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.     

The synthesis of [26,27-C]dihydroxyvitamin D3, a tracer for positron emission tomography (PET)

1α,25-Dihydroxyvitamin D₃, an endogenous ligand with the highest affinity for the vitamin D receptor (VDR), was labeled with ¹¹C for use in biological experiments. The radionuclide was incorporated via the reaction of [¹¹C]methyllithium on a methyl ketone precursor in tetrahydrofuran at −10 °C. Deprotection of the labeled intermediate yielded 2.5–3 GBq [26,27-¹¹C]1α,25-dihydroxyvitamin D₃ [¹¹C-1,25(OH)₂ D₃] with specific radioactivity averaging 100 GBq/μmol at the end of synthesis and HPLC purification. The entire process took 48 min from the end of radionuclide production. In vitro binding experiments in rachitic chick purified VDR demonstrated the high affinity binding of this novel tracer. Thus; ¹¹C-1,25(OH)₂ D₃ is available for in vivo distribution studies and may be suitable for the positron emission tomography (PET) determination of VDR levels and occupancy in animals and humans.     

Evaluation of C-2-substituted 19-nor-1α,25-dihydroxyvitamin D3 analogs as therapeutic agents for prostate cancer

1α,25-Dihydroxyvitamin D₃ (1α,25(OH)₂D₃) is known to inhibit the proliferation and invasiveness of prostate cancer cells. However, 1α,25(OH)₂D₃can cause hypercalcemia and is not suitable as a therapeutic agent. 19-Nor-vitamin D derivatives are known to be less calcemic when administered systemically. In order to develop more potent anti-cancer agents with less calcemic side effect, we therefore utilized ³H-thymidine incorporation as an index for cell proliferation and examined the antiproliferative activities of nine C-2-substituted 19-nor-1α,25(OH)₂D₃ analogs in the immortalized PZ-HPV-7 normal prostate cell line. Among the nine analogs we observed that the substitution with 2α- or 2β-hydroxypropyl group produced two analogs having antiproliferative potency that is approximately 500- to 1000-fold higher than 1α,25(OH)₂D₃. The ³H-thymidine incorporation data were supported by the cell counting data after cells were treated with 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ or 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ for 7 days. 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)₂D₃ and 19-nor-2β-(3-hydroxypropyl)-1α,25(OH)₂D₃ were also shown to be about 10-fold more active than 1α,25(OH)₂D₃ in cell invasion studies using prostate cancer cells. In conclusion, a substitution at the C-2 position of 19-nor-1α,25(OH)₂D₃ molecule with a hydroxypropyl group greatly increased the antiproliferative and anti-invasion potencies. Thus, these two analogs could be developed to be effective therapeutic agents for treating early and late stages of prostate cancer.     

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.     

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     

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

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

Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D₃ metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3′-diaminobenzidine (DAB) for hydrogen peroxide (H₂O₂) detection and phloroglucinol for lignin staining showed that BTH causes H₂O₂ accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D₃, and the hydroxylated metabolites 25-hydroxyvitamin D₃ [25(OH)D₃] and 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D₃, though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D₃ and 1α,25(OH)₂D₃ metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.     

Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues having a spiro-oxetane fused at the C2 position in the A-ring

Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D₃ (3a–d) bearing a spiro-oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d), respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a beneficial effect on the VDR affinity.     

Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)₂D₃ and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)₂D₃ enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)₂D₃ treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)₂D₃-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)₂D₃ and did not change that by 1,25(OH)₂D₃ plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)₂D₃-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na⁺,K⁺-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.     

Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells

The active form of vitamin D₃, 1,25(OH)₂D₃, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4⁺ effector T cells. The biological actions of 1,25(OH)₂D₃ are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)₂D₃ by itself regulates VDR expression in human CD4⁺ T cells. We found that activated CD4⁺ T cells have the capacity to convert the inactive 25(OH)D₃ to the active 1,25(OH)₂D₃ that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)₂D₃ does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4⁺ T cells. Furthermore, 1,25(OH)₂D₃ induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)₂D₃ stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)₂D₃-induced gene activation. In conclusion, our study shows that activated CD4⁺ T cells can produce 1,25(OH)₂D₃, and that 1,25(OH)₂D₃ induces a 2-fold up-regulation of the VDR protein expression in activated CD4⁺ T cells by protecting the VDR against proteasomal degradation.     

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

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

Synthesis and biological activity of 22-iodo- and (E)-20(22)-dehydro analogues of 1α,25-dihydroxyvitamin D3

Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1α,25-dihydroxyvitamin D₃. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1α-hydroxyvitamin D₃ compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by ¹H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1α,25-dihydroxyvitamin D₃ was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1α,25-dihydroxyvitamin D₃ and 22-iodo-1α,25-dihydroxyvitamin D₃ isomers were ca. ten times less potent than the natural hormone 1α,25-(OH)₂D₃ both in intestinal calcium transport and bone calcium mobilization.     

Vitamin D Receptor Gene Expression and Function in a South African Population: Ethnicity,Vitamin D and FokI

Polymorphisms of the vitamin D receptor gene (VDR) have been associated inconsistently with various diseases, across populations of diverse origin. The T(f) allele of the functional SNP FokI, in exon 2 of VDR, results in a longer vitamin D receptor protein (VDR) isoform, proposed to be less active. Genetic association of VDR with disease is likely confounded by ethnicity and environmental factors such as plasma 25(OH)D₃ status. We hypothesized that VDR expression, VDR level and transactivation of target genes, CAMP and CYP24A1, depend on vitamin D, ethnicity and FokI genotype. Healthy volunteers participated in the study (African, n = 40 and White, n = 20). Plasma 25(OH)D₃ levels were quantified by LC-MS and monocytes cultured, with or without 1,25(OH)₂D₃. Gene expression and protein level was quantified using qRT-PCR and flow cytometry, respectively. Mean plasma 25(OH)D₃ status was normal and not significantly different between ethnicities. Neither 25(OH)D₃ status nor 1,25(OH)₂D₃ supplementation significantly influenced expression or level of VDR. Africans had significantly higher mean VDR protein levels (P<0.050), nonetheless transactivated less CAMP expression than Whites. Genotyping the FokI polymorphism by pyrosequencing together with HapMap data, showed a significantly higher (P<0.050) frequency of the CC genotype in Africans than in Whites. FokI genotype, however, did not influence VDR expression or VDR level, but influenced overall transactivation of CAMP and 1,25(OH)₂D₃-elicited CYP24A1 induction; the latter, interacting with ethnicity. In conclusion, differential VDR expression relates to ethnicity, rather than 25(OH)D₃ status and FokI genotype. Instead, VDR transactivation of CAMP is influenced by FokI genotype and, together with ethnicity, influence 1,25(OH)₂D₃-elicited CYP24A1 expression. Thus, the expression and role of VDR to transactivate target genes is determined not only by genetics, but also by ethnicity and environment involving complex interactions which may confound disease association.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Culture requirements for optimal expression of 1α,25-dihydroxyvitamin D3-enhanced thyrotropin secretion

Summary An effect of the hormone, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on hormone secretion by normal rat pituitary cells was investigated in vitro. Based on previous findings using GH₄C₁ cells, dispersed anterior pituitary cell cultures were prepared and maintained in serum-free conditions for up to 6 d. Under these circumstances, there was no effect of 1,25(OH)₂D₃ to alter medium or cell-associated levels of thyrotropin (TSH), prolactin (PRL), or growth hormone (GH). Cultures maintained under these conditions had lower medium and cell-associated hormone levels and lesser responses to agonists than cultures maintained in serum-supplemented medium. In the presence of 10% charcoal-treated fetal bovine serum, treatment with 10⁻⁸ M 1,25(OH)₂D₃ for 24 h selectively increased TRH (10⁻¹⁰ to 10⁻⁷ M)-induced TSH secretion (P<0.001), with maximal enhancement observed at 10⁻⁹ M TSH-releasing hormone (TRH). Enhancement of TSH secretion by 1,25(OH)₂D₃ was detected after 15 min exposure to TRH. There was no effect on agonist-induced PRL or GH secretion or on cell-associated hormone levels. The effect was evident after 24 h treatment with 1,25(OH)₂D₃, and decreased thereafter. Several other steroid hormones had no effect on 10⁻⁹ M TRH-induced TSH secretion. These data contrast with the effect of 1,25(OH)₂D₃ in GH cells. They suggest that 1,25(OH)₂D₃ may act selectively in the normal pituitary to modulate TSH secretion.