Figure 6.

Arachidonic acid binds Smo and synergizes with 20(S)-OHC. For all panels, * indicates p ≤ 0.05; *** indicates p ≤ 0.0001; ns indicates p > 0.05. Error bars indicate SEM. A–A’. Light2 cells were treated with increasing concentrations of arachidonic acid. Normalized baseline reporter activity is shown relative to the vehicle-treated ShhN-stimulated level of reporter activity, set to 100%, shown in A’. A’. Light2 reporter cells were stimulated with ShhN conditioned media, 20(S)-OHC (10 µM) or SAG (100 nM) plus increasing arachidonic acid. Enhancement is shown relative to the vehicle-treated, Smo agonist-stimulated level of reporter activity set to 100% for each agonist. Significance was determined using a two-way ANOVA. B. Light2 cells were treated with 20(S)-OHC (10 µM) +/−MAFP (5 µM). MAFP-treated cells were treated with arachidonic acid, as indicated. The line indicates the baseline 20(S)-OHC stimulated level, set to 100%. Significance was determined relative to this control using a one-way ANOVA. C. Arachidonic acid was coupled to agarose beads using click chemistry and beads were incubated with lysate from SmoYFP-expressing cells in the presence of 20(S)-OHC or vehicle. Binding was competed with cold arachidonic acid. The endocannabinoid N-stearoyldopamine (SD) failed to compete Smo from arachidonic acid beads. The experiment was performed two times. A representative blot is shown. D–D’. Light2 cells were stimulated with 20(S)-OHC (10 µM) and treated with increasing concentrations of cyclopamine and arachidonic acid. D’. X marks show the calculated IC₅₀ for cyclopamine at each arachidonic acid concentration tested. Each X represents one independent experiment done in triplicate. For all reporter assays, experiments were repeated two or three times in triplicate and all data pooled. E. Binding of fluorescent BODIPY-cyclopamine to Smo was monitored by flow cytometry. The experiment was performed three times. A representative experiment is shown. F. A model for cPLA2α modulation of Smo signaling. Initiation of Smo signaling by a CRD oxysterol agonist activates cPLA2α, resulting in lysophospholipid (green) and arachidonic acid (orange) production. Arachidonic acid is proposed to target the TM domain of active Smo to enhance ciliary translocation and bolster signal output. Smo activated by the 7TM agonist SAG induces PLA2, but does not require PLA2-generated lipids for optimal ciliary translocation and high-level signaling.