Hedgehog pathway modulation by multiple lipid binding sites on the smoothened effector of signal response

Abstract

Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

Figure 1

Distinct structural determinants required for regulation of Smo by Ptch and by endogenous and exogenous ligands. (A) Schematic diagram of Hh pathway components showing multiple regulatory mechanisms that impinge on Smo, including Hh antagonism of Ptch-mediated suppression, direct modulation by cyclopamine and related small molecule agonists and antagonists, activation by oxysterols, and a requirement for cholesterol. (B) Luciferase activity in Smo^−/− mouse embryonic fibroblasts (MEFs) cotransfected with Gli-luciferase reporter plasmids and cDNAs encoding wild-type (wt) mSmo or the E522K or ΔCRD mutants following treatment with: control or ShhN conditioned medium (red and green bars, respectively); 20(S)-OHC (10 µM, blue bars); SAG (200 nM), purmorphamine (2.5 µM), or ShhN conditioned medium with KAAD-cyclopamine (KAADcyc, 300 nM) (black bars). Error bars represent standard deviations (n = 3 independent transfections per data point). A summary of the experiment is shown; complete data set is presented in Fig. S1A. (C) Ciliary localization of Myc-tagged wt or ΔCRD Smo constructs was examined in fixed NIH3T3 cells stained with antibodies against acetylated tubulin (red) or the Myc epitope (green); nuclei were visualized with DAPI (blue). Small panels to the right of selected images display shifted overlays of the acetylated tubulin and Myc channels. See Fig. S1B for quantification. (D) Quantification of wt Smo and Smo ΔCRD protein in transfected Smo^−/− MEFs over a range of expression levels using Cterminal fusions to Renilla luciferase. X-axis indicates % of Smo cDNA transfected into each well, while Y-axis indicates Renilla measurement, normalized to a control secreted alkaline phosphatase (SEAP) construct; replicates and error bars are as in panel (B). See also Figure S1 and Table S1.

Figure 2

The Smo CRD is required for modulation by oxysterols and indirectly affects Ptch1-mediated regulation. (A) Basal (non-ShhN-stimulated) Gli-luciferase activity in NIH3T3 cells transfected with i the indicated cDNA constructs in the absence (red) or presence (orange) of cotransfected Ptch1 (25% DNA for Smo wt and ΔCRD; 5% DNA for the constitutively active SmoA1 construct). (B) Gli-luciferase assay in Smo^−/− MEFs revealed that SmoA1 activity is induced by oxysterols (20(S)-OHC + 22(S)-OHC, 5 μM each) upon suppression of its basal activity by co-expression of Ptch1. (C) 8xGli-luciferase assay in Smo^−/− MEFs transfected with wt or ΔCRD Smo, in the presence or absence of overexpressed mPtch1, following treatment with control or ShhN conditioned medium, SAG (200 nM), 20(S)-OHC (10 µM), or 20(S)-OHC + 22(S)-OHC (5 µM each). Comparable Ptch1-mediated suppression was demonstrated by transfecting increased wt Smo cDNA to compensate for the higher ΔCRD Smo basal activity. Error bars represent standard deviations (n = 3 independent transfections per data point). See also Table S1.

Figure 3

The Smo CRD directly binds oxysterols. (A) Detergent-solubilized membranes from HEK293FT cells transfected with Myc-tagged wt Smo, ΔCRD, or ΔCT deletion mutants were incubated with control or 20(S)-yne affinity resin in the presence of 50 µM 20(S)-OHC competitor or vehicle, as indicated in the table. After washing, bound protein was eluted and analyzed by immunoblotting (“bound”). The ΔCRD and ΔCT mutants bound less efficiently than wt Smo to 20(S)-yne matrix; analysis of 3.3× equivalents of bound material nevertheless revealed specific binding for ΔCT, but not ΔCRD. (B) A similar experiment as in (A) with Myc-tagged wt mFz7 or a mFz7-mSmoNT chimera. (C) Experiments as above, but using concentrated conditioned medium (no added detergent) from 293S-GnT1− cells infected with BacMam virus encoding Protein C-tagged mSmoNT-3CS or mFz7 CRD constructs. In this and all subsequent blots the migration of molecular weight markers (in kDa) is indicated to the left. Results are representative of multiple independent experiments. See also Figure S2.

Figure 4

A conserved CRD lipid-binding interface in Smo and Fz CRDs. (A) Structure of a complex between XWnt8 and the mFz8 CRD as reported by Garcia and colleagues (Janda et al., 2012) (orange and blue, respectively; PDB code 4F0A) showing the Wnt lipid adduct (red) and the location of CRD site 1 (yellow) and site 2 (purple) residues. In the image at right, a magnified view of the Fz8 CRD site 1 is presented to highlight residues that contact the lipid. See sequence alignment in Fig. S3. (B) In vitro binding assay as in Fig. 2A and 2B with the indicated Myc-tagged Smo constructs transfected into HEK293FT cells. Site 1 and 2 mutants are indicated in yellow and purple to correspond to the structural model. Low and high exposures of the same input and bound fractions are also provided, and arrows above and below the 100-kDa marker indicate the positions of the immature ER-resident and fully glycosylated post-ER forms of wt Smo. Note that this assay sensitively detects specific 20(S)-OHC binding to low amounts of post-ER Smo, as seen for wt Smo diluted 33-fold in a control membrane extract and for the site 2 mutant Y89A; no specific binding, however is observed for the site 1 WGL mutant, which exhibits similar post-ER levels (see “input” panel). (C) Effect of the site 1 WGL mutation on oxysterol binding to Myc-tagged mFz7-mSmoNT chimera. See also Figure S3.

Figure 5

Naturally occurring oxysterols modulate Smo activity via CRD engagement. (A) Schematic diagram of sterol biosynthetic pathways leading to the formation of 7-keto-25-OHC and 7-keto-27-OHC (in red). Enzyme names are indicated in black italics; solid and dashed arrows represent experimentally verified or proposed biosynthetic steps, respectively. Abbreviations: cholesterol 7α-hydroxylase (CYP7A1), sterol 27-hydroxylase (CYP27A1), cholesterol 25-hydroxylase (CH25H). For comparison, the structure of the non-cellular 20(S)-OHC is shown in blue. (B) Gli-luciferase activity in Hh pathway-responsive NIH3T3 Shh-LIGHT2 cells was measured following treatment with the indicated sterols either alone (blue) or in combination with ShhN conditioned medium (green). (C) Concentration-response analysis of Gli-luciferase activity in Shh-LIGHT2 cells treated with 7-keto-27-OHC alone (red), in combination with 22(S)-OHC (5 µM, blue), or with a threshold concentration of SAG (1 nM, black). Statistical significance (Student’s t-test): p<0.01 (*); p<0.001 (**). (D) Ciliary accumulation of endogenous Smo in NIH3T3 cells treated with control or ShhN conditioned medium, or 7-keto-27-OHC; the inactive oxysterol 7α-27-OH-3-one serves as a negative control. Fixed cells were analyzed by immunofluorescence using antibodies against Smo (green) or acetylated tubulin (red) with DAPI counterstain (blue). Shifted overlays of Smo and acetylated tubulin stains are displayed as small panels to the right of selected images. See Fig. S4F for image quantification. (E) Binding of wt Smo to immobilized 20(S)-yne was tested as in Fig. 2A, using the indicated sterols as competitors (all at 50 µM except 20(S)-yne, at 100 µM). Sterols newly identified in this study as Smo modulators are shown in red, while inactive analogs are shown in black. For comparison, 20(S)-OHC and related derivatives are shown in blue. (F) Gliluciferase assay in Smo^−/− MEFs transfected with the indicated Smo and Ptch1 constructs and stimulated with control (red) or ShhN conditioned medium (green), 100 nM SAG (black), or 5 µM 7-keto-27-OHC (blue). Error bars for A, B, C, and F represent standard deviations (n = 3 independent culture wells or transfections per data point). See also Figure S4 and Tables S1–S2.

Figure 6

The Smo activation state is regulated by multiple distinct sterol effects. (A) NIH3T3 cells were transfected with Gli-luciferase reporter plasmids and stimulated with ShhN conditioned medium in combination with the indicated exogenous sterols (at 100 µM) following endogenous sterol depletion (with methyl-β-CD plus lovastatin as described in the methods section). (B) Ciliary accumulation of endogenous Smo in ShhN-stimulated NIH3T3 cells following depletion of endogenous sterols (as described in panel A) and subsequent rescue with exogenous cholesterol. See Fig. S5 for image quantification. (C) In Smo^−/− MEFs transfected with the indicated Smo constructs, Gli-luciferase activity induced by ShhN stimulation was measured under mock-treated (red) or sterol-depleted (blue) conditions as in (A). (D) In NIH3T3 cells, endogenous sterol depletion (“depleted”) reduced Gli-luciferase activity induced by treatment with ShhN conditioned medium, SAG (500 nM), or purmorphamine (2.5 µM), as compared to mock-depleted controls (“mock”). Error bars in all luciferase assays represent standard deviations (n = 3 independent transfections per data point). See also Figure S5 and Table S1.

Figure 7

Summary model indicating multiple sterol modulatory effects in Hh signal transduction at the level of mSmo as well as their binding determinants. Oxysterols (blue) activate Smo via CRD engagement (hypothetical structural model of human Smo CRD, with mutations affecting oxysterol binding indicated in blue), while cyclopamine and related anticancer drugs (orange) bind to a pocket in the extracellular half of the heptahelical domain. Mutations that affect binding and/or signaling inhibition by cyclopamine and its mimics GDC-0449 and NVP LDE-225 are displayed in orange; cholesterol (purple) may bind directly to an allosteric site facing the membrane, as has been shown for other GPCRs. See also Figure S6.