0
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found

      Effects of ‘Non-Calcaemic’ Vitamin D Analogues on 24-Hydroxylase Expression in MG-63 Osteoblast-Like Cells

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Background: New ‘non-calcaemic’ analogues of 1,25-dihydroxyvitamin D<sub>3</sub> (1,25(OH)<sub>2</sub>D<sub>3</sub>) are entering the clinical arena and some of them have been shown to have differential effects in bone. This may have a bearing on the evolution of bone lesions in uraemic patients receiving vitamin D therapies. A potential mechanism for differential effects of analogues lies in their target cell inactivation. Methods: Using a human osteoblastic cell line, MG-63, three analogues, 22-oxacalcitriol (OCT), 19-nor-1,25-dihydroxyvitamin D<sub>2</sub> (paricalcitol) and 1α,25-dihydroxydihydrotachysterol<sub>2</sub> (1,25(OH)<sub>2</sub>DHT<sub>2</sub>), were compared with 1,25(OH)<sub>2</sub>D<sub>3</sub> for (1) their affinity for the vitamin D receptor (VDR) by competitive displacement of tritiated 1,25(OH)<sub>2</sub>D<sub>3</sub> from calf thymus VDR; (2) effects on 24-hydroxylase mRNA expression using comparative RT-PCR, and (3) rates of metabolism, using high performance liquid chromatography, over a 24-hour time course. Results: Relative VDR-binding affinities (IC<sub>50</sub>) were 1,25(OH)<sub>2</sub>D<sub>3</sub> (100%), OCT (25%), paricalcitol (14%) and 1,25(OH)<sub>2</sub>DHT<sub>2</sub> (0.3%). A ≧3-fold increase in 24-hydroxylase mRNA expression was observed for all compounds at 2 h peaking at 7- to 8-fold above control levels by 12 h, with no significant difference between the analogues and 1,25(OH)<sub>2</sub>D<sub>3</sub>. Differences in their rates of metabolism were observed [calculated t½ values = OCT (1.2 h) > paricalcitol (2.3 h) > 1,25(OH)<sub>2</sub>D<sub>3</sub> (2.6 h) > 1,25(OH)<sub>2</sub>DHT<sub>2</sub> (3.4 h)], with OCT having a significantly shorter half-life. Conclusion: In MG-63 cells these analogues up-regulate 24-hydroxylase mRNA expression with similar potency, in each case accelerating ligand inactivation, despite significant differences in VDR affinity. VDR affinity did not correspond to either 24-hydroxylase mRNA expression or the rates of ligand disappearance, suggesting cellular metabolism is one of several factors that determine the analogue specificity of these agents in bone.

          Related collections

          Most cited references 11

          • Record: found
          • Abstract: found
          • Article: not found

          Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme.

          Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-(3)H]D3 substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system. These products exactly comigrated with authentic standards for 24-oxo-25-OH-D3, 23(S),25-(OH)2D3, 24(R),25-(OH)2D3, and 24-oxo-23(S),25-(OH)2D3. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)2D3 nor any of the other 25-OH-D3 metabolites. The products were isolated and subjected to a normal-phase amino HPLC for further separation, purification, and characterization. Comigration on two HPLC systems, periodate cleavage reactions, and NaBH4 reduction established clearly the identity of these metabolites. Incubation of recombinant h-P450cc24 with 25-OH-[3 alpha-3H]D3 led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D3. Treatment of putative 24,25,26,27-tetranor-23-OH-[3 alpha-3H]D3 with acetic anhydride changed its migration on amino HPLC to a less polar position, indicating acetylation of a hydroxyl group(s). These data demonstrate conclusively that h-P450cc24 is a multicatalytic enzyme catalyzing most, if not all, of the reactions in the C-24/C-23 pathway of 25-OH-D3 metabolism. It is likely that this enzyme by itself converts 25-OH-D3 and 1,25-(OH)2D3 to one of its final excretion products.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) promoter.

             D Kerry,  P Dwivedi,  C N Hahn (1996)
            Transcription 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(-11) M). The contribution of both VDREs was hormone-concentration dependent from 10(-10) to 10(-12) M, 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.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: not found

              Metabolic studies using recombinant escherichia coli cells producing rat mitochondrial CYP24 CYP24 can convert 1alpha,25-dihydroxyvitamin D3 to calcitroic acid.

               T Sakaki,  N Sawada,  Y Nonaka (1999)
              Previously we expressed rat 25-hydroxyvitamin D3 24-hydroxylase (CYP24) cDNA in Escherichia coli JM109 and showed that CYP24 catalyses three-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3 [Akiyoshi-Shibata, M., Sakaki, T., Ohyama, Y., Noshiro, M., Okuda, K. & Yabusaki, Y. (1994) Eur. J. Biochem. 224, 335-343]. In this study, we demonstrate further oxidation by CYP24 including four- and six-step monooxygenation towards 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3, respectively. When the substrate 25-hydroxyvitamin D3 was added to a culture of recombinant E. coli, four metabolites, 24, 25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3, 24-oxo-23, 25-dihydroxyvitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3 were observed. These results indicate that CYP24 catalyses at least four-step monooxygenation toward 25-hydroxyvitamin D3. Furthermore, in-vivo and in-vitro metabolic studies on 1alpha,25-dihydroxyvitamin D3 clearly indicated that CYP24 catalyses six-step monooxygenation to convert 1alpha,25-dihydroxyvitamin D3 into calcitroic acid which is known as a final metabolite of 1alpha,25-dihydroxyvitamin D3 for excretion in bile. These results strongly suggest that CYP24 is largely responsible for the metabolism of both 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.
                Bookmark

                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2003
                August 2003
                12 September 2003
                : 94
                : 4
                : p62-p73
                Affiliations
                Departments of aEndocrinology and bExperimental Medicine and Nephrology and cClinical Biochemistry, St. Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK
                Article
                72519 Nephron Physiol 2003;94:p62–p73
                10.1159/000072519
                12972708
                © 2003 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 6, References: 43, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/72519
                Categories
                Original Paper

                Comments

                Comment on this article