Cholesterol Biosynthesis from Lanosterol

Abstract

The 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624–14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5′-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at −177, −172, −125, and −20), NF-Y (at −88 and −51), and SREBP-1 or ADD1 (at −33). Deletion analysis of the Dhcr7 gene promoter (−1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the −179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (−33/−22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at −125) and NF-Y (CCAAT box at −88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals. The 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624–14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5′-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at −177, −172, −125, and −20), NF-Y (at −88 and −51), and SREBP-1 or ADD1 (at −33). Deletion analysis of the Dhcr7 gene promoter (−1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the −179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (−33/−22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at −125) and NF-Y (CCAAT box at −88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals. 7-dehydrocholesterol reductase adipocyte determination and differentiation-dependent factor 1 lipoprotein-deficient serum fetal bovine serum sterol regulatory element SRE-binding protein nuclear factor Y electrophoretic mobility shift assay sonic hedgehog Smith-Lemi-Opitz syndrome polymerase chain reaction base pair(s) hydroxymethyl glutaryl The 7-dehydrocholesterol reductase (Dhcr7,1 EC1.3.1.21) catalyzes the reduction of the Δ7-double bond of sterol intermediates, which is the terminal reaction, in the pathway of cholesterol biosynthesis from lanosterol (1Gibbon G.F. Goad L.J. Goodwin T.W. Nes W.R. J. Biol. Chem. 1971; 246: 3967-3976Abstract Full Text PDF PubMed Google Scholar, 2Lee J.N. Paik Y.-K. J. Biochem. Mol. Biol. 1997; 30: 370-377Google Scholar, 3Bae S.-H. Paik Y.-K. Biochem. J. 1997; 326: 609-616Crossref PubMed Scopus (74) Google Scholar). Studies of this enzyme have recently drawn our attention due to its implication in both cholesterol metabolism and mammalian developmental biology (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Porter J.A. Young K.E. Beachy P.A. Science. 1996; 11: 255-259Crossref Scopus (1126) Google Scholar, 6Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Science. 1998; 5: 1603-1607Crossref Scopus (785) Google Scholar). In particular, mutations of Dhcr7 has been involved in genetic disease such as Smith-Lemi-Opitz syndrome (SLOS) (7Fitzky B.U. Witsch-Baumgartner M. Erdel M. Lee J.N. Paik Y.-K. Glossmann H. Utermann G. Moebius F.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8181-8186Crossref PubMed Scopus (334) Google Scholar, 8Waterham H.R. Wijburg F.A. Hennekam R.C. Vreken P. Poll-The B.T. Dorland L. Duran M. Jira P.E. Smeitink J.A. Wevers R.A. Wanders R.J. Am. J. Hum. Genet. 1998; 63: 329-338Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 9Wassif C.A. Maslen C. Kachilele-Linjewile S. Lin D. Linck L.M. Conner W.E. Steiner R.D. Porter F.D. Am. J. Hum. Genet. 1998; 63: 55-62Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), which is an autosomal recessive disorder with various developmental abnormalities. SLOS is characterized by elevated 7-dehydrocholesterol in serum (10Smith D.W. Lemli L. Opitz J.M. J. Pediatr. 1964; 64: 210-217Abstract Full Text PDF PubMed Scopus (558) Google Scholar, 11Tint G.S. Irons M. Elias E.R. Batta A.K. Frieden R. Chen T.S. Salen G. N. Engl. J. Med. 1994; 13: 107-113Crossref Scopus (688) Google Scholar, 12Irons M. Elias E.R. Tint G.S. Salen G. Frieden R. Buie T.M. Ampola M. Am. J. Med. Genet. 1994; 1: 347-352Crossref Scopus (89) Google Scholar, 13Tint G.S. Seller M. Hughes-Benzie R. Batta A.K. Shefer S. Genest D. Irons M. Elias E. 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This connection between cholesterol and the developmental program in mammals was established based on the critical role of cholesterol in autoprocessing of sonic hedgehog (shh) protein (5Porter J.A. Young K.E. Beachy P.A. Science. 1996; 11: 255-259Crossref Scopus (1126) Google Scholar, 6Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Science. 1998; 5: 1603-1607Crossref Scopus (785) Google Scholar, 19Porter J.A. Ekker S.C. Park W.J. von Kessler D.P. Young K.E. Chen C.H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 12: 21-34Abstract Full Text Full Text PDF Scopus (439) Google Scholar), a morphogen, which binds to, patched protein in developing the central nervous system and limbs. For example, the mouse mutated in shh showed SLOS-like phenotype (20Chiang C. Litingtung Y. Lee E. Young K.E. Corden J.L. Westphal H. Beachy P.A. Nature. 1996; 383: 407-413Crossref PubMed Scopus (2599) Google Scholar,21Lanoue L. Dehart D.B. Hinsdal M.E. Maeda N. Tint G.S. Sulik K.K. Am. J. Med. Genet. 1997; 73: 24-31Crossref PubMed Scopus (105) Google Scholar). Recently, cDNAs encoding Dhcr7 from mouse (22Moebius F.F. Fitzky B.U. Lee J.N. Paik Y.-K. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1899-1902Crossref PubMed Scopus (194) Google Scholar), rat (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), human (7Fitzky B.U. Witsch-Baumgartner M. Erdel M. Lee J.N. Paik Y.-K. Glossmann H. Utermann G. Moebius F.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8181-8186Crossref PubMed Scopus (334) Google Scholar), and Arabidopsis thaliana (23Lecain E. Chenivesse X. Spagnoli R. Pompon D. J. Biol. Chem. 1996; 271: 10866-10873Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) have been isolated. Dhcr7 mRNA was expressed mainly in liver and its regulation appeared to be controlled by tissue sterol contents as well as some cholesterogenic inhibitors (2Lee J.N. Paik Y.-K. J. Biochem. Mol. Biol. 1997; 30: 370-377Google Scholar, 4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, molecular cloning and the nature of sterol-mediated transcriptional control of the Dhcr7 gene in any species have not been reported previously. Furthermore, the relative importance of multiple motifs at the 5′-flanking region in transcriptional regulation of the Dhcr7 gene is also not known. In order to characterize the 5′-flanking region and the response of multiple regulatory elements to sterols, we isolated the 5′-flanking region of the rat Dhcr7 gene and the complete genomic sequence (GenBankTM accession numberAF279892). 2J. N. Lee, S.-H. Bae, and Y.-K. Paik, submitted for publication. In this paper, we describe the promoter activity and the sterol-mediated regulation of this gene by various mutation analyses, with emphasis on the proximal promoter (−179 to +1), which controls transcriptional induction of the gene in response to starvation of cellular sterols. A rat Promoter FinderTM DNA Walking Kit was purchased from CLONTECH (Palo Alto, CA). The following materials were purchased from manufacturers, as indicated. Hyper x-ray film, [α-32P]dCTP (3000 Ci/mmol), [γ-32P]ATP (3000 Ci/mmol), poly(dI-dC) ·poly(dI-dC), Amersham Pharmacia Biotech; DNA sequencing reagents, PerkinElmer Life Sciences; restriction endonucleases, T4-DNA ligase, T4-polynucleotide kinase, and Klenow fragment of DNA polymerase I, New England Biolabs; RNasin ribonuclease inhibitor and avian myeloblastosis virus-reverse transcriptase,Promega; lipoprotein-deficient serum (LPDS), Sigma; Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), gentamycin, and l-glutamine, Life Technologies; sterols including cholesterol and various oxysterols including 25-, 20-, 19-, 4-, 7α-, 7β-hydroxycholesterols, 6-keto, and 7-keto-sterols, Steraloid. The sources of the following drugs or agents are also indicated. AY-9944, Dr. D. Dvornik at Wyth-Ayerst, Princeton; and LovastatinTM, Dr. Y-K. at was isolated by Kit was in a and to Pharmacia with of the at for in the were rat Dhcr7 for the were with at were by Hyper x-ray with an at The that appeared in the were with a system and to the of mRNA the of this rat Dhcr7 promoter was from DNA in the rat Promoter to the The to the for rat and of the promoter were in the The were and for the with nested in the region of Dhcr7 and were and with an DNA sequencing The Dhcr7 promoter region was also by from genomic To Dhcr7 the 5′-flanking region of the gene was the as The was by Klenow enzyme of a DNA fragment to by the site of the was with and to the of the Dhcr7 and with DNA polymerase to In the was with and and to and and were by cloning by a The was by a of genes the in a site of and were by DNA The various were by the Dhcr7 promoter DNA as a with polymerase and to The mutated Dhcr7 promoter were as to The of the for the mutation of the of the Dhcr7 promoter are with the mutated in The reaction were with restriction and the The DNA sequence of was by and were in 1640 with 1 and in a at were to of were with of control Dhcr7 gene and of was of I DNA and were and to to the The were on for and were to a with as sterol starvation or as sterol for with the medium, were and were for activity for and the were with and in of 1 were and the to the of for the activity of were to the DNA were from Dhcr7 gene and with or of ADD1 protein in A 1 1 and of at for in a of the of the reaction, of a and was the with DNase I, the DNA were by a and on a sequencing with a sequencing ADD1 to including the was overexpressed in and from the as previously T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). hepatoma nuclear were to the of Res. 11: PubMed Scopus Google Scholar). were by the assay and at For were [γ-32P]ATP by were in A in the presence of 1 bovine serum of the was with of nuclear or of recombinant ADD1 in for at was on a with 1 and for For binding of were the of The DNA sequence of were as site of Dhcr7 site of Dhcr7 site of Dhcr7 box binding and We have previously reported (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) that were on a sterol starvation by in for both mRNA and the enzyme activity of Dhcr7 were vivo and To this of sterol-mediated Dhcr7 induction also be in in analysis was Dhcr7 mRNA from and that been in or in 1 mRNA from the Dhcr7 gene was specifically in However, this induction by sterol depletion was in Dhcr7 mRNA was in a were sterol starvation were with the cholesterogenic enzyme and reductase and In to this the Dhcr7 gene was by of with 1 sterol-mediated regulatory in the Dhcr7 gene. To this we isolated and the 5′-flanking region of this gene in A upstream fragment of the site from the rat genomic was and to regulatory The site was previously The sequence including its 5′-flanking and of is The proximal region (−179 to is and contains well factor binding sites are binding to Sp1 and binding to NF-Y and and an SRE1/E box binding to SREBP-1 or ADD1 regulatory sites are as motifs in genes involved in cholesterol M.K. J.N. J.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. P.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, G. G. R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. P.A. J. Lipid Res. 1998; Full Text Full Text PDF PubMed Google Scholar, R. S. Biochem. Res. 1998; PubMed Scopus (20) Google Scholar, L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was box the promoter region of the the presence of multiple sites as previously The site by the of is at an which is from the in Dhcr7 Dhcr7 gene to be by a This 5′-flanking region of the Dhcr7 promoter to also contains well characterized regulatory elements that may control of this gene. and and and binding are many and sites in the Dhcr7 promoter and of to sterol-mediated we have on the proximal region for analysis on Dhcr7 gene transcription. To the transcriptional regulatory in rat Dhcr7 gene and to this region is by sterols, a nested series of various of the 5′-flanking with a of the first of were by or restriction The for this was as to was by DNA of various the gene and for promoter activity a assay as were and following with sterols To activity in cells, we the with a sterol-regulated were expressed as by the of the sterol starvation or sterol For the analysis of the regulation of the Dhcr7 were due to as with that of not The promoter activity of the at was at for the from in sterol starvation The promoter activity was at the activity of in the promoter activity of the was sterol was the the presence of sterol-mediated regulatory elements such as the SRE1/E box at in the 5′-flanking region Deletion of and and caused significant in promoter activity or sterol-mediated of Dhcr7 promoter activity. However, of between and caused a reduction in both the promoter activity relative to the region and the sterol-mediated was a in the of sterol-mediated between This the presence of in this region and which to the of This is by the that the constructs, and a sterol-mediated to that the at and are important proximal for Dhcr7 promoter activity in response to sterols. of the fragment sterol-regulated which activity of the gene activity in the of sterol that of promoter suggesting the of promoter activity in this A of was also observed for were or we have on to showed the activity on sterol depletion in the 5′-flanking region of the rat Dhcr7 gene for analysis of sterol of promoter of genes of the Dhcr7 promoter in and were with one of the genes or and with a for an were in in the presence of were as a of activity of to the activity of and were to for in The relative activity of in was and in and are the from To which may be in of the Dhcr7 were for in the presence of in was to be the the oxysterols However, the of Dhcr7 inhibitor or reductase inhibitor to the depletion in of and in that been with The of or was to this are with of mRNA and of to the or of Dhcr7 promoter activity suggesting a role for In reductase a of Dhcr7 promoter in a of for inhibitors in To characterize binding to the Dhcr7 a rat of human was DNase I footprinting and were the DNA fragment and a binding to ADD1. in a recombinant ADD1 binds specifically to the proximal promoter specifically in a and its binding to the was by the of of a in the sequence elements such as SRE1/E box involved in sterol response of Dhcr7 was protected by overexpressed recombinant ADD1. A to was this protected region the SRE1/E box The binding of overexpressed ADD1 to the Dhcr7 promoter was also observed by The to SRE1/E binds to ADD1 in binding was in the presence of a of such as and sequence However, binding was not observed for any of in that the for binding to the overexpressed ADD1 are the between and which are with To any binding for Sp1 and NF-Y to the proximal region of Dhcr7 was the this region in the presence or of at binds to Sp1 and at −88 binds to NF-Y A that proximal to as binding sites for both Sp1 and and NF-Y binding to the and in the rat Dhcr7 was with nuclear of as The DNA to to were by 1 DNA DNA with as in the binding reaction a of the box or box as as in at the site or site that the upstream region of the Dhcr7 promoter is both necessary and sufficient for optimal sterol-regulated transcription. To functional between the identified cis-elements in the Dhcr7 which are to be involved in the sterol-mediated we a series of multiple mutations this region and the to a gene. was and for sterol-regulated by the or of mutations the region to relative to the site are and the of gene activity from or is reported as of the sites site and site caused in sterol-mediated response However, mutation of the site or the first site caused a reduction of sterol regulatory of the proximal site and the region of SRE1/E box or the of sites caused in sterol regulatory that and are to the sterol-mediated regulation of the Dhcr7 The in that the between and are functional of the rat Dhcr7 promoter and as the element genes J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar). of Dhcr7 gene by sterol starvation and in to the SRE1/E box such as Sp1 and may function in with SREBP-1 to the sterol-regulatory response of the Dhcr7 gene L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google X. M.R. X. C. J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). To Dhcr7 was by SREBP-1 T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). in the activity of activity in a to were with The activity of the Dhcr7 promoter by was the sequence and was suggesting that Sp1 binding to or NF-Y binding to is necessary for of the To the and potential mechanism for this activation, we analysis to which regulatory element or is critical for by SREBP-1 various in elements of the Dhcr7 promoter were with promoter for the SREBP-1 was from an the mutation of and SRE1/E box 13Tint G.S. Seller M. Hughes-Benzie R. Batta A.K. Shefer S. Genest D. Irons M. Elias E. Salen G. J. Lipid Res. 1995; 36: 89-95Abstract Full Text PDF PubMed Google and 14Alley T.L. Gray B.A. Lee S.H. Scherer S.W. Tsui L.C. Tint G.S. Williams C.A. Zori R. Wallace M.R. Am. J. Hum. Genet. 1995; 56: 1411-1416PubMed Google Scholar) in significant reduction and of Dhcr7 promoter activity as with the However, mutation of in a significant reduction of the was This is also with the in B. A of in which was by with that SREBP-activated Dhcr7 promoter the element in However, site for Sp1 may to the Dhcr7 promoter activity in response to of multiple regulatory elements of Dhcr7 promoter in SREBP-activated was with the were in 1640 with with are of the from SREBP-1 relative to control are The of this was to multiple motifs in the proximal promoter region are involved in sterol-mediated regulation of the Dhcr7 gene. of and mutations in the Dhcr7 we have identified and elements involved in the sterol-mediated which of Sp1 NF-Y and SREBP-1 SRE1/E of the Dhcr7 promoter with to sterol-mediated gene was important for Dhcr7 is involved in the reaction of cholesterol control of gene by the was mutations of Dhcr7 genes were to be with regulation of its gene be critical for The in this at with to Dhcr7 gene rat Dhcr7 gene be by sterols including the cholesterol In particular, induction of Dhcr7 promoter by sterol starvation was by this sterol of the presence of cholesterol biosynthesis inhibitors sterol-mediated Dhcr7 gene regulation to of at and a box at in to the SRE1/E box sequence In the critical in box was to be of the mutation of this in caused in sterol of box as with in I or of box for example, and box of Dhcr7 are in a are analysis also that the rat Dhcr7 promoter region contains and a suggesting that has to of reductase and human in the cholesterol biosynthesis pathway and a of genes G. G. R.L. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Genet. Full Text PDF Scopus Google Scholar). the that of Dhcr7 by of or to a that the sterol the Dhcr7 as by in the of Dhcr7 promoter by sterol starvation multiple elements that are to the and the rat of which may have some in as well as in both NF-Y and Sp1 to in a in sterol-mediated the in activation, the latter acts in A may be the of and the gene which the SREBP-1 and co-regulator Sp1 for S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M.K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Recently, the by SREBP-1 and Sp1 has also been on an in vitro and Sp1 has been to with in vitro in the of DNA P.A. D. S. J. R. 1998; 12: PubMed Scopus Google Scholar, K. M. 1999; PubMed Scopus Google Scholar). is that an between NF-Y and Sp1 plays a role in the sterol regulation of rat Dhcr7 from of In Dhcr7 is the first enzyme in the post-lanosterol pathway a sterol-mediated gene coordinated control of cholesterol biosynthesis between in the pathway reductase and and in the pathway. We M. B. and for of the