The Hedgehog (Hh) signal transduction pathway (BOX 1)
has conserved functions throughout metazoan develop-
ment, controlling patterning, growth and cell migration.
It is also expressed widely in adults, regulating tissue
homeostasis in the gastrointestinal tract1, nervous sys-
tem2, skin and blood3. Misregulation of Hh signalling is
at the root of several developmental defects4,5, and can
lead to tumorigenesis in adults6–9. Over the past 12 years,
accumulating evidence suggests that lipids might regu-
late this important signal-transduction pathway at many
levels. However, the identity of these lipids, their exact
function and the logic of their use remain mysterious.
The first clue that lipids might have a special function
in Hh signalling came from the identification of a unique
processing mechanism that results in the covalent link-
age of Hh to cholesterol. Hh is synthesized as a 45 kDa
pro-protein that undergoes autocatalytic cleavage by an
intein-like mechanism. The C-terminal domain of Hh,
which resembles a self-splicing intein domain, catalyses
its own removal and replacement by cholesterol. This
cleavage results in a 19 kDa N-terminal fragment that
is covalently linked at its C terminus to cholesterol10,11.
The Hh N-terminal fragment is also palmitoylated on
a Cys residue near its N terminus12 by the endoplasmic
reticulum (ER) transmembrane protein skinny hedge-
hog13–16 (SKI, also known as sightless, rasp or central
missing; FIG. 1a). Both lipid modifications are required
for full signalling activity in vivo12,13,17–20. Lipid-modified
Hh has a high affinity for cell membranes. Indeed, it is
targeted to raft-lipid microdomains in both vertebrate and
invertebrate cells21–23. The mechanisms that allow the
secretion, release and spread of such a molecule have
been the subject of intense interest.
This review considers the historical findings that
hint at important roles for lipids in Hh trafficking and
signalling, and will discuss at length work that has
begun to outline the specific functions of lipoproteins
and signalling cholesterol derivatives in regulating this
Secretion and trafficking of Hh
Hh secretion. Dispatched is required for Hh secretion24.
Dispatched contains 12 transmembrane domains and
is related to the resistance-nodulation division (RND)
family of bacterial proton-driven pumps25. Bacterial
proteins of the RND family use a proton gradient to
transport multiple small lipophilic molecules across the
membrane bilayer26. The two other metazoan members
of this family include the Hh receptor Patched, and the
protein encoded by the Niemann–Pick type C1 (NPC1)
disease gene, which promotes cholesterol efflux from
late endosomes. The functions of both Patched and
NPC1 will be discussed later in this review. Members
of the RND family, including Patched, Dispatched and
NPC1, contain two related copies of a signature domain
with six transmembrane-spanning regions. Mutations
in Dispatched which disturb conserved residues that are
important for the function of bacterial transporters, also
prevent Hh release25, consistent with the hypothesis that
Dispatched can transport a small molecule across the
A fragment of the signature RND domain, called a
sterol-sensing domain, is also shared with other proteins
that are involved in sterol metabolism. The sterol-sensing
domain of HMGCoA reductase (the rate-limiting enzyme
in cholesterol biosynthesis) regulates its stability in
Max Planck Institute of
Molecular Cell Biology
01307 Dresden, Germany.
(Protein introns). Enzymatically
active domains that splice
themselves out of the
precursor protein, ligating the
protein fragments (the
‘exteins’) on either side. Inteins
can also ligate exteins in trans
as well as in cis, and this has
been exploited to modify
proteins in vitro.
Small phase-separated regions
of the cell membranes that are
rich in cholesterol and
sphingolipids. Their affinity for
specific transmembrane and
lipid-linked proteins, and their
ability to cluster to form higher
order structures have been
proposed to be important for
the regulation of signalling and
Multiple roles for lipids in the
Hedgehog signalling pathway
Abstract | The identification of endogenous sterol derivatives that modulate the Hedgehog
(Hh) signalling pathway has begun to suggest testable hypotheses for the cellular biological
functions of Patched, and for the lipoprotein association of Hh. Progress in the field of
intracellular sterol trafficking has emphasized how tightly the distribution of intracellular
sterol is controlled, and suggests that the synthesis of sterol derivatives can be influenced by
specific sterol-delivery pathways. The combination of this field with Hh studies will rapidly
give us a more sophisticated understanding of both the Hh signal-transduction pathway and
the cell biology of sterol metabolism.
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response to cholesterol. The sterol-sensing domain
of SCAP (sterol-regulatory-element-binding protein
(SREBP)) cleavage-activating protein) responds to
cholesterol levels by altering membrane trafficking and
the cleavage of the membrane-associated transcrip-
tion factor SREBP, which regulates the transcription of
genes that are involved in sterol metabolism27. whether
Dispatched itself responds to sterol levels is not known,
and its precise function in Hh release has not yet been
Hh spreading. How can a lipid-modified protein spread
so widely through tissue? Signalling-competent Hh can
be isolated from tissue-culture-cell supernatants in high-
molecular-weight multimer complexes between 158 and
4,000 kDa (from 6 to at least 160 times the monomer
size). Monomer-sized complexes do not signal as effi-
ciently. The formation of the highest molecular-weight
complexes depends on both palmitoylation of the
N terminus and cholesterol modification of the C term-
inus (FIG. 1b). whether these complexes contain other
proteins besides Hh is unknown18,19,23,28,29.
In Drosophila melanogaster larvae, lipoprotein parti-
cles might help mobilize Hh. Biochemical fractionation
of imaginal discs from D. melanogaster larvae shows
that, although most lipid-modified Hh will form pel-
lets with cell membranes, Hh molecules that remain
in the supernatant are almost entirely associated with
lipoprotein particles30. It will be interesting to determine
whether the cholesterol-dependent Hh multimers that
are secreted by tissue-culture cells might reflect the
association of Hh with serum-derived lipoproteins, or
whether multimer formation is a completely distinct
mechanism for Hh release.
Lipoproteins comprise a phospholipid monolayer
that surrounds a core of esterified cholesterol and
triglyceride (FIG. 1c). Insect lipoproteins, called lipo-
phorins, are scaffolded by Apolipophorin31–33 — a
protein that is evolutionarily related to the vertebrate
apolipoprotein B (REF. 34). Lipid modifications, such
as the addition of cholesterol, palmitate and glycosyl
phosphatidylinositol (GPI), that target proteins to the
exoplasmic face of the plasma membrane should fit
equally well into the outer phospholipid monolayer
of lipoproteins (FIG. 1c). Indeed, D. melanogaster lipo-
phorin particles also bind to the morphogen molecule
wingless, which is palmitoylated twice, and to several
RNA interference-mediated knockdown of
lipophorin restricts the range of Hh signalling in
D. melanogaster imaginal discs: it reduces the activation
of long-range target genes but leaves short-range target-
gene activation unaffected. In lipophorin-deficient discs,
Hh protein is released but accumulates to an abnorm-
ally high level in short-range cells30. Thus, the role of
lipophorin in Hh release is not clear. It is possible that
incomplete knockdown of lipophorin produces only an
intermediate phenotype. Alternatively, two mechanisms
for Hh release might operate in wing discs: a long-range
mechanism that depends on lipophorin and a short-
range mechanism that is lipophorin-independent.
whether any of the mammalian Hh proteins bind to
low-density lipoprotein (LDL) or high-density lipo-
protein (HDL)-type particles is unknown, although this
would be interesting to investigate.
Cholesterol modification clearly has an important
influence on Hh trafficking. The 19 kDa N-terminal
Hh domain can be artificially generated in the absence
of cholesterol modification by the simple expedient of
stop codon insertion or C-terminal domain dele-
tions11. This altered protein, termed HhN, is secreted
in a Dispatched-independent manner24, does not form
multimeric complexes18,19,23,28,29, and is distributed dif-
ferently in both producing and receiving cells17,18,29.
Although HhN has been reported to spread further,
it does not seem to signal as efficiently as cholesterol-
modified Hh10,18,24,29,36. The increased range of spread-
ing might suggest that the normal function of Hh
cholesterol modification is to promote the interaction
of Hh with the cell membrane — this interaction could
prevent dissociation from receiving tissue and delay Hh
movement. However, this simple explanation seems
unlikely because the anchors probably interact, either
with each other (when Hh is organized as micellar
multimers) or with the outer phospholipid monolayer
of a lipoprotein (FIG. 1).
Box 1 | The Hedghog signalling pathway
Hedgehog (Hh) signals by binding to Patched (PTC), a protein with 12 transmembrane
domains that, as with Dispatched, is related to the resistance-modulation division
family of bacterial proton-driven transporters. In the absence of Hh (see figure, left
panel), Patched represses the activity of the G-protein-coupled receptor Smoothened
(SMO). Repression is associated with reduced Smoothened stability and depletion of
the protein from either the plasma membrane (in Drosophila melanogaster) or from
the primary cilium (in vertebrates)66,102–104. In the absence of Smoothened signalling,
GLI family transcription factors (Cubitus interruptus (Ci) in D. melanogaster) are
multiply phosphorylated by protein kinase A (PKA), glycogen synthase kinase (GSK)
and casein kinase (CSK). Phosphorylation targets GLI (Ci) for processing by the
proteasome, converting the full-length transcriptional activator (CiAct) to a shorter
transcriptional repressor (CiR). When Patched-mediated repression is relieved by Hh
binding (see figure, right panel), Smoothened moves to the primary cilium or to the
plasma membrane. In D. melanogaster, it has been shown that Smoothened becomes
multiply phosphorylated105,106, which changes the conformation of its cytoplasmic tail
and promotes dimerization107. Activated Smoothened then inhibits phosphorylation of
GLI (Ci) proteins, thereby preventing degradation of the repressor form and allowing
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A craniofacial abnormality that
results from failure to fuse the
left and right palatal shelves at
the midline during
embryogenesis. It can be
caused by several
environmental and genetic
factors, including defects in
Sonic Hh signalling.
Interaction with heparan sulphate proteoglycans
(HSPGs) provides a likely explanation for the continu-
ing association of Hh micelles or Hh–lipoprotein com-
plexes with tissue. Lipid-modified Hh does not enter
tissue that cannot synthesize heparan sulphate37–39.
Recent work suggests that lipoproteins interact physi-
cally with HSPGs in D. melanogaster wing discs35. Hh
that has interacted with lipoproteins through lipid
anchors might therefore be restricted to tissue through
these lipoprotein–heparan sulphate interactions. This
would be consistent with the observation that only lipid-
modified Hh is dependent on HSPGs in order to asso-
ciate with tissue. Direct binding of Hh to HSPGs might
also provide tissue affinity. In this case, Hh multimeri-
zation might also promote HSPG binding by increasing
the local concentration of heparan sulphate-binding
sites on Hh.
The role of cholesterol in Hh signalling
As illustrated in BOX 1, Patched-mediated repression of
Smoothened signalling is essential to keep the Hh path-
way inactive in the absence of Hh ligand. Smoothened
repression in both D. melanogaster and in mammals
correlates with changes in its subcellular localization. In
flies, the stability and phosphorylation of Smoothened
are also altered by Patched-mediated repression.
However, the mechanism by which Patched alters
Smoothened trafficking and phosphorylation is not
understood in detail. It is unlikely that Patched directly
binds to Smoothened; sub-stoichiometric amounts of
Patched are sufficient to repress Smoothened function
in tissue-culture cells40.
Mammalian Smoothened signalling activity can
be regulated by binding to exogenous small lipophilic
molecules that are structurally related to sterols.
Cyclopamine, a steroidal alkaloid derived from the wild
California grass Veratrum californicum (FIG. 2), binds to
and inactivates mammalian Smoothened41–43. By con-
trast, various small molecule agonists compete with
cyclopamine to bind to Smoothened and activate signal-
ling44,45. These findings have led to the hypothesis that a
structurally related endogenous ligand for Smoothened
might exist, and that Patched might regulate its avail-
ability. However, none of these exogenous agonists or
antagonists has been shown to affect the activity of
D. melanogaster Smoothened. If there are lipophilic
ligands for D. melanogaster Smoothened, these might
differ from their mammalian counterparts.
The repressive activity of Patched is dependent on
the conserved residues that it shares with RND trans-
porters46. These transporters use a proton gradient to
flux small lipophilic molecules across the bilayer26,47.
The protein most closely related to Patched is encoded
by the NPC1 gene, another member of the RND trans-
porter family. NPC1 is required for the mobilization
of LDL cholesterol from late endosomes, and can act
as a proton-driven pump when expressed in bacteria.
Based on these analogies, a plausible hypothesis is that
Patched regulates the availability of a Smoothened
ligand (possibly sterol-related) by pumping it across
Insights from disease models. The importance of sterols
in regulating Smoothened signalling has also been
suggested by developmental defects that have been
observed in diseases of distal cholesterol biosynthe-
sis4,48,49. Smith–Lemli–Opitz syndrome (SLO) results
from mutations in 7-dehydrocholesterol (7-DHC)
reductase. Patients with SLO accumulate the immediate
cholesterol precursor 7-DHC (FIG. 2) and have reduced
levels of cholesterol. Fibroblasts that have been isolated
from patients with SLO exhibit reduced Hh-pathway
activity when deprived of exogenous LDL cholesterol4.
In principle, this might result from either reduced
cholesterol or from a negative effector that is derived
from 7-DHC, or both. However, lowered cholesterol
availability is likely to be at least in part responsible.
Removal of LDL cholesterol and the loss of endogenous
synthesis of cholesterol from wild-type fibroblasts also
inhibits Smoothened activity4. Surprisingly, knocking
down 7-DHC reductase in Xenopus activates rather
than inhibits Hh signalling50.
Recent studies of mouse mutants for both INSIG1
(insulin-induced gene-1) and INSIG2 have suggested
that the accumulation of cholesterol precursors might
also exert negative effects on the Hh pathway51. INSIG1
and INSIG2 proteins are transmembrane proteins of the
ER that restrict both the biosynthesis and uptake of chol-
esterol when cholesterol is abundant. Removing both
INSIG1 and INSIG2 increases cholesterol biosynthesis,
but also elevates the levels of cholesterol precursors such
as 7-DHC. Although these mice resemble SLO mice in
the elevation of 7-DHC (REF. 52), they have increased
rather than decreased levels of cholesterol51. INSIG1
INSIG2 double-knockout mice share some of the pheno-
typic abnormalities of SLO, including a cleft palate4,51.
Figure 1 | Proposed vehicles for Hedgehog release. a | Hedgehog (Hh; green) is
covalently linked to cholesterol (red) and palmitate (blue). b | Interaction of the lipid
moieties (such as cholesterol and palmitate) with each other drives the formation of Hh
multimers. c | Lipoproteins consist of an outer phospholipid monolayer (beige) that
surrounds a core of esterified cholesterol (EC) and triglyceride (TG). Hh binds to
lipoprotein particles through the insertion of lipid moieties (cholesterol and palmitate)
into the outer phospholipid monolayer.
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This seems to indicate that the build-up of precursors,
rather than reduced cholesterol levels, can cause some
of the defects seen in SLO. It will be interesting to spe-
cifically examine whether the Hh signalling pathway is
altered in these mice.
Vitamin D3 negatively regulates Hh signalling. Recently,
the identification of specific endogenous cholesterol
derivatives that influence mammalian Smoothened
activity has confirmed that the pathway can be both
positively and negatively influenced by signalling
sterols, perhaps at different steps. work from the
Pepplenbosch laboratory has identified an endogenous
sterol derivative, vitamin D3 (FIG. 2), that seems to
repress Smoothened signalling activity when applied
to C3H/10T1/2 fibroblasts or to developing zebrafish
embryos53. Transfection of Smoothened increased
vitamin D3 binding to yeast cells, which suggests
that vitamin D3 might bind to Smoothened. This study
also identified a Smoothened inhibitor of moderate
activity in C3H/10T1/2 fibroblast supernatants in which
Patched expression has been increased by transfection.
Production of this inhibitor is reduced by pravastatin
and rescued by mevalonate, which indicates that it
could be a sterol derivative. However, in vivo, vitamin
D3 must be derived from nutritional sources or by uv
irradiation of 7-DHC in the skin54. Nutritionally derived
vitamin D3 is carried in lipoproteins, whereas the pro-
duct of de novo synthesis in the skin travels through
the circulation in complexes with vitamin-D-binding
protein (vDBP)55. Thus, if vitamin D3 is an endogenous
Smoothened repressor, then it cannot be produced
de novo from cells that produce Patched. The fact that
endogenous sterol synthesis is required for the release
of the Patched-dependent Smoothened inhibitor in
culture precludes vitamin D3 as the Smoothened inhibi-
tor, but points to another related molecule produced
by these cells.
The twice-hydroxylated derivative of vitamin D3,
1,25-(OH)2-D3 (FIG. 2), is a steroid hormone with impor-
tant functions in maintaining calcium homeostasis, and
in the control of cell differentiation and proliferation in
many tissues56. It is not clear how hydroxylation
affects the activity of vitamin D3 with respect to the
Hh pathway. Nevertheless, it is interesting to note that
1,25-(OH)2-D3 inhibits the growth of many tumours
in which the Hh pathway is active57–60. Furthermore,
7-DHC — the immediate precursor of vitamin D3 — is
the cholesterol precursor that accumulates to high levels
in SLO syndrome. However, it remains controversial
whether elevated 7-DHC levels, as opposed to reduced
cholesterol levels, cause the defects seen in SLO.
Reduced cholesterol could inhibit the production of
positively acting cholesterol derivatives, as described
in the next section.
Oxysterols positively regulate Hh signalling. Cholesterol
can be hydroxylated at different positions by enzymes
located in the ER and in the mitochondria (FIG. 2). In con-
strast to vitamin D3, hydroxylated cholesterol derivatives
positively influence Hh signalling in two separate systems:
Figure 2 | The structures of sterol-related molecules. The structure of cholesterol is
shown (top left), with the numbering system used to indicate the different carbon atoms.
The enzyme that converts the immediate cholesterol precursor 7-dehydrocholesterol
(7-DHC; top right) to cholesterol is missing in patients with Smith–Lemli–Opitz
syndrome. The molecules that stimulate Hedgehog (Hh) signalling include oxysterol
derivatives of cholesterol and the Smoothened agonist SAG, a synthetic small molecule.
The structures of hydroxylated sterols (20-hydroxycholesterol (20-OHC); 22-OHC;
24-OHC; 25-OHC) and 1,25-(OH)2-D3 are shown. Molecules that inhibit Hh signalling
include the natural steroid hormone vitamin D3 and the plant steroidal alkaloid
cyclopamine. Whether hydroxylation at C1 and C25 influences the activity of vitamin D3
with respect to Hh signalling is unknown.
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mesenchymal stem cells and medulloblastoma (MB) cell
lines. The combination of 20- and 22-hydroxycholesterol
(20-OHC and 22-OHC; see FIG. 2) promotes the differ-
entiation of bone and inhibits the differentiation of adi-
pose tissue from mesenchymal stem cells61. Recent work
from the Beachy and Parhami laboratories has shown
that osteogenic differentiation in response to oxysterols
requires Smoothened signalling and that the addition
of 20-OHC and 22-OHC, but not 7-OHC (cholesterol),
increases the activation of Hh target genes62.
work from the Scott laboratory focused on the
role of oxysterols in Hh signalling in the MB cell line
PZp53Med (REF. 63). These cells do not express Patched
and therefore constitutively activate Hh target genes and
proliferation64. Blocking distal cholesterol biosynthesis
with triparanol, an inhibitor of 3β-hydroxysterol-∆24
reductase, inhibits both proliferation and transcription
of a Hh signalling reporter construct in PZp53Med cells.
Signal transduction and proliferation are restored by
adding cholesterol, but addition of specific oxysterols
is at least tenfold more potent. 20-OHC and 22-OHC
are effective at activating Hh reporters, 24-OHC and
25-OHC also have significant activity, whereas 7-OHC
does not affect the activation of Hh reporters. Oxysterols
increase Hh signalling in MB cells and mesenchymal
How do oxysterols function to stimulate Hh sig-
nalling? Oxysterols, unlike the Smoothened agonist
SAG (FIG. 2), do not compete with cyclopamine to bind
to Smoothened. Cyclopamine addition is sufficient to
block oxysterol-mediated activation62. Oxysterols are
unlikely to displace a negative regulatory ligand from
Smoothened, but rather function upstream of this step.
One possibility might be that oxysterols inhibit Patched
activity. However, if oxysterols acted only by inhibiting
Patched then they should not further increase constitu-
tive signalling in Patched-deficient cells. This depends on
the cell type or experimental conditions. Although addi-
tion of oxysterol does not increase Hh reporter activity
in Ptc1–/–-mouse embryonic fibroblasts62, it does result in
a threefold increase in Hh signalling in PZp53Med cells63,
which do not express Patched64.
It is interesting to note that these experiments were
done using different amounts of added serum (0.5%
in REF. 64 compared with 5% in REF. 62). Serum is a
source of lipoproteins, and lipoprotein internalization
increases oxysterol synthesis65. It is possible that dif-
ferent basal rates of endogenous oxysterol production
determine whether the addition of exogenous oxy-
sterols further stimulates Hh signalling in the absence
of Patched. It seems that oxysterols probably do not
control Smoothened activity by inhibiting Patched,
but rather act at a subsequent step. Consistent with this
hypothesis, addition of oxysterols mimics the effects of
Patched loss-of-function on Smoothened trafficking
without affecting Patched levels or localization66.
How might oxysterols modulate Smoothened local-
ization? Oxysterols are potent ligands that bind to and
regulate different proteins. They bind to the liver X
receptor (LXR), a nuclear receptor that regulates the
transcription of genes that are involved in cholesterol
homeostasis67. However, LXR agonists do not activate
Hh target genes in mesenchymal stem cells62, which
suggests that oxysterols do not function through LXR
to modulate Hh signalling. Oxysterols also bind to a
family of conserved oxysterol-binding protein (OSBP)-
related proteins (ORPs), some of which regulate mem-
brane trafficking68,69. It would be interesting to find
out whether any of these proteins might influence
Smoothened localization. Oxysterol binding to INSIG
promotes the interaction of INSIG with SCAP and
retention of the SCAP–SREBP complexes in the ER70.
Do oxysterols regulate Smoothened trafficking by
binding to an analogous Smoothened-interacting pro-
tein? Investigating the mechanism of oxysterol action
should provide a new entry point to understanding
Smoothened trafficking and activation.
Sterol trafficking and the Hh pathway
The ability of specific sterol derivatives to positively or
negatively regulate Smoothened signalling raises the
interesting question of how the uptake, synthesis and
intracellular distribution of these molecules is control-
led and whether Patched might somehow influence
these processes. This section discusses how oxysterols
and vitamin D3 are produced and distributed (also
reviewed in REFS 27,56,71,72) and attempts to relate this
information to the Hh pathway.
Intracellular transport of sterols and their derivatives.
Cellular cholesterol is derived from endogenous synthe-
sis in the ER and from internalized lipoproteins. Cells
actively develop and maintain significantly different
cholesterol concentrations in different membrane com-
partments. Many vesicular and non-vesicular mecha-
nisms exist to actively transport cholesterol between
different membrane compartments and regulate its dis-
tribution within the cell. The first requirement for the
synthesis of oxysterols is the delivery of cholesterol to
intracellular sites where relevant enzymes are localized.
The enzymes that produce oxysterols have been isolated
from the ER and Golgi (cholesterol-25-hydroxylase and
cholesterol-24-hydroxylase) and from the mitochondria
(cholesterol-27-hydroxylase)73 (FIG. 3a). The site of 20-
OHC and 22-OHC production is unknown and the
enzymes have yet to be cloned, although consistent
enzymatic activity has been detected in mitochondria
from adrenal cells.
Recent data suggest that biosynthesis of some sterol
derivatives is dependent on specific intracellular cho-
lesterol trafficking machineries. High-level synthesis of
25-OHC and 27-OHC is dependent on NPC1-dependent
cholesterol mobilization65. NPC1 activity in late endo-
somes is necessary to mobilize the cholesterol that is
derived from internalized LDL and from the plasma
membrane to other cellular compartments. NPC1
expression in bacteria can promote influx of fatty acids
and acriflavine, but not cholesterol74. whether choles-
terol is the real substrate of NPC1 in vertebrate cells is
unclear; it is possible that NPC1 promotes the efflux
of cholesterol indirectly, by changing the concentration of
other lipids in the endosomes.
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When starved of nutrients, the
nematode worm C. elegans
progresses to a unique larval
form called a dauer. Dauers are
long-lived and resistant to
environmental insults but do
not reproduce. When
conditions are favourable,
dauer larvae re-enter
The molecules that subsequently deliver endosome-
derived cholesterol to different subcellular locations, such
as mitochondria, the plasma membrane or the ER, are
not completely clear. The steroidogenic acute regulatory
protein (START) domain protein MLN64, which binds
to cholesterol and is also present on NPC1-containing
endosomes, has been suggested to have a role in transfer-
ring cholesterol from the endosomal membrane to other
membranes75. OSBP and other ORPs have also been
suggested as candidates for the transport of cholesterol
to other membrane compartments76. In NPC1-deficient
cells, cholesterol accumulates in late endosomes and its
delivery to the plasma membrane or mitochondrial and
ER compartments is reduced77–79.
under normal conditions, cholesterol that is derived
from LDL uptake is delivered to sites of oxysterol syn-
thesis in the ER and mitochondria. Oxysterols then
function through LXR to reduce cellular cholesterol
levels by activating the transcription of genes that are
involved in cholesterol efflux80,81. NPC1-mutant cells
do not synthesize oxysterols in response to LDL uptake,
and therefore accumulate abnormally high levels of total
cellular cholesterol — this phenotype can be rescued by
the addition of oxysterols65. whether a particular NPC1-
mutant protein can promote cholesterol trafficking to
sites of oxysterol synthesis is uncorrelated with its ability
to deliver cholesterol to sites of cholesterol esterification
in the ER65. This suggests that different NPC1-dependent
mechanisms might promote cholesterol trafficking to
these different locations (FIG. 3a).
NPC1-dependent cholesterol transport to mito-
chondria is also essential for the generation of a sub-
set of steroid hormones (FIG. 3a). In D. melanogaster,
NPC1 is required for synthesis of the steroid hormone
ecdysone82,83, which regulates the transition between
different developmental stages in this organism. In
Caenorhabditis elegans, NPC1 homologues are needed
for synthesis of sterol-derived hormones that regulate
dauer formation84. In vertebrates, synthesis of neuro-
steroids is reduced in NPC1 mutants85, but the levels
of circulating steroid hormones such as testosterone,
progesterone and corticosterone are not affected86.
The steroid hormones corticosterone and aldoster-
one rely on an NPC1-independent mechanism for the
delivery of their cholesterol precursor. The main source
of components for the synthesis of these hormones is the
selective uptake of cholesterol esters from HDL through
the scavenger receptor B1 (SRB1)87,88. Cholesterol esters
absorbed by the selective uptake pathway are delivered
to lipid droplets, and the release of cholesterol for trans-
port to mitochondria depends on de-esterification by
hormone-inducible lipase (HLIP)89–91.
How might Patched and Hh influence the availability
of regulatory sterol derivatives? In principle, Patched and
Hh might regulate production, intracellular trafficking or
turnover of these molecules. The observation that specific
lipoprotein-dependent delivery pathways, such as selec-
tive uptake, can influence the efficiency with which sterol
derivatives are synthesized suggests an intriguing possible
role for the association of Hh with lipoproteins: could
the presence of Hh on lipoproteins influence the use of
sterols or sterol derivatives present in these particles?
The mechanisms that support hydroxylation of
25-OH-D3 provide an illustration of the importance
of facilitated delivery of sterol to specific subcellular loca-
tions (FIG. 3b). vitamin D3 itself is a prohormone; the active
form that maintains calcium homeostasis, 1,25-(OH)2-
D3, is formed by hydroxylation — first at C25 in the liver,
and subsequently at C1 in various tissues, including the
kidney56. Hydroxylation of 25-OH-D3 to form 1,25-
(OH)2-D3 is enhanced by specific cellular mechanisms that
internalize 25-OH-D3 and deliver it to the 1-hydroxylase.
Circulating 25-OH-D3–vDBP complexes are internalized
by the lipoprotein receptor-related protein Megalin92. The
cytoplasmic tail of Megalin binds to two proteins with
affinity for 25-OH-D3: intracellular D-binding protein-1
(IDBP1) and IDBP3 (REF. 93). vDBP is degraded in endo-
somes and 25-OH-D3 moves across the endosomal mem-
brane by an unknown mechanism and binds to IDBP3,
which promotes delivery to the mitochondria where the
1-hydroxylase is located. IDBP1 increases delivery of 1,25-
(OH)2-D3 to the vitamin D receptor (vDR), a nuclear hor-
mone receptor that mediates many of the transcriptional
outputs of vitamin D3 signalling94–96.
Models for Patched-mediated Smoothened repression.
Patched could function similarly to NPC1 by promot-
ing the efflux of cholesterol, but it could also function
by directing the delivery of cholesterol to different
subcellular compartments — inhibiting the produc-
tion of positive regulators or increasing the synthesis
Figure 3 | intracellular transport of sterols and sterol derivatives. a | Niemann–Pick
type C1 (NPC1) protein promotes cholesterol (C) trafficking from late endosomes to sites
of oxysterol and esterified cholesterol (EC) synthesis on mitochondria and the
endoplasmic reticulum (ER). Blue circles depict lipoproteins. b | 25-OH-D3 is internalized
with vitamin-D-binding protein (VDBP) by Megalin. Binding of intracellular D-binding
protein-3 (IDBP3) to 25-OH-D3 promotes transport to mitochondria, where 25-OH-D3 is
hydroxylated to 1,25-(OH)2-D3. IDBP1 promotes the delivery of 1,25-(OH)2-D3 to its
receptor (VDR) in the nucleus.
442 | juNE 2008 | vOLuME 9
© 2008 Nature Publishing Group
Nature Reviews | Molecular Cell Biology
The many different proteins of
the ABC transporter family use
ATP to transport small
molecules across the plasma
membrane. ABCA1 has an
important function in reverse
cholesterol transport —
effluxing cellular cholesterol to
HDL particles. ABCG5 and G8
efflux cholesterol and plant
sterols out of gut cells and into
the gut lumen, regulating
dietary sterol uptake.
of an inhibitor (FIG. 4). Hh signalling is reduced in tissue-
culture cells by drugs, such as progesterone and u18666A,
that mimic the NPC1 loss-of-function phenotype and
cause cholesterol accumulation in late endosomes97.
Although NPC1 does not seem to influence Hh signalling
in vertebrates, these data suggest an important function
for intracellular cholesterol trafficking in regulating Hh
signalling. Patched is found in the same late endosomal
compartment as its relative NPC1 — indeed, these two
proteins co-localize in tissue-culture cells97. It would be
interesting to compare the accumulation of different sterol
metabolites in cells with different levels of Patched activity.
An alternative possibility is that Patched might translocate
the small lipophilic inhibitor itself (FIG. 4b).
If vitamin D3 is a physiological regulator of Hh sig-
nalling in vivo, it must be acquired exogenously. Known
routes of acquisition include the lipoprotein-mediated
delivery of nutritional vitamin D3 and Megalin-medi-
ated internalization of vitamin D3 in complex with
vDBP55. Hh can be internalized by Megalin98,99, and
the holoprosencephaly observed in Megalin mutants
suggests that Hh signalling might be defective100. It
would be interesting to investigate whether Hh might
antagonize uptake of vitamin D3 by Megalin. Although
it seems likely that vitamin D3, as with cholesterol,
would require some cellular machinery to promote its
efflux from endosomes, no such machinery has yet been
identified. It would be interesting to see whether Patched
might have such a function (FIG. 4c). Also, in the case of
lipoprotein-mediated vitamin D3 delivery, the presence
or absence of Hh on lipoproteins might have the capacity
to regulate their use.
The levels of intracellular oxysterols will also depend
on the rate at which they are metabolized or removed
from cells. How does this normally occur? Esterification
is one method for the sequestration of oxysterols in
lipid droplets. Cells can also actively remove oxysterols
through ATP-binding cassette (ABC) transporter-mediated
efflux across the plasma membrane101. It is currently
unclear how or whether the mechanisms that promote
metabolism or efflux of oxysterols might be regulated.
Hydroxylation of 1,25-(OH)2-D3 at the C24 position
inactivates this molecule, at least with respect to signalling
through the vDR, thereby controlling intracellular levels
of the active ligand. Regulation of signalling sterol turn-
over is another possible control point with the potential
to be influenced by the Hh pathway.
Conclusions and future perspectives
Hh activates its signal transduction pathway by binding
to Patched, and preventing Patched from repressing the
G-protein-coupled receptor Smoothened. In the absence
of Hh, Patched-mediated Smoothened repression occurs
by an unknown mechanism that involves alterations
in Smoothened trafficking and stability. Recent work
has shown that Smoothened signalling activity can be
modulated by sterol derivatives. vitamin D3, and perhaps
another related sterol derivative, act negatively to repress
Smoothened activity. Specific oxysterol derivatives
activate the pathway upstream of Smoothened, possibly
by regulating Smoothened trafficking. Patched might
therefore function by regulating the availability of such
molecules, and its similarity to bacterial proton-driven
transporters and to NPC1 suggests that it might promote
the transmembrane transport of a small lipophilic mole-
cule. It will be interesting to examine whether Patched
has transporter activity, as NPC1 does, and to identify
Advances in our knowledge of the uptake and intra-
cellular trafficking of sterols and sterol derivatives
provides a framework which can be used to analyse
the biological function of Patched and Hh, and also to
suggest new avenues for research. Lipophilic molecules
that regulate Smoothened activity might be sought in
the lipoprotein particles with which Hh associates and
is internalized. Investigating the effects of Patched on the
trafficking of sterols and their derivatives might also yield
important insights into the Hh signalling pathway.
Figure 4 | Possible models for Patched-mediated Smoothened repression.
a | Patched (PTC) acts like other resistance-nodulation division family members as a
transporter to mobilize cholesterol (C) across the plasma membrane to allow specific
delivery to sites where a Smoothened (SMO) inhibitor (yellow star) can be synthesized
from it. Binding of the inhibitor to Smoothened represses its activity. Internalized
lipoproteins are shown as blue circles. b | A lipophilic compound that inhibits
Smoothened is already present in internalized lipoproteins. Patched acts as a transporter
and mobilizes this molecule across the membrane and into the cell, making it available to
repress Smoothened. c | Cellular cholesterol is efficiently transported to sites where
Smoothened agonists (purple star) such as 22-hydroxycholesterol (22-OHC) are
synthesized, but only in the absence of Patched activity. d | When Patched is present,
intracellular cholesterol transport is redirected, reducing the synthesis of 22-OHC and/
or promoting synthesis of a Smoothened inhibitor. ER, endoplasmic reticulum.
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I am grateful to T. Kurzchalia and the members of my
laboratory for critical comments on this work.
apolipoprotein B | 7-DHC reductase | Dispatched | IDBP1 |
Hedgehog | INSIG1 | INSIG2 | LXR | Megalin | MLN64 | NPC1 |
OSBP | Patched | SCAP | SKI | Smoothened | SRB1 | SREBP
Suzanne Eaton’s homepage:
all linkS are acTive in THe online Pdf
NATuRE REvIEwS | molecular cell biology
vOLuME 9 | juNE 2008 | 445
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