Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling

Abstract

Oxysterols bind the seven-transmembrane protein Smo (Smo) and potently activate vertebrate Hedgehog (Hh) signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, whether oxysterols are important for normal vertebrate Hh signaling and whether antagonizing oxysterols can inhibit the Hh pathway. We developed azasterols that block Hh signaling by binding the oxysterol-binding site of Smo. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smo and is completely separable from the site bound by other small-molecule modulators, located within the heptahelical bundle of Smo. Smo mutants in which oxysterol binding is abolished no longer respond to oxysterols and cannot be maximally activated by the Hh ligand. Our results show that oxysterol binding to vertebrate Smo is required for normal Hh signaling and that targeting the oxysterol-binding site is an effective strategy to inhibit Smo.

Figure 1

22-azacholesterol inhibits vertebrate Hh signaling (a) Structure of 22(S)-azacholesterol (22-NHC, 1) and 20(S)-hydroxycholesterol (20(S)-OHC). (b) Shh Light II cells were treated with various concentrations of Shh, in the presence of increasing amounts of 22-NHC, and Hh pathway activation was measured by luciferase assay. Error bars represent standard deviation (n=4 independent experiments). 22-NHC inhibits Hh pathway activation by Shh but does not significantly change the EC50 of Shh. (c) As in (b) but Hh signaling was activated by various concentrations of the 20-OHC analog, 20-OHC-Pent. 22-NHC inhibits Hh pathway activation by 20-OHC-Pent, without significantly changing the EC50. (d) As in (b) but Hh signaling was activated by various concentrations of SAG. 22-NHC does not inhibit Hh pathway activation by SAG but decreases the EC50 for SAG. (e) Smo−/− MEFs were rescued by stable expression of mSmo or the constitutively active mutant mSmoM2. Transcription of the Hh target gene, Gli1, was measured by Q-PCR in the absence or presence of 22-NHC (20 μM) or SANT1 (2 μM). Error bars indicate standard deviation (n=3 independent experiments). 22-NHC does not inhibit SmoM2. (f) Shh Light II cells were stimulated with Shh, in the presence of increasing amounts of 22-NHC, with the addition of cylopamine or cyclopamine-KAAD. Hh pathway activity was assayed as in (b) (g) As in (f) but with addition of SANT1, GDC0449 or itraconazole.

Figure 2

22-NHC binds Smo at the oxysterol-binding site (a) Schematic of 22-NHC and 20-OHC affinity matrices. 22-NHC and 20-OHC are covalently attached to agarose beads via a polyethylene glycol (PEG) linker. Control beads carry only the PEG linker. (b) 22-NHC beads were incubated with detergent extracts of 293T cells expressing mSmo-Cherry, in the presence of the indicated concentrations of competitor compounds. The beads were washed and bound protein was eluted, separated by SDS-PAGE and immunoblotted with anti-Cherry antibodies. A portion of the extract was analyzed in parallel, to show input. MSmo binds specifically to 22-NHC beads, and binding is not competed by other Smo inhibitors. (c) As in (b) but with the addition of the inactive oxysterol 7-OHC, or the active oxysterols, 20-OHC and 20-OHC-Pent. Binding of mSmo to 22-NHC beads is competed in a dose-dependent manner by 20-OHC and 20-OHC-Pent, but not by 7-OHC. (d) As in (b) but with the addition of 20(S)-OHC-Pent and 20(R)-OHC-Pent. Binding of mSmo to 22-NHC beads is competed by the active S diastereomer but not by the inactive R diastereomer. (e) As in (b) but using 20-OHC beads, in the presence of free 22-NHC or 20-OHC. Binding of mSmo to 20-OHC beads is competed in a dose-dependent manner by free 22-NHC and 20-OHC. The full immunoblots for this figure are shown in Supplementary figure 11.

Figure 3

Oxysterols and 22-NHC bind the extracellular cysteine-rich domain (CRD) of vertebrate Smo (a) Secreted HA-tagged mSmoCRD and detergent extracts of 293T cells expressing mSmo-Cherry were tested for binding to 22-NHC beads, in the presence of free 22-NHC. MSmoCRD binds 22-NHC beads, similar to mSmo. (b) MSmoCRD binding to 22-NHC beads was assayed as in (a), in the presence of 7-OHC, 20-OHC or 20-OHC-Pent. Only the active sterols 20-OHC and 20-OHC-Pent compete mSmoCRD binding to 22-NHC beads, while the inactive 7-OHC does not. (c) As in (b) but in the presence of the diastereomers 20(S)-OHC-Pent and 20(R)-OHC-Pent. Only the active S diastereomer competes mSmoCRD binding to 22-NHC beads. (d) MSmoCRD and mSmo binding to 20-OHC beads was assayed as in (c). Both mSmoCRD and mSmo bind 20-OHC beads and are competed by 20(S)-OHC-Pent, but not 20(R)-OHC-Pent. The binding affinities of mSmoCRD and mSmo to 20-OHC beads are similar. The full immunoblots for this figure are shown in Supplementary figure 11.

Figure 4

Structural requirements for oxysterol activation of Smo (a) Structures of 20-OHC analogs used in this study (compounds 7–13). All analogs except 20-OHC-Me (7) have a C-20 stereocenter, and pure S and R diastereomers were isolated and assayed separately. (b) Shh Light II cells were treated with varying concentrations of the oxysterols 20(S)-OHC, 20(S)-OHC-Pent, 20(S)-OHC-PentSat and 20(S)-OHC-Bu, followed by measuring Hh pathway activity by luciferase assay. The inactive oxysterol, 7-OHC, was used as negative control. Error bars represent standard deviation (n=4 independent experiments). (c) NIH-3T3 cells were incubated overnight with the indicated oxysterols (10 μM). Cells were then fixed and processed for immunofluorescence with rabbit anti-Smo antibodies (to detect endogenous Smo) and mouse anti-acetylated tubulin antibodies (to visualize primary cilia). SAG (1 μM) and 7-OHC (10 μM) were used as positive and negative control, respectively. The graph shows the percentage of Smo-positive cilia. At least 150 cilia were analyzed per condition. Error bars represent the sub-sampling standard deviation of the fraction of positive cilia (see Online Methods). (d) As in (c), but with box plots showing the fluorescence intensity of endogenous Smo at cilia. For each condition, the Smo signal was normalized to the intensity of the 20(S)-OHC treatment. The lower and upper bounds of each box represent the 25th and 75th percentile of the distribution of Smo intensity at cilia, while the horizontal line represents the median intensity across the entire population of cilia.

Figure 5

Oxysterol binding to Smo is required for high level Hh signaling (a) Alignment of a portion of CRDs of mouse Smo (mSmo), chicken Smo (gSmo), Xenopus Smo (xSmo), zebrafish Smo (zfSmo), Drosophila Smo (DrSmo) and mouse Fz8 (mFz8). The stretch indicated with red lines contains 5 residues that, in mFz8, contact the Xwnt8 palmityl moiety (Q71, F72, P74, L75 and I78). (b) Cherry-tagged mSmo, mSmoL112D, mSmoW113Y or mSmoS114Y were tested for binding to 22-NHC and 20-OHC beads, in the presence or absence of 20-OHC (100 μM). MSmoL112D and mSmoW113Y do not bind 22-NHC and 20-OHC beads. The full immunoblot is shown in Supplementary figure 11. (c) Smo−/− MEFs rescued with mSmo, mSmoL112D, mSmoW113Y or mSmoS114Y were incubated overnight with DMSO control, SAG (1 μM), 20-OHC (10 μM) or Shh. The graph shows the percentage of Smo-positive cilia. Error bars represent the sub-sampling standard deviation of the fraction of positive cilia (see Online Methods). Between 131–207 cilia were analyzed per condition. MSmoL112D and mSmoW113Y have a defective response to 20-OHC and Shh. (d) As in (c), but with box plots showing Smo fluorescence intensity at cilia. For each condition, the Smo signal was normalized to the intensity of the SAG treatment for the respective cell line. (e) As in (c), but cells were processed for Q-PCR, to measure Gli1 transcription. For each treatment, Gli1 levels were normalized to the level induced by SAG in the respective cell line. Error bars show standard deviation (n=3 independent experiments). MSmoL112D and mSmoW113Y do not respond to 20-OHC and have a reduced responsiveness to Shh.

Figure 6

Conserved and divergent aspects of Smo signaling (a) Smo−/− MEFs, stably expressing mSmo or the cilia-localized chimera mFz7^mSmoICD were incubated with control media, 20-OHC (10 μM), Shh, or wnt3a. The cells were processed for Q-PCR, to measure Gli1 transcription. Error bars show standard deviation (n=3 independent experiments). MFz7^mSmoICD did not rescue Hh signaling in Smo−/− cells, irrespective of the presence of Wnt3a. (b) As in (a), but with stable expression of the chimera rMAcChR^mSmoICD, which is recruited to cilia by treatment with acetylcholine (AcCh, 100 μM). RMAcChR^mSmoICDdoes not rescue Hh signaling, in the presence or absence of AcCh. (c) As in (a) but with stable expression of low levels of DrSmo^mSmoICD. DrSmo^mSmoICD is constitutively active, and is not further activated by 20-OHC (10 μM), Shh, or SAG (400 nM). In contrast, DrSmo is inactive in Smo−/− MEFs. (d) As in (c), but with addition of 20 μM forskolin (FSK), to block Hh signaling downstream of Smo. Signaling by both mSmo and DrSmo^mSmoICD is blocked by FSK. (e) Schematic of the mSmo protein and of the location of Sites A and B. For each site, activators are in blue, while inhibitors are in red. (f) Regulation of vertebrate Smo. Inhibition of Ptch by Shh results in Smo Site A activation; it is unclear if Site B is also activated by Shh. The oxysterol 20-OHC, which binds to Site B in the extracellular domain of Smo, potentiates Site A activation. Active Smo then signals to the cytoplasm.