lower, suggesting that the 6-s-cis locked or steroid-like form was preferred over the extended 6-s-trans conformer to promote these rapid effects of the hormone. We conclude that the agonist effects of 1alpha,25(OH)2D3 in osteoblasts at the cellular membrane level seem to be determined by some structural features of the molecule which may be crucial for its interaction with a putative membrane receptor in the cell surface.

Calcitriol↗
PubMed1997

Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues and their 20-epi homologues with the vitamin D receptor.

An important focus of structure-function studies of synthetic ligands for the vitamin D receptor (VDR) concerns the chiral center at carbon 20 of the steroid side chain; 20-epi analogues are 100-10, 000 times more potent transcriptionally than the natural hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3). We have compared the binding properties of three pairs of analogues either with a natural (N) or 20-epi (E) orientation. In intact cells, 45-60% of VDR.N-analogue complexes, but only 5-20% of VDR.E-analogue complexes, dissociated over a 3-h interval. The two groups of ligands induced distinct changes in VDR conformation as revealed by protease clipping assays. Mapping of ligand-VDR binding activity by deletions indicated that amino acids 420-427 were important for high affinity of VDR.N-analogue complexes, but not for VDR.E-analogue complexes. Site-directed mutagenesis revealed that residues 421 and 422 were essential for 1alpha,25-(OH)2D3-induced conformational changes, high affinity of 1alpha,25-(OH)2D3 for VDR, and transcriptional activity, but not for binding of its 20-epi analogue. In contrast, deletion of residues 396-427 abolished binding of 1alpha,25-(OH)2D3, but binding of its 20-epi analogue was still detectable. The results suggest that the ligand-binding domain of VDR has multiple and different contact sites for the two families of side chain-modified ligands, resulting in VDR.ligand complexes with different half-lives and transcriptional activities.

Amino Acid Sequence↗
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

Vitamin D receptor expression in chicken muscle tissue and cultured myoblasts.

Muscle has long been recognized as a target tissue for 1,25-dihydroxy-vitamin D3 (1,25[OH]2D3). Evidence of the presence of VDR is provided here, thus supporting the existence of a receptor-mediated mechanism of action of 1,25(OH)2D3. Vitamin D receptor (VDR) expression is evidenced by detection of VDR-mRNA, through reverse transcription and polymerase chain reaction (RT/PCR), in chicken muscle and muscle cells (myoblasts) as well as in a variety of tissues such as intestine, kidney, heart and brain. VDR presence is also demonstrated by Southern blot of PCR products with a specific VDR-cDNA probe and by immunocytochemistry carried out on myoblasts and cardiac myocytes. Localization of VDR is mainly nuclear and more faintly detected in the cytosol.

Animals↗
PubMed1997

24R,25-dihydroxyvitamin D3: an essential vitamin D3 metabolite for both normal bone integrity and healing of tibial fracture in chicks.

We tested the hypothesis that 24R,25-dihydroxyvitamin D3 [24R,25-(OH)2D3] is an essential vitamin D metabolite for the development of normal bone integrity and the healing of fractures. The natural 24R,25-(OH)2D3 and its synthetic epimer 24S,25-dihydroxyvitamin D3 [24S,25-(OH)2D3] were tested alone or in combination with 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3], on normal bone development and other related variables of the Ca2+ homeostasis system [serum Ca2+, 25-hydroxyvitamin D3 (25OHD3), 24,25-(OH)2D3, and 1alpha,25-(OH)2D3 levels] in chicks. Mechanical testing of torsional strength was carried out on the femur. 24R,25-(OH)2D3 (80 nmol/kg diet) alone was sufficient for normal bone growth and integrity similar to that achieved by the vitamin D3-replete controls. Next, chicks were fed a 25OHD3-replete diet (75 nmol/kg diet) for 8 days after hatching, and then 25OHD3 was withdrawn to minimize any residual circulating metabolites before the imposition of standardized tibial fractures 14 days later. Vitamin D metabolites were administered for 2 weeks to determine their effects on the mechanical properties of healed tibia. 24S,25-(OH)2D3 combined with 1alpha,25-(OH)2D3 or 1alpha,25-(OH)2D3 alone resulted in poor healing [strength values of 0.158 +/- 0.011 and 0.123 +/- 0.009 Nm (Newton x meter), respectively] compared with that in the 25OHD3-treated control group (0.374 +/- 0.029 Nm). In contrast, the fractured tibia of the birds fed 24R,25-(OH)2D3 in combination with 1alpha,25-(OH)2D3 showed healing equivalent to that in the control group, with strength values of 0.296 +/- 0.043 Nm. These results suggest that when 24R,25-(OH)2D3 is present at normal physiological concentrations, it is an essential vitamin D3 metabolite for both normal bone integrity and healing of fracture in chicks.

24,25-Dihydroxyvitamin D 3↗
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↗
PubMed1997

Three-fold induction of renal 25-hydroxyvitamin D3-24-hydroxylase activity and increased serum 24,25-dihydroxyvitamin D3 levels are correlated with the healing process after chick tibial fracture.

To investigate the possible biological actions of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3), a tibial fracture-healing model was established in White Leghorn chicks. Three-week-old White Leghorn chicks fed a vitamin D3-replete diet were divided into four groups (control, anesthetized, sham, and fractured). On varying days after tibial fracture (F) or sham manipulation (S), renal 25(OH)D3-1 alpha-hydroxylase and 25(OH)D3-24-hydroxylase (24-hydroxylase) activities and serum Ca2+ concentrations were measured. Metofane anesthesia was found to have no effect on the activity of either of the hydroxylases; the activities of the hydroxylases in the control, anesthetized, and sham-operated birds were similar. By 10 days after tibial fracture, the renal 24-hydroxylase activity increased more than 3-fold in F (1.33 +/- 0.07 pmol/mg of protein) as compared with S (0.42 +/- 0.03 pmol/mg of protein) (p < 0.0001). A time-dependent study of the renal 24-hydroxylase activity during the fracture repair process revealed a slow increase from the first day after fracture, a higher activity at 8 days, which peaked at 10-11 days, which is consistent with the formation of the callus. The 24-hydroxylase activity then returned to the same level as the sham group 14 days after fracture. There was no significant difference in serum Ca2+ levels between the F and S groups over the 3-week postfracture period. Serum levels of vitamin D3 metabolites were also measured during the fracture healing process: a 3.4x increase of the 24,25(OH)2D3 level in the fractured group (3.64 +/- 1.16 nM) was observed as compared with the control groups (1.08 +/- 0.49 nM) at 10 days after fracture (p = 0.068). No significant differences were observed in the plasma levels of 25(OH)D3 or 1 alpha, 25(OH)2D3 between the group with a fracture and the controls. Exposure of primary chick kidney cells in culture to serum obtained from chicks with a tibial fracture for 20 h resulted in an approximately 40% increase in the activity of the 24-hydroxylase as compared with cells exposed to serum from control birds. These results suggest that 24,25(OH)2D3 is involved in the early process of fracture repair and that there is some form of physiological communication between the fractured bone and the kidney so as to increase the renal 24-hydroxylase and the circulating concentration of this metabolite.

24,25-Dihydroxyvitamin D 3↗