Repression of Smoothened by Patched-Dependent (Pro-)Vitamin D3 Secretion (original) (raw)
Figure 3
Ptch1 Inhibits Smo Intercellularly
(A) Detaching and mixing of two populations of fluorescently labelled cells resulted in a homogenous mixture with intimate cell–cell contacts. The procedure is summarized in the left panel. Original magnification is 400×. Intercellular capacity of Ptch1 to inhibit Smo was assessed by mixing reporter cells overexpressing Smo and a Gli reporter with Ptch1-overexpressing donor cells. The observed decrease in Gli reporter activity could not be inhibited by the addition of 1 μg/ml 5E1 Shh-blocking antibody. Exogenously added Shh (1 μg/ml) was capable of abolishing the Ptch1-mediated inhibition. Transfecting donor cells with Ptch1 siRNA reversed the inhibitory effect (blue bars). MDA-MB-231 breast carcinoma cells (hatched bars) showed a response similar to the C3H/10T1/2 fibroblasts'. Donor cells were transfected, washed (16 h post transfection), mixed with reporter cells, and lysed after 6 to 8 h. Shown are RLU values corrected for an internal CMV Renilla luciferase control, expressed as percent differences relative to control-transfected donor cells (vector DNA or scrambled control siRNA). ( n ≥ 4; *, p < 0.05; ***, p < 0.005). Depicted is the mean ± SEM.
(B) Fusion of reporter cells with Ptch1 overexpressing donor cells showed a decrease in Gli reporter activity comparable in magnitude to the inhibition observed in mixed cells. Right panel: treating fluorescently labelled cells with PEG1500 4 h after mixing resulted in multiple nuclei per cell and composite cell colours, indicating successful cell fusion. Magnification is 1,000×. Statistics and incubation times are as in (A).
(C) To assess the specificity of Ptch1 acting on Smo, reporter cells expressing the Ptch1-insensitive mutant SmoM2 (as indicated on the _y_-axis) were mixed with donor cells. The loss of inhibition indicates the specificity of Ptch1 acting through Smo in the mix-and-match setup. Statistics and incubation times are as in (A).
(D) Shown is a model for the proposed intercellular action of Ptch1 inhibition following the results obtained in the mix-and-match experiments.