Annexin II is the membrane receptor that mediates the rapid actions of 1alpha,25-dihydroxyvitamin D(3)

1alpha,25-Dihydroxyvitamin D(3) has been shown to exert its effects by both genomic (minutes to hours) and rapid (seconds to minutes) mechanisms. The genomic effects are mediated by interaction with the nuclear vitamin D receptor. We show that the vitamin D analog, [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate, is specifically bound to a protein (molecular weight 36 kDa) in the plasma membrane of rat osteoblastlike cells (ROS 24/1). The plasma membrane protein labeled with the bromoacetate analog was identified as annexin II by sequence determination and Western blot. Partially purified plasma membrane proteins (PI 6.9-7.4) and purified annexin II exhibited specific and saturable binding for [(3)H]-1alpha, 25-dihydroxyvitamin D(3). Antibodies to annexin II inhibited [(14)C]-1alpha,25-dihydroxyvitamin D(3) bromoacetate binding to ROS 24/1 plasma membranes, immunoprecipitated the ligand-protein complex, and inhibited 1alpha,25-dihydroxyvitamin D(3)-induced increases in intracellular calcium in ROS 24/1 cells. The results indicate that annexin II may serve as a receptor for rapid actions of 1alpha, 25-dihydroxyvitamin D(3).     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Antiproliferative, low-calcemic, fluorinated sulfone analogs of 1alpha,25-dihydroxyvitamin D3: chemical synthesis and biological evaluation

Novel fluorinated sulfone analogs of the hormone 1alpha,25-dihydroxyvitamin D(3) have been designed and synthesized in order to study the biological effects of fluorine incorporation at the terminus of the C,D-ring side chain. Although biologically active 26,27-hexafluorinated 1alpha,25-dihydroxyvitamin D(3) analogs have been synthesized previously, this investigation reports the first successful fluorinated series in which trifluoromethyl sulfone analogs present a favorable biological profile. This study shows that two new analogs featuring incorporation of a synthetically simple single trifluoromethyl sulfone group have significantly increased antiproliferative activity while causing desirably low in vivo calciuria relative to that of calcitriol. Incorporation of additional fluorines, as in a perfluorobutyl analog, results in a loss of antiproliferative activity.     

The stereochemical configuration of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone

Four possible diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were chemically synthesized and compared with the natural metabolite by high-pressure liquid chromatography. The four synthetic diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone could be separated into three peaks by high-pressure liquid chromatography. The naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum and in vitro incubation of chick kidney homogenates comigrated with 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. The four diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were tested against naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone to determine their relative competition in the 1 alpha,25-dihydroxyvitamin D3-specific cytosol receptor binding assay for 1 alpha,25-dihydroxyvitamin D3. 23(S)25(S)-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone was the best competitor followed by 23(R)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone and 23(R)25(S)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone, and 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was the poorest competitor. Natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum had almost the same binding affinity as that of 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. These data unequivocally demonstrate that the stereochemistry of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone has the 23(S) and 25(R) configuration.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

Increased intestinal chromatin template activity. Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes.

1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase. The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol. This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney. The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours. Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo. To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes. After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone. This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol. The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3. It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.     

Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1alpha,25-dihydroxyvitamin D3

The synthesis of new analogues of 1alpha,25-dihydroxyvitamin D3 containing a carbamate function at the A-ring fragment has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by regioselective enzymatic alkoxycarbonylation of A-ring enyne 3 and subsequent treatment with ammonia, amines, amino alcohols, and amino acids. Biological studies to evaluate the potency of all five of these carbamate analogues were performed and demonstrated very low binding affinity for the vitamin D receptor compared with 1alpha,25-dihydroxyvitamin D3. Moreover, all the carbamate analogues were less active than 1alpha,25-dihydroxyvitamin D3 in inhibiting cell proliferation or stimulating cell differentiation. Of all the five analogues, the 3-O-carbamoyl-1alpha,25-(OH)2-D3 analogue 10a was the most potent one in vitro. However, all investigated carbamate analogues demonstrated lower calcemic effects in vivo than the parent compound.     

Mechanistic studies to evaluate the enhanced antiproliferation of human keratinocytes by 1alpha,25-dihydroxyvitamin D(3)-3-bromoacetate, a covalent modifier of vitamin D receptor, compared to 1alpha,25-dihydroxyvitamin D(3).

1alpha,25-Dihydroxyvitamin D(3)-3-bromoacetate (1, 25(OH)(2)D(3)-3-BE), an affinity labeling analog of 1alpha, 25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), displayed stronger antiproliferative activities than 1,25(OH)(2)D(3) at 10(-10)-10(-6) M dose levels in cultured human keratinocytes (CHK). Additionally, preincubation of the cells with 10(-6) M 1,25(OH)(2)D(3), followed by treatment with various doses of 1,25(OH)(2)D(3)-3-BE, resulted in a significantly stronger antiproliferative activity by the mixture than individual reagents at every dose level. To search for a mechanism of this observation, we determined that [(14)C]1, 25(OH)(2)D(3)-3-BE covalently labeled human recombinant 1alpha, 25-dihydroxyvitamin D(3) receptor (reVDR) swiftly (<1 min) with a 1:1 stoichiometry and induced conformational changes (in VDR) that are different from 1,25(OH)(2)D(3), by limited tryptic digestion. Furthermore, a protein band, corresponding to reVDR, was specifically labeled by [(14)C]1,25(OH)(2)D(3)-3-BE in CHK extract, indicating that VDR is the main target of [(14)C]1, 25(OH)(2)D(3)-3-BE. The above-mentioned observations suggest that a rapid covalent labeling of VDR in CHK might alter the interaction between the holo-VDR and 1,25(OH)(2)D(3)-controlled genes. Furthermore, we observed that 1,25(OH)(2)D(3)-3-BE significantly decreased the binding of VDR to human osteocalcin vitamin D responsive element (hOCVDRE), as well as the dissociation rate of VDR from hOCVDRE, compared with 1,25(OH)(2)D(3) in COS-1 cells, transiently transfected with a VDR construct. Additionally, 1, 25(OH)(2)D(3)-3-BE was found to be more potent in inducing 1alpha, 25-dihydroxyvitamin D(3)-24-hydroxylase (24-OHase) promoter activity and mRNA expression in keratinocytes. The accumulation of 24-OHase message was also prolonged by the analog. Collectively these results indicated that rapid covalent labeling of VDR in keratinocytes (by 1, 25(OH)(2)D(3)-3-BE) might result in the conversion of apo-VDR to a holo-form, with a conformation that is different from that of the 1, 25(OH)(2)D(3)-VDR complex. This resulted in an enhanced stability of the 1,25(OH)(2)D(3)-3-BE/VDR-VDRE complex and contributed to the amplified antiproliferative effect of 1,25(OH)(2)D(3)-3-BE in keratinocytes.     

1 alpha, 25-dihydroxyvitamin D3 specific regulation of growth, morphology, and fibronectin in a human osteosarcoma cell line

The ability of the hormonally active vitamin D metabolite, 1 alpha, 25-dihydroxyvitamin D3, to affect cell growth, morphology and fibronectin production has been examined using the MG-63 human osteosarcoma cell line. Hormone treatment reduced cell growth rate, saturation density and [3H]thymidine incorporation. Inhibition was specific for 1 alpha, 25-dihydroxyvitamin D3 relative to other vitamin D metabolites (1 alpha, 25-dihydroxyvitamin D3 greater than 25-dihydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3 greater than D3), antagonized by high concentrations of serum and readily reversed by removal of 1 alpha, 25-dihydroxyvitamin D3 from the culture medium. Hormone treatment also increased cell associated alkaline phosphatase activity up to twofold and altered morphology such that treated cells were more spread out on the culture dish and contained more cytoplasmic processes. Significantly, 1 alpha, 25-dihydroxyvitamin D3 increased cellular and medium concentrations of fibronectin, a glycoprotein known to be involved in cellular adhesiveness. MG-63 cells contain a specific 1 alpha, 25-dihydroxyvitamin D3 receptor which may mediate these responses.     

Syntheses and biological evaluation of novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues

Novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues were efficiently synthesized and their biological activities were evaluated. 2alpha-Methyl-1alpha,25-dihydroxyvitamin D3 (2), whose unique biological activities were previously reported, was modified to 2alpha-alkyl (ethyl and propyl) and 2alpha-hydroxyalkyl (hydroxymethyl, hydroxyethyl, and hydroxypropyl) analogues 3-7 by elongation of the alkyl chain and/or introduction of a terminal hydroxyl group. 2alpha-Hydroxypropyl-1alpha,25-dihydroxyvitamin D3 (7) exhibited an exceptionally potent calcium-regulating effect and a unique activity profile.     

Enhanced biological activity of 1alpha,25-dihydroxy-20-epi-vitamin D3, the C-20 epimer of 1alpha,25-dihydroxyvitamin D3, is in part due to its metabolism into stable intermediary metabolites with significant biological activity

1alpha,25-dihydroxy-20-epi-vitamin D3 (1alpha,25(OH)2-20-epi-D3), the C-20 epimer of the natural hormone 1alpha,25(OH)2D3, is several fold more potent than the natural hormone in inhibiting cell growth and inducing cell differentiation. At present, the various mechanisms responsible for the enhanced biological activities of this unique vitamin D3 analog are not fully understood. In our present study we compared the target tissue metabolism of 1alpha,25(OH)2D3 with that of 1alpha,25(OH)2-20-epi-D3 using the technique of isolated perfused rat kidney. The results indicated that the C-24 oxidation pathway plays a major role in the metabolism of both compounds in the rat kidney. However, it was noted that the concentrations of two of the intermediary metabolites of 1alpha,25(OH)2-20-epi-D3, namely, 1alpha,24(R),25(OH)3-20-epi-D3 and 1alpha,25(OH)2-24-oxo-20-epi-D3 in the kidney perfusate, exceeded the concentrations of the corresponding intermediary metabolites of 1alpha,25(OH)2D3. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3 induces the conformation of the vitamin D receptor similar to that induced by its parent analog and is nearly as potent as its parent in inducing transactivation of a gene construct containing the human osteocalcin vitamin D-responsive element. We conclude that 1alpha,25(OH)2-20-epi-D3 by itself is not metabolically stable when compared to 1alpha,25(OH)2D3, but it acquires its metabolic stability because of the reduced rate of catabolism of its intermediary metabolites. Furthermore, 1alpha,25(OH)2-24-oxo-20-epi-D3, the stable bioactive intermediary metabolite plays a significant role in generating the enhanced biological activities ascribed to 1alpha,25(OH)2-20-epi-D3.     

Selective analogs of 1alpha,25-dihydroxyvitamin D3 for the study of specific functions of Vitamin D

New analogs of 1alpha,25-dihydroxyvitamin D(3) synthesized in our research group that show selective activity in vivo are presented along with supporting biological results. Compounds that act preferentially on intestine are 2-(3'-propylidene-19-nor-(20S or 20R))-1alpha,25-dihydroxyvitamin D(3) and 2-methylene-19-21-dinor-1alpha,25-dihydroxyvitamin D(3). Compounds that act anabolically to induce bone formation are 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD), the 2alpha-methyl derivative, the 26,27-dimethyl derivative, and the 26-dimethylene derivative. Compounds that act preferentially on parathyroid glands are 2-methylene-19-nor-1alpha-hydroxy-homopregnacalciferol, the 20S-bishomo derivative and the 2-methylene-19,26,27-trinor-1alpha,25-dihydroxyvitamin D(3). These latter compounds do not elevate serum calcium until doses of the order of >300 microg/kg body weight are used, while parathyroid hormone levels are suppressed at much lower doses. Some of these novel analogs may ultimately be useful as new and safer therapeutic agents. Regardless of their clinical utility, they represent valuable research tools that can be used to study the specific functions of the Vitamin D hormone in vivo.     

Methyl substitution of the 25-hydroxy group on 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) reduces potency but allows bone selectivity

The discovery of 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) as a bone selective and bone anabolic form of vitamin D has stimulated an investigation of structure/function of bone selectivity. Four new 2-substituted-19-norvitamin D analogs 3-6 have been developed to study the structure-activity relationship at C-25. As predicted, removing the 25-hydroxy group (compound 3) from the very potent analog 2MD and its 2-methyl derivatives (5 and 6) dramatically reduces in vitro activities, but biological potency is nearly fully restored in vivo likely due to in vivo 25-hydroxylation. The introduction of a methyl group at C-25 (compound 4) that blocks in vivo 25-hydroxylation reduces biological activity both in vitro and in vivo. However, analog 4 retains bone selectivity making it interesting as a possible therapeutic for bone loss diseases.     

Studies on the mechanism of action of 1 alpha, 24-dihydroxyvitamin D3. II. Specific binding of alpha, 24-dihydroxyvitamin D3 to chick intestinal receptor

The binding of vitamin D3 analogues to the chick intestinal cytosol receptor was studied. In intestinal cytosol fraction, receptor proteins having the sedimentation constant of 2.5 S and 3.7 S to which 1 alpha,25-dihydroxyvitamin D3 binds were present, and the latter was specific for the compound. The binding of 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3 to the receptor was also observed, while very weak binding was seen in the case of 24(R)25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. The binding affinity of 1 alpha,24(R)-dihydroxyvitamin D3 to the 3.7 S receptor was 1.3 times as high as that of 1 alpha,25-dihydroxyvitamin D3, whereas those of 1 alpha,24(S)-dihydroxyvitamin D3, 1 alpha-hydroxyvitamin D3 and 25-hydroxyvitamin D3 were 10, 304 and 652 times lower than 1 alpha,25-dihydroxyvitamin D3, respectively. The dissociation constant of the receptor-1 alpha,25-dihydroxyvitamin D3 complex at 0 degrees C was 3.0 x 10(-11) M, and the dissociation constants were calculated to be 2.4 x 10(-11) M and 2.7 x 10(-10) M for the complexes with 1 alpha,24(R)-dihydroxyvitamin D3 and 1 alpha,24(S)-dihydroxyvitamin D3, respectively.     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in vitamin D receptor-ablated mice in vivo

The metabolism of 1alpha,25-dihydroxyvitamin D(3) was studied in vitamin D receptor-ablated mice following the administration of a physiological dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). The degradation of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) in the vitamin D receptor null mutant mice was substantially reduced compared to the wild-type control mice. At 24 h postadministration of radiolabeled 1alpha,25-dihydroxyvitamin D(3) more than 50% of the radioactivity was recovered unmetabolized, whereas in wild-type mice nearly all of the 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3) was degraded. In wild-type mice three polar metabolites other than 1alpha,25-dihydroxyvitamin D(3) were detected and identified on straight-phase and reverse-phase high-performance liquid chromatography as 1alpha,24(R),25-trihydroxy-[26,27-(3)H]vitamin D(3), 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3), and (23S, 25R)-1alpha,25-dihydroxy-[(3)H]vitamin D(3)-26,23-lactone. Only one metabolite was detected in the plasma and kidneys of vitamin D receptor null mutant mice at 3 h following an intrajugular dose of 1alpha,25-dihydroxy-[26,27-(3)H]vitamin D(3). This metabolite was clearly identified as 1alpha,25(S),26-trihydroxy-[26,27-(3)H]vitamin D(3) by comigration on two HPLC systems and periodate cleavage reaction. At 6, 12, and 24 h postinjection 1alpha,24(R), 25-trihydroxy-[26,27-(3)H]vitamin D(3) was also detected at low levels in plasma, kidneys, and liver of some but not all mutant mice. The presence of 25-hydroxyvitamin D(3)-24-hydroxylase mRNA in the kidneys of these vitamin D receptor null mutant mice was confirmed by ribonuclease protection assay.     

Metabolic pathways from 1 alpha,25-dihydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization

The present study was carried out in order to elucidate the metabolic pathway from 1 alpha,25-(OH)2D3 to 1 alpha,25-(OH)2D3-26,23-lactone. For that purpose, we stereospecifically synthesized the vitamin D3 derivatives 1 alpha,23(S),25-(OH)3D3, 1 alpha,23(S),25(R),26-tetrahydroxyvitamin D3, and 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-lactol. The in vitro metabolism of these compounds was examined in kidney homogenates and intestinal mucosa homogenates from 1 alpha,25-(OH)2D3-supplemented chicks. The naturally occurring 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was produced (in increasing amounts) from 1 alpha,25-(OH)2D3, 1 alpha,25(R),26-(OH)3D3, 1 alpha,23(S),25-(OH),D3, 1 alpha,23(S),25(R),26-(OH)4D3, and 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol. These results indicated that there are two possible metabolic pathways from 1 alpha,25-(OH)2D3 to 1 alpha,23(S),25(R),26-(OH)4D3: the major one is by way of 1 alpha,23(S),25-(OH)3D3 and the minor one is by way of 1 alpha,25(R),26-(OH)3D3. 1 alpha,23(S),25(R),26-Tetrahydroxyvitamin D3 is further metabolized to 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone via 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactol. In the course of our studies, a new biosynthetic vitamin D3 metabolite was isolated in pure form. This metabolite was identified as 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol by UV spectrophotometry and mass spectrometry. Furthermore, we establish in this report that the lactonization of 1 alpha,23,25,26-(OH)4D3 and 1 alpha,25-(OH)2D3-26,23-lactol occurs in a stereo-retained and stereo-selective fashion.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

Gene expression profiles in dendritic cells conditioned by 1alpha,25-dihydroxyvitamin D3 analog

Inhibition of dendritic cell (DC) maturity is an important immunomodulatory effect of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) and related analogs (D(3) analogs). The mechanisms underlying 1alpha,25(OH)(2)D(3)-mediated DC modulation are Vitamin D receptor (VDR)-dependent and likely involve direct or indirect regulation of multiple genes. Gene expression profiles of bone marrow-derived DCs (BMDCs) generated in the absence or presence of a potent D(3) analog were analyzed using microarray technology. Results for D(3) analog-conditioned DCs were also compared with glucocorticoid-conditioned BMDCs and with BMDCs conditioned with D(3) analog and glucocorticoid combined. Of approximately 12,000 gene products assayed, 52% were considered to have detectable expression in unconditioned BMDCs. Based on relative expression levels, 5.3% of these expressed genes were "silenced" or "suppressed" in D(3) analog-conditioned BMDCs and 2.1% were "augmented". In addition, 1.7% of gene products undetectable in control BMDCs were "induced" by D(3) analog. Functional grouping of modulated genes demonstrated important effects of D(3) analog on immunoreceptors, on chemokines and chemokine receptors, on growth factors/cytokines and related receptors, and on neuroendocrine hormones and related receptors. Many of these gene products were unaffected or differently regulated by glucocorticoid suggesting specific VDR-mediated regulatory effects. Confirmation of microarray analysis results for two differentially regulated chemokines (MIP-1alpha and RANTES) was obtained by RT-PCR and ELISA. The methodology provides novel insights into DC gene regulation by 1alpha,25(OH)(2)D(3) agonists.