Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-Dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3☆
Abstract
The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7–10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 × 106 cells) in HL-60 cells was noted with a 12-h exposure to 10−9m 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1α-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon γ did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.
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Cited by (20)
The 25(OH)D<inf>3</inf>/1α,25(OH)<inf>2</inf>D<inf>3</inf>-24R-hydroxylase: A catabolic or biosynthetic enzyme?
2001, SteroidsThe kidney is the major source of the circulating dihydroxylated metabolites of vitamin D, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3]. The enzymes which catalyze the production of these two dihydroxylated vitamin D metabolites are the 25(OH)D3-1α-hydroxylase (1α-hydroxylase) and –24R-hydroxylase (24R-hydroxylase), respectively. While there is no controversy regarding the fundamental importance of the 1α-hydroxylase in the production of the steroid hormone 1α,25(OH)2D3, the biologic significance of the 24R-hydroxylase has been the subject of ongoing discussion. Some hold that it is strictly catabolic, leading to side chain oxidation and cleavage of 25-hydroxylated vitamin D sterols, and others hold that it plays a biosynthetic role in the production of 24R,25(OH)2D3 which has biologic activities distinct from those of 1α,25(OH)2D3. The 24R-hydroxylase has properties in common with other multicatalytic steroidogenic enzymes: (1) the enzyme carries out multiple oxidative and carbon-carbon bond cleavages; (2) it utilizes two natural substrates; (3) its regulation varies depending on the cell or tissue in which it occurs. The purpose of this paper is to review the current literature relevant to the characteristics of the 24R-hydroxylase and its regulation in the context of other multicatalytic steroid hydroxylases in order to provide a perspective regarding its possible function as both a catabolic and activating enzyme in the vitamin D endocrine system.
The secosteroid hormone, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1α,25(OH)2D3 and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1α,25(OH)2D3 is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1α,25(OH)2D3-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1α,25-dihydroxy-3-epi-vitamin-D3. We performed the present study first to examine in detail the metabolism of 1α,25(OH)2D3 in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1α,25(OH)2D3 in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1β-3H] 1α,25(OH)2D3 and demonstrated that these cells metabolize 1α,25(OH)2D3 mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1α,25(OH)2D3 derived via the C-24 oxidation pathway namely, 1α,24(R),25-trihydroxyvitamin D3, 1α,25-dihydroxy-24-oxovitamin D3 and 1α,23(S),25-trihydroxy-24-oxovitamin D3 [1α,23(S),25(OH)3-24-oxo-D3] were almost as potent as 1α,25(OH)2D3 in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1α,23(S),25(OH)3-24-oxo-D3 which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1α,25(OH)2D3 to examine further its effects on these cells. Our results indicated that 1α,23(S),25(OH)3-24-oxo-D3 was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1α,25(OH)2D3 is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1α,25(OH)2D3 in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1α,25(OH)2D3 were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1α,25(OH)2D3 may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1α,25(OH)2D3.
Prostaglandin E<inf>2</inf> regulates vitamin D receptor expression, vitamin D- 24-hydroxylase activity and cell proliferation in an adherent human myeloid leukemia cell line (Ad-HL60)
1999, Prostaglandins and Other Lipid MediatorsThe effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 μM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were ∼23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 ± 0.17, 72 h PGE2-treated cells 12.10 ± 1.90 pmol/h/106 cells), and an increase in receptors for the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 μM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.
The active form of vitamin D3 [1α,25-dihydroxyvitamin-D3 (1α,25(OH)2D3)] modulates the proliferation and differentiation of hematopoietic cells. Analogs of 1α,25(OH)2D3 that have greater potency may have the potential as adjuvant therapy for high-risk patients in remission for acute myelogenous leukemia (AML) and myelodysplastic syndromes. A new generation of 11 analogs of 1α,25(OH)2D3 has been synthesized, and we examined their effects on the human leukemic cell line HL-60. This cell line provides a sensitive monitor of activity of the 1α,25(OH)2D3 analogs. All the compounds were potent, producing a 50% clonal inhibition (ED50) in the range of 10-8 to 10-11 mol/L; nine of the 11 analogs had ED50s at concentrations that were at least 10-fold lower than those for the parental 1,25(OH)2D3. The most active compound [cmpd LA, (22R)-1α,25-(OH)2-16,22,23-triene-D3] had an ED50 of 2 × 10-11 mol/L; it was also tested on clonogenic cells from patients with AML, and it achieved an ED50 of approximately 6 × 10-11 mol/L, while 1α,25(OH)2D3 produced an ED50 of approximately 10-8 mol/L on the same population of cells. Five different cell surface markers were examined on HL-60 cells exposed to the 1α,25(OH)2D3 analogs: HLA-DR and CD11b were induced by all of the compounds; CD13 was induced by six of the 12 compounds, including 1,25(OH)2D3; CD14 was strongly induced by all compounds; and CD38 was induced rather weakly by nine of 12 analogs. WAF1/CIP1/p21, a cyclin-dependent kinase inhibitor (CDKI), which is important in blocking the cell cycle, was examined by Western blot and was found to be induced by all of the compounds, suggesting a possible mechanism by which these analogs inhibit leukemic growth. The induction of WAF1 occurred at concentrations of vitamin D analogs as low as 10-10 mol/L. This structure-function study showed that a new series of 1α,25(OH)2D3 analogs was active in clonal inhibition, as well as induction of differentiation and WAF1 expression of HL-60 cells. The key structural motifs included C-16 double bond, double and/or triple bonds in the side chain, lengthening of the side chain, 20-epi-conformation of the side chain, replacement of six hydrogens at the end of the side chain with fluorines, and the removal of C-19. Consideration should be given to further in vivo testing of toxicity and efficacy to move toward a clinical trial, especially in a setting of minimal residual disease.
Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D <inf>3</inf> 24-hydroxylase (CYP24) promoter
1996, Journal of Biological ChemistryTranscription of the CYP24 gene is induced by 1,25-(OH)2D3 through a vitamin D receptor-dependent process. The functional activities of three possible vitamin D response elements (VDREs), located on the antisense strand of the rat CYP24 promoter, were investigated by transient expression of native and mutant promoter constructs in COS-1, JTC-12, and ROS 17/2.8 cells. A putative VDRE with a half-site spacing of 6 base pairs at −249/−232 (VDRE-3) did not contribute to 1,25-(OH)2D3 induced expression in the native promoter, although activity has been reported when the element was fused to the heterologous thymidine kinase promoter. Two VDREs with half-site spacings of 3 base pairs at −150/−136 and −258/−244 (VDRE-1 and VDRE-2, respectively), showed transcriptional synergism in COS-1 cells when treated with 1,25-(OH)2D3 (10−7 to 10−11M). The contribution of both VDREs was hormone-concentration dependent from 10−10 to 10−12M, with VDRE-1 demonstrating greatest sensitivity to 1,25-(OH)2D3. Transactivation by VDRE-1 was always greater than VDRE-2, but the converse was observed for the binding of vitamin D receptor-retinoid X receptor complex by each VDRE in gel mobility shift assays. The synergy observed between VDRE-1 and VDRE-2 may have important implications in cellular responses to different circulating levels of 1,25-(OH)2D3.
Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24-hydroxylase promoter
1995, Journal of Biological ChemistryThe interaction between the two vitamin D response elements (DRE) located at −154 to −134 base pairs (bp) and −262 to −238 bp from the transcription initiation site has been studied using reporter gene assays and binding assays by electrophoretic gel shift measurements. 3 half-sites separated by 3 bp were found necessary for transactivation by the −154 to −125 DRE, while 2 half-sites separated by 3 bp were needed for the DRE at −262 to −238 to function. However, the two DREs together provided maximal activity. The 93-bp fragment separating the two DREs was not required and could be deleted. The most effective binding by receptor was found with the two complete DREs (dissociation constant (Kd) = 13.7 pM), although each DRE bound to the receptor and nuclear accessory factor with about 5 nMKd. The two DREs (a total of 5 half-sites) apparently account for most if not all of the transactivation of the rat 24-hydroxylase by 1,25-dihydroxyvitamin D3. This system represents the most powerful of the DREs reported to date.
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This work was supported by USPHS Grants CA26,038, CA-33,936, CA-03,273, and AM-14750.
- 1a
H.R. was supported by the Deutsche Forschungsgemeinschaft (Re 613/1-1).
- 1b
H.P.K. has a Career Development Award from NIH and is a member of the Jonsson Cancer Center.