7-Dehydrocholesterol reductase activity is independent of cytochrome P450 reductase

Article abstract of DOI:10.1016/j.jsbmb.2011.06.011

7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450 reductase (POR) as its redox partner. To test this hypothes

Full text of DOI:10.1016/j.jsbmb.2011.06.011

Journal of Steroid Biochemistry & Molecular Biology 127 (2011) 435–438
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry and Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Short communication
7-Dehydrocholesterol reductase activity is independent
of cytochrome P450 reductase
Ling Zou, Li Li, Todd D. Porter∗
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0596, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
7-Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis. The enzyme
utilizes NADPH as a source of electrons and has been reported to require NADPH-cytochrome P450
reductase (POR) as its redox partner. To test this hypothesis, microsomes were prepared from the livers
of mice in which hepatic cytochrome P450 reductase expression was extinguished during maturation.
These microsomes contained negligible levels of POR but had 2.5-fold greater DHCR7 activity than did
microsomes from wild-type mice. Consistent with this greater activity, immunoblot analysis of DHCR7
expression indicated that DHCR7 protein levels were elevated 2-fold in POR-null microsomes. Addition of
POR to these microsomes provided no stimulation of DHCR7 activity, confirming the lack of a role for POR
in DHCR7 activity. Because the original observation that POR was necessary for DHCR7 activity was based,
in part, on antibody inhibition studies with POR antibody, the ability of an antibody to the full-length POR
protein to inhibit DHCR7 activity and cytochrome c reductase activity was tested; the antibody had no
effect on DHCR7 activity but decreased cytochrome c reductase activity (a POR-catalyzed reaction) by 50%.
Immunoblot analysis further demonstrated no cross-reactivity between POR and DHCR7 with antibodies
to either protein. We conclude that cytochrome P450 reductase is not involved in 7-dehydrocholesterol
reductase activity.
Received 10 May 2011
Received in revised form 24 June 2011
Accepted 25 June 2011
Keywords:
7-Dehydrocholesterol reductase
NADPH-cytochrome P450 reductase
Cholesterol synthesis
Smith-Lemli-Opitz Syndrome
Liver microsomes
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
cytochrome P450 reductase is more widely recognized for its role
in the reduction of cytochrome P450 in the oxidation of xenobiotics
7-Dehydrocholesterol reductase (DHCR7) is a 54 kDa microso-
mal enzyme that catalyzes the final step in cholesterol synthesis,
the reduction of the sterol C(7-8) double bond, using NADPH as an
electron source. Mutations in the DHCR7 gene that impair or inac-
tivate the enzyme result in Smith-Lemli-Opitz Syndrome (SLOS), a
disease characterized by developmental and neurological deficits
due to cholesterol deficiency [1]. Human and rat DHCR7 cDNAs
were cloned in 1998 and 1999, respectively [2,3], and the enzymes
were expressed in yeast and shown to be active in microsomal
preparations. A subsequent study with rat liver microsomes [4]
presented several lines of evidence that DHCR7 required NADPH-
cytochrome P450 reductase (POR) for activity; this included a
marked inhibition of DHCR7 activity in microsomes by addition
of antibody to POR, as well as a reconstitution of DHCR7 activ-
ity by addition of purified POR to protease-treated microsomes
and to partially purified DHCR7 preparations. Although NADPH-
and steroids, POR is required for cholesterol synthesis, where it is
the requisite electron donor to lanosterol demethylase (CYP51A1)
[5] and the principal, but not exclusive, electron donor to squalene
monooxygenase [6]. POR is ubiquitously expressed in mammalian
cells and also in yeast.
DHCR7 has not yet been purified to homogeneity or expressed in
a host lacking POR (e.g., Escherichia coli), so the requirement for POR
for activity has not been unequivocally established. Moreover, the
absence of a recognizable NADPH binding domain in this protein,
or the closely related reductase, ꢀ14-sterol reductase (TM7SF2)
has further clouded the issue. The recent availability of hepatic
POR-null mice [7] provided an opportunity to examine the require-
ment for this electron transfer protein in mammalian microsomes
that lack this reductase. Our results argue strongly that POR is not
required for DHCR7 activity.
2. Materials and methods
7-Dehydrocholesterol reductase activity was measured by fol-
lowing the conversion of ergosterol to brassicasterol as described
[8] in liver microsomes prepared from wild-type and hepatic
POR-null mice [7]. Incubations (500 ␮l) contained 100 ␮g of
microsomal protein and 60 ␮M ergosterol solubilized in 5% methyl-
Abbreviations:
DHCR7, 7-dehydrocholesterol reductase; POR, NADPH-
cytochrome P450 oxidoreductase; GC/MS, gas chromatography/mass spectrometry;
TM7SF2, ꢀ14-sterol reductase.
∗
Corresponding author. Tel.: +1 859 257 1137; fax: +1 859 257 7564.
E-mail address: tporter@uky.edu (T.D. Porter).
0960-0760/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2011.06.011

436
L. Zou et al. / Journal of Steroid Biochemistry & Molecular Biology 127 (2011) 435–438
␤-cyclodextrin (Sigma–Aldrich, St. Louis, MO) and were carried
out for 60 min at 37 ◦C. Lipids were saponified (1 M ethanolic
NaOH) for 1 h at 90 ◦C, extracted twice with hexane, deriva-
tized, and identified and quantified by GC/MS at the UK Mass
Spectrometry facility, focusing on mass ions 363 (ergosterol),
380 (brassicasterol), and 394 (stigmasterol, internal standard).
POR-mediated cytochrome c reductase activity was measured on
an HP8453 UV/Vis spectrophotometer at 550 nm at room tem-
perature. Sterols were obtained from the following suppliers:
7-dehydrocholesterol (Sigma–Aldrich); ergosterol, brassicasterol,
7␣-hydroxycholesterol, and stigmasterol (Steraloids, Inc., New-
port, RI). Cytochrome c was from Sigma–Aldrich; purified rat
cytochrome P450 reductase was purchased from ENZO Life Sci-
ences, Plymouth Meeting, PA.
Polyclonal antibodies to an N-terminal peptide (Santa
Cruz Biotechnology, Santa Cruz, CA) or an internal segment
(Sigma–Aldrich) of human DHCR7, to full-length rat POR (ENZO Life
Sciences), and a monoclonal antibody to ␤-actin (Sigma–Aldrich)
were used to detect these proteins on 8% SDS-polyacrylamide
gel immunoblots on nitrocellulose (Bio-Rad, Hercules, CA) using
fluorescent-labeled secondary antibodies (GE Healthcare, Pitts-
burgh, PA). Image intensity was quantified on a GE Typhoon 9400
Imager.
3. Results
As shown in Fig. 1, 7-dehydrocholesterol reductase activity,
measured as the conversion of ergosterol to brassicasterol [8], was
prominent in microsomes prepared from the livers of mice in which
cytochrome P450 reductase expression is extinguished during mat-
uration [7]. This activity was consistently 2–3-fold greater than
that found in microsomes from the corresponding control (wild-
type) mice when evaluated on the basis of microsomal protein
concentration, time-course, or substrate concentration. As noted
by Shefer et al. [8], the substrate ergosterol was inhibitory to activ-
ity, with maximal activity at ∼50 ␮M. DHCR7 protein content in
microsomes correlated with activity, and was approximately 2-fold
higher in POR-null microsomes; POR protein was undetectable in
these preparations.
To determine if POR was stimulatory to DHCR7 activity, puri-
fied rat POR was added to POR-null microsomes and DHCR7 and
cholesterol 7␣-hydroxylase activity was monitored by GC/MS. As
shown in Fig. 2, DHCR7 activity did not change in the presence
of 50 nM POR (inset). This addition of POR restored cholesterol
7␣-hydroxylase activity catalyzed by CYP7A1, a POR-dependent
microsomal enzyme, indicating that the added reductase was func-
tional in this system.
To address the possibility that antibody to POR is inhibitory
to DHCR7 (as found by Nishino and Ishibashi [4]), a commer-
cial polyclonal antibody to the full-length POR protein was added
to wild-type microsomes and DHCR7 activity was measured. As
shown in Fig. 3A, POR antibody had no effect on the conversion of
ergosterol to brassicasterol. This antibody was effective in inhibit-
ing a POR-catalyzed reaction, the reduction of cytochrome c, by
about 50%, as shown in Fig. 3B. POR antibody did not cross-react
with DHCR7, as evidenced by the immunoblot of Fig. 4, and poly-
clonal antibodies to an internal segment or the N-terminal segment
(data not shown) of DHCR7 did not detect POR in wild-type micro-
somes, arguing that these two enzymes do not share common
epitopes.
Fig. 1. Activity of DHCR7 in wild-type and hepatic POR-null mouse liver micro-
somes. Microsomal protein content (n = 2), time (n = 2), and substrate concentration
(n = 3) were varied and brassicasterol formation from ergosterol was monitored.
Standard conditions were 100 ␮g of microsomal protein and 60 ␮M ergosterol for
60 min at 37 ◦C. DHCR7 protein content was measured by immunoblotting, with
20 ␮g of microsomal protein per lane, n = 3. All data presented as mean range;
*significantly different from wild-type, p < 0.05, Student’s t-test.
4. Discussion
The present results argue strongly against a role for cytochrome
P450 reductase in 7-dehydrocholesterol reductase activity. The

L. Zou et al. / Journal of Steroid Biochemistry & Molecular Biology 127 (2011) 435–438
437
metabolism [7,11]. Although some POR can be detected immuno-
logically [6], in mature POR-null mouse liver it is less than 5% of
wild-type levels and may be derived from nonparenchymal cells.
In the microsomes used in these studies POR protein was not evi-
dent (Fig. 1) and no NADPH (POR)-dependent CYP2E1-catalyzed
activity could be detected [12], making it unlikely that any residual
cytochrome P450 reductase was responsible for the DHCR7 activity
found here. Indeed, the loss of POR expression severely impacts the
activity of all known POR-dependent enzymes [7,11].
The loss of POR expression also disables cholesterol and bile
acid synthesis, as both pathways are dependent upon microso-
mal cytochromes P450. While this lowers plasma cholesterol and
triglyceride levels it paradoxically increases hepatic triglyceride
levels [7,11], perhaps due at least in part to the loss of bile
acid-mediated stimulation of FXR [13]. Cholesterol synthesis is
blocked at CYP51 (lanosterol demethylase), resulting in the accu-
mulation of 24-dihydrolanosterol [6]. As might be expected, the
loss of cholesterol synthesis modestly upregulates gene expres-
sion in the cholesterolgenic pathway [9,10], although enzyme
protein and activity levels have not been systematically evalu-
ated in these livers. Indeed, we noted [6] that HMG-CoA reductase
and squalene monooxygenase enzyme levels were decreased by
half in POR-null mouse livers, which we attributed to secondary
post-translational down-regulation of these enzymes in response
to elevated levels of 24-dihydrolanosterol; lanosterol and 24-
dihydrolanosterol have been shown to enhance the degradation
of HMG-CoA reductase [14,15] but not squalene monooxygenase
[16]. In contrast to HMG-CoA reductase and squalene monooxyge-
nase, 7-dehydrocholesterolreductaseproteinlevelswereincreased
several-fold in POR-null mouse liver in the present study (Fig. 1),
consistent with the modest increase seen in mRNA level for
this enzyme [9,10]. Because DHCR7 is downstream of lanosterol
demethylase it is not surprising that it is not subject to post-
translational suppression by accumulating 24-dihydrolanosterol in
POR-null liver. It is clear from this and other recent studies [e.g.,
15,16] that the post-translational regulation of cholesterol synthe-
sis is considerably more complex than previously appreciated.
The study by Nishino and Ishibashi [4] noted that antibody to
POR inhibited DHCR7 activity. We were unable to replicate this
finding with a commercial polyclonal antibody to the full-length
reductase, although it was effective in inhibiting a marker activ-
ity for POR, the reduction of cytochrome c. The antibody used
by Nishino and Ishibashi was raised to the trypsin-released cat-
alytic fragment of rat POR and was inhibitory to the rat POR and
DHCR7 enzymes, whereas our antibody was raised to the com-
plete rat enzyme but was tested against mouse DHCR7 and POR.
It is possible that species differences may explain the different
inhibitory results obtained, as their antibody showed greater effec-
tiveness in inhibition of cytochrome c reductase activity than we
obtained with our antibody with mouse liver microsomes. We
Fig. 2. Addition of POR to POR-null microsomes does not stimulate DHCR7 activity.
Two micrograms of purified rat POR (0.026 nmol) was added to POR-null micro-
somes (50 nM final concentration) and DHCR7 and cholesterol 7␣-hydroxylase
activity was measured. The inset bar graph shows DHCR7 activity (mean range,
n = 2); the chromatograms depict the 456 m/e− ion abundance eluting at 24.7 min
characteristic for 7␣-hydroxycholesterol in POR-null and POR-supplemented POR-
null microsomes.
2-fold increase in DHCR7 activity in liver microsomes from hep-
atic POR-null mice matched the 2-fold increase in immunoreactive
DHCR7 protein, even while POR protein levels were reduced below
detectable levels. This increase in DHCR7 activity and protein is con-
sistent with the 1.3- to 1.9-fold increase in DHCR7 gene expression
that results from the loss of POR expression in this tissue [9,10]. The
lack of increase in DHCR7 activity when purified POR was added to
POR-null microsomes further argues that POR does not serve as an
alternative or supplemental electron donor to DHCR7.
The selective suppression of cytochrome P450 reductase expres-
sion in mouse liver results in a number of metabolic changes in this
tissue, most notably the loss of cytochrome P450-mediated drug
DHCR7 activity
POR activity
0.15
1.0
0.8
0.6
0.4
0.2
0.0
0.10
*
0.05
0.00
wild-type
+POR Ab
Untreated
+POR Ab
Fig. 3. Antibody to POR inhibits POR but not DHCR7. Polyclonal antibody to POR was added to liver microsomes from wild-type mice (1 ␮g/␮g microsomal protein) and
DHCR7 activity (left) and cytochrome c reduction (right) was measured, mean range, n = 2; *p < 0.05, Student’s t-test.

438
L. Zou et al. / Journal of Steroid Biochemistry & Molecular Biology 127 (2011) 435–438
Fig. 4. Antibodies to DHCR7 and POR do not cross-react. Liver microsomes (20 ␮g of protein) from two wild-type and two hepatic POR-null mice were fractionated by
SDS-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose for immunodetection with polyclonal antibodies to an internal segment of DHCR7 or full-length
POR. M, molecular weight markers at 95, 72, and 52 kDa.
considered the possibility that POR and DHCR7 share antigenic
epitopes, and that inhibition of DHCR7 by antibody to POR was
due to direct interaction with DHCR7. However, we could find no
evidence of cross-reaction between the POR antibody and DHCR7
protein in microsomes (Fig. 4). In addition, antibody to DHCR7
did not cross react with microsomal POR protein. It remains pos-
sible that other antibodies might recognize epitopes shared by
these two proteins, and might account for cross inhibition of activ-
ity. It also must be considered that the antibody used by Nishino
and Ishibashi recognized both proteins due to contamination of
the trypsin-released POR antigen with trypsin-released DHCR7
protein.
We are unable to provide an explanation for why partially
purified fractions of DHCR7 required the addition of purified,
detergent-solubilized POR for activity in the study by Nishino
and Ishibashi, but would note that the DHCR7 assay used by
this group followed 7-dehydrocholesterol disappearance by mon-
itoring absorbance at 280 nm by HPLC, whereas we monitored
brassicasterol formation from ergosterol by GC/MS. Substrate dis-
appearance is generally considered a less reliable means to measure
enzyme activity, and does not exclude other mechanisms by which
the substrate might be removed. We attempted to measure 7-
dehydrocholesterol disappearance by GC/MS in wild-type and
POR-null microsomes but were unable to detect significant changes
in substrate levels by this semi-quantitative technique; similarly,
it is not possible to measure cholesterol formation due to the high
levels of cholesterol in microsomes. It remains formally possible
that DHCR7 uses different redox mechanisms (e.g., direct binding
of NADPH vs electron transfer from POR) for different substrates
(i.e., 7-dehydrocholesterol vs ergosterol), but we are not aware of
any oxidoreductases that discriminate redox sources by substrate.
Ultimately this issue can best be resolved by reconstitution stud-
ies with the purified native or recombinant DHCR7 enzyme in a
defined lipid milieu.
References
[1] F.D. Porter, Smith-Lemli-Opitz syndrome: pathogenesis, diagnosis and man-
agement, Eur. J. Hum. Genet. 16 (2008) 535–541.
[2] F.F. Moebius, B.U. Fitzky, J.N. Lee, Y.K. Paik, H. Glossmann, Molecular cloning and
expression of the human delta7-sterol reductase, Proc. Natl. Acad. Sci. U.S.A. 95
(1998) 1899–1902.
[3] S.H. Bae, J.N. Lee, B.U. Fitzky, J. Seong, Y.K. Paik, Cholesterol biosynthesis from
lanosterol. Molecular cloning, tissue distribution, expression, chromosomal
localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-
Lemli-Opitz syndrome-related protein, J. Biol. Chem. 274 (1999) 14624–14631.
[4] H. Nishino, T. Ishibashi, Evidence for requirement of NADPH-cytochrome P450
oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system,
Arch. Biochem. Biophys. 374 (2000) 293–298.
[5] J. Trzaskos, S. Kawata, J.L. Gaylor, Microsomal enzymes of cholesterol biosyn-
thesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome
P-450 from hepatic microsomes, J. Biol. Chem. 261 (1986) 14651–14657.
[6] L. Li, T.D. Porter, Hepatic cytochrome P450 reductase-null mice reveal a second
microsomal reductase for squalene monooxygenase, Arch. Biochem. Biophys.
461 (2007) 76–84.
[7] J. Gu, Y. Weng, Q.Y. Zhang, H. Cui, M. Behr, L. Wu, W. Yang, L. Zhang, X.
Ding, Liver-specific deletion of the NADPH-cytochrome P450 reductase gene:
impact on plasma cholesterol homeostasis and the function and regulation of
microsomal cytochrome P450 and heme oxygenase, J. Biol. Chem. 278 (2003)
25895–25901.
[8] S. Shefer, G. Salen, A. Honda, A.K. Batta, L.B. Nguyen, G.S. Tint, Y.A. Ioannou,
R. Desnick, Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase:
substrate specificity, competitive and non-competitive inhibition, and phos-
phorylation/dephosphorylation, J. Lipid Res. 39 (1998) 2471–2476.
[9] X.J. Wang, M. Chamberlain, O. Vassieva, C.J. Henderson, C.R. Wolf, Relationship
between hepatic phenotype and changes in gene expression in cytochrome
P450 reductase (POR) null mice, Biochem. J. 388 (2005) 857–867.
[10] Y. Weng, C.C. DiRusso, A.A. Reilly, P.N. Black, X. Ding, Hepatic gene expression
changes in mouse models with liver-specific deletion or global suppression
of the NADPH-cytochrome P450 reductase gene. Mechanistic implications for
the regulation of microsomal cytochrome P450 and the fatty liver phenotype,
J. Biol. Chem. 280 (2005) 31686–31698.
[11] C.J. Henderson, D.M. Otto, D. Carrie, M.A. Magnuson, A.W. McLaren, I. Rosewell,
C.R. Wolf, Inactivation of the hepatic cytochrome P450 system by conditional
deletion of hepatic cytochrome P450 reductase, J. Biol. Chem. 278 (2003)
13480–13486.
[12] L. Li, T.D. Porter, Chlorzoxazone hydroxylation in microsomes and hepatocytes
from cytochrome P450 oxidoreductase-null mice, J. Biochem. Mol. Toxicol. 23
(2009) 357–363.
[13] T.D. Porter, S. Banerjee, E.I. Stolarczyk, L. Zou, Suppression of cytochrome P450
reductase (POR). Expression in hepatoma cells replicates the hepatic lipidosis
observed in hepatic POR-null mice, Drug Metab. Dispos. 39 (2011) 966–973.
[14] Y. Lange, D.S. Ory, J. Ye, M.H. Lanier, F.F. Hsu, T.L. Steck, Effectors of rapid
homeostatic responses of endoplasmic reticulum cholesterol and 3-hydroxy-
3-methylglutaryl-CoA reductase, J. Biol. Chem. 283 (2008) 1445–1455.
[15] B.L. Song, N.B. Javitt, R.A. DeBose-Boyd, Insig-mediated degradation of HMG
CoA reductase stimulated by lanosterol, an intermediate in the synthesis of
cholesterol, Cell Metab. 1 (2005) 179–189.
Acknowledgments
We thank Drs. Xinxin Ding and Jun Gu for providing the liver
tissue used in these studies; generation and maintenance of this
mouse line was supported in part by NIH Grant ES-07462. Ms.
Lauren Smith carried out the initial studies, and Dr. Jack Good-
man provided advice and guidance on the mass spectrometric
analysis.
[16] S. Gill, J. Stevenson, I. Kristiana, A.J. Brown, Cholesterol-dependent degradation
of squalene monooxygenase, a control point in cholesterol synthesis beyond
HMG-CoA reductase, Cell Metab. 13 (2011) 260–273.