CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

Abstract

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can lead to congenital malformations and diseases including cancer. Although cholesterol and several oxysterol lipids have been shown to play crucial roles in HH activation, the molecular mechanisms governing their regulation remain unresolved. Here, we identify Canopy4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that modulates levels of membrane sterol lipids. Cnpy4^-/- embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway in vitro and elevates membrane levels of accessible sterol lipids, such as cholesterol, an endogenous ligand involved in HH activation. Our data demonstrate that CNPY4 is a negative regulator that fine-tunes HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

Fig. 1. Developmental defects in Cnpy4^–/– hindlimbs.

a Dorsal view of control and Cnpy4 mutant hindlimbs at embryonic day (E)18.5 (top row) and E14.5 (middle row). The majority of Cnpy4 mutant hindlimbs exhibit either an extra digit anteriorly (red asterisk) or a transformation of digit 1 from biphalangeal to triphalangeal (yellow asterisk). Whole mount in situ hybridization for Sox9 at E12.5 (bottom row) indicates an extra digit and an enlarged digit 1 primordium. The top table summarizes the phenotype frequency in mutant hindlimbs; less frequent phenotypes are shown in Supplementary Fig. 1a, b. The scale bars represent 500 μm. b Single molecule in situ hybridization (RNAscope) of Cnpy4 in the hindlimbs of control and Cnpy4 mutant embryos at E12.5. The scale bars represent 200 μm (top) and 40 μm (bottom). c Protein levels in lysates of MEF cells from control and mutant embryos were detected using the indicated antibodies by Western blot analysis. All experiments were performed three independent times with similar results.

Fig. 2. Absence of Cnpy4 leads to hyperactivation of HH-related gene expression and signaling.

a Shh in situ hybridization and lacZ expression of Cnpy4;Gli1^lacZ in hindlimb buds at embryonic day (E)10.5, E11.5, and E12.5. Samples at E11.5 show an enlarged Shh domain (lines) and samples at E12.5 have ectopic expression of both Shh and Gli1 (arrowheads) in Cnpy4 mutants. The scale bars represent 500 μm. b, c Luciferase reporter assay in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA and stimulated with SMO agonist (SAG), recombinant SHH (b), 20(S)-hydroxycholesterol (20(S)-HC), or 24(S), 25-exposycholesterol (24(S), 25-EC) (c). Data were normalized to the average value of control siRNA-treated cells stimulated with DMSO or vehicle. Data represent the mean ± SD (n = 9 from three biological and three technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ***p < 0.001 (p_siCnpy4+SHH = 0.0004), ****p < 0.0001. d, e qRT-PCR assessment of Gli1 expression in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA and stimulated with SAG, recombinant SHH (d), 20(S)-HC or 24(S), 25-EC (e). Data represent the mean ± SD (n = 12 from three biological and four technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ****p < 0.0001. All experiments were performed a minimum of three independent times with similar results.

Fig. 3. CNPY4 intersects the HH pathway at the level of SMO.

a Immunofluorescence-based staining of primary cilia (acetylated tubulin, red), SMO (SMO, green), and nuclei (DAPI, blue) in ciliated NIH3T3 cells treated with control or Cnpy4 siRNA. The scale bar represents 10 μm. Cilia scale bar represents 1 μm. b Quantification of the percentage of ciliated NIH3T3 cells, as assessed by acetylated tubulin immunofluorescence. Data represent the mean ± SEM (n = 152 siCtrl cells and n = 110 siCnpy4 cells from three biological replicates). Significance was calculated using a two-sided unpaired Welch’s t-test with ns p > 0.05 (p = 0.5286). c Quantification of the length of cilia in NIH3T3 cells. Measurements were made in FIJI using the acetylated tubulin channel. Data represent the mean ± SEM (n = 77 cells from three biological replicates). Significance was calculated using a two-sided unpaired Welch’s t-test with ***p < 0.001 (p = 0.0002). d–g Luciferase reporter assay in ciliated Ptch1 (d), Sufu (e), or Smo (f, g) null MEFs treated with control (gray bars) or Cnpy4 (blue bars) siRNA. Smo null MEFs were stimulated with either SAG (f) or SHH (g). h, i Luciferase reporter assay in ciliated NIH3T3 cells treated with control (gray bars) or Cnpy4 (blue bars) siRNA stimulated with SAG (h) or SHH (i) in the presence of SMO antagonist (SANT-1). Data for d–i were normalized to the average value of control siRNA treated cells stimulated with DMSO where applicable. Data represent the mean ± SD (n = 9 from three biological and three technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ns p > 0.05 (p_{siCtrl+SAG+SANT1} = 0.0503), **p < 0.005 (p_{siCnpy4+SHH+SANT1} = 0.0040), ****p < 0.0001. Experiments were performed a minimum of three independent times with similar results.

Fig. 4. CNPY4 modulates levels of accessible cholesterol.

a Immunofluorescence-based staining of accessible cholesterol (PFO*-AF647, red) and nuclei (DAPI, blue) in NIH3T3 cells treated with control or Cnpy4 siRNA. Boxed areas are magnified on the right. The scale bar represents 10 μm. b, c FACS analysis of NIH3T3 cells treated with control or Cnpy4 siRNA (b) or control and Cnpy4^–/– MEFs (c) stained with PFO*-AF647 for accessible cholesterol. Data were normalized to the average value of control siRNA treated or control cells. Data represent the distribution with the median (solid line) and the first and third quartiles (dashed lines) indicated (n = 244,550 siCtrl NIH3T3 cells, 291,375 siCnpy4 NIH3T3 cells, 92,183 control MEF cells, and 74,848 for Cnpy4^–/– MEF cells from two independent experiments with two biological replicates each). Significance was calculated using a two-sided unpaired Welch’s t-test with ****p < 0.0001. d, e Luciferase reporter assay in ciliated NIH3T3 cells, incubated with methyl-β-cyclodextrin (MCβD) and lovastatin prior to treatment with control (gray bars) or Cnpy4 (blue bars) siRNA treatment and stimulation with SMO agonist (SAG) (d) or recombinant SHH (e). Data were normalized to the average value of control siRNA treated cells stimulated with vehicle. Data represent the mean ± SD (n = 9 from three biological and three technical replicates). Significance was calculated using a two-sided Mann–Whitney non-parametric test with ns p > 0.05 (p_{siCtrl+SAGvs.siCnpy4+SAG} = 0.7984; p_{siCtrl+SAGvs.siCnpy4+SHH} = 0.9069), **p < 0.005 (p_siCnpy4+SHH = 0.0022), ***p < 0.001 (p_siCnpy4+SAG = 0.0002). Experiments were performed three independent times with similar results.

Fig. 5. CNPY4 is an ER-resident protein that elevates membrane levels of accessible cholesterol.

a Immunofluorescence-based staining of CNPY4 (Flag, green), organelle markers (indicated antibody, red), and nuclei (DAPI, blue) in COS-7 cells transiently transfected with Flag-tagged human CNPY4. The scale bar represents 10 μm. b Immunofluorescence-based staining of primary cilia (ARL13B-GFP, green), accessible cholesterol (PFO*-AF647, red), and nuclei (DAPI, blue) in ciliated NIH3T3 cells stably expressing ARL13B-GFP, treated with control or Cnpy4 siRNA. The scale bar represents 10 μm. Cilia scale bar represents 1 μm. c Quantification of PFO* fluorescence intensity at the cilia and in the rest of the cell as described in the “Methods” section. Data represent the mean ± SEM (n = 104 siCtrl cells and n = 116 for siCnpy4 cells from three independent experiments). d Schematic illustrating proposed model of CNPY4 modulation of HH activation. CNPY4, an ER-resident protein, regulates the ability of unbound sterols synthesized in the ER to be expressed at cell membrane, thus controlling the activation of SMO.