Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process

Abstract

The Hedgehog (Hh) signaling pathway controls growth, cell fate decisions, and morphogenesis during development. Damage to Hh transduction machinery can lead to birth defects and cancer. The transmembrane protein Smoothened (Smo) relays the Hh signal and is an important drug target in cancer. Smo enrichment in primary cilia is thought to drive activation of target genes. Using small-molecule agonists and antagonists to dissect Smo function, we find that Smo enrichment in cilia is not sufficient for signaling and a distinct second step is required for full activation. This 2-step mechanism--localization followed by activation--has direct implications for the design and use of anticancer therapeutics targeted against Smo.

Fig. 1.

Smo localization in cilia is not sufficient to activate Hh target genes. Smo-GFP (with or without Shh) and SmoM2-GFP transfected into NIH 3T3 cells accumulate in cilia to similar degrees (A); however, SmoM2-GFP is a substantially better inducer of a Gli-responsive transcriptional reporter (B). (A) Single-plane confocal images of NIH 3T3 cells transfected with the indicated constructs show the ciliary marker acetylated tubulin (red) detected by immunofluorescence, Smo-GFP (green) detected by GFP fluorescence, and nuclei (blue) detected by DAPI staining. All 3 channels are shown as a single overlay, with the green (Smo) layer shifted relative to the other channels (shifted overlay) for easier visualization. (B) NIH 3T3 cells were cotransfected with a Gli-luciferase transcriptional reporter and either an empty vector control or the same Smo constructs as used in (A). The mean (± SEM) luciferase reporter activity was measured after treatment with or without Shh for 24 h.

Fig. 2.

Smo inhibitors have different effects on Smo accumulation in primary cilia. SANTs 1 and 2 inhibit Shh-induced Smo enrichment in primary cilia (A and B) in a dose-dependant manner, but cyclopamine has no effect (A and C), even though all 3 inhibit Shh-induced Ptc1 protein accumulation (D). NIH 3T3 cells were treated with Shh for 7 h in the presence or absence of SANT1, SANT2, or cyclopamine and then stained with DAPI to show nuclei (blue) and antibodies against acetylated tubulin (red) and Smo (green) (A–C) or lysed for immunoblotting with antibodies against Ptc1 or Smo (D). Panel (A) shows confocal images (shifted overlays) of cells treated with the indicated agents. Panels (B and C) show graphs of the mean (± SEM) intensity of Smo fluorescence at cilia in the presence of Shh and the indicated concentrations of SANTs 1 and 2 (B) or cyclopamine (C). Panel (D), with sets of bars aligned below the corresponding Smo band, shows quantitation of the total Smo signal, the top Smo signal, and the bottom Smo signal from the anti-Smo immunoblot.

Fig. 3.

Constitutive Smo accumulation in the cilia of Ptc1^−/− cells can be reversed with SANTs1 and 2, but not with cyclopamine. MEFs from Ptc1^−/− mice were treated with SANT1, SANT2, or cyclopamine for 4 h and then stained with DAPI to show nuclei (blue), anti-Smo (green), or anti-acetylated tubulin (red). (A) Shifted overlays of confocal images of Ptc1^−/− cells treated as indicated. (B) The mean (± SEM) Smo fluorescence at cilia under the indicated conditions, calculated based on images of the type shown in (A).

Fig. 4.

Cyclopamine can induce and SANT1 can block the accumulation of Smo in primary cilia. (A) Confocal images (shifted overlays) of NIH 3T3 cells treated for 7 h with the indicated combinations of SANT1 (100 nM), cyclopamine (5 μM), or SAG (100 nM). (B) The mean Smo fluorescence at cilia increases with increasing doses of cyclopamine from 2 different sources (#1 and #2), but not with the inactive alkaloid tomatidine. (C) Cyclopamine concentrations required to induce Smo in cilia of untreated NIH 3T3 cells (black circles) are similar to those required to inhibit the Shh-stimulated Gli-luciferase reporter (red squares) in ShhL2 cells. (D) Confocal images (shifted overlay) of cilia (arrows) from live NIH 3T3 cells transfected with tdTomato-tagged Smo (red) and treated for 50 min with BODIPY-cyclopamine (green) alone (Top) or in combination with an 100-fold excess of unlabeled cyclopamine (Bottom). (E and F) SANT1 can inhibit the accumulation of Smo in cilia of NIH 3T3 cells induced by either 5 μM cyclopamine (E) or 100 nM SAG (F). All points in the graphs show mean (± SEM) values.

Fig. 5.

Full activation of Smo requires ciliary transport coupled to a second activation step. Full activation of cytoplasmic Smo (Smo1) first requires its transport to cilia (Smo2), followed by a second, rate-controlling activation step in cilia that converts Smo2 to Smo3, the state capable of engaging downstream signaling machinery that ultimately leads to activation of the Gli proteins. Solid black arrows denote the individual steps. Transport to cilia (Smo1→Smo2) is controlled by entry and exit steps, designated R1 and R2; the activation step (Smo2→Smo3) is controlled by R3; and the exit of Smo3 from cilia is controlled by R4. The proposed site of action of various regulators of Hh signaling regulators is denoted above the kinetic scheme by light gray arrows.