1. The document discusses how a Chlamydomonas lipid that inhibits the Sonic hedgehog (Shh) signaling pathway was identified as ergosterol endoperoxide, a sterol with a similar structure to 7-dehydrocholesterol (7DHC) which accumulates in Smith-Lemli-Opitz syndrome (SLOS). 2. Ergosterol endoperoxide was purified from Chlamydomonas extracts and shown to inhibit Shh signaling at the level of Smoothened (Smo). 3. Depleting cells of 7DHC using methyl-β-cyclodextrin enhanced Shh activation by a pathway agonist,

1. A steroid impact on Sonic hedgehog signaling
Navdar Sever1
, Randall K. Mann1
, Libin Xu2
, William J. Snell3
, Carmen I. Hernandez-Lara3
, Ned A. Porter2
, and Philip A. Beachy1
1
Institute for Stem Cell and Regenerative Medicine, Departments of Biochemistry and Developmental Biology, and HHMI, Stanford University
2
Department of Chemistry, Vanderbilt University
3
Department of Cell Biology, University of Texas Southwestern Medical School at Dallas
2. Purification of Chlamydomonas lipid that
inhibits Shh pathway
3. Chlamydomonas lipid was identified as the
endoperoxide derivative of ergosterol, which
has an identical ring structure to 7DHC, a
sterol that accumulates in SLOS
Many of the developmental malformations in Smith-
Lemli-Opitz syndrome (SLOS) occur in tissues and
structures whose embryonic patterning depends on
signaling by the Sonic hedgehog (Shh) family of secreted
proteins1
. Binding of Shh to the transporter-like protein
Patched (Ptc) relieves the repression of the 7TM protein
Smoothened (Smo) and leads to its accumulation in
primary cilia; response to the Shh signal is compromised
in mutant cells from mouse models of SLOS and in normal
cells pharmacologically depleted of sterols. Moreover,
the plant-derived steroidal alkaloid cyclopamine can
directly bind to and inhibit Smo, phenocopying mutations
in Shh.
These and several other observations have led to a model
where Ptc acts by affecting the availability of a lipidic
small molecule modulator of Smo ciliary localization and
activity. Starting with lipids from the ciliated unicellular
organism, Chlamydomonas, a rich source of Hh pathway-
modulating lipids, we have identified an inhibitory sterol
with mammalian analogues that accumulate to especially
high levels in SLOS.
We thank P. Aronov for assistance
with LC-MS, and C. Liu for
assistance with NMR. This research
was supported in part by funding
from NIH. N.S. is a Siebel Scholar.
P.A.B. is an HHMI investigator.
1. Chlamydomonas extract inhibits Shh
pathway at the level of Smo
4. 7-DHC derived oxysterols inhibit Shh
pathway
Teratogenic effects of steroidal compounds
and inhibition of sterol metabolism
Binns W et al. 1964
cyclopamine jervine
Maternal ingestion
of V. californicum
Roux C et al. 2000
AY9944-
treated rat fetus
cholesterol7-DHC
DHCR7
American Journal of Medical Genetics Part C:
Seminars in Medical Genetics 2012
SLOS
cholesterol7-DHC
DHCR7
Genetic mutation
HO
Primary cilia are found in many organisms
and required for Shh signaling
R Rohatgi, and M P Scott Nature Cell Biol 2007;9:1005-1009
Chlamydomonas
ba c
pCEFL pCEFL-
Smo
pCEFL-
SmoA1
Smo-/-
MEFs
0 5 10 15 20 25 30 35 40 45
Time (min)
0
20
40
60
80
100
RelativeAbundance
41.6
35.7
34.3
100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
m/z
0
20
40
60
80
100
RelativeAbundance
395.33
C28H43O
[M+H-H2O2]+
429.34
C28H45O3
[M+H]+
377.32
C28H41
[M+H-H2O2-H2O]+
874.69
C56H92O6N
[2M+NH4]+
gLC-MS base peak chromatogram
hMass spectrum
at 34.3 min
*
H2O-MeOH
(1:9)
MeOH-
DCM (9:1)
* * *
d e
1 3 5 7 9 1113151719 21 242628303234 36 384042--- MeOH
Phenyl HPLC
( ACN + 0.1% FA)fSilica (DCM:MeOH 95:5) C18 flash
7-DHC
7-DHCEP
ergosterol
EEP
O2, hν,
Rose bengal
HO
R
HO
R
O
O
R =
Chlamydomonas EEP
Synthetic EEP
+ShhN
HO
O
O
HO
HO
O
HO
O
O
OH
DHCEO 2a
+ShhN
HO
O
O
d
MCD
HO
HO
HOHOOH OH
5. Extraction of 7-DHC by methyl-β-
cyclodextrin (MCD) potentiates Smo agonist
(SAG)
a
αCD
CDβ
MCD
HPCD
+SAG1.5
αCD
CDβ
MCD
HPCD
b
+ShhN
c
αCD
CD
β
M
CD
HPCD
no
CD
Decreasing levels of cellular
sterols in mutant cells from
mouse models of SLOS as well
as normal cells were
previously shown to correlate
with diminished
responsiveness to the Shh
signal. Here we describe the
purification and identification
of ergosterol endoperoxide as
a novel Smo antagonist.
Similar sterols derived from
7-DHC have been observed in
SLOS models. We found that
depleting endogenous 7-DHC
with MCD treatment
enhances Shh activation by a
pathway agonist. Conversely,
exogenous addition of
DHCEO, a naturally occurring
B-ring oxysterol derived from
7-DHC, blocked Shh signaling.
Our current findings raise the
possibility that sterols that
accumulate in SLOS may
further compromise the
response to the Shh signal.