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27-Hydroxylation of 7- and 8-dehydrocholesterol in Smith–Lemli–Opitz syndrome: a novel metabolic pathway
Steroids 68 (2003) 497–502
27-Hydroxylation of 7- and 8-dehydrocholesterol in Smith–Lemli–Opitz syndrome: a novel metabolic pathway Christopher A. Wassif a , Jinghua Yu b , Jisong Cui b , Forbes D. Porter a , Norman B. Javitt c,∗ a b
National Institute of Child Health and Human Development, National Institute of Health Bethesda, Maryland, MD 20892, USA Department of Atherosclerosis, Endocrinology, and Metabolic Research, Merck Research Laboratories, Rahway, NJ 07065, USA c Department of Pediatrics and Medicine, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 13 December 2002; received in revised form 7 April 2003; accepted 6 May 2003
Abstract Smith–Lemli–Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydrocholesterol reductase. Low to absent enzyme activity accounts for the accumulation of both 7-dehydrocholesterol and 8-dehydrocholesterol in plasma and other tissues. Since oxysterols can participate in the regulation of cholesterol homeostasis, we examined the possibility that they are formed from these dehydrocholesterol intermediates. In patients with SLOS, we found serum levels of 27-hydroxy-7-dehydrocholesterol ranging from 0.1 to 0.25 M and evidence for circulating levels of 27-hydroxy-8-dehydrocholesterol (0.04–0.51 M). Picomolar quantities of 27-hydroxy-7-dehydrocholesterol were identified in normal individuals. Biologic activities of 27-hydroxy-7-dehydrocholesterol were found to include inhibition of sterol synthesis and the activation of nuclear receptor LXR␣ but not that of LXR. These activities occurred at concentrations found in plasma and presumably at those existing in tissues. Thus, patients with SLOS have increased levels of metabolites derived from intermediates in cholesterol synthesis that are biologically active and may contribute to the regulation of cholesterol synthesis in vivo. © 2003 Elsevier Inc. All rights reserved. Keywords: Oxysterols; Cholesterol synthesis; Nuclear receptors; Mutations
1. Introduction The studies of Kandutsch and coworkers [1,2] led to the recognition that oxysterols lower the rate of cholesterol synthesis. As inhibitors of cholesterol synthesis, mole for mole oxysterols were found to have more than 100-fold greater potency than cholesterol [3]. Although a wide variety of oxysterols are biologically active, recent interest [4,5] has focused on 24S-hydroxycholesterol, 25-hydroxycholesterol and 25R,26-hydroxycholesterol (also referred to as 27-hydroxycholesterol), which are equipotent with 25-hydroxycholesterol as inhibitors of cholesterol synthesis [3,6]. Of the genes that code for the syntheses of these three oxysterols, CYP 27 is the most widely expressed in tissues [4,5] and is known to have a broad substrate specificity for many endogenous C27 sterols [7,8]. Previous studies done in vitro found that 27-hydroxy metabolites of 7-dehydrocholesterol occur [9]. Our findings establish their occurrence in vivo, thus establishing the existence of ∗
Corresponding author. Tel.: +1-212-263-6588; fax: +1-212-263-8282. E-mail address: [email protected] (N.B. Javitt).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0039-128X(03)00090-4
a metabolic pathway that bypasses cholesterol as the end product.
2. Experimental Frozen serum samples from 10 patients with SLOS were taken for analysis. The age and clinical severity score of each patient, based on the criteria recommended by Kelly and Hennekam [10], are given in Table 1 together with the data obtained in this study. After the addition of 100 ng of d4-27-hydroxycholesterol prepared as described previously [11] to 1.0 ml of plasma, the hydroxysterols were separated from cholesterol using silica cartridges [12]. The hydroxycholesterol fraction was further purified by thin-layer chromatography on silica gel G using a solvent system of chloroform:acetone (4:1, v/v). After separation of the dihydroxycholesterol fraction (Rf = 0.82) from the trihydroxycholesterol fraction (Rf = 0.47), dihydrocholesterols were eluted from the silica with methanol. After solvent removal, the trimethylsilyl ethers were formed by reaction with BSTA with 1% TMCS
498
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Table 1 Concentration of 27-hydroxysterolsa in SLOS and control plasma Patient
Age years
Clinical severity
27-Hydroxy cholesterol (M)
27-Hydroxy cholesta5,8-dienediol (M)
27-Hydroxy cholesta5,7-dienediol (M)
1 2 3 4 5 6 7 8 9 10
4.1 7.3 22.9 8.6 2.7 4.3 0.2 22.2 1.5 12.4
44 39 28 39 11 28 11 22 31 22
0.18 0.16 0.34 0.32 0.32 0.24 0.28 0.68 0.29 0.61
0.51 0.07 0.14 0.37 0.14 0.04 0.11 0.04 0.26 0.30
0.25 0.03 0.07 0.15 0.09 0.02 0.03 0.01 0.08 0.13
M
Control 1 2 3 a Systematic names are cholesta-5,7-diene-3,27-diol.
pM
0.41 0.26 0.38 cholest-5-ene-3,27-diol
None None None
(27-hydroxycholesterol),
(Pierce). Because 7-DHC is known to undergo structural modification to yield Vitamin D on exposure to ultraviolet light, procedures were done in laboratory hoods that were shielded from sunlight. Artificial fluorescent lighting in the laboratory was turned off and tanks for thin-layer chromatography were placed in laboratory cabinets.
cholesta-5,8-diene-3,27-diol
2.0 2.4 1.0 (27-hydroxy-5,8-dienediol),
A Shimadzu GC-17A gas chromatograph and GCMSQP5000 gas chromatograph mass spectrometer was used for the analysis of all hydroxysterols. The TMS ethers were injected in the split mode onto a capillary column (ZB 1701, Phenomenex, Torrance, CA) with an initial temperature of 240 ◦ C and temperature programming at 1.5 ◦ C/min
Fig. 1. GLC-MS analysis of SLOS plasma. The trimethylsilyl ether derivatives were prepared from an eluate (Rf = 0.52) of the sterol fraction of plasma after thin-layer chromatography on silica gel using a solvent system of chloroform:acetone (4:1). Because TLC does not separate 27-hyroxycholesterol from 27-hydroxy-7-dehydrocholesterol and 27-hydroxy-8-dehydrocholesterol, they are obtained as a single eluate free of cholesterol (Rf = 0.79) and cholest-5-ene-3,7␣,27-triol (Rf = 0.22). To detect 27-hydroxycholesterol, the ion m/z = 456 (molecular ion = m/z 546–590) and the (−) 15 ion, m/z = 441 were programmed. For 27-hydroxy-7- and 8-dehydrocholesterol the corresponding m/z = 454 and (−) 15 ion, m/z 439 were programmed. As depicted in the top panel, three peaks were detected at 22.0, 22.5 and 23.8 min. The middle panel indicates that the first peak has the ions characteristics of 27-hydroxycholesterol. The bottom panel indicates the third peak has the ions characteristic of 27-hydroxy-7-dehydro- and 8-dehydrocholesterol. The 2nd peak, 22.5 min, is the putative 27-hydroxy-8-dehydrocholesterol (see text). Thus, the single eluate from the TLC plate contains the 27-hydroxy metabolites of cholesterol and 7- and 8-dehydrocholesterol.
C.A. Wassif et al. / Steroids 68 (2003) 497–502
for 30 min. Both the SCAN and SIM modes were used for analysis. In the SIM mode, m/z 456 and 441 were used to detect of 27-hydroxycholesterol and m/z 454 and 439 were used to detect 27-hydroxy-7-dehydrocholesterol and 27-hydroxy-8-dehydrocholesterol. An authentic standard of 27-hydroxy-7-dehydrocholesterol was prepared from 27-hydroxycholesterol diacetate using a previously described procedure [13]. In cell culture studies, 7-dehydrocholesterol was quantified using 2 g of coprostanol (Sigma) as an internal standard. 2.1. Cell culture SLOS fibroblasts were grown (37 ◦ C, 5% CO2 ) to confluence in DMEM supplemented with 10% fetal bovine serum. The cells were then harvested and plated in T-75 flasks at a concentration of 1.0 × 106 cells per flask in growth serum. After 16 h, the medium was changed to McCoy’s 5A medium supplemented with lipoprotein-deficient serum. 27-Hydroxycholesterol, 27-hydroxy-7-dehydrocholesterol,
499
and 2-hydroxypropyl-- cyclodextrin were added to the medium at the specified concentrations before addition to the cells. Two SLOS fibroblast cells lines (A2SLO and 4350SLO) that are known null mutations (A2SLO: IVS 8-1G > C/IVS 8-1G > C, 4350SLO: IVS 8-1G > C/W151X) and therefore have only 7-dehydroxycholesterol as the metabolic end product were also utilized to evaluate the biologic activity of 27-hydroxy-7-dehydrocholesterol. For ligand affinity studies, the fluorescence resonance energy transfer-based coactivator association assay (FRET) was used as previously described in detail [14].
3. Results Isotope ratio mass spectrometry studies monitoring the ions m/z 456 and 454, characteristic of 27-hydroxy metabolites of cholesterol, and either 7-DHC or 8-DHC, respectively, detected in the sera of SLOS patients two
Fig. 2. GLC-MS analysis of cholesta-5,7-diene-3,27-diol (27-hydroxy-7-dehydrocholesterol). The top panel depicts the retention time (23.8 min) of the standard, cholesta-5,7-diene-3,27-diol prepared by a known method. The complete mass spectral pattern of the standard is shown in the middle panel and the mass spectral pattern of the peak obtained from a pooled plasma sample from patients with SLOS is shown in the bottom panel. The molecular ion m/z = 544 is shown together with an m/z = 455 fragment, which represents the expected (−90 silyl ether) and is within the 1 mass unit allowance of the instrument, particularly since the true molecular ion is closer to m/z = 544.3. A very prominent m/z = 439 peak is seen in both the middle and lower panels and represents the loss of methyl group (−15) from the m/z = 454 fragment.
500
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m/z 454 peaks with retention times at 22.5 and 23.9 min, respectively (Fig. 1). These retention times are slightly greater than that of 27-hydroxycholesterol (22.0 min). Based on the knowledge that both 7-DHC and 8-DHC elute with or shortly after cholesterol, we reasoned that the 27-hydroxysterol metabolites might have a similar retention pattern. Pooling the samples to obtain a full spectrum scan (Fig. 2) established that the peak at 23.9 min was not significantly different from that of an authentic standard of 27-hydroxy-7-dehydrocholesterol. We are not aware of a method for the preparation of 27-hydroxy-8-dehydrocholesterol. However, using a procedure developed to convert 7-DHC to 8-DHC [15], we obtained from 27-hydroxy-7-dehydrocholesterol a small amount of putative 27-hydroxy-8-dehydrocholesterol with a m/z 454 derivative and a retention time of 22.5 min. Combining these results, with the knowledge that 8-DHC levels are increased in SLOS, we suspect that the peak at 22.5 min is in fact 27-hydroxy-8-dehydrocholesterol. Table 1 indicates the age and clinical severity score [10] in ten SLOS patients together with the concentration of 27-hydroxy-7-dehydrocholesterol in plasma. Levels ranged from 0.01 to 0.25 M and from 0.04 to 0.51 M for the presumed 27-hydroxy-8-dehydrocholesterol. Picomolar amounts of 27-hydroxy-7-dehydrocholesterol were found in unaffected individuals. All the SLOS patients in this study showed normal to elevated levels of 27-hydroxycholesterol. This is consistent with the results reported by Bjorkhem et al. [16] which showed that increased levels of 27-hydroxycholesterol are present in serum from SLOS patients. Fig. 3 illustrates the effect of 27-hydroxy-7-dehydrocholesterol and 27-hydroxycholesterol on sterol synthesis in
Fig. 4. FRET assay of LXR␣ and LXR activation by 27-hydroxy7-dehydrocholesterol and 27-hydroxycholesterol. Top: in two separate experiments (open and closed diamond) 27-hydroxy-7-dehydrocholesterol activated LXR␣ (open and closed diamond) but failed to activate LXR (open and closed squares); bottom: 27-hydroxycholesterol activates both LXR␣ and LXR (closed square and closed diamond).
SLOS fibroblast cell lines. Both compounds appear equipotent on a molar basis in down-regulating sterol synthesis. Using the FRET assay we found that 27-hydroxy-7dehydrocholesterol is a specific ligand for LXR␣ (Fig. 4, top). Activation of LXR␣ by 27-hydroxy-7-dehydrocholesterol was found to be dose dependent, reaching a maximal activation at between 1 and 5 M. 27-Hydroxy-7-dehydrocholesterol did not activate LXR. In the same study, as expected, 27-hydroxycholesterol was found to activate both LXR␣ and LHX (Fig. 4, bottom).
4. Discussion
Fig. 3. Suppression of sterol synthesis by 27-hydroxy-7-dehydrocholesterol and 27-hydroxycholesterol. 7-DHC and cellular protein were measured in SLOS skin fibroblasts grown in cholesterol-deficient media with the addition of cyclodextrin carrier (open diamond), 27-hydroxycholesterol or 27-hydroxy-7-dehydrocholesterol (closed rectangle and closed diamond, respectively). 27-Hydroxycholesterol and 27-hydroxy-7-dehydrocholesterol inhibited 7-DHC synthesis to the same extent at similar concentrations. No change in cell viability was noted at higher concentrations of the sterols.
The existence of 27-hydroxy metabolites of 7- and 8-dehydrocholesterol was established by in vitro studies using a rat mitochondrial preparation [9]. The present studies indicate their presence in the plasma of patients with SLOS at concentrations more than 1000-fold greater than in three normal individuals. Presumably, the marked increase in these metabolites reflect the much higher concentrations of 7- and 8-dehydrocholesterol in tissues owing to reduced to absent activity of 7-dehydrocholesterol-7-reductase in patients with SLOS [17]. These novel metabolites can be classified together with other endogenous “oxysterols” that are known to have varying biologic effects ranging from both lowering the rate of transcription and accelerating the rate of degradation of HMG Co A reductase [18–21] to functioning as ligands for nuclear receptors such as LXR␣ and  [22,23]. In these studies, we evaluated the potency of
C.A. Wassif et al. / Steroids 68 (2003) 497–502
27-hydroxy-7-dehydrocholesterol compared with 27-hydroxcholesterol with respect to cholesterol synthesis and as a ligand for the LXR nuclear receptors. In preliminary studies (data not shown) using normal fibroblasts, we found that much of the 27-hydroxy-7dehydrocholesterol added to the medium was metabolized to 27-hydroxycholesterol. Therefore, we utilized cells lacking 7-dehydrocholesterol-7-reductase activity and determined the rate of 7-DHC synthesis. Since oxysterols affect mevalonic acid production and are not known to be late-stage inhibitors of cholesterol synthesis, 7-DHC is a reasonable surrogate for cholesterol. Under conditions of the assay, no difference in potency between 27-hydroxycholesterol and 27-hydroxy-7-dehydrocholesterol was found. Although oxysterols are known to be 100-fold more potent than cholesterol as inhibitors of cholesterol synthesis in vitro [3], it is difficult to evaluate their physiologic role with respect to cholesterol synthesis in vivo. In patients with SLOS it is evident that both 27-hydroxycholesterol [16] and 27-hydroxy-7- and 8-dehydrocholesterol levels are much greater than normal. Therefore, it is reasonable to suspect that they are a determinant of the low rates of cholesterol production that were found in patients with SLOS using metabolic balance techniques [24]. However, it is also known from in vitro studies that although 7-DHC does not affect the level of HMG Co A reductase mRNA in mouse liver, it does lower enzyme activity by accelerating the rate of degradation of the protein [25]. Since levels of 7-DHC in tissues and plasma are also much greater in patients with SLOS compared to normal [17], it is reasonable to think that it is a determinant of the rate of cholesterol production in vivo. Although it is beyond the scope of this study to evaluate the relative roles of increased levels of 7- and 8-dehydrocholesterol and its 27-hydroxy metabolites in regulating cholesterol production in vivo, considering how to modulate each of these possible determinants can provide novel approaches to enhancing cholesterol production in patients with SLOS. Compared to 27-hydroxycholesterol, 27-hydroxy-7-dehydrocholesterol was found to have a specific effect on the activation of only the LXR␣ at all concentrations used (0.01 and 1 M). Given that serum concentrations of 27-hydroxy-7-dehydrocholesterol ranged from 0.01 to 0.25 M in our SLOS patients, it is plausible that this oxysterol is physiologically active. The data also provide some insight into the structural aspects of the ligand binding to LXR␣ and LXR. In a study designed to determine the structural requirements of LXR␣ and LXR ligands, Janowski et al. [23] identified a single synthetic oxysterol, 5,6-24(S),25-diepoxysterol, which was LXR␣ selective. Because 24(S),25-diepoxysterol is an equipotent LXR␣ and LXR ligand, they suggested that either addition of the oxygen of the 5,6-epoxide could stabilize the interaction with LXR␣ or alteration in the B-ring geometry could perturb binding to LXR. We
501
have shown that the latter is most likely correct as the 27-hydroxy-7-dehydrocholesterol B-ring contains an additional double bond and hence results in a geometric change in the structure of the molecule. This change in the molecular geometry most likely precludes it from binding to LXR. LXR␣ has a major role in regulating the physiological response to dietary cholesterol [26]. Based on our findings, it is interesting to speculate that some patients with SLOS may have less than normal rates of intestinal cholesterol absorption.
Acknowledgements This work was supported in part by NIH grant HL 63304 and by the Jerome and Joan Jakubovitz Foundation. References [1] Kandutsch AA, Chen HW. Regulation of sterol synthesis in cultured cells by oxygenated derivatives of cholesterol. J Cell Physiol 1975;85:415–24. [2] Chen HW, Kandutsch AA, Waymouth C. Inhibition of cell growth by oxygenated derivatives of cholesterol. Nature 1974;251:419–21. [3] Parish EJ, Parish SC, Li S. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by side-chain oxysterols and their derivatives. Crit Rev Biochem Mol Biol 1999;34:265–72. [4] Javitt NB. Biologic role(s) of the 25(R),26-hydroxycholesterol metabolic pathway. Biochim Biophys Acta 2000;1529:136–41. [5] Russell DW. Oxysterol biosynthetic enzymes. Biochim Biophys Acta 2000;1529:126–35. [6] Esterman AL, Baum H, Javitt NB, Darlington GJ. 26-Hydroxycholesterol: regulation of hydroxymethylglutaryl-CoA reductase activity in Chinese hamster ovary cell culture. J Lipid Res 1983;24:1304–9. [7] Ohyama Y, Masumoto O, Usui E, Okuda K. Multi-functional property of rat liver mitochondrial cytochrome P-450. J Biochem (Tokyo) 1991;109:389–93. [8] Okuda K, Masumoto O, Ohyama Y. Purification and characterization of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylase from female rat liver mitochondria. J Biol Chem 1988;263:18138–42. [9] Honda A, Salen G, Shefer S, Batta AK, Honda M, Xu G, et al. Bile acid synthesis in the Smith–Lemli–Opitz syndrome: effects of dehydrocholesterols on cholesterol 7␣-hydroxylase and 27-hydroxylase activities in rat liver. J Lipid Res 1999;40:1520–8. [10] Kelley RI, Hennekam RAM. The Smith–Lemli–Opitz syndrome. J Med Genet 2000;37:321–35. [11] Javitt NB, Kok E, Cohen B, Burstein S. Cerebrotendinous xanthomatosis: reduced serum 26-hydroxycholesterol. J Lipid Res 1982;23:627–30. [12] Hahn C, Reichel C, von Bergmann K. Serum concentration of 7 alpha-hydroxycholesterol as an indicator of bile acid synthesis in humans. J Lipid Res 1995;36:2059–66. [13] Kim HS, Wilson WK, Needleman DH, Pinkerton FD, Wilson DK, Quiocho FA, et al. Inhibitors of sterol synthesis. Chemical synthesis, structure, and biological activities of (25R)-3 beta,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one, a metabolite of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one. J Lipid Res 1989;30:247–61. [14] Zhou G, Cummings R, Li Y, Mitra S, Wilkinson HA, Elbrecht A, et al. Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer. Mol Endocrinol 1998;12:1594–604.
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[15] Fieser LF, Ourrison G. Cholesterol and companions IV. Oxidation of 7 sterols with selenium dioxide. J Am Chem Soc 1953;75:4404–14. [16] Bjorkhem I, Starck L, Andersson U, Lutjohann D, von Bahr S, Pikuleva I, et al. Oxysterols in the circulation of patients with the Smith–Lemli–Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J Lipid Res 2001;42:366–71. [17] Tint GS, Irons M, Elias ER, Batta AK, Frieden R, Chen TS, et al. Defective cholesterol biosynthesis associated with the Smith– Lemli–Opitz syndrome. N Engl J Med 1994;330:107–13. [18] Faust JR, Luskey KL, Chin DJ, Goldstein JL, Brown MS. Regulation of synthesis and degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by low density lipoprotein and 25-hyddroxycholesterol in UT-1 cells. Proc Natl Acad Sci USA 1982;79:5205–9. [19] Tanaka RD, Edwards PA, Lan SF, Fogelman AM. Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in avian myeloblasts. Mode of action of 25-hydroxycholesterol. J Biol Chem 1983;258:13331–9. [20] Chin DJ, Gil G, Faust JR, Goldstein JL, Brown MS, Luskey KL. Sterols accelerate degradation of hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase encoded by a constitutively expressed cDNA. Mol Cell Biol 1985;5:634–41.
[21] Dawson PA, Metherall JE, Ridgway ND, Brown MS, Goldstein JL. Genetic distinction between sterol-mediated transcriptional and posttranscriptional control of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J Biol Chem 1991;266:9128–34. [22] Peet DJ, Janowski BA, Mangelsdorf DJ. The LXRs: a new class of oxysterol receptors. Curr Opin Genet Dev 1998;8:571–5. [23] Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey EJ, et al. Structural requirements of ligands for the oxysterol liver X receptors LXRalpha and LXRbeta. Proc Natl Acad Sci USA 1999;96:266–71. [24] Steiner RD, Linck LM, Flavell DP, Lin DS, Connor WE. Sterol balance in the Smith–Lemli–Opitz syndrome: reducation in whole body cholesterol synthesis and normal bile acid production. J Lipid Res 2000;41:1437–47. [25] Fitzky BU, Moebius FF, Asaoka H, Waage-Baudet H, Xu L, Xu G, et al. 7-Dehydrocholesterol-dependent proteolysis of HMG-CoA reductase suppresses sterol biosynthesis in a mouse model of Smith–Lemli–Opitz/RSH syndrome. J Clin Invest 2001;108:905–15. [26] Millat LJ, Bocher V, Fruchart J-C, Staels B. Liver X receptors and control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis. Biochim Biophys Acta 2003;1631:107–18.
27-Hydroxylation of 7- and 8-dehydrocholesterol in Smith–Lemli–Opitz syndrome: a novel metabolic pathway Christopher A. Wassif a , Jinghua Yu b , Jisong Cui b , Forbes D. Porter a , Norman B. Javitt c,∗ a b
National Institute of Child Health and Human Development, National Institute of Health Bethesda, Maryland, MD 20892, USA Department of Atherosclerosis, Endocrinology, and Metabolic Research, Merck Research Laboratories, Rahway, NJ 07065, USA c Department of Pediatrics and Medicine, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 13 December 2002; received in revised form 7 April 2003; accepted 6 May 2003
Abstract Smith–Lemli–Opitz syndrome (SLOS) is attributable to mutations in the gene coding for 7-dehydrocholesterol reductase. Low to absent enzyme activity accounts for the accumulation of both 7-dehydrocholesterol and 8-dehydrocholesterol in plasma and other tissues. Since oxysterols can participate in the regulation of cholesterol homeostasis, we examined the possibility that they are formed from these dehydrocholesterol intermediates. In patients with SLOS, we found serum levels of 27-hydroxy-7-dehydrocholesterol ranging from 0.1 to 0.25 M and evidence for circulating levels of 27-hydroxy-8-dehydrocholesterol (0.04–0.51 M). Picomolar quantities of 27-hydroxy-7-dehydrocholesterol were identified in normal individuals. Biologic activities of 27-hydroxy-7-dehydrocholesterol were found to include inhibition of sterol synthesis and the activation of nuclear receptor LXR␣ but not that of LXR. These activities occurred at concentrations found in plasma and presumably at those existing in tissues. Thus, patients with SLOS have increased levels of metabolites derived from intermediates in cholesterol synthesis that are biologically active and may contribute to the regulation of cholesterol synthesis in vivo. © 2003 Elsevier Inc. All rights reserved. Keywords: Oxysterols; Cholesterol synthesis; Nuclear receptors; Mutations
1. Introduction The studies of Kandutsch and coworkers [1,2] led to the recognition that oxysterols lower the rate of cholesterol synthesis. As inhibitors of cholesterol synthesis, mole for mole oxysterols were found to have more than 100-fold greater potency than cholesterol [3]. Although a wide variety of oxysterols are biologically active, recent interest [4,5] has focused on 24S-hydroxycholesterol, 25-hydroxycholesterol and 25R,26-hydroxycholesterol (also referred to as 27-hydroxycholesterol), which are equipotent with 25-hydroxycholesterol as inhibitors of cholesterol synthesis [3,6]. Of the genes that code for the syntheses of these three oxysterols, CYP 27 is the most widely expressed in tissues [4,5] and is known to have a broad substrate specificity for many endogenous C27 sterols [7,8]. Previous studies done in vitro found that 27-hydroxy metabolites of 7-dehydrocholesterol occur [9]. Our findings establish their occurrence in vivo, thus establishing the existence of ∗
Corresponding author. Tel.: +1-212-263-6588; fax: +1-212-263-8282. E-mail address: [email protected] (N.B. Javitt).
0039-128X/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0039-128X(03)00090-4
a metabolic pathway that bypasses cholesterol as the end product.
2. Experimental Frozen serum samples from 10 patients with SLOS were taken for analysis. The age and clinical severity score of each patient, based on the criteria recommended by Kelly and Hennekam [10], are given in Table 1 together with the data obtained in this study. After the addition of 100 ng of d4-27-hydroxycholesterol prepared as described previously [11] to 1.0 ml of plasma, the hydroxysterols were separated from cholesterol using silica cartridges [12]. The hydroxycholesterol fraction was further purified by thin-layer chromatography on silica gel G using a solvent system of chloroform:acetone (4:1, v/v). After separation of the dihydroxycholesterol fraction (Rf = 0.82) from the trihydroxycholesterol fraction (Rf = 0.47), dihydrocholesterols were eluted from the silica with methanol. After solvent removal, the trimethylsilyl ethers were formed by reaction with BSTA with 1% TMCS
498
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Table 1 Concentration of 27-hydroxysterolsa in SLOS and control plasma Patient
Age years
Clinical severity
27-Hydroxy cholesterol (M)
27-Hydroxy cholesta5,8-dienediol (M)
27-Hydroxy cholesta5,7-dienediol (M)
1 2 3 4 5 6 7 8 9 10
4.1 7.3 22.9 8.6 2.7 4.3 0.2 22.2 1.5 12.4
44 39 28 39 11 28 11 22 31 22
0.18 0.16 0.34 0.32 0.32 0.24 0.28 0.68 0.29 0.61
0.51 0.07 0.14 0.37 0.14 0.04 0.11 0.04 0.26 0.30
0.25 0.03 0.07 0.15 0.09 0.02 0.03 0.01 0.08 0.13
M
Control 1 2 3 a Systematic names are cholesta-5,7-diene-3,27-diol.
pM
0.41 0.26 0.38 cholest-5-ene-3,27-diol
None None None
(27-hydroxycholesterol),
(Pierce). Because 7-DHC is known to undergo structural modification to yield Vitamin D on exposure to ultraviolet light, procedures were done in laboratory hoods that were shielded from sunlight. Artificial fluorescent lighting in the laboratory was turned off and tanks for thin-layer chromatography were placed in laboratory cabinets.
cholesta-5,8-diene-3,27-diol
2.0 2.4 1.0 (27-hydroxy-5,8-dienediol),
A Shimadzu GC-17A gas chromatograph and GCMSQP5000 gas chromatograph mass spectrometer was used for the analysis of all hydroxysterols. The TMS ethers were injected in the split mode onto a capillary column (ZB 1701, Phenomenex, Torrance, CA) with an initial temperature of 240 ◦ C and temperature programming at 1.5 ◦ C/min
Fig. 1. GLC-MS analysis of SLOS plasma. The trimethylsilyl ether derivatives were prepared from an eluate (Rf = 0.52) of the sterol fraction of plasma after thin-layer chromatography on silica gel using a solvent system of chloroform:acetone (4:1). Because TLC does not separate 27-hyroxycholesterol from 27-hydroxy-7-dehydrocholesterol and 27-hydroxy-8-dehydrocholesterol, they are obtained as a single eluate free of cholesterol (Rf = 0.79) and cholest-5-ene-3,7␣,27-triol (Rf = 0.22). To detect 27-hydroxycholesterol, the ion m/z = 456 (molecular ion = m/z 546–590) and the (−) 15 ion, m/z = 441 were programmed. For 27-hydroxy-7- and 8-dehydrocholesterol the corresponding m/z = 454 and (−) 15 ion, m/z 439 were programmed. As depicted in the top panel, three peaks were detected at 22.0, 22.5 and 23.8 min. The middle panel indicates that the first peak has the ions characteristics of 27-hydroxycholesterol. The bottom panel indicates the third peak has the ions characteristic of 27-hydroxy-7-dehydro- and 8-dehydrocholesterol. The 2nd peak, 22.5 min, is the putative 27-hydroxy-8-dehydrocholesterol (see text). Thus, the single eluate from the TLC plate contains the 27-hydroxy metabolites of cholesterol and 7- and 8-dehydrocholesterol.
C.A. Wassif et al. / Steroids 68 (2003) 497–502
for 30 min. Both the SCAN and SIM modes were used for analysis. In the SIM mode, m/z 456 and 441 were used to detect of 27-hydroxycholesterol and m/z 454 and 439 were used to detect 27-hydroxy-7-dehydrocholesterol and 27-hydroxy-8-dehydrocholesterol. An authentic standard of 27-hydroxy-7-dehydrocholesterol was prepared from 27-hydroxycholesterol diacetate using a previously described procedure [13]. In cell culture studies, 7-dehydrocholesterol was quantified using 2 g of coprostanol (Sigma) as an internal standard. 2.1. Cell culture SLOS fibroblasts were grown (37 ◦ C, 5% CO2 ) to confluence in DMEM supplemented with 10% fetal bovine serum. The cells were then harvested and plated in T-75 flasks at a concentration of 1.0 × 106 cells per flask in growth serum. After 16 h, the medium was changed to McCoy’s 5A medium supplemented with lipoprotein-deficient serum. 27-Hydroxycholesterol, 27-hydroxy-7-dehydrocholesterol,
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and 2-hydroxypropyl-- cyclodextrin were added to the medium at the specified concentrations before addition to the cells. Two SLOS fibroblast cells lines (A2SLO and 4350SLO) that are known null mutations (A2SLO: IVS 8-1G > C/IVS 8-1G > C, 4350SLO: IVS 8-1G > C/W151X) and therefore have only 7-dehydroxycholesterol as the metabolic end product were also utilized to evaluate the biologic activity of 27-hydroxy-7-dehydrocholesterol. For ligand affinity studies, the fluorescence resonance energy transfer-based coactivator association assay (FRET) was used as previously described in detail [14].
3. Results Isotope ratio mass spectrometry studies monitoring the ions m/z 456 and 454, characteristic of 27-hydroxy metabolites of cholesterol, and either 7-DHC or 8-DHC, respectively, detected in the sera of SLOS patients two
Fig. 2. GLC-MS analysis of cholesta-5,7-diene-3,27-diol (27-hydroxy-7-dehydrocholesterol). The top panel depicts the retention time (23.8 min) of the standard, cholesta-5,7-diene-3,27-diol prepared by a known method. The complete mass spectral pattern of the standard is shown in the middle panel and the mass spectral pattern of the peak obtained from a pooled plasma sample from patients with SLOS is shown in the bottom panel. The molecular ion m/z = 544 is shown together with an m/z = 455 fragment, which represents the expected (−90 silyl ether) and is within the 1 mass unit allowance of the instrument, particularly since the true molecular ion is closer to m/z = 544.3. A very prominent m/z = 439 peak is seen in both the middle and lower panels and represents the loss of methyl group (−15) from the m/z = 454 fragment.
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m/z 454 peaks with retention times at 22.5 and 23.9 min, respectively (Fig. 1). These retention times are slightly greater than that of 27-hydroxycholesterol (22.0 min). Based on the knowledge that both 7-DHC and 8-DHC elute with or shortly after cholesterol, we reasoned that the 27-hydroxysterol metabolites might have a similar retention pattern. Pooling the samples to obtain a full spectrum scan (Fig. 2) established that the peak at 23.9 min was not significantly different from that of an authentic standard of 27-hydroxy-7-dehydrocholesterol. We are not aware of a method for the preparation of 27-hydroxy-8-dehydrocholesterol. However, using a procedure developed to convert 7-DHC to 8-DHC [15], we obtained from 27-hydroxy-7-dehydrocholesterol a small amount of putative 27-hydroxy-8-dehydrocholesterol with a m/z 454 derivative and a retention time of 22.5 min. Combining these results, with the knowledge that 8-DHC levels are increased in SLOS, we suspect that the peak at 22.5 min is in fact 27-hydroxy-8-dehydrocholesterol. Table 1 indicates the age and clinical severity score [10] in ten SLOS patients together with the concentration of 27-hydroxy-7-dehydrocholesterol in plasma. Levels ranged from 0.01 to 0.25 M and from 0.04 to 0.51 M for the presumed 27-hydroxy-8-dehydrocholesterol. Picomolar amounts of 27-hydroxy-7-dehydrocholesterol were found in unaffected individuals. All the SLOS patients in this study showed normal to elevated levels of 27-hydroxycholesterol. This is consistent with the results reported by Bjorkhem et al. [16] which showed that increased levels of 27-hydroxycholesterol are present in serum from SLOS patients. Fig. 3 illustrates the effect of 27-hydroxy-7-dehydrocholesterol and 27-hydroxycholesterol on sterol synthesis in
Fig. 4. FRET assay of LXR␣ and LXR activation by 27-hydroxy7-dehydrocholesterol and 27-hydroxycholesterol. Top: in two separate experiments (open and closed diamond) 27-hydroxy-7-dehydrocholesterol activated LXR␣ (open and closed diamond) but failed to activate LXR (open and closed squares); bottom: 27-hydroxycholesterol activates both LXR␣ and LXR (closed square and closed diamond).
SLOS fibroblast cell lines. Both compounds appear equipotent on a molar basis in down-regulating sterol synthesis. Using the FRET assay we found that 27-hydroxy-7dehydrocholesterol is a specific ligand for LXR␣ (Fig. 4, top). Activation of LXR␣ by 27-hydroxy-7-dehydrocholesterol was found to be dose dependent, reaching a maximal activation at between 1 and 5 M. 27-Hydroxy-7-dehydrocholesterol did not activate LXR. In the same study, as expected, 27-hydroxycholesterol was found to activate both LXR␣ and LHX (Fig. 4, bottom).
4. Discussion
Fig. 3. Suppression of sterol synthesis by 27-hydroxy-7-dehydrocholesterol and 27-hydroxycholesterol. 7-DHC and cellular protein were measured in SLOS skin fibroblasts grown in cholesterol-deficient media with the addition of cyclodextrin carrier (open diamond), 27-hydroxycholesterol or 27-hydroxy-7-dehydrocholesterol (closed rectangle and closed diamond, respectively). 27-Hydroxycholesterol and 27-hydroxy-7-dehydrocholesterol inhibited 7-DHC synthesis to the same extent at similar concentrations. No change in cell viability was noted at higher concentrations of the sterols.
The existence of 27-hydroxy metabolites of 7- and 8-dehydrocholesterol was established by in vitro studies using a rat mitochondrial preparation [9]. The present studies indicate their presence in the plasma of patients with SLOS at concentrations more than 1000-fold greater than in three normal individuals. Presumably, the marked increase in these metabolites reflect the much higher concentrations of 7- and 8-dehydrocholesterol in tissues owing to reduced to absent activity of 7-dehydrocholesterol-7-reductase in patients with SLOS [17]. These novel metabolites can be classified together with other endogenous “oxysterols” that are known to have varying biologic effects ranging from both lowering the rate of transcription and accelerating the rate of degradation of HMG Co A reductase [18–21] to functioning as ligands for nuclear receptors such as LXR␣ and  [22,23]. In these studies, we evaluated the potency of
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27-hydroxy-7-dehydrocholesterol compared with 27-hydroxcholesterol with respect to cholesterol synthesis and as a ligand for the LXR nuclear receptors. In preliminary studies (data not shown) using normal fibroblasts, we found that much of the 27-hydroxy-7dehydrocholesterol added to the medium was metabolized to 27-hydroxycholesterol. Therefore, we utilized cells lacking 7-dehydrocholesterol-7-reductase activity and determined the rate of 7-DHC synthesis. Since oxysterols affect mevalonic acid production and are not known to be late-stage inhibitors of cholesterol synthesis, 7-DHC is a reasonable surrogate for cholesterol. Under conditions of the assay, no difference in potency between 27-hydroxycholesterol and 27-hydroxy-7-dehydrocholesterol was found. Although oxysterols are known to be 100-fold more potent than cholesterol as inhibitors of cholesterol synthesis in vitro [3], it is difficult to evaluate their physiologic role with respect to cholesterol synthesis in vivo. In patients with SLOS it is evident that both 27-hydroxycholesterol [16] and 27-hydroxy-7- and 8-dehydrocholesterol levels are much greater than normal. Therefore, it is reasonable to suspect that they are a determinant of the low rates of cholesterol production that were found in patients with SLOS using metabolic balance techniques [24]. However, it is also known from in vitro studies that although 7-DHC does not affect the level of HMG Co A reductase mRNA in mouse liver, it does lower enzyme activity by accelerating the rate of degradation of the protein [25]. Since levels of 7-DHC in tissues and plasma are also much greater in patients with SLOS compared to normal [17], it is reasonable to think that it is a determinant of the rate of cholesterol production in vivo. Although it is beyond the scope of this study to evaluate the relative roles of increased levels of 7- and 8-dehydrocholesterol and its 27-hydroxy metabolites in regulating cholesterol production in vivo, considering how to modulate each of these possible determinants can provide novel approaches to enhancing cholesterol production in patients with SLOS. Compared to 27-hydroxycholesterol, 27-hydroxy-7-dehydrocholesterol was found to have a specific effect on the activation of only the LXR␣ at all concentrations used (0.01 and 1 M). Given that serum concentrations of 27-hydroxy-7-dehydrocholesterol ranged from 0.01 to 0.25 M in our SLOS patients, it is plausible that this oxysterol is physiologically active. The data also provide some insight into the structural aspects of the ligand binding to LXR␣ and LXR. In a study designed to determine the structural requirements of LXR␣ and LXR ligands, Janowski et al. [23] identified a single synthetic oxysterol, 5,6-24(S),25-diepoxysterol, which was LXR␣ selective. Because 24(S),25-diepoxysterol is an equipotent LXR␣ and LXR ligand, they suggested that either addition of the oxygen of the 5,6-epoxide could stabilize the interaction with LXR␣ or alteration in the B-ring geometry could perturb binding to LXR. We
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have shown that the latter is most likely correct as the 27-hydroxy-7-dehydrocholesterol B-ring contains an additional double bond and hence results in a geometric change in the structure of the molecule. This change in the molecular geometry most likely precludes it from binding to LXR. LXR␣ has a major role in regulating the physiological response to dietary cholesterol [26]. Based on our findings, it is interesting to speculate that some patients with SLOS may have less than normal rates of intestinal cholesterol absorption.
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