Smoothened
SMO belongs to the Class F (Frizzled, FZD) of GPCRs and of the 11
members of this class 10 are FZD paralogues, the other is SMO
(Kozielewicz et al., 2020). SMO is a seven-pass transmembrane (7TM)
protein, essential to the Hh signalling pathway critical in normal
animal development, e.g. activation of the Hh pathway is required for
the differentiation of neural progenitor cells into motor neuron
progenitors, as well as pathological malignancies, including
medulloblastoma, the most common paediatric brain tumour.
The Hh signalling pathway is regulated in primary cilia where the
transporter protein Patched 1 (PTCH1) and SMO are co-located (Rohatgi,
Milenkovic, Corcoran & Scott, 2009). PTCH1 is the receptor for Hh
ligands, e.g. sonic hedgehog (SHH), and in the absence of ligand
inhibits SMO and the transduction of Hh signals across the plasma
membrane (Kong, Siebold & Rohatgi, 2019). When Hh ligands bind to PTCH1
inhibition is released and SMO transmits the Hh signal across the
membrane. PTCH1 is structurally related to the sterol transporting
protein NPC1 (Lange & Steck, 1998; Pfeffer, 2019), it has 12
transmembrane domains and two sterol binding cavities, and inactivation
of PTCH1 by Hh ligands is suggested to allow sterols to accumulate in
the cilia sufficiently to activate SMO (Deshpande et al., 2019). The
identity of the activating sterol is a point of considerable debate.
Current theories suggest at least two sterol binding sites in the SMO
protein. Based on the crystal structure of mouse SMO stabilised in an
active state, Deshpande et al suggest one binding site within the 7TM
pocket of SMO and a second deep within the extracellular CRD, with a
third pocket for the sterol-like antagonist cyclopamine (Deshpande et
al., 2019). Deshpande et al argued that both sterol-binding pockets are
likely occupied by cholesterol but acknowledged that oxysterols may be
alternative occupants of these pockets (Deshpande et al., 2019). Other
crystal structures based on inactive SMO failed to identify a 7TM sterol
and emphasised cholesterol binding to the CRD as the critical event
controlled by PTCH1 (Byrne et al., 2016; Huang et al., 2016). Deshpande
et al and Kinnebrew et al have both proposed biophysical models to
explain how abundant cholesterol can behave as a signalling molecule
(Deshpande et al., 2019; Kinnebrew et al., 2019); there is a precedent
for this in the regulation of cholesterol synthesis (and uptake) via the
SCAP/SREBP-2 pathway (Goldstein, DeBose-Boyd & Brown, 2006). Deshpande
et al proposed a hydrophobic tunnel between TM5 and TM6 of active SMO
that opens on the inner leaflet of the membrane bilayer, this may enable
sterols in this leaflet to activate SMO without energetically costly
membrane de-solvation (Deshpande et al., 2019). Activation of SMO via
the 7TM-binding sterol may lead to a displacement of TM6, which is
further stabilised by sterol binding to the CRD (Deshpande et al.,
2019). On the other hand, Kinnebrew et al introduced the concept of
“accessible cholesterol” in membrane and cilia in their model
(Kinnebrew et al., 2019). They defined three pools of cholesterol, a
fixed pool essential to maintain membrane integrity, a sphingomyelin
sequestered pool of low accessibility, and an available pool to interact
with proteins and be transported to the endoplasmic reticulum (Kinnebrew
et al., 2019). The idea of multiple pools of cholesterol was discussed
earlier by Radhakrishnan et al and in respect to brain by Dietschy and
Turley (Dietschy & Turley, 2004; Radhakrishnan, Anderson & McConnell,
2000). According to Kinnebrew et al’s model, the pool of accessible
cholesterol in cilia, the subcellular compartment where PTCH1 and SMO
are located together, is particularly low. PTCH1 functions in this
compartment to selectively transport accessible cholesterol from the
cilia to intracellular or extracellular receptors, precluding its
binding to the CRD and TM-binding site of SMO (Kinnebrew et al., 2019).
Inactivation of PTCH1 will lead to an increase in accessible cholesterol
in both leaflets of the ciliary membrane leading to SMO activation
through cholesterol binding through both sterol binding sites (Kinnebrew
et al., 2019). While Deshpande et al acknowledged that oxysterols may be
alternative ligands to these binding sites (Deshpande et al., 2019),
Kinnebrew et al argued against this idea based on a CRISPR screen
targeting lipid-related genes exploiting the NIH/3T3 cell line
(Kinnebrew et al., 2019). While they identified many genes of the
cholesterol biosynthesis pathway as positive regulators of the Hh
pathway, they failed to find oxysterol synthesising genes to positively
regulate Hh signalling (Kinnebrew et al., 2019). However, in the absence
of excess cholesterol it is uncertain whether these cells generate
oxysterols under the conditions employed. Interestingly, genes encoding
enzymes for sphingomyelin biosynthesis supressed Hh signalling,
promoting the concept of a sphingomyelin sequestered inaccessible pool
of cholesterol (Kinnebrew et al., 2019).
Despite the study of Kinnebrew et al (Kinnebrew et al., 2019) and the
crystal structures showing cholesterol bound to SMO (Byrne et al., 2016;
Deshpande et al., 2019; Huang et al., 2016), there is also good evidence
that oxysterols activate the Hh signalling pathway, perhaps in a
fine-tuning mode akin to their regulation of cholesterol biosynthesis
via the INSIG/SCAP/SREBP-2 pathway (Gill, Chow & Brown, 2008).
Synthetic oxysterols known to bind to SMO and activate Hh signalling
include 20S-HC, 24S-HC, 25-HC, 24S,25-EC, 24-oxocholesterol (24-OC),
7β,(25R)26-diHC, 25-hydroxy-7-oxocholesterol (25H,7O-C),
(25R)26-hydroxy-7-oxocholesterol ((25R)26H,7O-C) but not 20R-HC, 7α-HC,
7-OC nor 19-HC (Corcoran & Scott, 2006; Dwyer, Sever, Carlson, Nelson,
Beachy & Parhami, 2007; Kim, Meliton, Amantea, Hahn & Parhami, 2007;
Myers et al., 2013; Nachtergaele et al., 2012; Nachtergaele et al.,
2013; Nedelcu, Liu, Xu, Jao & Salic, 2013; Qi, Liu, Thompson, McDonald,
Zhang & Li, 2019; Raleigh et al., 2018). Conversely,
3β,5α-dihydroxycholest-7-en-6-one, an oxysterol derived from 7-DHC and
identified in brain of a mouse model of SLOS where 7-DHC is abundant (Xu
et al., 2012), binds to SMO and blocks Hh signalling (Sever et al.,
2016). Of the above oxysterols 24S,25-EC is abundant in embryonic mouse
brain, particularly the ventral midbrain, while 24S-HC and 25-HC are
also present during development, but at concentrations about one order
of magnitude lower than 24S,25-EC (Theofilopoulos et al., 2013; Wang et
al., 2009). In the new-born mouse, 24S,25-EC is still the most abundant
oxysterol (Meljon et al., 2012), but in the adult mouse 24S-HC is by far
the most abundant oxysterol and 20S-HC is also present but a low level
(Meljon et al., 2012; Yutuc et al., 2020). SLOS phenocopies dysregulated
Hh signalling (Cooper et al., 2003), however, at least in the new-born
mouse the pattern of SMO-activating oxysterols in brain is similar to
the WT (Meljon, Watson, Wang, Shackleton & Griffiths, 2013). Of the
other oxysterols suggested to activate the Hh pathway through binding to
SMO, we have identified 24-OC in brain, but using a derivatisation
method where the 24,25-epoxy group isomerises to the 24-oxo so cannot be
sure of its exact origin of 24-OC , while 7β,(25R)26-diHC, (25R)26H,7O-C
and 25H,7O-C are present in SLOS plasma but essentially absent from
control plasma (Meljon et al., 2012; Meljon, Watson, Wang, Shackleton &
Griffiths, 2013).
Similar to the situation with cholesterol, there appear to multiple
binding sites for oxysterols on SMO. Oxysterols can bind to the same CRD
pocket as cholesterol (Byrne et al., 2016) and also within a 7TM pocket
(Qi, Liu, Thompson, McDonald, Zhang & Li, 2019; Raleigh et al., 2018).
24S,25-EC appears to bind to and activate SMO via both pockets, while
20S-HC, 7β,(25R)26-diHC, (25R)26H,7O-C and 25H,7O-C act exclusively
through the CRD. As discussed above, PTCH1 acts to repress SMO by
removing agonist ligands from the plasma membrane in proximity to SMO
and exploiting this concept Qi et al were able to extract 24S,25-EC and
also 24-OC, 24S-HC and 25-HC from purified PTCH1 (Qi, Liu, Thompson,
McDonald, Zhang & Li, 2019). PTCH1 and SMO function together in cilia,
and Raleigh et al found 24S,25-EC, 24-OC and 7β,(25R)26-diHC to be
enriched in cilia purified from embryonic sea urchin (Raleigh et al.,
2018). 7β,(25R)26-diHC and (25R)26H,7O-C are metabolically linked by
HSD11B enzymes (Figure 4) (Beck et al., 2019a; Hult et al., 2004;
Schweizer, Zurcher, Balazs, Dick & Odermatt, 2004) and both HSD11B1 and
HSD11B2 are expressed in brain (Holmes & Seckl, 2006). Interestingly,
HSD11B2 is expressed during development brain development (Heine &
Rowitch, 2009; Holmes et al., 2006), and will catalyse the reduction of
the 7β-hydroxy group to a 7-oxo while HSD11B1 catalyses the reverse
oxidation (Beck et al., 2019a; Beck et al., 2019b; Schweizer, Zurcher,
Balazs, Dick & Odermatt, 2004). Importantly, Hsd11b2 is enriched
in mouse models of medulloblastoma and HSD11B2 is enriched in
Hh-pathway associated human medulloblastoma (Raleigh et al., 2018).
Raleigh et al proposed a mechanism involving HSD11B2 and CYP27A1 by
which Hh agonists 7β,(25R)26-diHC and (25R)26H,7O-C could be formed from
7β-HC and, remarkably, pharmacological inhibition of HSD11B2 reduced Hh
signalling and tumour growth in mouse medulloblastoma (Raleigh et al.,
2018). The contribution of HSD11B2 to oncogenic Hh signalling suggests
that oxysterols produced by this enzyme are required for high level
pathway activity. Our interpretation of this data is that while
(25R)26H,7O-C and 7β,(25R)26-diHC are both agonists to SMO,
(25R)26H,7O-C must be more potent. Whether this is a consequence of
differences in SMO binding or simply accessibility of ligand to receptor
is unclear. While 20S-HC and 24S,25-EC have been identified in brain and
are bona fide neuro-oxysterols, neither 7β,(25R)26-diHC nor
(25R)26H,7O-C have been identified in brain or medulloblastoma, however
their precursors 7-OC and 7β-HC have (Griffiths et al., 2019a; Meljon et
al., 2019). Importantly, 7O-C and 7β-HC can traverse the blood brain
barrier and providing an extracerebral source of precursors for
(25R)26-hydroxylation by CYP27A1 (Iuliano et al., 2015).