1,25(OH)2D, the Preferred Substrate for CYP24
2002; Oxford University Press; Volume: 17; Issue: 1 Linguagem: Inglês
10.1359/jbmr.2002.17.1.179
ISSN1523-4681
AutoresGlenville Jones, Harriet S. Tenenhouse,
Tópico(s)Sexual Differentiation and Disorders
ResumoJournal of Bone and Mineral ResearchVolume 17, Issue 1 p. 179-180 Letter of the EditorFree Access 1,25(OH)2D, the Preferred Substrate for CYP24 Glenville Jones, Glenville Jones Departments of Biochemistry and Medicine Queen's University Kingston, Ontario, CanadaSearch for more papers by this authorHarriet S. Tenenhouse, Harriet S. Tenenhouse Departments of Pediatrics and Human Genetics McGill University, Montreal Children's Hospital RI Montreal, Quebec, CanadaSearch for more papers by this author Glenville Jones, Glenville Jones Departments of Biochemistry and Medicine Queen's University Kingston, Ontario, CanadaSearch for more papers by this authorHarriet S. Tenenhouse, Harriet S. Tenenhouse Departments of Pediatrics and Human Genetics McGill University, Montreal Children's Hospital RI Montreal, Quebec, CanadaSearch for more papers by this author First published: 24 October 2009 https://doi.org/10.1359/jbmr.2002.17.1.179Citations: 13AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat To the Editor: In a recent issue, Taniguchi et al.1 reported that the enzyme 25-hydroxyvitamin D3-24-hydroxylase, a cytochrome P450 also known as 1α,25-dihydroxyvitamin D3-24-hydroxylase (CYP24), has a much higher affinity for 25(OH)D3 than for 1α,25-(OH)2D3, leading the authors to conclude that 25(OH)D3 is the preferred substrate. This conclusion is completely opposite to the current dogma that CYP24 metabolizes 1α,25(OH)2D3 very efficiently, and that one of its major roles in the body is to catabolize the vitamin D hormone inside target cells.2, 3 The latter is based on numerous studies performed over the past 15 years from a number of laboratories, including a collaborative investigation of our own in isolated murine renal mitochondria.4 In that study, 24-hydroxylase activity was measured using [3H]-labeled 25(OH)D3 and 1α,25(OH)2D3 substrates, and both substrate and products of the reaction were measured by high pressure liquid chromatography (HPLC) analysis. We concluded from that data that the apparent Km of the 24-hydroxylase is at least 10-fold lower for 1α,25(OH)2D3 than for 25(OH)D3.4 These findings were corroborated in subsequent studies by two other groups, Shinki et al.5 and Burgos-Trinidad and DeLuca.6 To understand how Taniguchi et al.1 arrived at a completely opposite conclusion, we examined their methodology and results very carefully. We reached the conclusion that the methodology used to determine CYP24 activity, particularly with respect to the 1α,25(OH)2D3 substrate, was flawed and led to erroneous data and an erroneous conclusion. Our main criticism derives from the fact that the authors failed to measure both the substrate and total reaction products; therefore, they did not obtain an accurate estimate of enzyme activity and of the kinetic parameters, particularly for the 1α,25(OH)2D3 substrate. It is well known from the work of several groups that CYP24 is capable of the complete catabolism of 25(OH)D3 and 1α,25(OH)2D3 via a five-step reaction process that includes 24-hydroxylation, 24-oxidation, 23-hydroxylation, side chain cleavage, and subsequent production of the final degradative products, cholacalcioic acid and calcitroic acid.7-9 Thus, to assay CYP24 activity it is necessary to separate and quantitate all potential reaction products derived from either 25(OH)D3 or 1α,25(OH)2D3. Moreover, it is important to appreciate that under initial rate conditions, the relative proportion of reaction products will vary with substrate concentration. For example, at low substrate concentrations 24,25(OH)2D and 1α,24,25(OH)3D3, the first products of the reaction sequence, comprise a much smaller proportion of the total degradative products of 25(OH)D3 and 1α,25(OH)2D3, respectively, than at higher substrate concentrations. Thus, to accurately assess enzyme activity at each substrate concentration it is essential to measure all reaction products. Of equal importance is the knowledge that the side-chain cleavage products of 25(OH)D3 and 1α,25(OH)2D3 are not lipid soluble and must be recovered from the aqueous phase. These issues are particularly relevant to CYP24-mediated catabolism of 1α,25(OH)2D3 because it is degraded more rapidly than 25(OH)D3. Thus, the first product in the reaction sequence, 1α,24,25(OH)3D3, comprises a much smaller proportion of the total reaction products. The methodology of Taniguchi et al.1 did not take the above issues into account and the protocol as described discarded the water-soluble products following lipid extraction. Because the authors only measured the production of 24,25(OH)2D3 and 1α,24,25(OH)3D3 and failed to quantitate the more polar reaction products, they underestimated CYP24 activity, particularly at low substrate concentrations. Consequently, the Km values reported by Taniguchi et al.1 are not accurate. This is particularly true for 1α,25(OH)2D3 (Km = 20.9 μM vs. 20 nM as reported previously4), because at each substrate concentration, a greater proportion of the reaction products were not taken into account. Had Taniguchi et al.1 used radioactive 25(OH)D3 and 1α,25(OH)2D3 substrates, they would have detected the lipid-soluble reaction products in their metabolic extracts. However, by choosing to follow the metabolism of nonradioactive substrates using a relatively insensitive ultraviolet (UV) detector, these authors have limited their studies to reaction products obtained at high substrate concentrations. Furthermore, their methodology did not permit the detection of the aqueous side-chain cleavage products. To overcome these problems, we suggest that the authors reanalyze their raw data and measure CYP24 activity based on substrate disappearance. We predict that at nanomolar concentrations, the amount of remaining 1α,25(OH)2D3 substrate will be negligible because of its rapid catabolism to a variety of 24-oxidation products, including but not confined to 1α,24,25(OH)3D3. The above prediction is based on recent studies from one of our laboratories.10 We performed similar experiments with CYP24 in a mammalian cell line (human keratinocyte, HPK1A-ras) and used three different techniques for assessing product formation, namely radioactively labeled substrates, UV spectrophotometry,1 and a novel method based on liquid chromatography—mass spectrometry (LC-MS). The results indicate that the only detectable product derived from 1α,25(OH)2D3, in the 1-250 nM range, is calcitroic acid. As the substrate concentration is gradually raised, other C-24 oxidation intermediates accumulate, and only at substrate concentrations above 1 μM does 1α,24,25(OH)3D3 become the major product. Our critique of the experimental design of Taniguchi et al.1 and the new data10 is particularly relevant because it illustrates the complexity of the metabolic products of CYP24 and the necessity to design kinetic studies of CYP24 with great care. In addition, it shows the important physiological role of CYP24 in the catabolism of 1α,25(OH)2D3. Thus, the claim based on murine mitochondrial data made in 1988 by Tenenhouse et al.4 remains attractive and is further supported by later findings.2 These studies showed the following: (1) CYP24 prefers 1α,25(OH)2D3 over 25(OH)D3, (2) CYP24 gene expression is not confined to the kidney but can be observed in all vitamin D target cells where it is inducible by 1α,25(OH)2D3 in a vitamin D receptor (VDR)-dependent manner, suggesting that CYP24 protects target cells from overstimulation by the vitamin D hormone, (3) recombinant CYP24 protein executes all five catabolic reactions observed in the early studies, and (4) CYP24-null mice show an inability to catabolize a bolus dose of 1α,25(OH)2D3 and many die prematurely of hypercalcemia and nephrocalcinosis.11 The article by Taniguchi et al.1 fails to discuss these important issues. Rather, their Discussion section focuses on a minor issue: a putative inhibitory factor in rodent mitochondria (believed for many years to be vitamin D-binding protein [DBP]), which sequesters 25(OH)D3 and raises the Km for this substrate. In the process, the authors do not address the physiological consequences of their data. Instead they focus on the role of Tween 20 (Biorad, Richmond, CA, USA) in solubilizing the mitochondrial proteins without considering its effect on the lipid-soluble substrates, 25(OH)D and 1α,25(OH)2D3. Furthermore, the highly purified CYP24 assay system used is not physiological in that it removes two key proteins that influence substrate supply: DBP and megalin. There is no reference to these key proteins and their role in 25(OH)D3 entry into kidney cells. Finally, Taniguchi et al.1 fail to mention that DBP limits 25(OH)D uptake by most cells and by mitochondria in vivo and in vitro. The notion that the kidney might facilitate access of 25(OH)D3 to CYP24 (and its main mitochondrial target, CYP1α or CYP27B), a conclusion derived from studies with megalin-null mice,12 is also omitted. REFERENCES 1 Taniguchi T, Eto T-A, Shiotsuki H, Sueta H, Higashi S, Iwamura T, Okuda K-Y, Setoguchi T 2001 Newly established assay method for 25-hydroxyvitamin D3-24-hydroxylase revealed much lower Km for 25-hydroxyvitamin D3 than for 1α,25-dihydroxyvitamin D3. J Bone Miner Res 16: 57– 62. Wiley Online LibraryCASPubMedGoogle Scholar 2 Makin G, Lohnes D, Byford V, Ray R, Jones G 1989 Target cell metabolism of 1,25-(OH)2D3 to calcitroic acid: Evidence for a pathway in kidney and bone involving 24-oxidation. Biochem J 262: 173– 180. CrossrefCASPubMedWeb of Science®Google Scholar 3 Jones G, Strugnell S, DeLuca HF 1998 Current understanding of the molecular actions of vitamin D. Physiol Rev 78: 1193– 1231. CrossrefCASPubMedWeb of Science®Google Scholar 4 Tenenhouse HS, Yip A, Jones G 1988 Increased renal catabolism of 1α,25-dihydroxyvitamin D3 in murine X-linked hypophosphatemic rickets. J Clin Invest 81: 461– 465. CrossrefCASPubMedWeb of Science®Google Scholar 5 Shinki T, Jin CH, Nishimura A, Nagai Y, Ohyama Y, Noshiro M, Okuda K, Suda T 1992 Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1α,25-dihydroxyvitamin D3 in rat kidney but not in intestine. J Biol Chem 267: 13757– 13762. CASPubMedWeb of Science®Google Scholar 6 Burgos-Trinidad M, DeLuca HF 1991 Kinetic properties of 25-hydroxyvitamin D- and 1,25-dihydroxyvitamin D-24-hydroxylase from chick kidney. Biochim Biophys Acta 1078: 226– 230. CrossrefCASPubMedWeb of Science®Google Scholar 7 Beckman MJ, Tadikonda P, Werner E, Prahl J, Yamada S, DeLuca HF 1996 Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme. Biochemistry 35: 8465– 8472. CrossrefCASPubMedWeb of Science®Google Scholar 8 Sakaki T, Sawada N, Nonaka Y, Ohyama Y, Inouye K 1999 Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24. CYP24 can convert 1α,25-dihydroxyvitamin D3 to calcitroic acid. Eur J Biochem 262: 43– 48. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 9 Akiyoshi-Shibata M, Sakaki T, Ohyama Y, Noshiro M, Okuda K, Yabusaki Y 1994 Further oxidation of hydroxycalcidiol by calcidiol 24-hydroxylase. A study with the mature enzyme expressed in Escherichia coli. Eur J Biochem 224: 335– 343. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 10 Kaufmann M, Masuda S, Jones G 2001 LC-MS based enzyme kinetic analysis of CYP24 confirms that 1α,25-(OH)2D3 is a physiologically relevant substrate. 23rd Annual Meeting of the American Society of Bone Mineral Research, Phoenix, AZ, October 12–16, 2001. J Bone Miner Res 16: S1;S313. Google Scholar 11 St-Arnaud R, Arabian A, Travers R, Barletta F, Raval-Pandya M, Chapin K, Depovere J, Mathieu C, Christakos S, Demay MB, Glorieux FH 2000 Deficient mineralization of intra-membranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxy vitamin D and not to the absence of 24,25-dihydroxyvitamin D. Endocrinology 141: 2658– 2666. CASPubMedWeb of Science®Google Scholar 12 Leheste JR, Rolinski B, Vorum H, Hilpert J, Nykjaer A, Jacobsen C, Aucouturier P, Moskaug JO, Otto A, Christensen EI, Willnow TE 1999 Megalin knockout mice as an animal model of low molecular weight proteinuria. Am J Pathol 155: 1361– 1370. CrossrefCASPubMedWeb of Science®Google Scholar Citing Literature Volume17, Issue1January 2002Pages 179-180 ReferencesRelatedInformation
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