Transformation of vitamin D3 to 1α,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains

To enzymatically synthesize active metabolites of vitamin D₃, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D₃ to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] via 25-hyroxyvitamin D₃ [25(OH)D₃]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D₃ to 25(OH)D₃ and 1,25(OH)₂D₃ in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D₃/l culture and 0.17 mg 1,25(OH)₂D₃/l culture. A related compound, vitamin D₂, could be also converted to 25-hydroxyvitamin D₂ and 1,25-dihydroxyvitamin D₂ using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D₃ in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D₃ and at C-1 of 25(OH)D₃ originates from cytochrome P-450.Correspondence to: J. Sasaki     

Immunological modulation by 1α,25-dihydroxyvitamin D3 in patients with squamous cell carcinoma of the head and neck

Prior studies showing that treatment of head and neck squamous cell carcinoma (HNSCC) patients with 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] stimulated intratumoral immune infiltration were extended to analysis of cytokine profiles in the periphery and in oral tissues. Most prominent was the disparity between cytokine levels in plasma and in either pathologically normal oral tissue or HNSCC tissue from patients that were untreated or treated with 1,25(OH)(2)D(3). Levels of IL-6 and IL-10, but not IL-2, IFN-γ or TNF-α, tended to be increased in the plasma of HNSCC patients and 1,25(OH)(2)D(3) further increased plasma levels of all of these cytokines. While these cytokines tended to be increased in HNSCC tissue, 1,25(OH)(2)D(3) resulted in variable cytokine responses that showed a general tendency toward further increased levels. Levels of IL-8 and VEGF were increased in plasma and tissue of untreated HNSCC patients, and were further increased in plasma, but not in tissues, of patients treated with 1,25(OH)(2)D(3). Levels of IL-1α and IL-1β were similar in plasma of controls and HNSCC patients, but were increased in HNSCC tissues. In contrast to that seen in plasma where 1,25(OH)(2)D(3) increased levels of IL-1α and IL-1β, this was not seen in tissue following 1,25(OH)(2)D(3) treatment. These results show a discordant relationship between systemic and intratumoral cytokine profiles and suggest a tendency of 1,25(OH)(2)D(3)to increase a multitude of cytokines within tumor tissue.     

The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion

The biologically active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)₂D₃, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E₂.     

Circulating 1α,25-dihydroxyvitamin D in the chicken: Enhancement by injection of prolactin and during egg laying

In order to investigate possible modulation of vitamin D metabolism by prolactin, circulating 1α,25-dihydroxyvitamin D (1α,25-(OH)₂D) was measured by radioreceptor assay in chicks given injections of prolactin for five days. At a dose of 100 μg/day, the lactogenic hormone elicited a two-fold increase in plasma 1α,25-(OH)₂D. This effect may explain the known action of prolactin in producing hypercalcemia and could be physiologically important in birds. The laying hen represents a physiologic state in which calcuim absorption is known to be stimulated and prolactin has been reported to be elevated. Assay of serum 1α,25-(OH)₂D in the laying hen demonstrates a nine-fold enhancement over non-laying controls. Since this marked increase during egg laying is at least partially mimicked by injecting prolactin, a possible causative relationship between elevated prolactin and 1α,25-(OH)₂D is suggested.     

Evidence of vitamin D and interferon-β cross-talk in human osteoblasts with 1α,25-dihydroxyvitamin D3 being dominant over interferon-β in stimulating mineralization

It is well established that 1α-25-dihydroxyvitamin D3 (1,25D3) regulates osteoblast function and stimulates mineralization by human osteoblasts. The aim of this study was to identify processes underlying the 1,25D3 effects on mineralization. We started with gene expression profiling analyses of differentiating human pre-osteoblast treated with 1,25D3. Bioinformatic analyses showed interferon-related and -regulated genes (ISG) to be overrepresented in the set of 1,25D3-regulated genes. 1,25D3 down-regulated ISGs predominantly during the pre-mineralization period. This pointed to an interaction between the vitamin D and IFN signaling cascades in the regulation of osteoblast function. Separately, 1,25D3 enhances while IFNβ inhibits mineralization. Treatment of human osteoblasts with 1,25D3 and IFNβ showed that 1,25D3 completely overrules the IFNβ inhibition of mineralization. This was supported by analyses of extracellular matrix gene expression, showing a dominant effect of 1,25D3 over the inhibitory effect of IFNβ. We identified processes shared by IFNβ- and 1,25D3-mediated signaling by performing gene expression profiling during early osteoblast differentiation. Bioinformatic analyses revealed that genes being correlated or anti-correlated with interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) were associated with osteoblast proliferation. In conclusion, the current study demonstrates a cross talk between 1,25D3 and IFNβ in osteoblast differentiation and bone formation/mineralization. The interaction is complex and depends on the process but importantly, 1,25D3 stimulation of mineralization is dominant over the inhibitory effect of IFNβ. These observations are of potential clinical relevance considering the impact of the immune system on bone metabolism in conditions such as rheumatoid arthritis.     

The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.     

Two stages of the differentiation of HL-60 cells induced by 1α,25 dihydroxyvitamin D3; Commitment by 1α,25 dihydroxyvitamin D3 and promotion by DMSO

The differentiation of HL-60 cells induced by 1,25 dihydroxyvitamin D₃ was found to be separated into two stages, i.e. commitment and promotion. Most of the HL-60 cells were committed to monocyte/macrophage lineage by pretreatment with 1,25 dihydroxyvitamin D₃ (5–50 ng/ml) for 18–24 hr. The promotion in the second stage was inducer and lineage independent; treatment with 1.25% DMSO for 2 or 3 days promoted the differentiation of the committed HL-60 cells by 1,25 dihydroxyvitamin D₃ into monocyte/macrophage lineage, but not granulocyte lineage.Abbreviations used NEA nonspecific esterase activity - NBT nitroblue tetrazolium - DMSO dimethylsulfoxide - RA retinoic acid - TPA 12-O-tetradecanoylphorbol-13-acetate     

Studies on the mode of action of vitamin D—XIV. Quantitative assessment of the structural requirements for the interaction of 1α,25-dihydroxyvitamin D3 with its chick intestinal mucosa receptor system

The structural requirements for the interaction of the 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] molecule with its chick intestinal mucosa receptor system have been quantitatively evaluated. This analysis was carried out using structural analogs of 1α,25-dihydroxyvitamin D₃ in a competitive binding assay with a reconstituted chromatin-cytosol system from rachitic chick intestinal mucosa. In this assay the steroid-cytosol receptor complex binds avidly to the chromatin. The results for the melabolites and analogs were expressed on a linear scale of relative competitive index (RCI) where the RCI of 1α,25(OH)₂D₃ is defined as 100. These studies demonstrated that the 1α-and 25-hydroxyl functions were the most critical groups to the binding process: their absence reduced the RCI to 0.5 and 0.4 respectively. The 3β-hydroxyl, although important, is somewhat less critical since the RCI was reduced to only 5.7. When all three of these hydroxyls are present, the receptor will tolerate alterations in the side chain at carbon-24. Thus shortening the side chain of 1α,25(OH)₂D₃ by only one methylene group reduced the RCI to 46. When the 25-hydroxylated side chain is present, modification of the A-ring at C-1, C-3, C-4, C-10 and C-19 all markedly reduce the RCI. Competitors with both A-ring and side chain modifications were found to have almost no ability to interact with the receptor system. Collectively these results support the view that the intestinal cytosol-chromatin receptor system has a very high degree of specificity for its hormonal ligand which reflect bonding-interactions at multiple (carbons 1, 3, 4, 10, 19, 24, and 25) sites on the 1α.25(OH)₂D₃ molecule. A model is presented for this interaction which emphasizes the key role of a standard length side chain with a 25-OH functional group.     

Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D3 in the chemoprevention of DMBA-induced mammary carcinogenesis in rats

The present study demonstrates the anti-tumor effects of combined supplementations of dietary fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ (vitamin D₃) on 7,12-dimethylbenz(α)anthracene (DMBA)-induced rat mammary carcinogenesis. Female Sprague–Dawley rats at 50 days of age were treated with 7,12-dimethylbenz(α)anthracene (DMBA; 0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Both fish oil (rich in EPA and DHA) and vitamin D₃ were administered orally at a dose of 0.5 ml/day/rat and 0.3 μg/100 μL propylene glycol twice a week respectively and continued to 35 weeks after DMBA administration. Fish oil in combination with vitamin D₃ resulted in a significant reduction in incidence, multiplicity and volume of mammary tumors. These supplementation also inhibited DMBA-induced mammary 7-methylguanine DNA adducts formation, which was measured by HPLC-fluorescence assay (at four sequential time points; ANOVA, F = 42.56, P < 0.0001). Immunohistochemical analysis revealed that the effect of fish oil and vitamin D₃ occurred through suppression of cell proliferation (BrdU-LI: P < 0.0001). Fish oil and vitamin D₃ together also reduced the mRNA expression of iNOS (84%, P < 0.05). In view of their natural availability, non-toxicity and acceptability; combined supplementation of fish oil and vitamin D₃ might be effective for chemoprevention of mammary carcinogenesis.     

Transforming growth factor-β1 modulates the effect of 1α,25-dihydroxyvitamin D3 on leukemic cells

Summary The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-β1 (TGF-β1) were studied by examining cell surface antigens and macrophage-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-β1 with 1α,25-dihydroxyvitamin D₃ [1α, 25(OH)₂D₃] induced more Leu-M3 (CD14)-positive cells (approximately 80%) than 5×10⁻⁸ M 1α,25(OH)₂D₃ alone did (30 to 50%), although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-α (TNF-α) with TGF-β1 and 1α,25(OH)₂D₃ was found to potentiate the expression of these surface antigens. Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CD11b) antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells after the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-β1 and 1α,25(OH)₂D₃ might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-β superfamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.     

A new insight into the role of rat cytochrome P450 24A1 in metabolism of selective analogs of 1α,25-dihydroxyvitamin D₃

We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D? (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D? (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D? (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D? (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.